Michael Fromm

Abstract: Abstract Prerequisite of epithelial transport is a paracellular barrier function, which seals the tissue against an uncontrolled leak flux. Moreover, a selective paracellular permeability has been shown to be crucial for physiological epithelial transport function. Claudins are tetraspan tight junction proteins which play a major role for paracellular ion permeability across epithelia. The multigene family consists of 24 members and several splice variants which show distinct tissue-specific expression profiles. Moreover in diseases associated with a loss of barrier function such as forms of inflammatory bowel disease, expression of claudins is altered. Functional characterization of single claudins revealed specific contribution to barrier properties in epithelia. This review gives an overview on the exploration of molecular structure and barrier function along intestine and nephron, which share mechanisms of selective restriction of the paracellular pathway, but also exhibit distinct organ-specific characteristics.

Abstract: In tubular epithelia, barrier function varies in a segment-specific way. The aim of this study was to correlate the presence of tight junction proteins and paracellular barrier properties along rat intestine. Tissue segments of duodenum, jejunum, ileum, and colon were stripped of submucosal cell layers and mounted in Ussing chambers for impedance spectroscopy to measure epithelial resistance (R (epi)). In parallel, expression of tight junction proteins was analysed by Western blots and immune fluorescence confocal microscopy. Colon showed highest R (epi), followed by duodenum, jejunum, and ileum. In small intestine, common transepithelial resistance (R (trans) or TER) overestimated true R (epi) by approximately 60%. In colon, strongest expression of "tightening" claudins 1, 3, 4, 5, and 8 was detected. In accordance with R (epi) the most proximal of the small intestinal segments, duodenum exhibited highest expression of "tightening" claudins and lowest expression of claudins mediating permeability, namely claudin-2, -7, and -12, compared to jejunum and ileum. These results correspond to the specific role of the duodenum as the first segment facing the acidic gastric content.

Abstract: Sealing of the paracellular cleft by tight junctions is of central importance for epithelia and endothelia to function as efficient barriers between the extracellular space and the inner milieu. Occludin and claudins represent the major tight junction components involved in establishing this barrier function. A special situation emerges at sites where three cells join together. Tricellulin, a recently identified tetraspan protein concentrated at tricellular contacts, was reported to organize tricellular as well as bicellular tight junctions. Here we show that in MDCK cells, the tricellulin C-terminus is important for the basolateral translocation of tricellulin, whereas the N-terminal domain appears to be involved in directing tricellulin to tricellular contacts. In this respect, identification of homomeric tricellulin-tricellulin and of heteromeric tricellulin-occludin complexes extends a previously published model and suggests that tricellulin and occludin are transported together to the edges of elongating bicellular junctions and get separated when tricellular contacts are formed.

Abstract: Whether or not significant amounts of water pass the tight junction (TJ) of leaky epithelia is still unresolved, because it is difficult to separate transcellular water flux from TJ-controlled paracellular water flux. Using an approach without differentiating technically between the transcellular and paracellular route, we measured transepithelial water flux with and without selective molecular perturbation of the TJ to unequivocally attribute changes to the paracellular pathway. To this end, MDCK C7 cells were stably transfected with either claudin-2 or claudin-10b, two paracellular cation-channel-forming TJ proteins that are not endogenously expressed in this cell line. Claudin-2 is typical of leaky, water-transporting epithelia, such as the kidney proximal tubule, whereas claudin-10b is present in numerous epithelia, including water-impermeable segments of the loop of Henle. Neither transfection altered the expression of endogenous claudins or aquaporins. Water flux was induced by an osmotic gradient, a Na(+) gradient or both. Under all conditions, water flux in claudin-2-transfected cells was elevated compared with vector controls, indicating claudin-2-mediated paracellular water permeability. Na(+)-driven water transport in the absence of an osmotic gradient indicates a single-file mechanism. By contrast, claudin-10b transfection did not alter water flux. We conclude that claudin-2, but not claudin-10b, forms a paracellular water channel and thus mediates paracellular water transport in leaky epithelia.

Abstract: Abstract Aim: Intestinal pressure differences or experimental distension induce ion secretion via the enteric nervous system, the sensorial origin of which is only poorly understood. This study aimed to investigate sensorial inputs and the role of afferent and interneurones in mechanically activated submucosal secretory reflex circuits. Methods: Distension-induced rheogenic chloride secretion was measured as increase in short-circuit current 10 min after distension (DeltaI(SC)(10); distension parameters +/- 100 muL, 2 Hz, 20 s) in partially stripped rat distal colon in the Ussing-chamber in vitro. PGE(2) and PGI(2) were measured by radioimmunoassay. Results: DeltaI(SC)(10) was 2.0 +/- 0.2 mumol h(-1) cm(-2) and could be attenuated by lobeline, mecamylamine and dimethylphenylpiperazine, indicating an influence of nicotinergic interneurones. Additionally, a contribution of afferent neurones was indicated from the short-term potentiation of DeltaI(SC)(10) by capsaicin (1 mum). As evidence for its initial event, indomethacin (1 mum) inhibited distension-induced secretion and the release of PGI(2) was directly detected after distension. Furthermore, serotoninergic mediation was confirmed by granisetron (100 mum) which was functionally localized distally to PGI(2) in this reflex circuit, as granisetron inhibited an iloprost-induced I(SC), while indomethacin did not affect serotonin-activated ion secretion. Conclusions: Distension-induced active electrogenic chloride secretion in rat colon is mediated by a neuronal reflex circuit which includes afferent neurones and nicotinergic interneurones. It is initiated by distension-induced PGI(2) release from subepithelial cells triggering this reflex via serotoninergic 5-HT(3) receptor transmission. Functionally, this mechanism may help to protect against intestinal stasis but could also contribute to luminal fluid loss, e.g. during intestinal obstruction.

Abstract: The KCNE3 beta-subunit constitutively opens outwardly rectifying KCNQ1 (Kv7.1) K(+) channels by abolishing their voltage-dependent gating. The resulting KCNQ1/KCNE3 heteromers display enhanced sensitivity to K(+) channel inhibitors like chromanol 293B. KCNE3 was also suggested to modify biophysical properties of several other K(+) channels, and a mutation in KCNE3 was proposed to underlie forms of human periodic paralysis. To investigate physiological roles of KCNE3, we now disrupted its gene in mice. kcne3(-/-) mice were viable and fertile and displayed neither periodic paralysis nor other obvious skeletal muscle abnormalities. KCNQ1/KCNE3 heteromers are present in basolateral membranes of intestinal and tracheal epithelial cells where they might facilitate transepithelial Cl(-) secretion through basolateral recycling of K(+) ions and by increasing the electrochemical driving force for apical Cl(-) exit. Indeed, cAMP-stimulated electrogenic Cl(-) secretion across tracheal and intestinal epithelia was drastically reduced in kcne3(-/-) mice. Because the abundance and subcellular localization of KCNQ1 was unchanged in kcne3(-/-) mice, the modification of biophysical properties of KCNQ1 by KCNE3 is essential for its role in intestinal and tracheal transport. Further, these results suggest KCNE3 as a potential modifier gene in cystic fibrosis.

Abstract: BACKGROUND & AIMS: The epithelial sodium channel (ENaC) mediates electrogenic sodium absorption in distal colon. In patients with inflammatory bowel disease (IBD), ENaC induction is impaired, mainly through transcriptional suppression by proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha. Glucocorticoid therapy rapidly increases sodium absorption; we investigated the molecular mechanisms underlying the interaction among TNF-alpha, glucocorticoids, and ENaC induction. METHODS: ENaC-mediated sodium transport in glucocorticoid receptor (GR)-expressing HT-29/B6 cells and rat distal colon, under the influence of the synthetic glucocorticoid dexamethasone and TNF-alpha, was quantified in Ussing chambers. ENaC messenger RNA (mRNA) levels were monitored by real-time polymerase chain reaction. GR transactivation and expression were investigated by gene reporter, immunoblot, and confocal immunofluorescence microscopy analyses. The GR mRNA half-life was determined. Signaling pathways were characterized using mitogen-activated protein kinase inhibitors. RESULTS: Dexamethasone not only prevented TNF-alpha-mediated ENaC suppression but caused synergistic induction of ENaC-dependent sodium absorption in HT-29/B6-GR cells and rat distal colon. This synergy resulted from TNF-alpha-mediated increases in GR protein levels because of GR mRNA stabilization and subsequent GR transactivation by dexamethasone. As a consequence, transcription of the ENaC beta- and gamma-subunits was up-regulated, increasing ENaC-dependent sodium absorption. p38 Mitogen-activated protein kinase is required for this synergistic effect: p38 inhibition blocked the increase in GR protein expression and ENaC-dependent sodium absorption. CONCLUSIONS: TNF-alpha and dexamethasone induce ENaC, explaining the rapid and intense proabsorptive effect of glucocorticoid therapies.

Abstract: Paracellular ion transport in epithelia is mediated by pores formed by members of the claudin family. The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood. By expressing a high-conductance claudin isoform, claudin-2, in high-resistance Madin-Darby canine kidney cells under the control of an inducible promoter, we were able to quantitate claudin pore permeability. Claudin-2 pores were found to be narrow, fluid filled, and cation selective. Charge selectivity was mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.

Abstract: Our aim has been to characterize the molecular mechanisms regulating the expression of the channel-forming tight-junctional protein claudin-2 in response to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha), which is elevated, for example, in active Crohn's disease. TNFalpha caused an 89% decrease of the paracellular resistance in colonic HT-29/B6 cells, whereas transcellular resistance was unaltered. The claudin-2 protein level was increased by TNFalpha without changes in subcellular tight-junctional protein localization as revealed by confocal laser scanning microscopy. Enhanced gene expression was identified as the source of this increase, since claudin-2-specific mRNA and promoter activity was elevated, whereas mRNA stability remained unaltered. Specific inhibitors and phospho-specific antibodies revealed that the increased gene expression of claudin-2 after TNFalpha treatment was mediated by the phosphatidylinositol-3-kinase pathway. Thus, the up-regulation of claudin-2 by TNFalpha is attributable to the regulation of the expression of the gene, as a result of which epithelial barrier function is disturbed, for example, during chronic intestinal inflammation.

Abstract: BACKGROUND: Norovirus infection is the most frequent cause of infectious diarrhoea in the western world. This study aimed to characterise functionally and histomorphologically the diseased duodenum in human biopsies. METHODS: Norovirus infection was diagnosed by the Kaplan criteria and confirmed by PCR of stool samples. Duodenal biopsies were obtained endoscopically. In miniaturised Ussing chambers, short circuit current, flux measurements and impedance spectroscopy were performed. Histological analysis including apoptosis staining and characterisation of intraepithelial lymphocytes was performed. Tight junction proteins were quantified by immunoblotting. RESULTS: In norovirus infection, epithelial resistance decreased from (mean (SEM)) 24 (2) Omega cm(2) in controls to 10 (1) Omega cm(2). Mannitol flux increased from 113 (24) nmol h(-1) cm(-2) in controls to 242 (29) nmol h(-1) cm(-2). Microdissection revealed a villus surface area reduced by 47% (6.6%). Intraepithelial lymphocytes were increased to 63 (7) per 100 enterocytes, with an increased rate of perforin-positive cytotoxic T cells. Expression of tight junctional proteins occludin, claudin-4 and claudin-5 was reduced. The epithelial apoptotic ratio was doubled in norovirus infection. Furthermore, the basal short circuit current was increased in norovirus infection and could be reduced by bumetanide and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). CONCLUSIONS: Norovirus infection leads to epithelial barrier dysfunction paralleled by a reduction of sealing tight junctional proteins and an increase in epithelial apoptosis, which may partly be mediated by increased cytotoxic intraepithelial lymphocytes. Furthermore, active anion secretion is markedly stimulated. Thus, the diarrhoea in norovirus infection is driven by both a leak flux and a secretory component.

Abstract: In the distal colon, the epithelial sodium channel (ENaC) is rate limiting for sodium absorption. Progress in the molecular characterization of ENaC expression and trafficking in response to the mineralocorticoid aldosterone has been hampered, since no epithelial colonic cell line existed expressing functional ENaC stimulated by nanomolar aldosterone via mineralocorticoid receptor (MR). Here, we present a human colonic epithelial cell line inducibly expressing the MR (HT-29/B6-Tet-On-MR) which exhibits aldosterone-dependent ENaC-mediated sodium transport in the presence of the short-chain fatty acid butyrate. Butyrate was necessary for high-level expression of MR which allowed for aldosterone-dependent upregulation of beta- and gamma-ENaC expression. As butyrate alone was not capable of promoting ENaC-mediated sodium transport, aldosterone-induced GILZ (glucocorticoid-induced leucine zipper protein) was identified as a candidate factor increasing apical ENaC levels.

Abstract: Objective. In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal transport and barrier function. The mechanisms of cytokine action have usually been studied in cell culture models only; therefore the aim of this study was to establish an in vitro model based on native intestine to analyze distinct cytokine effects on barrier function, mucosal structure, and inherent regulatory mechanisms. Material and methods. Rat colon was exposed to tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) in Ussing chambers. Transepithelial resistance (R(t)) and (3)H-mannitol fluxes were measured for characterization of the paracellular pathway. Transcellular transport was analyzed by horseradish peroxidase (HRP) flux measurements. Expression and distribution of tight junction proteins were characterized in immunoblots and by means of confocal laser-scanning microscopy (LSM). Results. Colonic viability could be preserved for 20 h in a specialized in vitro set-up. This was sufficient to alter mucosal architecture with crypt surface reduction. R(t) was decreased (101+/-10 versus 189+/-10 Omega.cm(2)) with a parallel increase in mannitol permeability after cytokine exposure. Tight junction proteins claudin-1, -5, -7, and occludin decreased (45+/-10%, 16+/-7%, 42+/-8%, and 42+/-13% of controls, respectively), while claudin-2 increased to 208+/-32%. Occludin and claudin-1 translocated from the plasma membrane to the cytoplasm. HRP flux increased from 0.73+/-0.09 to 8.55+/-2.92 pmol.h(-1).cm(-2). Conclusions. A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFalpha and IFNgamma alters mucosal morphology and impairs epithelial barrier function by up-regulation of the paracellular pore-former claudin-2 and down-regulation of the barrier-builders claudin-1, -5, and -7. These alterations resemble changes seen in IBD and thus underline their prominent role in IBD pathogenicity.

Abstract: Klaus Hierholzer (1929-2007) dissected various functions influenced by steroids in the distal tubule and showed that aldosterone in low doses reversed the sodium and potassium transport defect in adrenalectomized rats, through a rapid activation of Na+,K+-ATPase. Subsequent studies addressed the role of 11-beta-hydroxysteroid oxidoreductase (11-HSD) and showed that the undisturbed functioning of 11-HSD is a prerequisite for selective mineralocorticosteroid regulation of epithelial transport. Another set of original experiments showed that 11-HSD was equally important in the distal colon, thus establishing that the large intestine acts in parallel with the distal nephron. Hierholzer, born in Konstanz on June 8, 1929, was laureated in medicine on May 25, 1954. Subsequently he worked at the Department of Pharmacology of the University of Freiburg, Cornell University with J. F. Pitts, the Department of Medicine of the University of Frankfurt-am-Main, the University of Copenhagen with H. H. Ussing, and the Institute of Physiology of the Freie Universitaet in Berlin where he became full professor and head of the Institute of Clinical Physiology in 1968. He held that position until 1998. He died in Allensbach in the family house on February 27, 2007. Hierholzer was a member of the Naturforscher Leopoldina Academy and of many other scientific societies, including the Academy of Science and Technology in Berlin, and received various awards including an honorary professorship at the University of Naples, the Bezold Medal, the Volhard Medal, the Schoeller/Junkman Award, and the Malpighi Medal (in memoriam). He published nearly 300 papers including various seminal books. Noteworthy also are his papers on the history of physiology of the kidney and acid-base balance. A total of 26 scientists who trained in his laboratory became professors.

Abstract: Listeria monocytogenes is a food-borne pathogen, which is able to induce diarrhea when residing in the intestine. We studied the effect of listeriolysin O (LLO), an extracellular virulence factor of L. monocytogenes, on intestinal transport and barrier function in monolayers of HT-29/B6 human colon cells using the Ussing technique to understand the pathomechanisms involved. Mucosal addition of LLO, but not a LLO mutant, induced a dose- and pH-dependent increase in short-circuit current (I(SC)). Sodium and chloride tracer flux and DIDS sensitivity studies revealed that I(SC) was mainly due to electrogenic chloride secretion. Barrier function was impaired by LLO, as assessed by transepithelial resistance (R(t)) and mannitol flux measurements. Intracellular signal transduction occurred through Ca(2+) release from intracellular stores and PKC activation. In conclusion, listeriolysin induces chloride secretion and perturbs epithelial barrier function, thus potentially contributing to Listeria-induced diarrhea.

Abstract: Solutes are transported across epithelial cell layers via transcellular and paracellular pathways. The transcellular pathway leads across the apical and basolateral cell membrane, whereas the paracellular pathway is directed through the tight junction. Tight junction proteins (claudins, occludin, tricellulin) can not only form barriers but also paracellular channels that are--in concert with membrane channels and transporters--regulated in a wide range in health and disease states. Thus, it is desirable to determine para- and transcellular resistance (R(para), R(trans)) separately. This cannot be achieved by conventional transepithelial resistance (TER) measurements. We present an impedance spectroscopic approach that is optimized for differentiation between these two pathways. The method is based on a transepithelial impedance measurement in specialized Ussing chambers, combined with a Ca(2+)-dependent modulation of R(para) through EGTA and flux measurements of a nonradioactive paracellular marker, fluorescein. The prerequisites are a paracellular marker that varies in parallel to 1/R(para), an experimental regime that alters R(para) without affecting R(trans), and exact knowledge of the resistance of subepithelial components. The underlying prerequisites and the applicability as a routine method were verified on cell lines of different tightness including HT-29/B6 colon cells and Madin-Darby canine kidney tubule cells C7 and C11.

Abstract: BACKGROUND AND AIMS: Impairment of the gastrointestinal mucosal barrier contributes to progression of HIV infection. The purpose of this study was to investigate the effect of highly active antiretroviral therapy (HAART) on the HIV-induced intestinal barrier defect and to identify underlying mechanisms. METHODS: Epithelial barrier function was characterised by impedance spectroscopy and [(3)H]mannitol fluxes in duodenal biopsies from 11 untreated and 8 suppressively treated HIV-infected patients, and 9 HIV-seronegative controls. The villus/crypt ratio was determined microscopically. Epithelial apoptoses were analysed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) and caspase-3 staining. Tight junction protein expression was quantified by densitometric analysis of immunoblots. Mucosal cytokine production was determined by cytometric bead array. RESULTS: Only in untreated but not in treated HIV-infected patients, epithelial resistance was reduced (13 (1) vs 23 (2) ohm cm(2), p<0.01) and mannitol permeability was increased compared with HIV-negative controls (19 (3) vs 9 (1) nm/s, p<0.05). As structural correlates, epithelial apoptoses and expression of the pore-forming claudin-2 were increased while expression of the sealing claudin-1 was reduced in untreated compared with treated patients and HIV-negative controls. Furthermore, villous atrophy was evident and mucosal production of interleukin 2 (IL2), IL4 and tumour necrosis factor alpha (TNFalpha) was increased in untreated but not in treated HIV-infected patients. Incubation with IL2, IL4, TNFalpha and IL13 reduced the transepithelial resistance of rat jejunal mucosa. CONCLUSIONS: Suppressive HAART abrogates HIV-induced intestinal barrier defect and villous atrophy. The HIV-induced barrier defect is due to altered tight junction protein composition and elevated epithelial apoptoses. Mucosal cytokines are mediators of the HIV-induced mucosal barrier defect and villous atrophy.

Abstract: In distal colon, the limiting factor for Na(+) absorption is represented by the epithelial sodium channel (ENaC). During absorption, high transepithelial Na(+) gradients are observed. In human colon and in HT-29/B6-GR cells, we investigated whether Na(+) back-leakage is prevented by paracellular sealing. Tissues and cells were incubated with corticosteroids. Barrier properties were analyzed in electrophysiological experiments. Subsequently, analysis of ENaC and tight junction protein expression, localization, and regulation was performed. In colon, nanomolar aldosterone induced sodium absorption via ENaC. Concomitantly, paracellular (22)Na(+) permeability was reduced by half and claudin-8 within the tight junction complex was nearly doubled. Real-time PCR validated an increase of claudin-8 transcripts. Two-path impedance spectroscopy following ENaC induction in HT-29/B6-GR revealed a specific increase of paracellular resistance. These results represent an important physiological implication: Na(+) absorption is paralleled by claudin-8-mediated sealing of the paracellular barrier to prevent Na(+) back-leakage, supporting steep Na(+) gradients in distal colon.

Abstract: The epithelium in inflamed intestinal segments of patients with Crohn's disease is characterized by a reduction of tight junction strands, strand breaks, and alterations of tight junction protein content and composition. In ulcerative colitis, epithelial leaks appear early due to micro-erosions resulting from upregulated epithelial apoptosis and in addition to a prominent increase of claudin-2. Th1-cytokine effects by interferon-gamma in combination with TNFalpha are important for epithelial damage in Crohn's disease, while interleukin-13 (IL-13) is the key effector cytokine in ulcerative colitis stimulating apoptosis and upregulation of claudin-2 expression. Focal lesions caused by apoptotic epithelial cells contribute to barrier disturbance in IBD by their own conductivity and by confluence toward apoptotic foci or erosions. Another type of intestinal barrier defect can arise from alpha-hemolysin harboring E. coli strains among the physiological flora, which can gain pathologic relevance in combination with proinflammatory cytokines under inflammatory conditions. On the other hand, intestinal barrier impairment can also result from transcellular antigen translocation via an initial endocytotic uptake into early endosomes, and this is intensified by proinflammatory cytokines as interferon-gamma and may thus play a relevant role in the onset of IBD. Taken together, barrier defects contribute to diarrhea by a leak flux mechanism (e.g., in IBD) and can cause mucosal inflammation by luminal antigen uptake. Immune regulation of epithelial functions by cytokines may cause barrier dysfunction not only by tight junction impairments but also by apoptotic leaks, transcytotic mechanisms, and mucosal gross lesions.

Abstract: High-resolution analysis of epithelial barrier function adds substantial information to that provided by conventional transepithelial electrical resistance (TER) measurements. This chapter describes three high-resolution techniques. First, two variants of impedance spectroscopy are delineated. One-path impedance spectroscopy discriminates vertically between serial pathways, namely resistances of the epithelial cell layer and of subepithelial tissues. As a typical application, measurements on human sigmoid colon biopsies from patients suffering from Crohn's disease are reported. Two-path impedance spectroscopy allows to discriminate between trans- and paracellular resistance, and the general principle of this technique is outlined. Second, the conductance scanning technique is presented, which discriminates horizontally between optically distinct parallel pathways over a wide range of spatial resolutions. Using this technique, it was shown that occludin--in contrast to the then prevailing opinion--is not irreplaceable to barrier function. Third, three-dimensional confocal fluorescence imaging for depicting transepithelial transport processes is introduced. Using this method the transepithelial translocation of bacteria which generate focal leaks was discovered.

Abstract: The tight junction protein claudin-10 is known to exist in two isoforms, resulting from two alternative exons, 1a and 1b (Cldn10a, Cldn10b). Here, we identified and characterized another four claudin-10 splice variants in mouse and human. One (Cldn10a_v1) results from an alternative splice donor site, causing a deletion of the last 57 nucleotides of exon 1a. For each of these three variants one further splice variant was identified (Cldn10a_v2, Cldn10a_v3, Cldn10b_v1), lacking exon 4. When transfected into MDCK cells, Cldn10a, Cldn10a_v1 and Cldn10b were inserted into the tight junction, whereas isoforms of splice variants lacking exon 4 were retained in the endoplasmic reticulum. Cldn10a transfection into MDCK cells confirmed the previously described increase in paracellular anion permeability. Cldn10a_v1 transfection had no direct effect, but modulated Cldn10a-induced organic anion permeability. At variance with previous reports in MDCK-II cells, transfection of high-resistance MDCK-C7 cells with Cldn10b dramatically decreased transepithelial resistance, increased cation permeability, and changed monovalent cation selectivity from Eisenman sequence IV to X, indicating the presence of a high field-strength binding site that almost completely removes the hydration shell of the permeating cations. The extent of all these effects strongly depended on the endogenous claudins of the transfected cells.

Abstract: Tight junctions form the paracellular barrier for ions and uncharged solutes not only in "tight" but also in "leaky" epithelia. In the premolecular era of tight junction research, this was believed to be achieved in a perfect or less perfect way, depending mainly on the amount of horizontally oriented tight junction strands. During the past decade it emerged that tight junction molecules, such as claudin-1 and many others, strengthen the barrier, while a few claudins, such as claudin-2 or -10, weaken it. This report focuses on three claudins: one channel former and two barrier builders. Claudin-2 represents the prototype of a paracellular, channel-forming, tight junction protein responsible for specific transfer of solutes across the epithelium without entering the cells. This channel is selective for small cations but nearly impermeable to anions and uncharged solutes of any size. In contrast, claudin-5, a tight junction protein typical for all endothelia but also found in some epithelia, was characterized as a potent barrier builder. Claudin-8, another barrier builder, was demonstrated to be regulated by Na(+) uptake in surface epithelial cells of human colon. Here, aldosterone enhanced Na(+) absorption by dual action: transcellularly by inducing the epithelial sodium channel and paracellularly by preventing back leakage of absorbed Na(+) by upregulating claudin-8.

Abstract: Tight junctions of epithelial and endothelial cells form selective barriers that regulate paracellular transport of solutes, immune cells, and drugs. Tight junctions consist of proteins that physically "seal" the tight junction but also form channels that allow for permeation between the cells, resulting in epithelial surfaces of different tightness. The tight junction proteins occludin, tricellulin, and at least 24 members of the claudin family are characterized by four transmembranal domains and two extracellular loops that, like teeth of a zipper, contact the appropriate loops from opposing cell membranes. Tight junctions are regulated in their molecular composition, ultrastructure, and function by intracellular scaffolding proteins and the cytoskeleton; such regulation serves normal, physiologic adaptation but also occurs in numerous diseases.

Abstract: Aldosterone is the principal hormonal regulator of sodium homeostasis in vertebrates. It exerts its actions through the mineralocorticoid receptor (MR) that regulates the transcription of specific target genes. In recent years, a number of MR target genes have been identified that are involved in the regulation of the epithelial sodium channel (ENaC), a key modulator of renal sodium absorption. Here we report the identification of cnksr3 as a direct MR target gene that is up-regulated in response to physiological concentrations of aldosterone. The cnksr3 promoter exhibits two functional aldosterone-responsive regions, which were bound by the MR as assessed by chromatin immunoprecipitation (ChIP). In vivo, CNKSR3 was highly expressed in the renal cortical collecting duct (CCD), the prime target segment of aldosterone-regulated sodium retention in the kidney. CCD cell lines stably overexpressing or silencing CNKSR3 were electrophysiologically analyzed and show that CNKSR3 expression correlated with and is required for ENaC-mediated transepithelial sodium transport. In parallel, CNKSR3 expression led to decreased MEK phosphorylation. We conclude that CNKSR3, a homologue of scaffold proteins involved in MAPK pathway regulation, is a direct target of MR and is required for the maintenance of transepithelial sodium transport in the kidney.-Ziera, T., Irlbacher, H., Fromm, A., Latouche, C., Krug, S. M., Fromm, M., Jaisser, F., Borden, S. A. Cnksr3 is a direct mineralocorticoid receptor target gene and plays a key role in the regulation of the epithelial sodium channel.

Abstract: BACKGROUND AND AIMS: The etiology of pouchitis after coloproctomucosectomy with ileal pouch-anal anastomosis in patients with ulcerative colitis is still unknown. Beside changes in luminal antigens, the immunological predisposition is assumed to be responsible. In previous electrophysiological studies, we showed that mucosal barrier and transport function in pouchitis is markedly reduced. Thus, the aim of the present study was to analyze barrier function on the molecular level. MATERIAL AND METHODS: Pouch biopsies of 36 ulcerative colitis patients were analyzed. Time points were (1) intraoperative immediately prior to ileal pouch-anal anastomosis (n = 13), (2) >1 year after ileostomy closure (pouch, n = 12), and (3) during pouchitis (n = 11). Control terminal ileum biopsies were obtained from eight patients undergoing hemicolectomy due to carcinoma. Expression of tight junction proteins was analyzed by Western blotting and confocal laser-scanning microscopy. To elucidate effects on epithelial barrier properties, impedance spectroscopy was performed in miniaturized Ussing chambers. RESULTS: In pouchitis, epithelial resistance was markedly reduced compared to non-inflamed pouch and control ileum. Expression of tight junction proteins claudin-1, 3, 4, 5, and 7 and occludin revealed differential expression regulation with the tightening tight junction protein claudin-1 being decreased and an increase of the pore-forming claudin-2, whereas other claudins remained constant. Morphometry indicated the mucosal surface to be unchanged. CONCLUSION: Pouchitis is characterized by a selective change of tight junction proteins in favor of opening the epithelial tight junction and, thus, the paracellular pathway, which contributes to the inflammatory process. This resembles changes in inflammatory bowel disease (IBD) and indicates IBD recurrence in pouchitis.

Abstract: Claudin-16 (paracellin-1) is a tight junction protein localized mainly in the thick ascending limb of Henle's loop and also in the distal nephron. Its defect causes familial hypomagnesaemia with hypercalciuria and nephrocalcinosis. This had been taken as an indication that claudin-16 conveys paracellular Mg(2+) and Ca(2+) transport; however, evidence is still conflicting. We studied paracellular ion permeabilities as well as effects of claudin-16 on the driving forces for passive ion movement. MDCK-C7 cells were stably transfected with wild-type (wt) and mutant (R146T, T233R) claudin-16. Results indicated that paracellular permeability to Mg(2+) but not to Ca(2+) is increased in cells transfected with wt compared to mutant claudin-16 and control cells. Increased basolateral Mg(2+) concentration activated a transcellular Cl(-) current which was greatly enhanced in cells transfected with wt and T233R claudin-16, as compared to R146T claudin-16-transfected or control cells. This current was triggered by the basolateral calcium-sensing receptor causing Ca(2+) release from internal stores, thus activating apical Ca(2+)-sensitive Cl(-) channels and basolateral Ca(2+)-sensitive K(+) channels. Immunohistochemical data suggest that the Cl(-) channel involved is bestrophin. We conclude that claudin-16 itself possesses only moderate paracellular Mg(2+) permeability but governs transcellular Cl(-) currents by interaction with apical Ca(2+)-activated Cl(-) channels, presumably bestrophin. As the transepithelial voltage generated by such a current alters the driving force for all ions, this may be the major mechanism to regulate Mg(2+) and Ca(2+) absorption in the kidney.

Abstract: Tricellulin is a tight junction protein localized in tricellular tight junctions (tTJs), the meeting points of three cells, but also in bicellular tight junctions (bTJs). To investigate its specific barrier functions in bTJs and tTJs, TRIC-a was expressed in low-level tricellulin-expressing cells, and MDCK II, either in all TJs or only in tTJs. When expressed in all TJs, tricellulin increased paracellular electrical resistance and decreased permeability to ions and larger solutes, which are associated with enhanced ultrastructural integrity of bTJs toward enhanced strand linearity. In tTJs in contrast, ultrastructure was unchanged and tricellulin minimized permeability to macromolecules but not to ions. This paradox is explained by properties of the tTJ central tube which is wide enough for passage of macromolecules, but too rare to contribute significantly to ion permeability. In conclusion, at low tricellulin expression the tTJ central tube forms a pathway for macromolecules. At higher expression, tricellulin forms a barrier in tTJs effective only for macromolecules and in bTJs for solutes of all sizes.

Abstract: BACKGROUND: Arcobacter butzleri causes watery diarrhea and bacteremia. Although, recently, more cases of diarrhea have been caused by Arcobacter species, very little is known about its pathogenesis, the identification of which is the aim of this study. METHODS: Human HT-29/B6 colonic epithelial monolayers were apically inoculated with A. butzleri. Transepithelial resistance and macromolecule fluxes were measured in Ussing chambers. Tight junction protein expression was analyzed by Western blotting, and subcellular distribution was analyzed by confocal laser-scanning microscopy. RESULTS: Infection of HT-29/B6 caused a decrease in transepithelial resistance to 30% and an increase in paracellular permeability to fluorescein (10.8+/-3.5 10(-6) cm/s vs. 1.8+/-0.6 10(-6) cm/s in control; P<.05) and dextran-4 kDa (0.036+/-0.005 10(-6) cm/s vs. 0.015+/-0.002 10(-6) cm/s in control; P<.01). This effect was time and dose dependent and was also caused by bacterial lysates showing heat and proteinase-K sensitivity. As structural correlate, expression of the tight junctional proteins claudin-1, -5, and -8 was reduced, and claudin-1 and -8 were redistributed off the tight junctional strands forming intracellular aggregates. Furthermore, A. butzleri induced epithelial apoptosis (3-fold). Conclusions: A. butzleri induces epithelial barrier dysfunction by changes in tight junction proteins and induction of epithelial apoptosis, which are mechanisms that are consistent with a leak flux type of diarrhea in A. butzleri infection.

Abstract: High dietary intake of fruits and vegetables is associated with a reduced disease risk. Therefore, clinical interest is growing in therapies based on dietary supplements and effects of food components. Immune-modulatory and barrier-protective effects have been described for the amino acid glutamine and the trace element zinc. In Caco-2-cells, zinc is necessary to maintain the expression of proteins like ZO-1 and occludin, and experimental evidence exists that glutamine has enterocyte-protective effects and modulates intestinal barrier function in stressed animals and humans. Polyunsaturated fatty acids (PUFA) improve paracellular permeability after IL-4 incubation. Enhancement of barrier properties by long-chain PUFA is discussed controversially, but a beneficial role preventing the redistribution of occludin and ZO-1 and reduction of epithelial resistance by IFN-gamma and TNF-alpha exists. In addition, a group of secondary plant compounds, the polyphenols, are supposed to be important in this respect. The flavonoid quercetin and its metabolite DHBA increased epithelial resistance of Caco-2-cells to 157 +/- 4% of control values, and DHBA up to 119 +/- 4% of control values, respectively. This is due to a 2.3 +/- 0.1-fold expression rate of the tight junction protein claudin-4.

Abstract: OBJECTIVE: Backwash ileitis (BI) has not been identified as a risk factor for pouchitis. The aim of this study was to investigate the barrier function of the ileoanal pouch depending on the presence of BI. The incidence of pouchitis in a population of ulcerative colitis patients with BI is also reported. MATERIAL AND METHODS: Biopsies were taken from 80 patients with ulcerative colitis: a) terminal ileum prior to pouch creation (pre-IAP); b) 16 months after ileostomy closure (intact pouch); and c) during pouchitis. Patients were stratified into the BI group and the non-BI (ØBI) group. Barrier function was determined in Ussing-chambers as epithelial resistance by impedance analysis and as mannitol permeability from (3)H-mannitol fluxes. Na(+)-glucose co-transport was measured as a change in short-circuit current (I(SC)) after addition of glucose. Relative risk of developing pouchitis was calculated by corrected chi(2) test. RESULTS: In 13/21 (BI/ØBI) pre-IAP patients, 23/37 (BI/ØBI) with an intact pouch, and 35/7 (BI/ØBI) with pouchitis, epithelial resistance in BI/ØBI was 13.5+/-1.6/14.3+/-0.9 Omega.cm(2) for pre-IAP, 12.7+/-1.3/16.8+/-1.2 Omega x cm(2) (p<0.05 BI versus ØBI) for the intact pouch, and 10.1+/-1.1/9.9+/-1.8 Omega x cm(2) for pouchitis (p<0.05 BI versus ØBI with an intact pouch). No differences were found for electrogenic chloride secretion and active Na(+)-glucose co-transport between BI/ØBI in the three groups. In patients with BI, pouchitis was more common (35 versus 7 patients, odds ratio 33.0 (95% CI 8.3-143.9; p<0.0001)). CONCLUSIONS: Ulcerative colitis patients with BI show impaired barrier function in the further course of the ileoanal pouch. Thus, BI has a long-term impact on epithelial barrier function.

Abstract: Claudins are essential components of the intercellular tight junction and major determinants of paracellular solute fluxes across epithelia and endothelia. Many members of this family display a distinct charge or size specificity, whereas others render the epithelium impermeable to transport. Due to intercellular localization, claudin-mediated transport processes are passive and driven by an electrochemical gradient. In epithelial tissues, claudins exhibit a temporal-spatial expression pattern corresponding with regional and local solute transport profiles. Whereas paracellular transport mechanisms in organs such as intestine and kidney have been extensively investigated, little is known about the molecular mechanisms determining solute transport in the peritoneum, and thus the determinants of peritoneal dialysis. Given the ubiquitous expression of claudins in endothelia and epithelia, it is predictable that claudins also contribute to pore formation and determination in the peritoneum, and that they are involved in solute flux. Therefore, we review the basic characteristics of claudin family members and their function as exemplified in renal tubular transport and give an outlook to what extent claudin family members might be of importance for solute reabsorption across the peritoneal membrane.

Abstract: BACKGROUND AND AIMS: The alpha(2)-gliadin-33mer has been shown to be important in the pathogenesis of coeliac disease. We aimed to study mechanisms of its epithelial translocation and processing in respect to transcytotic and paracellular pathways. METHODS: Transepithelial passage of a fluorescence-labelled alpha(2)-gliadin-33mer was studied in Caco-2 cells by using reverse-phase high-performance liquid chromatography, mass spectrometry, confocal laser scanning microscopy (LSM) and fluorescence activated cell sorting (FACS). Endocytosis mechanisms were characterised with rab-GFP constructs transiently transfected into Caco-2 cells and in human duodenal biopsy specimens. RESULTS: The alpha(2)-gliadin-33mer dose-dependently crossed the epithelial barrier in the apical-to-basal direction. Degradation analysis revealed translocation of the 33mer polypeptide in the uncleaved as well as in the degraded form. Transcellular passage was identified by confocal LSM, inhibitor experiments and FACS. Rab5 but not rab4 or rab7 vesicles were shown to be part of the transcytotic pathway. After pre-incubation with interferon-gamma, translocation of the 33mer was increased by 40%. In mucosal biopsies of the duodenum, epithelial 33mer uptake was significantly higher in untreated coeliac disease patients than in healthy controls or coeliac disease patients on a gluten-free diet. CONCLUSION: Epithelial translocation of the alpha(2)-gliadin-33mer occurs by transcytosis after partial degradation through a rab5 endocytosis compartment and is regulated by interferon-gamma. Uptake of the 33mer is higher in untreated coeliac disease than in controls and coeliac disease patients on a gluten-free diet.

Abstract: Quercetin is the most abundant flavonoid and is assumed to have positive effects on the gastrointestinal mucosa after dietary intake. The aim of the study was to analyze the influence of quercetin on intestinal barrier function using the human colonic epithelial cell line Caco-2. Transepithelial resistance (R(t)), tracer fluxes of [(3)H]-mannitol, 22Na+, and 36Cl- as well as electrogenic ion transport were determined in Ussing chambers. Expression of tight junction (TJ) proteins and mRNA was analyzed in Western blots and quantitative RT-PCR, respectively. Regulation of transcription was analyzed by reporter gene assay. Cellular distribution of TJ proteins was examined by confocal laser scanning microscopy (LSM). Apoptotic rate was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Quercetin induced a dose-dependent increase of R(t) persisting for >2 d. Daily addition of quercetin was able to perpetuate the effect, which was seen whether quercetin was added apically or to the basolateral compartment. Parallel to the R(t) increase, quercetin induced a strong increase of the TJ protein claudin-4 but not of other claudins. Confocal LSM showed a localization of claudin-4 in TJ. Apoptotic rate was not affected by quercetin. Consistent with these changes, fluxes of Na+ and Cl-, but not of mannitol, were reduced. Reporter gene assays revealed a stimulatory effect of quercetin on claudin-4 transcription. The flavonoid quercetin enhances barrier function via transcriptional expression regulation of the TJ protein claudin-4, which represents an important protective effect of this food component against barrier disturbance in intestinal inflammation.

Abstract: BACKGROUND & AIMS: Crohn's disease (CD) is a chronic inflammatory bowel disease. In this study, we have investigated sodium absorption via epithelial sodium channels (ENaC) in the macroscopically noninflamed colon in active CD. METHODS: Sodium transport via ENaC was investigated in Ussing chambers using biopsy specimens of sigmoid colon from controls and active CD limited to the small intestine. ENaC messenger RNA expression and subcellular localization were studied by real-time polymerase chain reaction and confocal microscopy. Effects of proinflammatory cytokines on ENaC and signaling via mitogen-activated protein kinases were investigated in rat distal colon. Therapeutic inhibition of mitogen-activated protein kinases was studied in CD biopsy specimens. RESULTS: Electrogenic sodium absorption via ENaC was strongly impaired in the macroscopically noninflamed CD colon because of reduced gamma-ENaC transcription, whereas subcellular localization of ENaC was not changed. In contrast to impaired epithelial sodium transport, epithelial barrier function was not altered in noninflamed CD colon, indicating that paracellular leak flux of ions did not contribute to decreased sodium absorption. Exposure of rat distal colon to tumor necrosis factor alpha led to reduced electrogenic sodium absorption because of impaired transcriptional gamma-ENaC induction, which resembled the changes found in CD. Tumor necrosis factor alpha effects were dependent on extracellular signal-regulated kinase 1/2 but not p38 or c-Jun-N-terminal kinase because inhibition of mitogen-activated protein kinase/extracellular regulated kinase (MEK)1/2 but not inhibition of p38 or c-Jun-N-terminal kinase prevented suppression of ENaC. Finally, therapeutic inhibition of MEK1/2 restored electrogenic sodium absorption in CD. CONCLUSIONS: In CD, macroscopically noninflamed colon contributes to diarrhea via impaired ENaC-mediated sodium absorption. Inhibition of extracellular signal-regulated kinase might serve as a potential therapeutic strategy for CD diarrhea.

Abstract: Endothelin-1 (ET-1), the most potent vasoconstrictor known to date, seems to be involved in the pathogenesis of primary open angle glaucoma. ET-1 was found in different tissues of the eye and in high concentrations in the aqueous humour. The effects of ET-1 are mediated by two receptors, ET-A receptor (ET-AR) and ET-B receptor (ET-BR), which are both expressed in bovine trabecular meshwork (TM). ET-1 induced contraction of TM predominantly by activation of ET-AR. This study analyzes the role of ET-BR in TM function and investigates the synthesis of ET-1 by human TM (HTM) cells. The effect of IRL-1620, a specific ET-BR agonist, on contractility of bovine TM (BTM) was investigated with a force length transducer system. The effect of this agonist on intracellular free Ca(2+) [Ca(2+)](i) was examined using the Ca(2+)-sensitive dye fura-2AM. The expression of the ET-AR and ET-BR was investigated in cultured HTM cells using Western blot and PCR methods. With PCR methods the expression of prepro-endothelin-1 (ppET-1) and isoforms of endothelin-1 converting enzyme (ECE-1) in cultured HTM cells was analyzed. Activation of ET-BR by IRL-1620 (5 x 10(-7)M) results in contraction of native BTM (41% of the carbachol value) and also in a transient increase in [Ca(2+)](i) in cultured BTM and HTM cells (365 and 273% of the basal level, respectively). IRL-1620 also induced contraction in native BTM under intra- and extracellularly Ca(2+)-free conditions. A clear signal for ET-AR at 40 kDa and ET-BR at 35 kDa could be detected in membrane lysates of cultured HTM cells. PCR analysis further confirmed the existence of ET-AR and ET-BR in these cells. Furthermore, RT-PCR revealed that neither ppET-1 nor one of the ECE-1 isoforms was expressed in cultured HTM cells. This study presents evidence for the expression of a functional ET-BR in bovine and human TM. Currently, there is no evidence for ET-1 synthesis in HTM cells. Further investigations are necessary to clarify the physiological function of ET-BR in TM and the source of ET-1 at this tissue.

Abstract: BACKGROUND: Giardia lamblia causes infection of the small intestine, which leads to malabsorption and chronic diarrhoea. AIM: To characterise the inherent pathomechanisms of G lamblia infection. METHODS: Duodenal biopsy specimens from 13 patients with chronic giardiasis and from controls were obtained endoscopically. Short-circuit current (I(SC)) and mannitol fluxes were measured in miniaturised Ussing chambers. Epithelial and subepithelial resistances were determined by impedance spectroscopy. Mucosal morphometry was performed and tight junction proteins were characterised by immunoblotting. Apoptotic ratio was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling staining. RESULTS: In giardiasis, mucosal surface area per unit serosa area was decreased to 75% (3%) of control, as a result of which epithelial resistance should increase. Instead, epithelial resistance of giardiasis biopsy specimens was decreased (19 (2) vs 25 (2) Omega cm(2); p<0.05) whereas mannitol flux was not significantly altered (140 (27) vs 105 (16) nmol/h/cm(2)). As structural correlate, reduced claudin 1 expression and increased epithelial apoptosis were detected. Furthermore, basal I(SC) increased from 191 (20) in control to 261 (12) microA/h/cm(2) in giardiasis. The bumetanide-sensitive portion of I(SC) in giardiasis was also increased (51 (5) vs 20 (9) microA/h/cm(2) in control; p<0.05). Finally, phlorizin-sensitive Na(+)-glucose symport was reduced in patients with giardiasis (121 (9) vs 83 (14) microA/h/cm(2)). CONCLUSIONS: G lamblia infection causes epithelial barrier dysfunction owing to down regulation of the tight junction protein claudin 1 and increased epithelial apoptoses. Na(+)-dependent d-glucose absorption is impaired and active electrogenic anion secretion is activated. Thus, the mechanisms of diarrhoea in human chronic giardiasis comprise leak flux, malabsorptive and secretory components.

Abstract: Extraintestinal pathogenic Escherichia coli (ExPEC) are usually harmless colonizer of the intestinal microflora. However, they are capable to translocate and cause life-threatening disease. Translocation of ExPEC isolates was quantified in colonic monolayers. Transepithelial resistance (R(t)) was monitored and local changes in conductivity analysed with conductance scanning. Confocal microscopy visualized the translocation route. Corroboratory experiments were performed on native rat colon. One translocating strain E. coli O4 was identified. This translocation process was associated with an R(t) decrease (36 +/- 1% of initial resistance) beginning only 2 h after inoculation. The sites of translocation were small defects in epithelial integrity (focal leaks) exhibiting highly increased local ion permeability. Translocation was enhanced by preincubation of monolayers with tumour necrosis factor-alpha or interleukin-13. Mutant strains lacking alpha-haemolysin lost the ability to induce focal leaks, while this effect could be restored by re-introducing the haemolysin determinant. Filtrate of a laboratory strain carrying the alpha-haemolysin operon was sufficient for focal leak induction. In native rat colon, E. coli O4 decreased R(t) and immunohistology demonstrated focal leaks resembling those in cell monolayers. E. coli alpha-haemolysin is able to induce focal leaks in colonic cell cultures as well as in native colon. This process represents a novel route of bacterial translocation facilitated by pro-inflammatory cytokines.

Abstract: BACKGROUND & AIMS: The epithelial sodium channel (ENaC) is the rate-limiting factor for colonic electrogenic sodium absorption. This study aimed to investigate ENaC regulation by butyrate, a short-chain fatty acid (SCFA) produced by intestinal bacteria. METHODS: ENaC was examined in HT-29/B6 cells and glucocorticoid receptor(GR)-transfected HT-29/B6 cells (HT-29/B6-GR) by reverse-transcription polymerase chain reaction, real-time polymerase chain reaction, and confocal microscopy. ENaC promoters were investigated by deletion/mutation analysis, electrophoretic mobility shift assays, and quantitative chromatin immunoprecipitation. Sodium transport of HT-29/B6-GR cells and rat distal colon was quantified in Ussing chambers. RESULTS: Butyrate up-regulated beta- and gamma-ENaC mRNA expression in HT-29/B6 cells and induced transcription from beta- and gamma-ENaC promoter constructs. The gamma-ENaC promoter could also be induced by the SCFA propionate but not by acetate. Deletion/mutation assays revealed that activation of the gamma-ENaC promoter depended on 2 GC boxes, which were shown to bind Sp1 and Sp3 in vitro. Although both transcription factors increased butyrate-mediated gamma-ENaC transcription upon overexpression, chromatin immunoprecipitation revealed that only Sp3 binds to the gamma-ENaC promoter in vivo and that Sp3 binding is enhanced by butyrate. Transcriptional ENaC induction by butyrate led to synthesis of gamma-ENaC subunits, but correct targeting of ENaC channels to the apical cell membrane was dependent on corticosteroid hormones. Finally, butyrate substantially increased electrogenic sodium absorption via ENaC in the presence of corticosteroid hormones in HT-29/B6-GR cells and in rat distal colon. CONCLUSIONS: Concerted action of SCFA and corticosteroid hormones is required for induction of ENaC and maintenance of intestinal electrogenic sodium absorption.

Abstract: BACKGROUND: Epithelial barrier function is impaired in Crohn's disease. AIM: To define the underlying cellular mechanisms with special attention to tight junctions. METHODS: Biopsy specimens from the sigmoid colon of patients with mild to moderately active or inactive Crohn's disease were studied in Ussing chambers, and barrier function was determined by impedance analysis and conductance scanning. Tight junction structure was analysed by freeze fracture electron microscopy, and tight junction proteins were investigated immunohistochemically by confocal laser scanning microscopy and quantified in immunoblots. Epithelial apoptosis was analysed in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and 4',6-diamidino-2-phenylindole staining. RESULTS: Patients with active Crohn's disease showed an impaired intestinal barrier function as indicated by a distinct reduction in epithelial resistance. As distribution of conductivity was even, focal epithelial lesions (eg, microerosions) did not contribute to barrier dysfunction. Instead, freeze fracture electron microscopy analysis showed reduced and discontinuous tight junction strands. Occludin and the sealing tight junction proteins claudin 5 and claudin 8 were downregulated and redistributed off the tight junction, whereas the pore-forming tight junctions protein claudin 2 was strongly upregulated, which constitute the molecular basis of tight junction changes. Other claudins were unchanged (claudins 1, 4 and 7) or not detectable in sigmoid colon (claudins 11, 12, 14, 15 and 16). Claudin 2 upregulation was less pronounced in active Crohn's disease compared with active ulcerative colitis and was inducible by tumour necrosis factor alpha. As a second source of impaired barrier function, epithelial apoptosis was distinctly increased in active Crohn's disease (mean (SD) 5.2 (0.5)% v 1.9 (0.2)% in control). By contrast, barrier function, tight junction proteins and apoptosis were unaffected in Crohn's disease in remission. CONCLUSION: Upregulation of pore-forming claudin 2 and downregulation and redistribution of sealing claudins 5 and 8 lead to altered tight junction structure and pronounced barrier dysfunction already in mild to moderately active Crohn's disease.

Abstract: Epithelial barrier function is determined by trans- and paracellular permeabilities, the latter of which is mainly influenced by tight junctions (TJs) and apoptotic leaks within the epithelium. The present article aims to present experimental evidence for a functional role of epithelial apoptoses by means of cell culture models as well as in tissues from patients with inflammatory bowel disease. It is shown that epithelial apoptoses are sites of elevated conductance within the intestinal epithelium and that proinflammatory cytokines like TNF-alpha upregulate both the apoptotic rate and single apoptotic conductivity, making cytokine-induced apoptosis functionally far more relevant than is spontaneous apoptosis. In ulcerative colitis and Crohn's disease (CD), but not in collagenous colitis, apoptotic rates are increased to about 5%, in mild-to-moderately inflamed colon specimens, where as the control apoptotic rate is about 2%. Thus, epithelial apoptoses lead to a loss of ions and water into the intestinal lumen, causing leak flux diarrhea and enabling small antigens of <4,000 Da in the intestinal lumen to enter the intestinal mucosa, thereby perpetuating inflammatory responses. In addition to TNF-alpha, interleukin (IL)-13 is an important inductor of epithelial apoptosis in Th2 immune responses. Therapeutically,TNF-alpha-antibodies (infliximab) can restore barrier function in Crohn's disease by downregulating epithelial apoptoses, while epithelial TJs are unaffected.

Abstract:

Abstract: TRPV4 is a calcium-permeable channel activated by extracellular hypotonicity, polyunsaturated fatty acids, phorbol esters, and heat. We show that TRPV4 is localized in the basolateral membrane of the mouse mammary cell line HC11. Activation of TRPV4 caused an increase in the intracellular Ca(2+) concentration through influx of extracellular Ca(2+), triggering two independent chains of events: 1) a rapid increase in transcellular conductance through the activation of apical large conductance calcium-activated (BK) potassium channels that were blockable by paxilline; 2) a slow increase in paracellular permeability for small solutes. The latter effect was accompanied by a down-regulation of the tight junctional proteins claudin -1, -3, -4, -5, -7, and -8 and by dramatic changes in tight junction morphology, including frequent large breaks in the tight junction strands. This dual modulation of epithelial permeability after TRPV4 activation may be involved in regulating the tonicity across mammary gland epithelia. TRPV4 activation may also be responsible for exudation and edema formation during inflammation processes.-Reiter, B., Kraft, R., Günzel, D., Zeissig, S., Schulzke, J-D., Fromm, M., Harteneck, C. TRPV4-mediated regulation of epithelial permeability.

Abstract: BACKGROUND AND AIMS: Bacterial overgrowth appears to play an important role in the pathogenesis of ileoanal pouches. Therefore, the capability of bacterial permeation and its determinants is of great interest. The aim of this study was to examine bacterial permeation in the ileoanal pouch and to correlate the results with the degree of inflammation, the epithelial resistance, the mucosal transport function, and the age of the ileoanal pouches. MATERIALS AND METHODS: Biopsies were taken from 54 patients before colectomy (n = 13; preileal pouch-anal anastomosis [IPAA]), and closure of ileostomy (n = 7; deviation), <1 year after closure of ileostomy (n = 8; intact pouch I), >1 year after closure of ileostomy (n = 16; intact pouch II), in the case of pouchitis (n = 11), and in 11 controls. Tissues were mounted in a miniaturized Ussing chamber. Escherichia coli was added to the mucosal side of the Ussing chamber, and the permeation was proven by serosal presence of E. coli. Epithelial and subepithelial resistance was determined by transmural impedance analysis. Active Na-glucose cotransport and active Cl secretion were measured. Specimens were analyzed by fluorescent in situ hybridization with oligonucleotide probes targeting the bacterial 16s ribosomal RNA. The bacteria in and on the tissue were enumerated. RESULTS: Bacterial permeation occurred in 2 of 13 pre-IPAA, 2 of 7 deviations, 0 of 8 intact pouch I, 9 of 16 intact pouch II, 5 of 11 pouchitis specimens, and 0 of 11 ileum controls. The frequency of bacterial permeation in the intact pouch II group is higher than in the intact pouch I group (P < 0.001). Epithelial resistance, mannitol fluxes, electrogenic chloride secretion, sodium-glucose cotransport of the bacterially permeated specimens versus nonpermeated of the intact pouch II group, and the pouchitis group and subepithelial resistance remained unchanged. Intramural bacteria could be detected by fluorescence in situ hybridization mainly in long-lasting pouches, but there was no correlation with bacterial permeation. CONCLUSIONS: The long-lasting ileoanal pouch is associated with increased bacterial permeability. This is not correlated with a disturbed function of the pouch mucosa but could be a precursor of pouchitis.

Abstract: PURPOSE: This study analyzes additional mechanisms behind the ocular hypotensive effect of prostaglandin F (PGF) receptor (FP receptor) agonists PGF2alpha and fluprostenol (fluprostenol-isopropyl ester [travoprost]), which reduce intraocular pressure (IOP) in patients with glaucoma probably by enhancing uveoscleral flow. The trabecular meshwork (TM) is actively involved in IOP regulation through contractile mechanisms. Contractility of TM is induced by endothelin (ET)-1, a possible pathogenic factor in glaucoma. The involvement of FP receptor agonists in the ET-1 effects on TM function was studied. METHODS: The effects of FP receptor agonists on contractility of bovine TM (BTM) were investigated using a force-length transducer. The effects of PGF2alpha on intracellular Ca2+ ([Ca2+]i) mobilization in cultured cells were measured using fura-2AM. The expression of the FP receptor protein was examined using Western blot analysis. RESULTS: The ET-1-induced (10(-8) M) contraction in isolated BTM was inhibited by PGF2alpha (10(-6) M) and fluprostenol (10(-6) M). This effect was blocked by FP receptor antagonists. Carbachol-induced contraction or baseline tension was not affected by PGF2alpha or fluprostenol. In cultured TM cells, ET-1 caused a transient increase in [Ca2+]i that was reduced by PGF2alpha. No reduction occurred in the presence of the FP receptor antagonist Al-8810. Western blot analysis revealed the expression of the FP receptor in native and cultured TM. CONCLUSIONS: FP receptor agonists operate by direct interaction with ET-1-induced contractility of TM. This effect is mediated by the FP receptor. Thus, FP receptor agonists may decrease IOP by enhancing aqueous humor outflow through the TM by inhibiting ET-1-dependent mechanisms.

Abstract: Integrity of colon epithelium is of crucial importance and, as small defects occur constantly, rapid repair (restitution) is essential. To investigate the mechanism of restitution, single-cell lesions were induced in mouse colonic surface epithelia by iontophoretic injection of Ca2+. Closure of the resulting defects was monitored using confocal laser scanning microscopy (CLSM), and functional sealing by electrophysiological techniques. Restitution was evaluated as the time constant tau of the exponential decrease in conductance of an induced leak and amounted to 0.28 min under control conditions. After 4 min, the leak was completely sealed. Repair was thus considerably faster than in previously investigated HT-29/B6 cells (tau=5.73 min). As in cultured cells, cytochalasin D delayed restitution in native colon epithelia (tau=0.69 min), indicating the involvement of actin in the healing process; however, no accumulation of actin surrounding the lesion was detected. Long-term incubation of epithelia with IFN-gamma alone or in combination with TNF-alpha increased tau to 0.49 and 0.59 min, respectively. In contrast to cultured cells, TNF-alpha alone did not affect restitution. A brief (<10 min) exposure to the sterile filtered supernatant of hemolytic E. coli O4 cultures did not affect the morphology of the epithelium, but delayed restitution. In CLSM studies, defects were still clearly visible 4 min after the onset of lesion induction. The supernatant of a nonhemolytic E. coli O4 mutant did not exhibit this effect. In conclusion, single-cell defects in native colon cause functional leaks that seal faster than in cell cultures. Proinflammatory cytokines and pathogenic bacteria delay restitution. This suggests a key role of very small lesions at the onset of pathogenic processes in the intestine.

Abstract: The epithelial sodium channel (ENaC) controls colonic sodium absorption. So far, investigation of ENaC was limited by an unexplained lack of steroid-dependent ENaC expression in cultured intestinal cells, which we aimed to resolve. HT-29/B6 cells constitutively expressed the alpha-ENaC subunit, while beta- and gamma-ENaC subunits could not be detected due to deficient basal as well as corticosteroid-induced transcription. This was due to a lack of expression of both activating and inhibiting isoforms of glucocorticoid receptor (GR-alpha, -beta) and mineralocorticoid receptor. Stable transfection of GR-alpha restored intestine-specific glucocorticoid upregulation of beta- and gamma-ENaC in HT-29/B6 cells, which was followed by intact targeting of ENaC channels to the apical cell membrane and dose-dependent induction of electrogenic sodium absorption. In conclusion, ENaC deficiency is due to a lack of steroid receptors and not the consequence of a crypt-like phenotype of cultured intestinal cells. By stable GR transfection we obtained a model, in which ENaC regulation can be studied.

Abstract: Claudin-16 (Cldn16) is selectively expressed at tight junctions (TJs) of renal epithelial cells of the thick ascending limb of Henle's loop, where it plays a central role in the reabsorption of divalent cations. Over 20 different mutations in the CLDN16 gene have been identified in patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), a disease of excessive renal Mg2+ and Ca2+ excretion. Here we show that disease-causing mutations can lead to the intracellular retention of Cldn16 or affect its capacity to facilitate paracellular Mg2+ transport. Nine of the 21 Cldn16 mutants we characterized were retained in the endoplasmic reticulum, where they underwent proteasomal degradation. Three mutants accumulated in the Golgi complex. Two mutants were efficiently delivered to lysosomes, one via clathrin-mediated endocytosis following transport to the cell surface and the other without appearing on the plasma membrane. The remaining 7 mutants localized to TJs, and 4 were found to be defective in paracellular Mg2+ transport. We demonstrate that pharmacological chaperones rescued surface expression of several retained Cldn16 mutants. We conclude that FHHNC can result from mutations in Cldn16 that affect intracellular trafficking or paracellular Mg2+ permeability. Knowledge of the molecular defects associated with disease-causing Cldn16 mutations may open new venues for therapeutic intervention.

Abstract: BACKGROUND AND AIMS: Diarrhoea is a frequent adverse effect of HIV protease inhibitors (PIs) which may be due to intestinal barrier disruption. We investigated whether tight junction dysregulation, apoptosis or necrosis are responsible for this epithelial damage. METHODS: Saquinavir, nelfinavir, and ritonavir were added to the mucosal or serosal side of HT-29/B6 colon cell monolayers. Transepithelial resistance was monitored for 72 h to assess epithelial barrier function. Apoptosis and necrosis were investigated by light and electron microscopy and quantified by nucleosome ELISA and LDH measurement, respectively. Tight junction components were analysed by Western blots of occludin and zonula occludens. Apoptosis induction in normal human intestinal epithelium was examined by measurement of poly(ADP-ribose) polymerase (PARP) cleavage in Western blots of mucosal tissue explants cultured with PIs for 24 h. RESULTS: HIV PIs decreased transepithelial resistance by more than 44% in HT-29/B6 monolayers. Histology revealed massive apoptotic body formation but no evidence for necrosis after PI treatment. Correspondingly, LDH release was lower than 0.2%/h of total LDH, independent of PI treatment, and nucleosomes were increased up to 22-fold after drug treatment versus control. Occludin and zonula occludens-1 expression in the membrane were not diminished. PARP cleavage increased in normal human intestinal tissue treated with PIs. CONCLUSIONS: PI-induced barrier disruption in intestinal epithelial cells is not due to necrosis or tight junction alterations, but to induction of massive apoptosis which may lead to leak-flux diarrhoea in vivo. Our findings suggest that induction of apoptosis by PIs could have potential for antitumour therapy.

Abstract: BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by a Th2 immune response with inflammation and epithelial barrier dysfunction. So far, Th2 cytokines have not been shown to directly influence epithelial barrier function. METHODS: Lamina propria mononuclear cells (LPMCs) were stimulated and interleukin (IL)-13 was measured by enzyme-linked immunosorbent assay. Functional IL-13 and IL-4 effects were studied on HT-29/B6 colonic epithelial cells in Ussing chambers and by conductance scanning. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. IL-13/IL-4 receptors were analyzed by reverse-transcription polymerase chain reaction and immunofluorescence. Western blotting combined with immunofluorescence was used to detect tight junction proteins. Furthermore, restitution velocity was measured. Finally, mucosal biopsy specimens from patients with UC were compared with cultured cells for these features. RESULTS: LPMCs from patients with UC produced large amounts of IL-13 (985 +/- 73 pg/mL), much more than from controls or patients with Crohn's disease. IL-13Ralpha1 and IL-4Ralpha receptors were present in HT-29/B6 cells and colonic epithelial cells of control patients and patients with UC. IL-13 had a dose-dependent effect on transepithelial resistance of HT-29/B6 monolayers (reduction to 60% +/- 4%), whereas IL-4 had no effect. This was due to an increased number of apoptotic cells (5.6-fold +/- 0.9-fold) and an increased expression of the pore-forming tight junction protein claudin-2 to 295% +/- 37%, both of which contributed equally. Finally, epithelial restitution velocity decreased from 15.1 +/- 0.6 to 10.6 +/- 0.5 microm/h after treatment with IL-13. Parallel changes were observed in human samples, with an increase in claudin-2 expression to 956% +/- 252%. CONCLUSIONS: IL-13 was identified as an important effector cytokine in UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity.

Abstract: Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.

Abstract: To clarify the potential role of endothelin-1 (ET-1) in the pathogenesis of glaucoma, the endothelin receptors expressed in bovine trabecular meshwork (TM) and ciliary muscle (CM) were identified. TM and CM strips were subjected to ET-1 as well as to specific endothelin receptor antagonists. In both tissues BQ123, a specific ET-A receptor antagonist, substantially inhibited ET-1-induced contraction. BQ788, a specific ET-B receptor antagonist, showed only moderate effects. Both ET receptor types were detected in bovine TM and CM using Western blot analysis. ET-1 produced an increase in intracellular calcium in cultured TM cells. This effect was inhibited by BQ123, but not by BQ788. Thus, although both receptors are present, the ET-A receptor appears to play the predominant role in mediating contraction in both the TM and CM, while the ET-B receptor seems to contribute little to the overall ET-1 effect.

Abstract: BACKGROUND AND AIMS: This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model. METHODS: Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (Re, epithelial resistance). Conductance scanning differentiated transcellular (Gc) and tight junctional conductance (Gtj). The pH-stat technique quantified gastric acid secretion. RESULTS: In occludin+/+ mice, Re was 23+/-5 Omega cm2 in jejunum, 66+/-5 Omega cm2 in distal colon and 33+/-6 Omega cm2 in gastric corpus and was not altered in heterozygotic occludin+/- or homozygotic occludin-/- mice. Additionally, [3H]mannitol fluxes were unaltered. In the control colon, Gc and Gtj were 7.6+/-1.0 and 0.3+/-0.1 mS/cm2 and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin-/- mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control Rt was 5.8+/-0.8 kOmega cm2 in urinary bladder and 33+/-6 Omega cm2 in stomach and not altered in occludin-/- mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin-/- mice. CONCLUSION: Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.

Abstract: Cadherin adhesion molecules are key determinants of morphogenesis and tissue architecture. Nevertheless, the molecular mechanisms responsible for the morphogenetic contributions of cadherins remain poorly understood in vivo. Besides supporting cell-cell adhesion, cadherins can affect a wide range of cellular functions that include activation of cell signalling pathways, regulation of the cytoskeleton and control of cell polarity. To determine the role of E-cadherin in stratified epithelium of the epidermis, we have conditionally inactivated its gene in mice. Here we show that loss of E-cadherin in the epidermis in vivo results in perinatal death of mice due to the inability to retain a functional epidermal water barrier. Absence of E-cadherin leads to improper localization of key tight junctional proteins, resulting in permeable tight junctions and thus altered epidermal resistance. In addition, both Rac and activated atypical PKC, crucial for tight junction formation, are mislocalized. Surprisingly, our results indicate that E-cadherin is specifically required for tight junction, but not desmosome, formation and this appears to involve signalling rather than cell contact formation.

Abstract: The diarrheal mechanisms in Aeromonas enteritis are not completely understood. In this study we investigated the effect of aeromonads and of their secretory products on ion secretion and barrier function of monolayers of human intestinal cells (HT-29/B6). Ion secretion was determined as a short-circuit current (I(SC)) of HT-29/B6 monolayers mounted in Ussing-type chambers. Transepithelial resistance (R(t)) served as a measure of permeability. A diarrheal strain of Aeromonas hydrophila (strain Sb) added to the mucosal side of HT-29/B6 monolayers induced a significant I(SC) (39 +/- 3 microA/cm(2)) and decreased the R(t) to approximately 10% of the initial value. A qualitatively identical response was obtained with sterile supernatant of strain Sb, and Aeromonas supernatant also induced a significant I(SC) in totally stripped human colon. Tracer flux and ion replacement studies revealed the I(SC) to be mainly accounted for by electrogenic Cl(-) secretion. Supernatant applied serosally completely abolished basal I(SC). The supernatant-induced I(SC) was inhibited by the protein kinase C inhibitor chelerythrine, whereas a protein kinase A inhibitor (H8) and a Ca(2+) chelator (BAPTA-AM) had no effect. Physicochemical properties indicated that the supernatant's active compound was an aerolysin-related Aeromonas beta-hemolysin. Accordingly, identical I(SC) and R(t) responses were obtained with Escherichia coli lysates harboring the cloned beta-hemolysin gene from strain SB or the aerA gene encoding for aerolysin. Sequence comparison revealed a 64% homology between aerolysin and the beta-hemolysin cloned from Aeromonas sp. strain Sb. In conclusion, beta-hemolysin secreted by pathogenic aeromonads induces active Cl(-) secretion in the intestinal epithelium, possibly by channel insertion into the apical membrane and by activation of protein kinase C.

Abstract: BACKGROUND AND AIMS: Barrier dysfunction is an important feature contributing to inflammation and diarrhoea in Crohn's disease (CD). Recently, tumour necrosis factor alpha (TNF-alpha) antibodies were recognised as effective in steroid refractory CD. The aim of this study was to characterise the effects of this therapy on the epithelial barrier. PATIENTS AND METHODS: Forceps biopsies were obtained from the sigmoid colon before and 14 days after TNF-alpha antibody therapy in 11 patients treated for chronic active CD (Crohn's disease activity index >150). Epithelial apoptoses were measured after terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) and 4',6-diamidino-2-phenylindole staining. Epithelial resistance was determined by alternating current impedance analysis in miniaturised Ussing chambers. Occludin, claudin 1, and claudin 4 expression was quantified in immunoblots. RESULTS: The epithelial apoptotic ratio was 2.1 (0.2)% in controls and increased to 5.3 (1.0)% in CD. TNF-alpha antibody therapy decreased the apoptotic ratio to 2.9 (1.0)% (normalised in 10 of 11 patients). In parallel, epithelial resistance was lower in CD than in controls (24 (3) v 42 (3) Omegaxcm(2)) and improved to 34 (3) Omegaxcm(2) after therapy. Occludin, claudin 1, and claudin 4 were not affected by TNF-alpha antibody therapy. In support of a functional role of epithelial apoptoses in CD, a similar decrease in resistance of -40% was observed when the apoptotic rate was selectively upregulated from 2.6% to 5.4% with camptothecin in HT-29/B6 cells. CONCLUSIONS: Epithelial apoptoses were upregulated in the colon in CD and restored to normal in 10 of 11 patients by TNF-alpha antibody therapy. This is the structural correlate of epithelial barrier dysfunction measured as epithelial resistance while expression of tight junction proteins did not contribute to this therapeutic effect.

Abstract: The epithelial Na+ channel (ENaC) provides the main absorptive pathway of the distal large intestine. This study aimed to characterize regulatory influences of cytokines in rat late distal colon. After 6 h incubation with either IL1beta, TNFalpha, IFNgamma, or combinations of TNFalpha and IFNgamma, ENaC was measured as electrogenic Na+ transport after 8 h induction by 3 nM aldosterone (JNa) in totally stripped specimens in the Ussing chamber. Subsequently, alpha-, beta-, and gamma-ENaC subunit mRNAs were analyzed by Northern blotting. The gamma-ENaC promoter was cloned and characterized by reporter gene assays. IL-1beta and TNFalpha, but not interferon-gamma, decreased JNa. In parallel, beta- and gamma-ENaC transcription was inhibited, whereas alpha-ENaC was unaffected. gamma-ENaC promoter activity was inhibited by IL-1beta and TNFalpha but not by IFNgamma. We conclude that the pro-inflammatory cytokines IL-1beta and TNFalpha inhibit electrogenic sodium absorption in rat distal colon by mRNA expression regulation of the beta- and gamma-ENaC subunits.

Abstract: BACKGROUND & AIMS: The main limiting factor for sodium absorption in distal colon is the amiloride-sensitive epithelial sodium channel (ENaC). This study aimed to characterize mechanisms involved in the dysregulation of ENaC expression in ulcerative colitis (UC). METHODS: Epithelial preparations from surgically removed inflamed and control sigmoid colons were used. Active electrogenic Na(+) transport (J(Na)) was determined after 8-hour aldosterone stimulation in Ussing-chambers (corrected for the altered epithelial/subepithelial resistance ratio). Subsequently, ENaC alpha-, beta-, and gamma-subunits were analyzed immunohistochemically and in Western and Northern blots (corrected for the inflammatory increase in subepithelial protein content). To study gene regulation, the promoters of beta- and gamma-ENaC were analyzed in reporter gene assays. RESULTS: In controls, aldosterone stimulated J(Na) and induced ENaC beta- and gamma-subunit expression, whereas this response was virtually abolished in UC. Preservation of surface epithelium in UC was indicated by unchanged ENaC alpha-subunit expression, which points also against a mere immaturity or epithelial cell loss. Inhibition of electrogenic sodium transport as well as beta- and gamma-ENaC mRNA expression could be mimicked in control colon by in vitro preexposure for 8 hours to tumor necrosis factor alpha and interferon gamma. Promoter analysis revealed that down-regulation of beta- and gamma-ENaC gene expression was primarily induced by tumor necrosis factor alpha. CONCLUSIONS: We conclude that, in UC, elevated proinflammatory cytokines selectively impair beta- and gamma-ENaC expression, which contributes to diarrhea by reducing colonic sodium absorption.

Abstract: The polarized morphology of epithelial cells depends on the establishment and maintenance of characteristic intercellular junctions. The dramatic morphological changes observed in apoptotic epithelial cells were ascribed at least in part to the specific fragmentation of components of adherens junctions and desmosomes. Little, however, is known about tight junctions during apoptosis. We have found that after induction of apoptosis in epithelial cells, tight junction proteins undergo proteolytic cleavage in a distinctive manner correlated with a disruption of tight junctions. The transmembrane protein occludin and, likewise, the cytoplasmic adaptor proteins ZO-1 and ZO-2 are fragmented by caspase cleavage. In addition, occludin is cleaved at an extracellular site by a metalloproteinase. The caspase cleavage site in occludin was mapped C-terminally to Asp(320) within the C-terminal cytoplasmic domain. Mutagenesis of this site efficiently blocked fragmentation. In the presence of caspase and/or metalloproteinase inhibitors, fragmentation of occludin, ZO-1 and ZO-2 was blocked and cellular morphology was almost fully preserved. Interestingly, two members of the claudin family of transmembrane tight junction proteins exhibited a different behavior. While the amount of claudin-2 protein was reduced similarly to occludin, ZO-1 and ZO-2, claudin-1 was either fully preserved or was even increased in apoptotic cells.

Abstract: Assembly of tight junctions at the most apical part of the lateral cell membrane is a key event in the differentiation of polarized epithelial cells. Claudin-2, a transmembrane protein involved in tight junction strand formation, has turned out to play a crucial role for the paracellular barrier function by opening pores for small cations. Physiological and pathological variations of epithelial barrier function are accompanied by differential expression of tight junction proteins. Therefore, we characterized molecular mechanisms regulating claudin-2 gene expression. Genomic DNA containing the transcription start point of human claudin-2 was isolated and functionally characterized by reporter gene assays. Activity of the claudin-2 promoter was elevated in mouse mammary epithelial C57 cells expressing Wnt-1. LEF-1, a nuclear effector of the Wnt signaling pathway which is involved in the regulation of cell differentiation and polarization, was found to bind directly to the claudin-2 promoter as revealed by electrophoretic mobility shift assays. Expression of LEF-1 and beta-catenin both enhanced claudin-2 promoter activity. This increase was reduced after mutation of LEF-1 binding sites within the claudin-2 promoter. Furthermore, claudin-2 promoter activity was found to be enhanced by the TCF-4/beta-catenin transcription complex. Therefore, we conclude that gene expression mediated by the promoter of the human tight junction protein claudin-2 is regulated by factors involved in Wnt signaling. Moreover, a functional crosstalk between Wnt signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins could be demonstrated in the regulation of claudin-2 promoter-mediated gene expression.

Abstract: Colitis in interleukin-2-deficient (IL-2(-/-)) mice resembles ulcerative colitis in humans. We studied epithelial transport and barrier function in IL-2(-/-) mice and used this model to characterize mechanisms of diarrhea during intestinal inflammation. (22)Na(+) and (36)Cl(-) fluxes were measured in proximal colon. Net Na(+) flux was reduced from 4.0 +/- 0.5 to 0.8 +/- 0.5 micromol.h(-1).cm(-2), which was paralleled by diminished mRNA and protein expression of the Na(+)/H(+) exchanger NHE3. Net Cl(-) flux was also decreased from 2.2 +/- 1.6 to -2.7 +/- 0.6 micromol.h(-1).cm(-2), indicating impaired Na(+)-Cl(-) absorption. In distal colon, aldosterone-induced electrogenic Na(+) absorption was 6.1 +/- 0.9 micromol.h(-1).cm(-2) in controls and was abolished in IL-2(-/-) mice. Concomitantly, mRNA expression of beta- and gamma-subunits of the epithelial sodium channel (ENaC) was reduced. Epithelial barrier was studied in proximal colon by impedance technique and mannitol fluxes. In contrast to ulcerative colitis, epithelial resistance was increased and mannitol fluxes were decreased in IL-2(-/-) mice. This was in accord with the findings of reduced ion transport as well as increased expression of tight junction proteins occludin and claudin-1, -2, -3, and -5. In conclusion, the IL-2(-/-) mucosa exhibits impaired electroneutral Na(+)-Cl(-) absorption and electrogenic Na(+) transport due to reduced mRNA and protein expression of NHE3 and ENaC beta- and gamma-subunit mRNA. This represents a model of early intestinal inflammation with absorptive dysfunction due to impaired transport protein expression/function while epithelial barrier is still intact. Therefore, this model is ideal to study regulation of transporter expression independent of barrier defects.

Abstract: To verify the relevance of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity in controlling breast-cancer cell growth, we have evaluated the correlation of 11beta-HSD2 expression and antiproliferative effects of glucocorticosteroids (GCs) on breast cancer cell proliferation. We cloned human 11beta-HSD2 cDNA into the expression vector pBK-CMV. The interspersing lac promoter region was deleted, achieving differential translational efficiency. The constructs were stably transfected into wild-type MCF-7 breast-cancer cells possessing almost no oxidative and no reductive 11beta-HSD activity. Low (times 7) and high (times 718) 11beta-HSD2 overexpression was achieved. We compared growth behavior of transfected cells In the presence of GCs to MCF-7 cells transfected with pBK-CMV alone (internal control). The antiproliferative effects of GCs were reversed and total cell growth boosted by overexpression of 11beta-HSD2; about 50 % of the increase in cell proliferation was attained by low 11beta-HSD2 overexpression, while high enzyme overexpression led to an increase in cell growth of about 120 %. Using direct evidence, this study shows 11beta-HSD2 to impair antiproliferative glucocorticosteroid effects, thus acting as an enzymatic shield aggravating breast-cancer cell growth. These results indicate a possible therapeutic role for 11beta-HSD inhibitors in the treatment of breast cancer.

Abstract: In the mammalian cochlea, tight junctional strands are visible on freeze fracture images of marginal cells and other inner ear epithelia. The molecular composition of the strial tight junctions is, however, largely unknown. We investigated the expression of integral tight junction-proteins, claudin-1 to -4, and occludin, in stria vascularis of the guinea-pig cochlea, as compared to kidney. Western blot analysis revealed a strong expression of claudin-4 and occludin in strial tissue, and confocal immunofluorescence microscopy demonstrated their presence in the tight junctions of the marginal cells. In addition, a moderate level of claudin-3 and claudin-1 was detected and both were located in the marginal tight junctions. Claudins-1, -3, and -4 are characteristic of epithelia with low paracellular permeability and claudin-4 is known to restrict the passage of cations through epithelial tight junctions. In the marginal cells, these claudins appear to be responsible for the separation of the potassium-rich endolymph from the sodium-rich intrastrial fluid. In contrast, Western blot analysis and confocal microscopy demonstrated that the marginal cell epithelium does not contain claudin-2, which forms a cation-selective pore in tight junctions. Its absence indicates a cation-tight paracellular pathway in the marginal cells.

Abstract: BACKGROUND AND AIMS: Little is known of the permeability of ileoanal pouches. Hence the aim of the present study was to determine changes in permeability and mucosal function after ileo-pouchanal anastomosis (IPAA) in patients with ulcerative colitis. MATERIALS AND METHODS: Biopsies were taken from 43 patients (male:female ratio 28:15; mean age 35.2 (12.5) years) prior to colectomy (ileum prior to pouch), prior to closure of ileostomy (deviation), and after closure of ileostomy (intact pouch) in the case of pouchitis, and from 14 healthy controls. Tissues were mounted in a miniaturised Ussing chamber. Epithelial and subepithelial resistance was determined by transmural impedance analysis. Active Na(+)-glucose cotransport was measured as change in short circuit current after stepwise addition of glucose, and active Cl(-) secretion was measured after stimulation with theophylline and prostaglandin E(2). RESULTS: Neither epithelial resistance nor mannitol fluxes were significantly altered compared with intact controls, indicating no barrier defect in pouchitis. Subepithelial resistances of intact pouches and pouchitis were increased compared with deviation (18.2 (1.6) and 24.3 (1.5) v. 13.6 (1.0) Omegaxcm(2)) consistent with an adaptive thickening of the subepithelial layer. In contrast, active Cl(-) secretion of pouchitis tissues was reduced versus intact pouch and controls (1.4 (0.3) v. 4.3 (0.7) and 4.6 (0.7) micromol/h/cm(2)), and Na(+)-glucose cotransport of pouchitis was reduced compared with intact pouch and controls (1.8 (0.5) v. 4.2 (0.8) and 8.8 (1.3) micromol/h/cm(2)). CONCLUSIONS: Ileal mucosa in pouchitis and terminal ileum prior to IPAA exhibit impaired secretory and absorptive transport functions whereas the epithelial barrier function remains unchanged. This differs from findings in ulcerative colitis. Thus the hypothesis that pouchitis represents a remanifestation of ulcerative colitis has to be questioned.

Abstract: OBJECTIVES: Characterization of the diarrhoea-inducing effect of altered cytokine production in HIV infection. METHODS: Monocyte-derived macrophages (MDM) were infected with macrophagetropic (SF162) and lymphocytotropic (IIIB) HIV-1 strains and cocultured with autologous peripheral blood mononuclear cells (PBMC). After 24 h the supernatants were collected and tested for their immunoreactive levels of cytokines by enzyme-linked immunosorbent assay. The effects of the supernatants and the respective recombinant human cytokines on barrier function of HT-29/B6 cells were determined. RESULTS: Infection of MDM with HIV-1 SF162 or IIIB led to increased production of tumour necrosis factor-alpha (TNFalpha), interleukin-1-beta, interferon-alpha and interferon-gamma after cell-cell contact with PBMC. Supernatants of infected cells decreased transepithelial resistance (R(t)), with higher effects on R(t) in HIV IIIB infection, which was due to higher cytokine concentrations. The effect was not due to cytotoxicity (negative LDH assay) or epithelial monolayer disruption [zonula occludens protein-1 (ZO-1) immunofluorescence staining]. The effect of HIV-1 IIIB coculture supernatants could be mimicked by the respective recombinant human cytokines. TNFalpha is an effector cytokine, because inhibition of TNFalpha by its soluble receptor decreased the effect of the supernatants on transepithelial resistance. Conductance scanning indicated the cytokine-induced barrier defect to be due to both, induction of epithelial apoptoses and tight junction alterations. CONCLUSIONS: Cell-cell interaction of HIV-infected macrophages with PBMC leads to a release of cytokines sufficient to alter intestinal epithelial barrier function. The main effect was mediated by TNFalpha inducing a leak-flux which may contribute to the diarrhoea by HIV per se (HIV-enteropathy).

Abstract: Tight junction proteins comprise a novel group of integral membrane proteins necessary for cell-to-cell contacts and responsible for the barrier function in epithelial and endothelial cells in various tissues. The tight junction membrane domain contains at least three distinct proteins, named occludin, claudin and junctional adhesion molecule. Claudins are products of a gene family consisting of more than 20 members. We investigated mRNA expression of occludin and 13 different claudins in neonatal foreskin, adult skin and cultivated HaCaT keratinocytes by the Northern blot technique, and performed immunohistochemical staining of adult skin for occludin, claudin 1 and claudin 2. Occludin, claudin 1 and claudin 3 mRNAs were expressed in human neonatal and adult keratinocytes as well as in HaCaT keratinocytes. All other tested claudins were negative. Immunohistochemical staining of adult skin was positive for occludin in the intercellular space of the granular layer, and for claudin 1 in the inter-cellular space of the spinosum layer and basal layer, but negative for claudin 2 in all skin layers. Claudin 1 was also positive in the outer root sheath of hair follicles. Our results indicate that occludin, claudin 1 and claudin 3 are involved in cell-to-cell contacts between keratinocytes in human epidermis, although their functional importance remains unknown.

Abstract: BACKGROUND & AIMS: Collagenous colitis is an inflammatory disease of unknown etiology with diarrhea as the leading symptom. The aim of this study was to examine the pathogenic mechanisms of this disease. METHODS: Biopsy specimens of the sigmoid colon were obtained endoscopically. Short-circuit current and (22)Na and (36)Cl fluxes were measured in miniaturized Ussing chambers. Alternating current impedance analysis discriminated epithelial from subepithelial resistance. Tight junction proteins occludin and claudin 1-5 were characterized in membrane fractions by Western blotting. Apoptotic ratio was determined by DAPI and TUNEL staining. RESULTS: In collagenous colitis, net Na(+) flux decreased from 8.8 +/- 1.8 to 0.2 +/- 1.5 and net Cl(-) flux from 11.2 +/- 3.0 to -3.0 +/- 2.7 micromol x h(-1) x cm(-2), indicating a pronounced decrease in NaCl absorption. The fact that short-circuit current increased from 1.5 +/- 0.4 to 3.9 +/- 0.8 micromol x h(-1) x cm(-2), together with the negative net Cl(-) flux, points to activation of active electrogenic chloride secretion. Subepithelial resistance increased from 7 +/- 1 to 18 +/- 2 Omega x cm(2) due to subepithelial collagenous bands of 48 +/- 8-microm thickness. Epithelial resistance was diminished from 44 +/- 3 to 29 +/- 2 Omega x cm(2), and this was accompanied by a decrease in occludin and claudin-4 expression. Neither mucosal surface area nor apoptotic ratio was altered in collagenous colitis. CONCLUSIONS: Reduced net Na(+) and Cl(-) absorption is the predominant diarrheal mechanism in collagenous colitis, accompanied by a secretory component of active electrogenic chloride secretion. The subepithelial collagenous band as a significant diffusion barrier is a cofactor. Down-regulation of tight junction molecules but not epithelial apoptoses is a structural correlate of barrier dysfunction contributing to diarrhea by a leak flux mechanism.

Abstract: Restitution of single-cell defects, a frequent event in epithelia with high turnover, is poorly understood. Morphological and functional changes were recorded, using intravital time-lapse video microscopy, confocal fluorescence microscopy, and conductance scanning techniques. After artificial single-cell loss from an HT-29/B6 colonic cell monolayer, the basal ends of adjacent cells extended. Concurrently, the local conductive leak associated with the defect sealed with an exponential time course (from 0.48 +/- 0.05 microS 2 min post lesion to 0.17 +/- 0.02 microS 8 min post lesion, n = 17). Between 3 and 10 min post lesion, a band of actin arose around the gap, which colocalized with a ring of ZO-1 and occludin. Hence, tight junction proteins bound to the actin band facing the gap, and competent tight junctions assembled in the adjoining cell membranes. Closure and sealing were inhibited when actin polymerization was blocked by cytochalasin D, delayed following decrease of myosin-ATPase activity by butanedione monoxime, and blocked after myosin light chain kinase inhibition by ML-7. The Rho-associated protein kinase inhibitor Y-27632 did not affect restitution. After loosening of intercellular contacts in low Ca(2+) Ringer solution, the time course of restitution was not significantly altered. Albeit epithelial conductivity was 12-fold higher in low Ca(2+) Ringer solution than in controls, under both conditions the repaired epithelium assumed the same conductivity as distant intact epithelium. In conclusion, epithelial restitution of single-cell defects comprises rapid closure by an actinomyosin 'purse-string' mechanism and simultaneous formation of a functional barrier from tight junction proteins also associated with the purse string.

Abstract: Interleukin-2-deficient (IL-2(-/-)) mice develop colitis with striking clinical and morphological similarities to ulcerative colitis. Since transport and barrier properties are impaired in ulcerative colitis, we studied transport and barrier functions in IL-2(-/-) mice in order to gain insight for the first time into the general pathomechanisms of disturbed transport and barrier function of the intestine during inflammation. Alternating current impedance analysis was used to determine tissue conductance in the inflamed proximal colon of IL-2(-/-) mice and to discriminate between pure epithelial and subepithelial conductance. Surprisingly, epithelial conductance was not increased but diminished in IL-2(-/-) mice compared to controls (20.2 +/- 1.3 versus 28.8 +/- 2.8 mS/cm(2)). Concomitantly, conductance of the subepithelial tissue layers was decreased in IL-2(-/-) mice as a result of edema and infiltration with inflammatory cells. In the distal colon, electrogenic Na(+) transport (J(Na)) mediated by the epithelial Na(+) channel (ENaC) was measured 8 h after stimulation with 3.10(-9) M aldosterone in vitro as the drop in I(SC) (short circuit current) after addition of 10(-4) M amiloride. In controls, J(Na) was 6.9 +/- 0.9 micromol x h(-1) x cm(-2), whereas it was abolished in IL-2(-/-) mice. In conclusion, the inflamed colon of IL-2(-/-) mice exhibits a severe disturbance in Na(+) uptake via the ENaC in the absence of a barrier defect. Thus, reduced expression of active absorptive transport and not a barrier defect is responsible for the diarrhea in this model of intestinal inflammation. This makes this model suitable for studying the general pathomechanisms of the inflammatory downregulation of intestinal transport proteins.

Abstract: Occludin is an integral membrane protein located at the tight junctions of epithelial cells. Multiple domains of occludin are involved in the regulation of paracellular permeability as well as in the targeting of the protein to the tight junction. In this study, different occludin variants were identified on the mRNA level. Four differentially spliced occludin-specific mRNA transcripts were detected. Expression of the resulting proteins revealed an altered subcellular distribution and a loss of co-localization with zonula occludens protein ZO-1 in the tight junction for two of the four splice variants. Our findings demonstrate that the fourth transmembrane domain of occludin is important for targeting occludin to the tight junction. Loss of the fourth transmembrane domain leads to a relocation of the C-terminal domain to the extracellular space. The structural diversity of natural occludin variants is further increased by an additional promoter and transcription start giving rise to an alternative exon 1. Gene expression mediated by this promoter is influenced by the pro-inflammatory cytokine tumor necrosis factor alpha.

Abstract: Tight junctions seal the paracellular pathway of epithelia but, in leaky tissues, also exhibit specific permeability. In order to characterize the contribution of claudin-2 to barrier and permeability properties of the tight junction in detail, we studied two strains of Madin-Darby canine kidney cells (MDCK-C7 and MDCK-C11) with different tight junctional permeabilities. Monolayers of C7 cells exhibited a high transepithelial resistance (>1 kOhms cm(2)), compared with C11 cells (<100 Ohms cm(2)). Genuine expression of claudin-1 and claudin-2, but not of occludin or claudin-3, was reciprocal to transepithelial resistance. However, confocal microscopy revealed a marked subjunctional localization of claudin-1 in C11 cells, indicating that claudin-1 is not functionally related to the low tight junctional resistance of C11 cells. Strain MDCK-C7, which endogenously does not express junctional claudin-2, was transfected with claudin-2 cDNA. In transfected cells, but not in vector controls, the protein was detected in colocalization with junctional occludin by means of immunohistochemical analyses. Overexpression of claudin-2 in the originally tight epithelium with claudin-2 cDNA resulted in a 5.6-fold higher paracellular conductivity and relative ion permeabilities of Na(+) identical with 1, K(+)=1.02, NMDG(+)=0.79, choline(+)=0.71, Cl(-)=0.12, Br(-)=0.10 (vector control, 1:1.04:0.95:0.94:0.85:0.83). By contrast, fluxes of (radioactively labeled) mannitol and lactulose and (fluorescence labeled) 4 kDa dextran were not changed. Hence, with regular Ringer's, Na(+) conductivity was 0.2 mS cm(-2) in vector controls and 1.7 mS cm(-2) in claudin-2-transfected cells, while Cl(-) conductivity was 0.2 mS cm(-2) in both cells. Thus, presence of junctional claudin-2 causes the formation of cation-selective channels sufficient to transform a 'tight' tight junction into a leaky one.

Abstract: Mechanisms to regulate closely fetal GC exposure are of considerable importance, as certain organs (kidney, brain) are adversely affected by excess GCs. 11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2) reduces transplacental passage of maternal GCs to the fetus. We hypothesized that 11beta-HSD2, if active in fetal kidney and colon, might allow local tissue modulation of GC access during the critical last trimester. We determined the presence, ontogeny and functionality of 11beta-HSD in the placenta and fetal, neonatal and adult kidney and colon in rats and rabbits and the cortisol:cortisone ratio in human amniotic fluid, which represents fetal urine. There was clear a 11beta-HSD2 expression in last trimester fetal colon, kidney and placenta in both rats and rabbits. This appeared of functional importance, since the potency of cortisol on fetal rabbit colonic sodium flux in the Ussing chamber was increased by 11beta-HSD inhibition. In human amniotic fluid, we found a decreasing ratio of cortisol:cortisone across the last trimester, suggesting an analogous onset of renal 11beta-HSD2 activity in the human fetal kidney. Local fetal tissue 11beta-HSD2 may modulate exposure to the deleterious effects of GCs upon target tissue maturation during sensitive periods of late gestation when fetal GC levels rise to prepare other organs (lung) for adaptations at birth.

Abstract: BACKGROUND & AIMS: In ulcerative colitis (UC), the epithelial barrier is impaired by erosion/ulcer-type lesions and epithelial apoptosis causing local leaks, and generalized tight junction alterations increasing the basal permeability. We quantified the contribution of these mechanisms to the increased colonic ion permeability. METHODS: Sigmoid colon was stripped, and the spatial distribution of current clamped across the viable epithelium was recorded by a microelectrode probe, using the conductance scanning method. Local leaks (circumscribed conductive peaks) were marked, and structural changes were studied in H&E-stained series sections. RESULTS: Overall conductivity increased from 8.4 +/- 0.7 mS/cm(2) (mean +/- SEM) in controls to 11.7 +/- 0.6 in specimens with mild inflammation (i.e., with intact epithelium) and 34.4 +/- 6.2 mS/cm(2) in moderate-to-severe inflammation (i.e., with visible epithelial lesions). Only in part this was caused by a generalized increase in basal conductivity (12.2 +/- 1.5 mS/cm(2) in moderate-to-severe UC vs. 8.3 +/- 0.7 in controls). More importantly, the spatial distribution of conductivity, which was even in controls, showed dramatic leaks in UC. Leaks found in mild inflammation without epithelial lesion turned out to be foci of epithelial apoptosis. In moderate-to-severe inflammation, leaks correlated with epithelial erosion/ulcer-type lesions or crypt abscesses. CONCLUSIONS: In early UC, but not in controls, seemingly intact epithelium comprises leaks at apoptotic foci. With more intensive inflammation, erosion/ulcer-type lesions are highly conductive, even if covered with fibrin. Local leaks contribute 19% to the overall epithelial conductivity in mild and 65% in moderate-to-severe inflammation.

Abstract: Solid State Nuclear Track Detectors (SSNTD's) are used in a wide range of applications such as Geological Dating, Environmental Sciences (radon), life Sciences (Radiobiology, Dosimetry...), as well as Nuclear and Astro-Physics. In order to be observable under a microscope, the nanometric latent damaged trails due to the slowing down of charged particles into the SSNTD have to be specifically etched. In our laboratory, we are studying this chemical action and propose models that enable simulations to be performed. In the literature, the basic model uses two distinct etch-rates that are considered constant, VB; the Bulk and VT the Track etch-rate. A little bit more sophisticated and realistic, a model with a variable track etch-rate was established, taking into account the variation of energy deposition along the particle's trajectory. Up to now, the known methods used for determining the response function of CR-39 are very time consuming and strenuous. The method we present here is based on the use of the confocal microscope, which provides three-dimensional track images. The obtained set of 3-D co-ordinates can be treated mathematically, giving, in the framework of the two etch-velocity model, the response function. With this new approach, tracks are analysed one by one; response functions are obtained for each track and can be compared to fundamental characteristics of the charged Particle-Matter interactions. Moreover, the method we propose is applied semi-automatically and could easily be automated in the near future.

Abstract: 1. The barrier function of colonic epithelia is challenged by apoptotic loss of enterocytes. In monolayers of human colonic HT-29/B6 cells, apoptosis induced by camptothecin was assessed by poly-(ADP-ribose)-polymerase (PARP) cleavage, histone ELISA and DNA-specific fluorochrome staining (with 4',6'-diamidino-2'-phenylindoladihydrochloride (DAPI)). Epithelial barrier function was studied in Ussing chambers by measuring transepithelial conductivity and unidirectional tracer fluxes. The ion permeability associated with single cell apoptoses was investigated with the conductance scanning technique. 2. The spontaneous rate of apoptotic cells was 3.5 +/- 0.3 % with an overall epithelial conductivity of 3.2 +/- 0.1 mS cm(-2). Camptothecin induced a time- and dose-dependent increase of apoptosis and permeability. With 20 microg ml(-1) of camptothecin for 48 h, apoptosis increased 4.1-fold to 14.3 +/- 1.5 % and the conductivity doubled to 6.4 +/- 1.0 mS cm(-2). 3. While 3H-mannitol flux increased 3.8-fold and 3H-lactulose flux increased 2.6-fold, the flux of 3H-polyethylene glycol 4000 remained unchanged. Hence, the higher permeability was limited to molecules < 4000 Da. 4. The local epithelial conductivity was higher at the sites of apoptosis than in non-apoptotic areas. With camptothecin the leaks associated with apoptosis became more numerous and more conductive, while in non-apoptotic areas the conductivity remained at control level. Hence, the camptothecin-induced increase in epithelial conductivity reflected the opening of apoptotic leaks and thus the results described, for the first time, epithelial permeability as a function of apoptosis only. 5. The conductivity of apoptotic leaks contributed 5.5 % to the epithelial conductivity of controls and 60 % to the conductivity of monolayers treated with 20 microg ml(-1) of camptothecin. Thus apoptosis increased the contribution of paracellular pathways to the overall epithelial permeability. Under control conditions the paracellular conductivity (G(para)) was smaller than the transcellular (G(trans)), but with 12 % apoptosis, G(para) exceeded G(trans). By definition, the epithelium became 'leaky'.

Abstract: BACKGROUND: The mechanism of the antisecretory effect of loperamide was investigated in cultured highly differentiated colon epithelial cells (HT-29/B6). METHODS: Chloride secretion was stimulated via cAMP by forskolin (FSK, 10(-5) M), via Ca2+ by the muscarinic agonist carbachol (CCh, 10(-4) M), and via protein kinase C by the phorbol ester PMA (5 x 10(-9) M). Stimulated Cl- secretion was quantified as short circuit current (I(SC)) of HT-29/B6 monolayers mounted in Ussing-type chambers. RESULTS: Loperamide (5 x 10(-5) M) inhibited I(SC) stimulated by FSK, CCh and PMA. The antisecretory action of loperamide was unaffected by preincubation with naloxone (10(-5) M). Furthermore, loperamide strongly inhibited basolateral 86Rb efflux. Like loperamide, the calmodulin antagonist trifluoperazine (10(-4) M) inhibited I(SC) induced by FSK, CCh or PMA. The Ca2+ channel blocker verapamil (5 x 10(-5) M), on the other hand, inhibited only PMA-stimulated I(SC),but had no effect on FSK or CCh-induced I(SC) CONCLUSIONS: Loperamide exerts a direct antisecretory action on chloride secretion of colon epithelial cells independently of the respective stimulatory signal transduction pathway. This antisecretory effect is not mediated by opiate receptors and reflects inhibition of basolateral K+ conductance.

Abstract: The 65 kDa protein occludin is a membrane-spanning part of the epithelial tight junction, which is the main barrier of the paracellular pathway. The function of occludin as part of tight junctions is still poorly understood and even less is known about the regulatory mechanisms that influence occludin gene expression. This study aimed to identify the sequences essential in cis for genomic regulation of tight junction formation and to investigate their funcional role in cytokine-dependent tight junction regulation. Using genome walking cloning of occludin-specific human genomic DNA sequences, a 1853 bp DNA fragment containing the transcription start point of occludin cDNA sequences was amplified and sequenced. Subcloning of this fragment in front of the luciferase reporter gene revealed strong expression of enzymatic activity after transfection of the human intestinal cell line HT-29/B6. With subsequent deletions of parts of the promoter fragment, its size was reduced to 280 bp that are necessary and sufficient to mediate promoter activity. Tumor necrosis factor alpha and another cytokine involved in inflammation, interferon gamma, reduced transepithelial resistance in HT-29/B6 cells, which was preceded by a decrease in occludin mRNA expression as revealed by northern blot analysis. Tumor necrosis factor alpha and interferon gamma diminished occludin promoter activity alone and even synergistically, suggesting a genomic regulation of alterations of the paracellular barrier. In conclusion, proinflammatory cytokines such as tumor necrosis factor alpha and interferon gamma can downregulate the expression of the transmembrane tight junction strand protein occludin, paralleling the barrier disturbance detected electrophysiologically. This could be an important mechanism in gastrointestinal diseases accompanied by barrier defects, for example inflammatory bowel diseases.

Abstract: Current opinion assumes epithelial integrity during spontaneous apoptotic cell death. We measured, for the first time, the local conductances associated with apoptoses and show leaks of up to 280 nS (mean 48 +/- 19 nS) in human intestinal epithelium. The results disprove the dogma that isolated cell apoptosis occurs without affecting the epithelial cell permeability barrier. After induction by tumor necrosis factor alpha (TNF-alpha) the apoptotic leaks were dramatically enhanced: not only was the frequency increased by threefold, but the mean conductance also increased by 12-fold (597+/-98 nS). Thus, apoptosis accounted for about half (56%) of the TNF-alpha-induced permeability increase whereas the other half was caused by degradation of tight junctions in nonapoptotic areas. Hence, spontaneous and induced apoptosis hollow out the intestinal barrier and may facilitate loss of solutes and uptake of noxious agents.

Abstract: AIM: In order to investigate the potential of Helicobacter pylori (HP) to induce dyspepsia, we performed a randomized prospective study on the long-term effect of HP-eradication on symptoms of HP-positive dyspeptic patients in whom other organic causes for dyspepsia were carefully ruled out. PATIENTS: 201 patients referred to our endoscopy unit with dyspeptic symptoms for at least six months entered the study. Patients with previous peptic ulcer were excluded. METHODS: After endoscopy of the upper alimentary tract and 13C-urea breath test, patients with active peptic ulcer, hiatal hernia, macroscopic evidence for esophagitis and negative HP-status were excluded. The remaining patients underwent abdominal sonography, H2-exhalation test with lactose, and 24-h pH monitoring in order to exclude other organic causes for dyspepsia. In 20 patients, dyspepsia was assumed to be due to HP-gastritis. Patients received eradication therapy and were controlled as assessed by the 13C-urea breath test six weeks and six months after completion of the therapy. Dyspeptic symptoms were monitored by means of a validated symptom score. RESULTS: Out of 20 patients with HP-gastritis the first eradication treatment was successful in 13, while seven patients remained HP-positive after antibiotic treatment. Six months after completion of therapy the symptoms of HP-eradicated patients improved considerably (score values 17.4 +/- 1.5 and 10.2 +/- 0.8, respectively, p < 0.01) whereas symptoms of patients with persistent infection remained unchanged (21.1 +/- 1.7 and 20.4 +/- 1.5, n.s.) and only improved after successful retherapy (20.4 +/- 1.5 and 11.7 +/- 2.1, p < 0.05). In total, 17 of 20 patients (85%) improved after successful eradication. Also, neutrophil infiltration in the gastric mucosa correlated to both dyspeptic symptoms before therapy (r = 0.85) and the decrease in symptom score after HP-eradication (r = 0.61). In contrast, the symptoms of eight patients with gastroesophageal reflux disease were not improved after eradication (20.0 +/- 1.1 and 18.2 +/- 1.0, n.s.) CONCLUSIONS: HP-infection per se contributes to dyspepsia. 17 of 20 (85%) HP-positive dyspeptic patients improved after HP-eradication, when other potential organic causes for dyspepsia had been ruled out. However, many patients did not completely recover but the symptoms only partly decreased which parallels the persistence of part of the inflammatory infiltration in the gastric mucosa. This emphasizes the importance of HP-gastritis as an organic disease causing dyspeptic symptoms.

Abstract: The epithelial barrier function of the large intestine resides in the trans- and paracellular pathways of the surface epithelium and crypts. Conventional transmural resistance and permeability measurements, however, yield only the resistance of the whole tissue and not that of its individual components. Combining conductance scanning techniques and impedance analysis, we determined the resistance of epithelial and subepithelial tissues, crypts and surface epithelium, and trans- and paracellular pathways of the mouse distal colon. The subepithelial tissue contributed 15% to the transmural resistance of 118+/-9 omega x cm2. In the epithelium proper the resistance of crypts (429+/-86 omega x cm2) exceeded that of the surface epithelium (132+/-15 omega x cm2). The paracellular resistance (3.2+/-0.4 k omega x cm2) of the surface epithelium was 23-fold higher than the transcellular resistance (137+/-16 omega x cm2), and thus the epithelium was classified as "medium tight". In order to investigate the trans- and paracellular resistances of the crypt epithelium as well, flat monolayers of HT-29/B6 cultured colon crypt cells were studied, which had a transepithelial resistance of 349+/-32 omega x cm2. With transcellular resistance (377+/-41 omega x cm2) tenfold lower than the paracellular resistance (3.9+/-1.3 k omega x cm2), this cryptal monolayer was also classified as "medium tight". Hence, considering the 1.2 times larger area of the crypt epithelium, the surface epithelium has a 4 times larger ion permeability than the crypt epithelium. However, the paracellular resistances are not different. Thus the lower transcellular resistance of the surface compared to the crypt epithelium suggests a higher density of ion channels in the apical membrane of surface cells.

Abstract: Aldosterone-induced sodium absorption is mediated by the epithelial Na(+) channel (ENaC). It is thought that the "early effect" is not based on genomic regulation of ENaC expression, because ENaC subunit transcription was reported to start later than Na(+) transport. We investigated electrogenic Na(+) absorption (J(Na)) and, in identical tissues, mRNA expression of ENaC subunits in early (EDC) and late (LDC) distal colon of the rat. In both segments, 8-h in vitro incubation with 3 nM aldosterone enhanced expression of beta- and gamma-ENaC mRNA and induced J(Na). J(Na) was 10 times higher in LDC than in EDC. alpha-ENaC mRNA was unchanged in EDC, whereas it decreased in LDC. In LDC, beta- and gamma-ENaC mRNA was induced 1 h after aldosterone addition, whereas J(Na) became apparent >1 h later. Downregulation of alpha-ENaC mRNA did not take part in acute regulation because it started after a lag time of 3 h. Time correlation of beta- and gamma-ENaC induction and J(Na) stimulation suggests that the early aldosterone effect on Na(+) absorption in distal colon is caused by transcriptional upregulation of beta- and gamma-ENaC expression.

Abstract: Occludin is an integral membrane protein with four transmembrane domains that is exclusively localized at tight junction (TJ) strands. Here, we describe the generation and analysis of mice carrying a null mutation in the occludin gene. Occludin -/- mice were born with no gross phenotype in the expected Mendelian ratios, but they showed significant postnatal growth retardation. Occludin -/- males produced no litters with wild-type females, whereas occludin -/- females produced litters normally when mated with wild-type males but did not suckle them. In occludin -/- mice, TJs themselves did not appear to be affected morphologically, and the barrier function of intestinal epithelium was normal as far as examined electrophysiologically. However, histological abnormalities were found in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, loss of cytoplasmic granules in striated duct cells of the salivary gland, and thinning of the compact bone. These phenotypes suggested that the functions of TJs as well as occludin are more complex than previously supposed.

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Abstract: Diarrhea and malabsorption due to intestinal dysfunction are common symptoms in HIV infection. The pathophysiologic mechanisms of these alterations are often not known, and the role of HIV per se is still controversially discussed. We measured the epithelial transport and barrier function by means of a miniaturized Ussing chamber system in the duodenum of HIV-infected patients in different disease stages, determined by the CD4 cell count in the serum as well as symptoms in patients with and without diarrhea. We could show that diarrhea induced by HIV per se is caused by a leak flux mechanism due to impaired epithelial barrier function. Antisecretory therapy does not seem to be useful in these patients, because we did not find increased active ion secretion. Along the course of the HIV infection, the epithelial transport and barrier function varies with HIV disease stage (expressed by CD4 cell status). In addition, an in vitro model was studied to characterize the effect of HIV-infected human immune cells on the epithelial barrier function using the human colonic epithelial cell line HT-29/B6. HIV infection of human immune cells induced an increase in cytokine release--for example, TNF-alpha, IL-1 beta, IFN-alpha, and IFN-gamma--downregulating the epithelial barrier function of the human colonic epithelial cell line HT-29/B6. Taken together we postulate a specific stage-dependent cytokine pattern released from HIV-infected immune cells in the mucosa, which, corresponding to the HIV disease stage, is responsible for the variation in epithelial function.

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Abstract: On the basis of recently observed high levels of iNOS expression that correlated with intestinal inflammation in interleukin-2-deficient [IL-2(-/-)] mice, it was postulated that nitric oxide may damage colonic epithelial cells or impair intestinal epithelial barrier function. This damage may result in an increased permeability of the colonic epithelium leading to high antigenic exposure of the intestinal immune system, which may perpetuate chronic inflammation. Our data demonstrate that high expression of iNOS in IL-2(-/-) mice is correlated with the length/weight ratio (L/W ratio), a widely accepted marker for intestinal inflammation. However, no reduction of epithelial resistance was observed, as would be expected in case of a damaged, leaky epithelium. Our results suggest that enhanced formation of NO in IL-2(-/-) mice does not cause impairment of epithelial barrier function.

Abstract: The signal transduction pathways of the induction of apoptosis in the gastrointestinal tract have in part been discovered. However, almost nothing is known about the functional influence of apoptotic signals on intestinal barrier function. In this study the effect of camptothecin-induced apoptosis in HT-29/B6 monolayers and the influence of apoptosis on epithelial barrier function were characterized. We demonstrated that camptothecin causes a decrease of transepithelial resistance and an increase in fluxes of the paracellular marker [3H]mannitol. Camptothecin increased the apoptotic rate and the conductance of single-cell apoptosis as measured by the conductance scanning technique. We conclude that in our model of HT-29/B6 cells camptothecin is a potent inductor of apoptosis that causes significant barrier defects measured by the Ussing chamber technique and the conductance scanning technique. Based on these results we are able to investigate the effect of other cytokines--TGF-beta, for instance, and its role in apoptotic conditions.

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Abstract: The barrier function of intestinal epithelia relies upon the continuity of the enterocyte monolayer and intact tight junctions. After incubation with tumor necrosis factor-alpha TNF-alpha, however, the number of strands that form the tight junctions decreases, and apoptosis is induced in intestinal epithelial cells. These morphological changes lead to a rise of transepithelial ion permeability, because the paracellular ion permeability increases and leaks associated with sites of apoptosis increase by number and magnitude. Thus apoptosis and degradation of tight junctions contribute to the increased permeability observed after exposure to TNF-alpha. These mechanisms explain clinical manifestations in the inflamed intestinal wall containing cytokine-secreting macrophages--for example, leak flux diarrhea and invasion of bacterial enterotoxins.

Abstract: Cytokines are supposed to be mediators in diarrhoeal diseases. The aim of this study is to characterize the effect of tumor necrosis factor-alpha (TNFalpha) on epithelial barrier function in the colonic epithelial cell line HT-29/B6. Active ion transport and barrier function were measured as short-circuit current and transepithelial electrical resistance (Rt), respectively. In parallel, freeze-fracture electron microscopy (EM) of tight junctions (TJ) and immunofluorescence microscopy of the zonula occludens protein-1 (ZO-1) were performed. Serosal addition of TNF(alpha) (100 ng/ml) decreased Rt by 81%. This effect was dose-dependent and could be mimicked by antibodies against the p55 form of the TNF receptor. Cytotoxic effects were excluded by a negative lactate dehydrogenase (LDH) assay. Immunofluorescence localization with anti-ZO-1 antibodies revealed no evidence for disruption of the monolayer after TNFalpha treatment. In freeze-fracture EM, TJ complexity was decreased by TNFalpha, as indicated by a decrease in the number of strands from 4.7 to 3.4. The tyrosine kinase blocker genistein and the protein kinase A inhibitor H-8 reduced the effect of TNFalpha. A combination of TNFalpha with interferon-gamma acted synergistically on the epithelial barrier. In conclusion, TNFalpha impairs epithelial barrier function by altering structure and function of the tight junction, which could be of pathogenic relevance in intestinal inflammation.

Abstract: BACKGROUND & AIMS: Mechanisms of diarrhea in ulcerative colitis (UC) are still unknown. Functional and structural characterization of epithelial barrier and transport properties in ulcerative colitis (UC) was performed. METHODS: Inflamed sigmoid colon epithelium from UC patients was studied by alternating current impedance analysis to determine the pure epithelial resistance as a measure of intestinal barrier function. Tight junction (TJ) structure was investigated by freeze-fracture electron microscopy. RESULTS: Although total wall resistance was reduced in UC by 50%, impedance analysis uncovered a much more pronounced barrier defect. Epithelial resistance decreased from 95 +/- 5 to 20 +/- 3 omega3. cm2, which in conventional analysis is masked by an increase in subepithelial resistance from 14 +/- 1 to 36 +/- 3 omega3. cm2 caused by inflammation. This was paralleled by a change in epithelial cell TJ structure in UC. Strand count decreased from 6.94 +/- 0.25 to 4.76 +/- 0.47 at the surface and from 7.26 +/- 0.31 to 5.46 +/- 0.37 in the crypts. Conclusions: The inflamed colonic mucosa in UC has an impaired barrier function that is much more pronounced than previously assumed. An altered TJ structure contributes to this barrier defect which, because of increased back leak, can reduce net ion transport. Thus, a leak-flux mechanism contributes to the diarrhea in UC.

Abstract: The potency of in vitro-added corticosteroids to stimulate electrogenic Na+ absorption (JNa, the Na+ absorptive short-circuit current blockable by 10(-4) M amiloride) was determined in rat late distal colon. JNa was determined 8 h after steroid addition from the drop in short-circuit current caused by 10(-4) M amiloride. The concentration dependency of JNa was obtained for seven corticosteroids and compared with that established for aldosterone. Apparent mineralocorticoid potencies as determined from apparent Michaelis-Menten constant (Km) values were as follows: aldosterone 1. 2 nM >> RU-28362 20 nM = deoxycorticosterone 20 nM > deoxycortisol 36 nM >/= dexamethasone 37 nM >> corticosterone 170 nM > cortisol 210 nM. These steroids exhibited Vmax values of 9-13 micromol. h-1. cm-2 and similar concentration dependencies. Hill coefficients were between 1.6 and 2.1, suggesting cooperative effects between activated receptors. We conclude that corticosteroids exhibit graded mineralocorticoid potency instead of a sharp partition into exclusive groups of mineralocorticoid and nonmineralocorticoid hormones. The low apparent Km value of RU-28362 for mineralocorticoid action and the need for high concentrations of the mineralocorticoid antagonist mespirenone to block this response indicated that JNa in a native mammalian epithelium can be mediated by the glucocorticoid receptor. Glucocorticoid receptor-specific amounts of RU-28362 in combination with mineralocorticoid receptor-specific amounts of aldosterone or of the mineralocorticoid antagonist spironolactone showed cooperative action, suggesting a heterodimeric activation of JNa by the glucocorticoid receptor and mineralocorticoid receptor.

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Abstract: Ussing chamber experiments with human intestinal tissue are impeded by the small size of forceps biopsy specimens. Therefore, a miniaturized container insert featuring low edge damage was designed with an exposure area of only 0.05 cm2. It allows measurement of short-circuit current (ISC) and transmural resistance (Rt) on endoscopically obtained biopsy specimens, as well as alternating current impedance analysis and conductance scanning. Comparison with larger specimens mounted in a conventional Ussing chamber without the insert (exposure area 0.54 cm2) was made using rat jejunum and rectum. No differences in ISC, Rt, or secretory response were found, indicating proper sealing and prevention of edge damage, as well as tissue viability in the container system. If biopsy samples obtained from human rectum were mounted in the insert, the local resistance near the edge was almost the same as the overall resistance (52.3 Omega.cm2). Epithelial and subepithelial resistances of human rectum were 43+/-1 Omega.cm2 and 10+/-1 Omega.cm2, respectively. In conclusion, we present a tool that allows reliable Ussing-type, impedance, and conductance scanning measurements to be made from intestinal biopsy specimens.

Abstract: OBJECTIVES: To characterize diarrhoeal mechanisms in HIV-infected patients, epithelial transport and barrier function of the duodenal mucosa was investigated in vitro. PATIENTS: Twenty-one HIV-seropositive patients (13 asymptomatic and eight with diarrhoea) and 12 controls from an urban referral-based tertiary care centre in Berlin who underwent duodenoscopy. METHODS: A new miniaturized Ussing chamber allowed measurements on duodenal forceps biopsies. Epithelial barrier function was characterized by alternating current impedance analysis, which allows differentiation of epithelial and subepithelial resistance and by 3H-lactulose and 3H-mannitol flux measurements. Na+-glucose cotransport was quantified as phlorizin-sensitive short circuit current (Isc) and active ion secretion by baseline and bumetanide-sensitive Isc. RESULTS: Duodenal biopsies from asymptomatic HIV-infected patients were no different from controls, whereas biopsies from HIV-infected patients with diarrhoea showed a decrease in epithelial resistance from 21.2+/-1.9 to 12.9+/-1.3 omega cm2 (P<0.01). Concomitantly, mucosal-to-serosal lactulose flux increased from 0.29+/-0.02 to 0.40+/-0.03 micromol (hcm2) (P<0.01). Phlorizin-sensitive Isc indicating Na+-glucose cotransport, as well as baseline and bumetanide-sensitive Isc indicating active electrogenic chloride secretion were not different between the three groups. CONCLUSIONS: A miniaturized Ussing device was developed for electrophysiological investigations of duodenal forceps biopsies, which allowed characterization of active ion transport mechanisms and epithelial barrier function. Duodenum of HIV-infected patients with diarrhoea showed no evidence for active ion secretion or Na+-glucose malabsorption, but showed an impaired epithelial barrier function, which could contribute to diarrhoea by a leak flux mechanism.

Abstract: This study tested the hypothesis that EGF has a protective effect on the intestinal barrier function in experimental TNBS-induced colitis. EGF was given intraperitoneally one hour before and 24 hours after induction of colitis. The rats were killed 48 hours after induction of colitis: The distal colon was resected and mounted into Ussing chambers. Flux measurements were performed for Na+ and mannitol, and epithelial and subepithelial resistances were determined. A semiquantitative histological score was used to grade acute and chronic inflammation. Compared to controls, TNBS caused a 3-fold increase in both fluxy, indicating enhanced paracellular permeability. There rates was a severe reduction of total and epithelial resistances indicating a dramatic defect of epithelial barrier function. EGF failed to improve the electrophysiologic and histologic parameters. Therefore, EFG has no protective effective in experimental TNBS colitis.

Abstract: The prerequisite for the development of a biohybrid artificial kidney, is a substrate for confluent growth of renal cells forming an epithelial monolayer without any leaks. Conventional cell culture supports cannot be adapted for this purpose, because they lack adequate mechanical properties and thermal stability. From two suitable materials, polysulfone and polyacrylonitrile, two permeable polymeric membranes have been produced that were, according to ISO 10993-5, not cytotoxic. Cloned Madin Darby Canine Kidney (MDCK) cells (an established renal cell line) were cultured on the surface of the plastic materials, and on conventional cell culture supports. With all materials, assays of mitochondrial and lactate dyhydrogenases exhibited similar proliferation and the viability of the MDCK cells. Transmission electron microscopy showed the expression of a normal morphology of kidney tubular cells. Perfect barrier function, consequent on the formation of intercellular junctions in a confluent tight epithelium, was visualized in electron micrographs, and quantified by measurement of the transepithelial resistance. The uniformity of the cells grown was demonstrated in samples by electron microscopy and in the whole epithelium by intravital impedance analysis. It was concluded that polymeric membranes produced from polysulfone or polyacrylonitrile are appropriate substrates in the design of biohybrid kidney devices.

Abstract: The aim of the present study was to determine the changes in permeability and mucosal function after ileo-pouchanal anastomosis (IPAA) in patients with ulcerative colitis. We examined 24 patients (m: f = 15:9; Age = 35.2 +/- 12.5) prior to colectomy (pre IPAA), prior to closure of ileostomy (pre CIS), after closure of ileostomy (post CIS), in case of pouchitis and 5 controls. With a miniaturizised Ussing-chamber electrophysiological parameters of permeability and resorptive isotope-fluxes for mannitol were measured. Total resistance could be differenciated into epithelial and subepithelial resistance by using alternating current impedance measurements. Active Na(+)-glucose-Cotransport was measured by detection of the maximal short-curcuit-current under stepwise addition of glucose and active Cl(-)-secretion was measured by the increase in short-curcuit-current after stimulation with Theophillin and PGE2. Epithelial resistance of pouchitis was slightly raised towards controls but did not differ from the others. Subepithelial resistance post CIS and pouchitis was significantly increased versus controls and pre CIS (21.2 +/- 22.4 vs. 10.7 +/- 13.3 omega cm2) Whereas Cl-secretion of pouchitis was significantly reduced vs. post CIS and controls (38.6 vs. 120.9 +/- 113.9 microA cm-2). In the same way Na(+)-glucose Cotransport of pouchitis was significantly reduced vs. post CIS and controls(41.8 vs. 138.4 +/- 264.4 microA cm-2). Increasing subepithelial resistance post CIS and in pouchitis is a hint for an adaptive thickening of the subepitheal layer. Concerning the functional analysis pouchitis and terminal ileum prior to IPAA show a reduced secretion and resorption whereas the mucosal barrier towards Mannitol remains unchanged.

Abstract: The aim of this study was to analyze the effect of enterally given zinc on the impaired epithelial barrier function in experimental TNBS colitis in rats. Rats in therapy group were zinc-fed in therapeutic dose. 48 hours after induction of TNBS colitis the rats were killed, the distal colon was resected and mounted into an Ussing chamber. The following electrophysiological measurements were carried out: 1. Flux measurements for Na+ and mannitol as a parameter of paracellular permeability, 2. Resistance measurements of the colon, distinguishing between pure epithelial resistance and resistance of the subepithelial tissue. In TNBS colitis we found a marked increase of both fluxes as a parameter of enhanced paracellular permeability (factor 3). Further there was a drastic reduction of total resistance, and especially of pure epithelial resistance indicating a massive epithelial barrier defect. In rats treated by zinc the increase of paracellular permeability was increased only by a factor of 2, and there was only a moderate decrease of resistance. For the first time we demonstrated zinc to have a protective effect on damaged mucosal barrier function in TNBS colitis. These results confirm the data collected in guinea pigs with malnutrition, whose intestinal integrity could be improved by zinc. Further studies are necessary in order to test whether this protective effect of zinc on mucosa barrier in experimental colitis may be relevant also in human ulcerative colitis.

Abstract: Tight junction morphology was analyzed in freeze fracture electron micrographs from biopsies at two locations along the surface-crypt axis in the jejunum of children with treated and untreated sprue and in control subjects. In control jejunum, strand number, meshwork depth, and total depth of the tight junction decreased from surface to crypt, consistent with the concept of the crypt being more permeable than the surface epithelium. In acute sprue, strand number was reduced in all regions along the surface-crypt axis, from 5.5+/-0.2 to 3.4+/-0.3 (surface) and from 4.7+/-0.2 to 3.6+/-0.1 (crypt). Meshwork depth was also reduced at all regions along the surface-crypt axis. Strand discontinuities were more frequent in acute sprue. Aberrant strands appeared below the main meshwork of crypt tight junctions in acute sprue. In asymptomatic children treated with the gluten-free diet, jejunal tight junctional structure only partially recovered. Strand number was restored to normal at the surface, but was still decreased in the crypts, from 4.7+/-0.2 to 3.9+/-0.3. We conclude that the epithelial barrier function of the small intestine is seriously disturbed by structural modifications of the tight junction in acute symptomatic celiac disease, thereby accounting for increased ionic permeability noted in a parallel study on identical specimens. This epithelial barrier defect may contribute to diarrhea in celiac disease by a "leak flux mechanism." In children with sprue treated with a gluten-free diet, barrier dysfunction was only partly recovered, suggesting a level of "minimal damage."

Abstract: 1. Aldosterone- and adrenaline-induced K+ secretion were investigated in rat late distal colon using conductance scanning and Ussing chamber techniques. K+ secretion was unmasked by the K+ channel blocker tetraethylammonium (TEA). Electrogenic Na+ absorption was inhibited by amiloride. Rb+ net fluxes consistently measured about 80% of K+ secretion estimated using change in short-circuit current (delta ISC) measurements. 2. Partial block of K+ absorption by mucosal ouabain did not change TEA-sensitive K+ secretion. Thus, K+ absorption and K+ secretion are not coupled. 3. Additivity of Rb+ fluxes as well as delta ISC caused by 3 nM aldosterone (6 h in vitro incubation) and, subsequently, adrenaline suggested additivity of aldosterone-induced and cAMP-mediated K+ secretion in the presence of amiloride. 4. Conductance scanning under control conditions revealed a small TEA-sensitive K+ conductivity in surface epithelium (0.3 +/- 0.2 mS cm-2) but not in crypts, as well as a small basal K+ secretion in surface epithelium (delta ISC = 0.3 mumol h-1 cm-2), which increased during sham incubation. 5. Aldosterone (3 nM, 6 h in vitro incubation) resulted, after correction for the basal K+ secretion, in a K+ secretion of delta ISC = 0.9 mumol h-1 cm-2. Aldosterone induced a TEA-sensitive conductivity of 1.1 +/- 0.3 mS cm-2 in surface epithelium, but not in crypts. 6. Adrenaline (5 microM) caused, in fresh tissue, a K+ secretion of delta ISC = 1.2 mumol h-1 cm-2 and equal conductivity changes in crypts (0.7 +/- 0.2 mS cm-2) and surface epithelium (0.7 +/- 0.1 mS cm-2). 7. We conclude that K+ secretion induced by aldosterone in physiological concentration is restricted to surface epithelium, whereas cAMP-mediated K+ secretion is located equally in crypts and surface epithelium.

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Abstract: Translocation of bacteria from the intestine causes local and systemic infection in severe acute pancreatitis. Increased intestinal permeability is considered a promoter of bacterial translocation. The mechanism leading to increased gut permeability may involve impaired intestinal capillary blood flow. The aim of this study was to evaluate and correlate early changes in capillary blood flow and permeability of the colon in acute rodent pancreatitis of graded severity. Edematous pancreatitis was induced by intravenous cerulein; necrotizing pancreatitis by intravenous cerulein and intraductal glycodeoxycholic acid. Six hours after induction of pancreatitis, the permeability of the ascending colon was assessed by the Ussing chamber technique; capillary perfusion of the pancreas and colon (mucosal and subserosal) was determined by intravital microscopy. In mild pancreatitis, pancreatic capillary perfusion remained unchanged (2.13 c 0.06 vs. 1.98 +/-0.04 nl x min(-1) x cap(-1) [control]; P = NS), whereas mucosal (1.59 +/-0.03 vs. 2.28 +/-0.03 nl x min(-1) x cap((-1))[control]; P <0.01) and subserosal (2.47 +/-0.04 vs. 3.74 +/-0.05 nl x min(-1) x cap((-1))[control]; P <0.01) colonic capillary blood flow was significantly reduced. Severe pancreatitis was associated with a marked reduction in both pancreatic (1.06 +/-0.03 vs. 1.98 +/-0.04 nl x min(-1) x cap(-1) [control]; P <0. 01) and colonic (mucosal: 0.59 +/-0.01 vs. 2.28 +/-0.03 nl x min(-1) x cap((-1))[control], P <0.01; subserosal: 1.96 +/-0.05 vs. 3.74 +/-0.05 nl x min(-1) x cap(-1) [control], P <0.01) capillary perfusion. Colon permeability tended to increase with the severity of the disease (control: 147 +/-19 nmol x thr(-1) x cm(-2); mild pancreatitis: 158 +/-23 nmol x hr(-1) x cm(-2); severe pancreatitis: 181 +/-33 nmol x hr(-1) x cm(-2); P = NS). Impairment of colonic capillary perfusion correlates with the severity of pancreatitis. A decrease in capillary blood flow in the colon, even in mild pancreatitis not associated with significant protease activation and acinar cell necrosis or impairment of pancreatic capillary perfusion, suggests that colonic microcirculation is especially susceptible to inflammatory injury. There was no significant change in intestinal permeability in the early stage of pancreatitis, suggesting a window of opportunity for therapeutic interventions to prevent the later-observed increase in gut permeability, which could result in improved intestinal microcirculation.

Abstract: Inflammatory bowel disease (IBD) and HIV infection can cause diarrhoea which is accompanied by elevated cytokine levels. To elucidate a pathogenic role of cytokines, their effect on ion secretion was studied in human distal colon using the Ussing technique. Interluekin 1beta (IL-1beta) dose dependently increased short-circuit current (ISC). An ISC maximum of 2.5+/-0.3 micromol. h-1.cm-2 was reached at 20 ng/ml within 43+/-4 min. 22Na+ and 36Cl- fluxes were not altered and residual flux increased by 2.4+/-1.0 micromol.h-1.cm-2 indicating that the IL-1beta-induced ISC is based on electrogenic bicarbonate secretion. IL-1beta had no effect on HT-29/B6 epithlial monolayers suggesting that IL-1beta does not act directly on the epithelium. Furthermore, in human colon the effect was not attenuated by removal of the submucosa (total stripping) pointing to a mediation step via subepithlial cells in the lamina propria. While tetrodotoxin and the 5-lipoxygenase inhibitor ICI-230487 had no effect, indomethacin completely blocked IL-1beta action. Prostaglandin determination by RIA revealed an increased production of PGE2. At half maximum effective concentrations an additive action of tumour necrosis factor alpha (TNF-alpha) could be demonstrated on IL-1beta-induced secretion. Interferon alpha (IFN-alpha), IFN-gamma, IL-6, and IL-8 had no seretory effect in human distal colon. None of the investigated cytokines altered the intestinal barrier function. By their secretory effects IL-1beta and TNF-alpha, but not IFN-alpha, IFN-gamma, IL-6, and IL-8, may contribute to diarrhoea in IBD and AIDS.

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Abstract: The barrier function of the intestinal wall plays a key role in body homeostasis and defense against noxious agents. Conventional Ussing chamber techniques determine the overall transmural resistance but do not differentiate epithelial and subepithelial tissues. The barrier function, however, resides in the epithelial cell layer only. Transmural impedance analysis can solve this problem, if adequate models are applied. We show that: (i) epithelial and subepithelial impedances are additive, (ii) the epithelium proper can be represented by a very general electrical model, which demonstrates short-circuiting at high frequencies (due to cell membrane capacitances), and (iii) the reactance of subepithelial tissue can be described phenomenologically. Using an empirical expression for description of the subepithelial impedance, the present method allows the determination of the epithelial and the subepithelial resistance. This was exemplified in rat ileum, which defied adequate impedance analysis so far. Of the transmural DC resistance of 61 +/- 5 omega.cm2 (n = 8) the subepithelial contribution was 28 +/- 2 omega.cm2 and the epithelial resistance was 33 +/- 4 omega.cm2.

Abstract: We compared the efficacy of three dual and two triple therapies for eradication of Helicobacter pylori (HP), and evaluated the influence of smoking and omeprazole pretreatment on HP eradication. 220 patients with proven HP infection (histology and 13C-urea breath test [UBT]) were randomly allocated to one of the following regimes: BMT (bismuth subsalicylate 600 mg t. i. d. for 28 days, metronidazole 400 mg t. i. d. and tetracycline 500 mg q. i. d. for ten days). OA (omeprazole 40 mg o. d. and amoxicillin 750 mq q. i. d. for 14 days), OC (omeprazole 40 mg o. d. and clarithromycin 500 mg b. i. d. for 14 days), OT (omeprazole 40 mg o. d. and tetracycline 500 mg q. i. d. for 14 days), OMC (omeprazole 40 mg o. d., metroinidazole 400 mg t. i. d. and clarithromycin 250 mg b. i. d. for seven days). Eradication was defined as negative UBT six weeks after completion of the therapy. In an "all-patients-treated" ("per-protocol") analysis, the eradication rates were: BMT, 91% (93%); OA, 84% (90%); OC, 74% (74%); OT, 24% (24%); and OMC, 90% (93%). Smoking impaired the success of OA and OT (p < 0.05), but the efficacy of the triple regimens was not affected. Omeprazole pretreatment did not influence eradication rates. Thus, highest eradication rates were achieved with the two triple therapies tested. However, OA, given at a daily antibiotic dose of 3 g amoxicillin for 14 d, was also highly effective. After failure of triple therapy, OA was successful in seven of ten patients (70%). The efficacy of OC was lower than that of the triple therapies (p < 0.05). In conclusion, metronidazole- and clarithromycin-based triple therapies are highly effective first line therapies. OA, given at a dose of 3 g per day over 14 days, should be considered as a possible second line therapy, e.g. in retherapy after failed triple therapy.

Abstract: Mucosal adaptation of the small intestine is morphologically restricted to only three different patterns, namely, atrophy, hyperplasia, and hyperregeneration. The hyperplastic mucosa in the experimental short bowel syndrome exhibits unchanged epithelial barrier properties and a differential functional adaptation with a 150% increase in Na-glucose cotransport but no change in electroneutral NaCl cotransport. In the hyperregeneratively transformed mucosa of the self-filling blind loop of rat jejunum, absorption is seriously impaired, as indicated by the 80% decrease in Na-glucose cotransport. To compensate for this, epithelial barrier function is upregulated by an increase in tight junction complexity to prevent leak flux of ions and substrates. In contrast, the hyperregeneratively transformed mucosa in celiac sprue shows reduced tight junction complexity. Possible candidates responsible for the heterogeneity of tight junction adaptation in these conditions could be cytokines, because tumor necrosis factor-alpha can specifically downregulate the tight junction, as indicated in the intestinal HT-29/B6 cell model.

Abstract: More than 50% of AIDS patients suffer from diarrhea, the cause of which is unexplained in 15-40%. In 60-85% a potential cause for diarrhea could be found but the pathogenic relevance often remains unclear. In this paper we review the current knowledge about the nonmalabsorptive mechanisms for diarrhea in HIV infection from a pathophysiologic point of view and we show evidence that the diarrhea in advanced HIV infection by HIV per se could be caused by a leak flux mechanism due to an epithelial barrier defect.

Abstract: A new method, conductance scanning, allows determination of local para- and transcellular conductivities in flat epithelia. Experiments were performed on kidney distal tubule cells, MDCK clone C11, which form monolayers on permeable supports. Above the apical surface, local voltage drops generated by a sinusoidal current clamp were recorded by means of a scanning microelectrode. Data were collected above cell centres and tight junctions. The scanning signal was always significantly higher above the tight junctions, but was uniformly distributed along the junctions. For determination of conductivities two procedures were applied. Method 1: the supraepithelial potential distribution was computed for given trans- and paracellular currents at all positions of the electrode. In a fit algorithm, the currents were varied until the calculated potential difference equalled the voltage measured. Method 2: after collecting scanning data in control Ringer's, intercellular space width was reduced by mucosal addition of 40 mM sucrose and a second set of data was obtained at decreased paracellular, but presumably unchanged transcellular, conductivity. From these data, trans- and paracellular conductivities were calculated. Results of both methods were in excellent agreement. Confluent MDCK-C11 monolayers exhibited a transepithelial conductivity of 13 mS/cm2. The transcellular pathway contributed 2.6 mS/cm2 (20%) and the paracellular pathway 10. 5 mS/cm2 (80%) to the total conductivity. Collapse of the lateral intercellular spaces decreased the paracellular conductivity to 4 mS/cm2 (60%). Confluent MDCK-C11 monolayers constitute true "leaky" epithelia with homogeneously distributed trans- and paracellular conductivities. In conclusion, conductance scanning fills a methodical gap, which hitherto impeded the functional characterization of tight junctions.

Abstract: An Ussing chamber was designed for impedance analysis of epithelial tissue and optimized for the use of high-frequency alternating current stimuli. Shielded voltage electrodes, located axially within the electric field of the Ussing chamber, minimized the reactive properties of the set-up. By vectorial subtraction, the small reactive contribution of the optimized Ussing chamber was completely compensated for. This method allowed for transmural impedance measurements in a frequency range of 1-65 kHz. For the first time, Nyquist plots of cultured intestinal cell monolayers (HT-29/B6) are presented. The epithelial monolayers in different stages of confluence showed the impedance locus as a semicircle, with the high frequency end close to the origin. These epithelial monolayers could be modeled by a simple RC-parallel circuit without a series resistance.

Abstract: BACKGROUND: Which protocol is optimal for the 13C-urea breath test (UBT) for Helicobacter pylori detection is controversial. This study aimed to characterize a very simple UBT protocol for the clinical routine (two-point-analysis performed with 75 mg 13C-urea and citric acid) with special consideration of 'false' UBT results. RESULTS; UBT was evaluated in reference to histology (Warthin-Starry). In mismatching results re-gastroscopy was performed. By UBT, 74 of 77 patients with H. pylori-positive histology were detected (sensitivity, 96%). The false-negative UBTs were due to low colonization densities during spontaneous H. pylori elimination or pyloric obstruction. Seven of 49 patients with negative histology had a positive UBT, but re-gastroscopy showed that all of them had a positive histology when multiple antral biopsy specimens were taken (UBT specificity, 100%). UBT correlated only weakly with H. pylori colonization density. No correlation was found between UBT and gastric neutrophil and lymphocyte infiltration. UBT reproducibility was excellent (93 of 94 in a 6-month period). Non-fasting conditions induced a shift to lower UBT results in H. pylori-positive and to higher UBT results in negative patients, resulting in 2 of 10 false-positive and 1 of 10 false-negative UBTs. CONCLUSION: This simple version of the urea breath test combines the highest sensitivity with excellent reproducibility. It is superior to histologic detection of H. pylori in the clinical routine and an optimal tool for monitoring H. pylori eradication. Fasting conditions are required for the test.

Abstract: Intestinal barrier failure and subsequent translocation of bacteria from the gut play a decisive role in the development of systemic infections in severe acute pancreatitis. Glutamine (GLN) has been shown to stabilize gut barrier function and to reduce bacterial translocation in various experimental settings. The aim of this study was to evaluate whether GLN reduces gut permeability and bacterial infection in a model of acute necrotizing pancreatitis. Acute necrotizing pancreatitis was induced in 50 rats under sterile conditions by intraductal infusion of glycodeoxycholic acid and intravenous infusion of cerulein. Six hours after the induction of pancreatitis, animals were randomly assigned to one of two groups: standard total parental nutrition (TPN) or TPN combined with GLN (0.5 g/kg(-1)/day(-1)). After 96 hours, the animals were killed. The pancreas was prepared for bacteriologic examination, and the ascending colon was mounted in a Ussing chamber for determination of transmucosal resistance and mannitol flux as indicators of intestinal permeability. Transmucosal resistance was 31% higher in the animals treated with GLN- supplemented TPN compared to the animals given standard TPN. Mannitol flux through the epithelium was decreased by 40%. The prevalence of pancreatic infections was 33% in animals given GLN-enriched TPN as compared to 86% in animals receiving standard TPN (P < 0.05). Adding GLN to standard TPN not only reduces the permeability of the colon but decreases pancreatic infections in acute necrotizing pancreatitis in the rat. This confirms previous reports that GLN decreases bacterial translocation by stabilizing the intestinal mucosal barrier. The present findings provide the first evidence suggesting that stabilizing the intestinal barrier can reduce the prevalence of pancreatic infection in acute pancreatitis and that GLN may be useful in preventing septic complications in clinical pancreatitis.

Abstract: Cholinergic stimulation triggers the secretion of apically stored, preformed mucin from goblet cells but the pathway of cAMP-stimulated mucin secretion is not known. In this study the effect of cholera toxin on mucin secretion in the human colonic goblet cell line HT-29/B6 was investigated and compared to the action of carbachol. PAS staining of mucin blotted onto nitrocellulose served to quantify the secretion of total mucin. Metabolic labelling was used to evaluate the secretion of newly synthesized mucin. The mucinous nature of the detected material was confirmed with an immunoblot employing a well-characterized polyclonal antibody reacting with MUC2-mucin. Cholera toxin caused a 116-fold increase of intracellular cAMP and strongly stimulated the secretion of both preformed and newly synthesized mucin for more than 20 h. Carbachol only triggered the release of preformed mucin immediately after addition. The secretory response to cholera toxin could be partly inhibited by the protein kinase A inhibitor H8 and the microtubule inhibitor colchicine. The action of carbachol was not affected by these agents. In conclusion, we demonstrate a direct cAMP-dependent effect of cholera toxin on mucin secretion by intestinal goblet cells. In contrast to carbachol, the action of cholera toxin involves de novo synthesis of mucin molecules and microtubule-mediated secretion. There seem to be distinct secretion pathways for muscarinic or cAMP-dependent stimulation of mucin secretion.

Abstract: The effect of cell swelling induced by hypotonic media was studied in segments of rat small intestine. In the Ussing chamber, exposure to a hypotonic medium caused a decrease in short-circuit current (Isc) and potential difference (Vms) in the jejunum, whereas the ileum responded with an increase in Isc and Vms. The transition from one pattern to the other was located about in the middle of the small intestine. Tissue conductance decreased in both segments, probably due to a reduction of paracellular shunt conductance induced by the cell swelling. Voltage scanning experiments revealed that the observed decrease in total tissue conductance in the ileum was caused solely by a decrease in local conductance in the villus region while the crypt conductance did not change, suggesting that the decrease in paracellular conductance of the crypts is compensated by an increase in cellular conductance. The response in both segments was dependent on the presence of Cl- and was blocked by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). It was not affected by the neurotoxin tetrodotoxin. In the jejunum the swelling-induced decrease in Isc was reduced in the presence of the cyclooxygenase inhibitor, indomethacin, or the lipoxygenase inhibitor, nordihydroguaiaretic acid. In the ileum the Cl- secretion induced by hypotonicity was blocked by the K+ channel blocker quinine and was reversed into a decrease in Isc when serosal Ca2+ was zero. We conclude that the observed volume regulatory changes are initiated in the jejunum by an eicosanoid-mediated opening of basolateral Cl- channels and in the ileum by a Ca2+-mediated opening of K+ channels which enhances apical Cl- efflux.

Abstract: Increased levels of tumor necrosis factor-alpha (TNF-alpha) have been found in, for example, inflammatory bowel disease (IBD) and human immunodeficiency virus (HIV) infection. To investigate a possible contribution of TNF-alpha to the pathogenesis of diarrhea in these diseases, ion transport of human distal colon was studied in the Ussing chamber in vitro. Serosal addition of TNF-alpha increased short-circuit current (Isc) of partially stripped tissues in a dose-dependent manner. Maximum Isc increase of 1.8 +/- 0.2 mumol.h-1.cm-2 was reached after 60 +/- 9 min at 200 ng/ml TNF-alpha. Bidirectional tracer flux measurements revealed that TNF-alpha induced an increase in 36 Cl serosal-to-mucosal flux, a decrease in 36Cl- mucosal-to-serosal flux, and a slight increase in K+ secretion indicated by an increased secretory 86Rb net flux. In the highly differentiated colonic epithelial cell line HT-29/B6, TNF-alpha had no effect on Isc, suggesting a mediation step located in the subepithelium. This supposition was supported by measurements on totally stripped human tissues, since removal of subepithelial layers by total stripping reduced the TNF-alpha effect by 40%. Experiments with tetrodotoxin (10(-6)M) indicated that the TNF-alpha effect was not mediated by the enteric nervous system. The specific 5-lipoxygenase blocker ICI-230487 (5 x 10(-8)M) also had no effect on TNF-alpha action. In contrast, inhibition of cyclooxygenase by indomethacin (10(-6)M inhibited the effect of TNF-alpha. Radioimmunoassay of prostaglandin E2 (PGE2) in the serosal bathing solution revealed an increase in PGE2 production/release after addition of TNF-alpha, which paralleled the Isc response. We conclude that TNF-alpha changed Cl- and K+ transport toward secretion in human colon. This effect was mediated by PGE2 produced by subepithelial cells. Thus TNF-alpha could be a mediator of diarrhea during intestinal inflammation, e.g., in IBD and HIV infection.

Abstract: Distension of rat rectal colon causes electrogenic Cl- secretion via the plexus submucosus Meissner. This study aimed to identify the neurotransmitter(s) of this reflex pathway. Distension was applied to partially stripped rat rectal colon in Ussing chambers. Baseline short-circuit current (Isc) increased and then slowly declined again within 30 min. The increase in Isc 10 min after distension (delta Isc10) was 1.8 +/- 0.3 mumol.h-1.cm-2. Atropine (1 microM) did not alter delta Isc10. Thus cholinergic neurons with muscarinic synapses were not involved. Tissues were then desensitized to vasoactive intestinal peptide (VIP) or substance P. This required continuous infusion of VIP or substance P into the chamber; otherwise, desensitization was only temporary due to rapid degradation of VIP or substance P. During substance P desensitization, distension still induced a secretory response (delta Isc10 not significant vs. control), whereas during VIP desensitization distension no longer had an effect. Furthermore, a polyclonal anti-VIP antiserum blocked 81% and the VIP antagonist [p-Cl-D-Phe6,Leu17]VIP blocked 89% of the distension-induced delta Isc10, supporting the results of the desensitization experiments. To localize the site of VIP action, tetrodotoxin (TTX) was used. The TTX effect on Isc during VIP stimulation was not different from its effect on baseline Isc. This is in accord with the concept that the VIP receptors are mainly located on the enterocytes. We conclude that VIP, but not substance P or acetylcholine (via muscarinic receptors), acts as a neurotransmitter in the distension-induced reflex pathway, causing Cl- secretion in rat rectal colon.

Abstract: In vivo electrogenic Na+ absorption (JeNa) in the human rectum is controlled by acute variation of aldosterone in nanomolar concentration range. In this study we report both the induction of JeNa in human rectum epithelium by nanomolar aldosterone added in vitro and the enzymatic control of glucocorticoid action on JeNa. JeNa was measured as amiloride-sensitive short-circuit current 8 h after addition of the respective steroid. Aldosterone (10 nM) caused JeNa of 5.7 +/- 1.4 mumol.h-1.cm-2. Cortisol in the same concentration did not induce significant JeNa. Because cortisol is readily inactivated by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), the true mineralocorticoid activity of cortisol was evaluated after inhibition of 11 beta-HSD by carbenoxolone. Carbenoxolone alone did not exhibit mineralocorticoid activity. If cortisol (10 nM) was given together with carbenoxolone (1 microM), the resulting JeNa (4.5 +/- 0.4 mumol.h-1.cm-2) was not significantly different from that after 10 nM aldosterone, indicating equal intrinsic mineralocorticoid activity of cortisol and aldosterone. The same mechanisms were found in rat late distal colon. Kinetic data of carbenoxolone at 10 nM cortisol resulted in a Michaelis constant of 0.3 microMs, maximal absorption of 8.4 mumol.h-1.cm-2, and a Hill coefficient of 1.8. The effects of carbenoxolone and glycyrrhetinic acid did not differ. We conclude that JeNa is under complete control of mineralocorticoid action. "Spontaneous" JeNa in the beginning of the in vitro period can be explained by elevated steroid levels before tissue removal.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Epithelial barrier function and ion transport was studied in coeliac sprue using a miniaturised Ussing device for measurements on diagnostic aspiration biopsy specimens from the jejunum of untreated or gluten free nourished sprue patients, or from healthy controls. Pure epithelial resistance (Re) indicating epithelial barrier function was determined by transmural alternating current impedance analysis. It was reduced by 56% in acute sprue mean (SEM) (9 (1) omega.cm2) compared with controls (20(2) omega.cm2). In gluten free nourished sprue patients Re was only partly recovered (15 (1) omega.cm2). Subepithelial resistance (Rsub) was also changed from 28 (1) omega.cm2- in control to 17 (1) omega.cm2 in acute sprue because of the change in mucosal architecture, but was unchanged in gluten free nourished sprue patients (29 (4) omega.cm2). In acute sprue, unidirectional Na+ and Cl- fluxes were increased in both directions as a consequence of the decreased resistance. However, short circuit current (ISC) as well as Na+ and Cl- net fluxes were not significantly different from control. Subsequently, the electrogenic Cl- secretory system was investigated. After maximal stimulation with theophylline and prostaglandin E1, a Cl(-)-dependent increase in ISC was obtained in the sprue mucosa and control jejunum. It showed saturation characteristics and was blockable by serosal bumetanide. When compared with control, neither Km nor Vmax of this electrogenic Cl- secretion was significantly changed in coeliac sprue. In conclusion, a miniaturised Ussing device was used for transport measurements on intestinal biopsy specimens. In acute coeliac disease, the epithelial barrier of the jejunum was seriously disturbed. The active electrogenic Cl- secretory transport system was present in the sprue mucosa, but was not activated in the Ussing chamber in vitro when compared with control jejunum.

Abstract: Intramembrane specializations of cultured bovine corneal endothelial cells were studied with thin section and freeze-fracture electron microscopy and related to the paracellular permeability and the transendothelial resistance (Rt) of the monolayers. The following intercellular junctions were found: single and discontinuous networks of tight junctions (TJ) which girdle the apico-lateral cell perimeter incompletely, gap junctions, and membrane undulations suggesting intermediate junctions. The macromolecular tracer ruthenium red penetrated into the lateral intercellular space beyond the level of the incomplete belt of TJ. Rt of these monolayers was 20.9 +/- 1.0 omega.cm2. Protamine induced a reversible increase of Rt to 118 +/- 5% of its control value. We conclude that incomplete belts of TJ may be the morphological counterpart of the high paracellular permeability of this monolayer and functionally and morphologically resemble those of their native endothelium. Cultured corneal endothelial cells are an excellent model for studying the influence of incomplete belts of TJ on paracellular permeability of cells.

Abstract: BACKGROUND: Yersinia enterocolitica is an important cause of diarrhea, but little is known about the underlying mechanisms. We therefore studied the impact of acute Y. enterocolitica infection on intestinal barrier function in a mouse model. METHODS: For this purpose CD-1 mice were infected with Y. enterocolitica (serotype 08; 6 x 10(7) viable bacteria), and alternating current impedance analysis was performed on days 1, 2, 3, 5, and 8 after infection. RESULTS: The infection resulted in a decrease in epithelial resistance from 18.0 +/- 0.9 omega.cm2 (controls) to 12.1 +/- 0.5 omega.cm2 (day 1, p < 0.001), from which the animals recovered by day 5. To locate this loss in barrier function, the horizontal distribution of local conductances was measured by voltage scanning, yielding two results. First, conductance was homogeneously distributed across the chamber area, excluding erosions or ulcers among the gross surface area and favoring tight junction opening as the source of barrier dysfunction. Second, the conductance of villus tips was compared with that of the intervillus region (consisting of lateral villus walls plus crypts). On day 1 the former was increased by 74% and the latter by 18%. Then, two other mechanisms of diarrhea were tested, namely malabsorption and secretion. First, the increase in ISC after the addition of 3-O-methylglucose, representing Na(+)-glucose cotransport, was shown not to be impaired. Second, bumetanide-inhibitable ISC, representing electrogenic Cl- secretion, also did not differ between controls and infected animals. CONCLUSIONS: Our data show that epithelial barrier dysfunction plays a role in Y. enterocolitica infection, while Na(+)-glucose cotransport and electrogenic Cl- secretion are unaltered.

Abstract: The corneal endothelium controls the hydration and nutrition of the avascular corneal stroma. To analyze the role of the tight junctions (TJ) for these functions, we examined human corneal endothelium by thin-section and freeze-fracture electron microscopy and by impedance analysis. On thin sections, tannic acid was seen to mark the external leaflet of the lateral plasma membrane also beyond the location of the TJ, indicating a significant macromolecular porosity of the TJ. On freeze-fracture images, the TJ surrounded the entire apicolateral plasma membrane but were found to be focally incomplete, suggesting a nonhomogeneous seal of the lateral intercellular space. Impedance analysis revealed a very leaky endothelial layer with a transendothelial resistance of 9.0 +/- 1.4 omega cm2. These findings are consistent with the hypothesized "pump-leak" model of the corneal endothelium: the TJ allow an effective dehydration of the corneal stroma, whereas the interruptions in the TJ network may be the morphological correlate for the passage of nutrients into the corneal stroma. Our data corroborate the assignment of the corneal endothelium to the group of "very leaky epithelia" that exhibit high transport rates of water and solutes against only minimal osmotic gradients.

Abstract: Increase in fetal surfactant synthesis and lung maturity is caused by the glucocorticoidal induction of enzymes required for phosphatidylcholine (PC) synthesis towards the end of gestation. The regulation of gestational age-dependent induction of PC synthesis by glucocorticoids is still unclear. Since 11-beta-hydroxysteroid dehydrogenase (11 beta-HSD) activity and its metabolising capacity for glucocorticoids have been suggested to play a central role in this regulation, we measured the gestational age-dependent changes in 11 beta-HSD and PC synthesizing enzymes in lung and liver of fetal rat. The activity of cholinephosphate cytidyltransferase (CCT; key enzyme in PC synthesis), choline phosphotransferase (CPT) and lysolecithin acyltransferase (LAT) were found to increase gradually in the lung towards the end of gestation, reached peak values at term followed by a decrease of activity reaching finally adult levels. Only CK activity exhibited constant levels until term followed by a slight increase after the birth. In comparison with the lung, the liver enzymes followed a similar pattern, but at a higher rate of activity except for CCT which was higher in the lung. The activity of 11 beta-HSD in fetal lung microsomes was detectable from day 20 and increased towards the end of gestation in the lung and liver of the rat. Oxidase activity was always found to exceed the reductase activity. The activity of 11 beta-HSD continued to increase after delivery and reached peak levels in adult animals in both organs. In order to test the hypothesis, whether 11 beta-HSD activity and PC synthesis are induced by increasing endogenous glucocorticoidal levels, we examined on day 19 of gestation the effect of dexamethasone (DEXA) on enzymatic activities (11 beta-HSD, CCT) and on [14C]choline incorporation in phosphatidylcholine in fetal lung organoid cultures. Additionally, changes in CCT activity in fetal lungs after maternal administration of DEXA were measured. DEXA accelerated 11 beta-HSD and CCT activities as well as [14C]choline incorporation. We conclude, that endogenous glucocorticoids induce PC synthesis as well as 11 beta-HSD activity in lung and liver of the fetal rat. Fetal PC synthesis is not altered by increasing 11 beta-HSD levels, because the increase of free serum corticosterone levels apparently exceeds the metabolising capacity of 11 beta-HSD towards term.

Abstract: There is no quantitative assignment of large intestinal electrogenic Na+ absorption to surface epithelium and crypts so far. We determined the spatial distribution of electrogenic Na+ absorption to crypts and surface epithelium of rat late distal colon using a modified voltage-scanning technique. Voltage deflections resulting from external 30-Hz current were sensed by an extracellular microelectrode stepping at 0.7 Hz above crypt openings or surface epithelium. Local conductances were calculated applying a planar model of electrical field distribution to surface epithelium and a electrostatic disk source model to the crypts. These models were confirmed by methodological experiments where the electrode position was varied in vertical and horizontal direction. Electrogenic Na+ absorption was detected by blocking apical Na+ channels by mucosal 0.1 mM amiloride. Under control conditions surface epithelium contributed 44% (2.0 +/- 0.2 mS/cm2) and crypts 56% (2.6 +/- 0.2 mS/cm2) to the total conductance of 4.6 +/- 0.4 mS/cm2. Electrogenic Na+ absorption was induced by 6 h in vitro incubation in a medium containing 3 nM aldosterone. This caused a short-circuit current (ISC) of 12.1 +/- 0.8 mumol.h-1.cm-2, which was paralleled by a 2.5-fold increase in surface epithelial conductance to 5.1 +/- 0.4 mS/cm2, whereas crypt conductance was not significantly altered (3.0 +/- 0.2 mS/cm2). Amiloride reversed ISC to -0.8 +/- 0.1 mumol.-1.cm-2 and decreased surface epithelium conductance to 2.3 +/- 0.3 mS/cm2 but again had no significant effect on crypt conductance (2.5 +/- 0.3 mS/cm2). Sham incubation (no hormones added) for 6 h neither induced electrogenic transport nor altered local epithelial conductances.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: In ulcerative colitis and polyposis coli, creation of an ileal pouch with ileoanal anastomosis after proctocolectomy has become a common surgical method. The aim of our study was to characterize the adaptation of the epithelial ion transport function in the pouch by using electrophysiologic techniques. Proctocolectomy and ileoanal anastomosis was performed in rats either with (pouch) or without (control) creation of an ileal J-pouch. To characterize the epithelial barrier function, impedance analysis was performed 6 months after surgery. Epithelial resistance was 29 +/- 2 omega.cm2 in controls and was unchanged in the pouch (28 +/- 4 omega.cm2; NS). In contrast, subepithelial resistance increased from 33 +/- 3 omega.cm2 to 54 +/- 5 omega.cm2 (P < 0.01) owing to work hypertrophy of the muscle layers in the pouch. To detect changes in net ion transport, ion tracer fluxes were measured in the Ussing chamber. In control ileum, absorptive net Na and Cl fluxes of similar magnitude were present, indicating that electroneutral NaCl absorption was the predominant ion transport system. Neither Isc nor net Na and Cl fluxes were significantly altered in the pouch. Glucose-coupled Na absorption was measured as the 3-o-methyl-glucose-induced increase in Isc. Km remained unaltered, while Vmax decreased from 7.5 +/- 2.1 mu eq.h-1 cm-2 in controls to 1.7 +/- 0.8 mu eq.h-1 cm-2 (P < 0.05) in the pouch. Then, maximal transport capacity for electrogenic Cl secretion was measured as the Cl-induced increase in Isc blockable by serosal bumetanide (in the presence of theophylline and prostaglandin E1).(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: A short-circuit current experiment on epithelial ion transport is described that is suitable for student classes in human and animal physiology. Segments of late distal colon from either pig or cow are obtained from the slaughterhouse depending on the animals' daily schedule. Initial tissue preparation already in the slaughterhouse, cold storage, and proper choice of bath solutions are essential prerequisites for success. Students monitor spontaneous transepithelial voltage and short-circuit current (Isc) by use of manually operated voltage clamp units. Two main transport mechanisms are studied, electrogenic Na+ absorption and Cl- secretion. Electrogenic Na+ absorption is studied by measuring the Isc drop after amiloride. Then Cl- secretion is stimulated by theophylline and subsequently inhibited by furosemide. In some experiments K+ secretion can be detected by the blocking effect of mucosal Ba2+. Response of tissues from pig and cow is qualitatively similar but quantitatively different. The equipment is sturdy and inexpensive, can be provided by most departmental workshops, and has been tested for 3 yr in regular lab courses. Observations made during these experiments are closely related to clinical states, such as secretory diarrhea, cystic fibrosis, and hyperaldosteronism, as well as to the mechanisms of clinically used diuretics.

Abstract: The modulation of the intracellular glucocorticoidal effect on surfactant synthesis of the fetal lung by the metabolic capacity of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) could be an important factor in lung maturation. The kinetic properties of microsomal 11 beta-HSD of the rat lung are characterized with respect to product inhibition, substrate specificity, effect of electrolytes or trace elements, and the dependence of the oxidase reductase (OR) ratio on incubation conditions. With NADP+ product inhibition of the reductase was demonstrated. The most common trace elements and electrolytes exhibited no effect on the activity of 11 beta-HSD. It is shown that the OR ratio was strongly dependent on assay conditions. With optimal assay conditions oxidase activity exceeds reductase activity in adult and fetal rat lung microsomes (OR ratio > 1). Thus, glucocorticoids are mainly metabolized to their inactive forms. The enzyme activity in the adult is about 10 times higher than in the fetal lung. The low enzyme activity in fetal lungs could be the reason why the glucocorticoidal effects on surfactant synthesis are not suppressed despite the predominance of oxidase activity.

Abstract: Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent Cl- secretion provides the ionic basis for secretory diarrhea. We quantified the spatial distribution of this process by measuring local ion conductance in crypts and surface epithelium or villi of rat late distal colon and ileum. By use of an improved voltage-scanning technique, the tissue was clamped to a 30-Hz sine-wave current and the electrical field above the respective structures was sensed by a stepping glass microelectrode. Under control conditions, crypts and surface epithelium contributed 61 and 39%, respectively, to the total ion conductance of distal colon. Theophylline (10 mM) increased crypt conductance (Gc) by 64% from 2.5 +/- 0.2 to 4.1 +/- 0.3 mS/cm2 and surface epithelium conductance (Gs) by 69% from 1.6 +/- 0.1 to 2.7 +/- 0.1 mS/cm2. These changes in local conductances were completely Cl- dependent, since theophylline had no effect when Cl- was replaced by gluconate. Similar results were obtained when Cl- secretion was elicited by prostaglandin E1 (1 microM) or by dibutyryl-cAMP (DBcAMP, 1 mM). After stimulation, the Cl- channel blocker 5-nitro-2-(3-phenyl-propylamino)benzoic acid (1 mM) decreased both Gc and Gs. In rat ileum, theophylline plus DBcAMP caused an increase in total conductance of 19% only because of its large paracellular conductance. The ratio of scanning signals above villi and intervillous spaces was unaffected, indicating that Cl- conductance is induced in both crypts and villi. We conclude that in distal large intestine cAMP-dependent Cl- secretion is not confined to crypts but is evenly performed also by surface cells. A similar distribution exists in small intestine.

Abstract: It has been possible to obtain in a mammalian epithelium of dietetically and surgically untreated animals a dose response of in vitro-added aldosterone (Aldo, 10(-10) to 10(-5) M) on electrogenic Na+ absorption (JeNa). JeNa was measured in the Ussing chamber on stripped rat late distal colon 8 h after in vitro addition of Aldo. Submaximal effects were obtained at 3 nM Aldo; after a lag time of 2 h, short-circuit current (Isc) increased to a maximum of 234 +/- 15 microA/cm2 and dropped after 0.1 mM amiloride to -18 +/- 3 microA/cm2, resulting in JeNa of 9.4 +/- 0.6 mumol.h-1 x cm-1. Net Na+ tracer fluxes and Isc exhibited parallel time courses, so that electroneutral Na+ transport was not induced in late distal colon by acute Aldo. A plot of JeNa vs. Na conductance revealed an electromotive force (ENa) of 126 +/- 1 mV for all Aldo concentrations tested. Kinetic data were as follows: Michaelis constant 1.2 nM, maximal velocity (Vmax) 10.5 mumol.h-1 x cm-2, and Hill coefficient 2.1. In contrast to the large effect in late distal colon, 3 nM Aldo caused JeNa of < 1 mumol.h-1 x cm-2 in early distal colon, proximal colon, and cecum. Antimineralocorticoid sensitivity and ENa did not vary with Aldo concentration or time of the experiment, consistent with a unique mechanism during the early and late response up to 8 h, as well as at mineralocorticoid and glucocorticoid Aldo concentrations. Acute Aldo in a range of 0.1-10 nM fully controls JeNa between zero and Vmax in late distal colon.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The adaptational changes of epithelial ion transport in the short bowel syndrome were studied. Ileal remnants of rats were investigated 8 weeks after 70% proximal small intestinal resection. Pure epithelial resistance measured by impedance analysis decreased from 27 +/- 1 to 21 +/- 1 omega.cm2, and polyethylene glycol 4000 fluxes increased from 2.5 +/- 0.3 to 3.6 +/- 0.3 nmol.h-1.cm-2, indicating increased permeability of the short bowel. Unidirectional flux measurements in control ileum showed absorptive net fluxes of Na+ and Cl- that were assigned to electroneutral NaCl absorption and a short-circuit current that was accounted for by the residual flux (HCO3- secretion). Neither NaCl absorption nor HCO3- secretion were altered in the short bowel. Also, electrogenic Cl- secretion, defined after maximal stimulation by theophylline and prostaglandin E1 was not changed in the short bowel. In contrast, electrogenic Na+/glucose cotransport increased in Vmax from 2.0 +/- 0.3 in controls to 5.0 +/- 1.0 mumol.h-1.cm-2 in the short bowel. Tight junction structure was studied by freeze-fracture electron microscopy. The number of horizontal strands was unchanged, whereas tight junction depth was slightly increased in the short bowel. Microvillus area of short bowels was increased by 20% in villus regions. Under the light microscope, villus height was increased by 30%. In conclusion, the short bowel mucosa undergoes adaptive responses to reduced overall absorptive area by increasing glucose-dependent electrogenic Na+ absorption to 250%, which is partly caused by increased villus and microvillus surface area. Electrogenic Cl- and HCO3- secretion and electroneutral NaCl absorption remained unchanged. The decreased epithelial resistance is caused by mucosal surface amplification.

Abstract: 1. Prairie dog gallbladders mounted in a Ussing-type chamber and bathed with symmetrical Ringer's solutions exhibited a transepithelial resistance (Rt) of 51 +/- 5 omega cm2, a lumen negative potential difference (Vms) of 11.5 +/- 0.7 mV and a short-circuit current (Isc) of 6.9 +/- 0.3 microEq/hr/cm2. 2. Radioisotopic ion flux experiments revealed that the basal Isc of 6.9 +/- 0.3 microEq/hr/cm2 was mostly accounted for by net Na+ absorption of 3.2 +/- 0.5 microEq/hr/cm2 and net Cl- secretion of 2.9 +/- 0.3 microEq/hr/cm2. 3. In HCO3- free Ringer's, net Na+ flux was virtually abolished, net Cl- flux decreased by 50% and Isc was reduced by 77%. 4. 10(-3) M mucosal amiloride and DIDS reduced Isc by 28 and 24%, respectively. 5. Mucosal NaCl diffusion potentials indicated that the paracellular pathway was cation selective. 6. Thin section electron micrographs showed a single cell population in this epithelium suggesting that net Na+ absorption and Cl- secretion may emerge from the same cells. 7. We conclude that prairie dog gallbladder epithelium is an electrogenic tissue and, in contrast to gallbladders of most other species, simultaneously but independently absorbs Na+ and secretes Cl-.

Abstract: HT-29, an undifferentiated human colon cell line, is known to differentiate when cultured without glucose. This study aimed to characterize ion transport in the clone HT-29/B6, which was selected from HT-29 cells differentiated by glucose-free culture. HT-29/B6 cells seeded onto filter membranes grew as polarized monolayers, mainly consisting of mucus-forming cells and exhibiting high transepithelial resistance. Short-circuit current (Isc) of unstimulated HT-29/B6 monolayers in Ussing chambers was 0.1 +/- 0.01 mumol.h-1.cm-2, and conductance was 2.0 +/- 0.2 mS/cm2. Serosal forskolin (FSK; 10(-5) M) induced a sustained Isc of 1.9 +/- 0.1 mumol.h-1.cm-2, associated with a rise of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Isc was identified as Cl- secretion by tracer studies and by the inhibitory effects of serosal bumetanide and Ba2+. The Cl- channel blockers NPPB and DPC diminished FSK-induced Isc at respective doses of 3 x 10(-4) and 10(-3) M, being effective from either side of the monolayer. Cl- secretion could be triggered by vasoactive intestinal peptide (10(-8) M), prostaglandin E1 (10(-6) M), and dibutyryl cAMP (10(-3) M) as well. In conclusion, HT-29/B6 cells grow as polarized monolayers, forming mucus and secreting Cl- in response to secretagogues. This clone may not only serve as a model for investigation of cellular mechanisms of intestinal Cl- secretion but may also be helpful to elucidate the contribution of mucus cells to this process.

Abstract:

Abstract: The short-term action of aldosterone in physiological concentration on net fluxes of Na, K, Cl, HCO3, osmolytes, and water was examined in the proximal colon and rectal colon of adrenalectomized (ADX) rats in vivo. The measuring time was 12 h, divided in eight periods of 90 min. (a) Aldosterone alone (6 nmol h-1 kg-1) did not stimulate transport in ADX rats. In these experiments plasma [K] increased to fatal values. A basal glucocorticoid substitution of 24 nmol h-1 kg-1 corticosterone caused plasma K to stay constant throughout the experiment, so that epithelial transport was not handicapped by non-specific effects of ADX, but this also did not restore the decreased transport of ADX rats to control values. Under these conditions (absence of aldosterone) in the rectal colon Na and H2O transport was zero, whereas in the proximal colon flux rates were depressed by between 30% and 50%. In contrast, basal glucocorticoid substitution of 18 nmol h-1 kg-1 corticosterone plus infusion of 6 nmol h-1 kg-1 aldosterone caused transport stimulation to values not significantly different from those of non-ADX controls. We conclude that after ADX, aldosterone at physiological concentrations increases transport if, as a prerequisite, a basal glucocorticoid substitution is provided. Transport of Na, K, and H2O is under the total control of aldosterone in the rectal colon but is only moderately altered in the proximal colon.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Protamine reversibly decreases cation permeability and alters the structure of Necturus gallbladder tight junctions. Conflicting results, however, have been published whether or not it also affects apical cell membrane permeability. We investigated this issue more systematically by measuring voltage (psi mc) and fractional resistance (fRa) of the apical membrane at varying concentrations of protamine, K+, and H+ in the bathing solution. At pH 7.6 and [K+] 2.5 mM, (Poler, M.S. and Reuss, L. (1987) Am. J. Physiol. 253, C662) 6 microM protamine caused psi mc to depolarize from -58 to -51 mV and fRa to decrease from 0.74 to 0.67. If we increased pH to 8.1 these effects were even more pronounced. At [K+] 2.5 mM, but not 4.5 mM, psi mc transiently hyperpolarized for about 5 min after adding protamine. Most importantly, if [K+] was 4.5 mM and pH was adjusted to 7.1 (Bentzel et al. (1987) J. Membr. Biol. 95, 9) no significant changes of psi mc and fRa occurred. In any case, at a supramaximal concentration of 200 microM, protamine did not further increase the paracellular response but produced decreasing psi mc and fRa. We conclude that 6 microM protamine decreases K+ conductance of the apical membrane, if it is already tuned high by high pH. At low control K+ conductance as observed at lower pH, protamine action is restricted to the paracellular pathway. Thus, conflicting results were due to different experimental conditions. At a solution pH of 7.1, 6 microM protamine fulfills criteria of a selective tool for reversibly altering structure and function of the tight junction in Necturus gallbladder.

Abstract: The endogenous opioid enkephalin drives ion transport towards absorption. To determine the site and mechanism of this effect, fractionated stripping of guinea-pig ileum was carried out. The muscularis propria, including myenteric plexus, was removed by partial stripping. The submucosa, including the submucosal plexus, plus the muscularis mucosae were removed by total stripping. For binding studies, epithelial cells were removed by the method of Weiser leaving the lamina propria mucosae with the mucosal plexus. Radio-receptor-assay with (3H)2-D-ala-5-D-leu-enkephalin revealed enkephalin binding sites in the submucosa plus muscularis mucosae (KD = 3.6 nmol l-1; Vmax = 7.3 fmol mg-1) and in the lamina propria mucosae (KD = 4.2 nmol l-1; Vmax = 5.1 fmol mg-1. The binding was stereospecific in both layers. No binding was detected on epithelial cells. In the Ussing chamber, partially stripped ileum exhibited spontaneous ISC which was abolished by addition of tetrodotoxin (TTX) or by total stripping indicating that this ISC was neuronally stimulated by the submucosal plexus. Electrogenic chloride secretion was identified as contributing to this ISC, since the TTX-sensitive part of ISC in the partially stripped ileum was lacking in Cl- and HCO3-free medium, reappeared after addition of Cl consistent with Michaelis-Menten kinetics (Km = 19 nmol l-1) and was reversed by serosal addition of bumetanide. In addition, enkephalin increased electroneutral NaCl-absorption as obtained by Na- and Cl-flux measurements. Enkephalin decreased this spontaneous neuronally stimulated electrogenic Cl-secretion in the partially stripped ileum, but had no effect in totally stripped ileum if ISC was stimulated at the cellular level by theophylline or PGE1. We conclude that ganglia located in the submucosal plexus regulate intestinal ion transport. Enkephalin acts by presynaptic inhibition via receptors on these neurons in the submucosa and/or via receptors on their neurites in the lamina propria mucosae.

Abstract: Ileal remnants 8 weeks after 70% proximal small intestinal resection were used as a model for the short bowel syndrome in man. For comparing active ion transport between control ileum and short bowel with the Ussing technique, the relative contribution of the subepithelial resistance has to be considered. Epithelial/subepithelial voltage divider ratios were determined in the Ussing chamber by positioning the tip of a microelectrode just below the epithelium. In control ileum, the ratio of total to epithelial voltage deflection was 1:0.56 +/- 0.03 (n = 48) and decreased to 1:0.42 +/- 0.01 (n = 67; p less than 0.001) under the short bowel condition. Thus, the factors by which a measured short-circuit current (Isc) underestimates the true electrogenic transport was 1.78 +/- 0.09 (n = 48) in control ileum and 2.36 +/- 0.08 (n = 67; p less than 0.001) in the short bowel. Glucose-dependent electrogenic Na absorption was defined using bathing media containing 48 mM 3-o-methyl-glucose as the decrease in Isc (delta Isc) after addition of 0.5 mM phlorizin. After correction for the respective contributions of the subepithelial resistance, delta Isc was -1.4 +/- 0.2 microEq.h-1.cm-2 (n = 13) in control ileum and -3.2 +/- 0.7 microEq.h-1.cm-2 (n = 11; p less than 0.01) in the short bowel. We conclude that the mucosa in the short bowel syndrome is characterized by an increase in glucose-dependent electrogenic Na absorption, probably as an adaptive response to the reduced overall absorptive area of the remaining intestine.

Abstract:

Abstract: Epithelial cell tight junction structure in self-filling blind loops of rat jejunum, a model for blind loop syndrome in humans, was analyzed morphometrically along the crypt-villus axis. In control jejunum, the number of strands and junctional depth, including meshwork depth, decreased from crypt to villus tip. In the blind loop, aberrant strands appeared below the meshwork, particularly in crypt cells. Consequently, total junctional depth was greater than in controls. Furthermore, strand number and junctional meshwork depth were increased in blind loops at the villus tip. It is that site along the crypt-villus axis which showed the most shallow junction in control jejunum. This structural change is paralleled by a three-fold increase in epithelial resistance as previously measured by alternating current impedance analysis. Relative Na over Cl permeability (PNa:Cl) was obtained from dilution potential measurements. PNa:Cl was 1.50:1 in control jejunum and 1.35:1 in the blind loop (n.s.). Considering the cation selectivity of the tight junction, the increase in epithelial resistance in blind loops cannot be attributed to a collapse of the lateral intercellular space but is due to changes in tight junctional permeability resulting from structural alteration. The blind loop syndrome represents a further example of diminished epithelial ion transport and concomitant decrease in tight junction permeability, thus supporting the general concept of regulation of the tight junction in response to active transport activity.

Abstract: The initial phase of in vitro experiments in Ussing-type chambers on large intestine is characterized by short-circuit currents (ISC) declining from high starting values to a lower plateau within 0.5 h. The origin of this "initial ISC-transient" was investigated by ISC measurements on partially stripped segments of rat rectal colon. Transport was pre-stimulated in vivo by keeping animals in barbiturate-anesthesia for 5 h prior to tissue preparation. This procedure caused by endogenous aldosterone-liberation amiloride-sensitive Na-absorption to become the predominant electrogenic transport. The initial ISC-transient was abolished by tetrodotoxin (TTX, 1 microM), indicating a neuronal mediation of this phenomenon. In order to identify the transport which was subject to neuronal control, the amiloride-sensitive Na-absorption was measured during electrical field stimulation (bipolar rectangular pulses: 5 Hz, 1 ms, +/- 6 mA). There was no difference to unstimulated controls. In contrast, the initial ISC-transient was dependent on Cl in the bath following Michaelis-Menten-kinetics (KM = 20 mM) and could be prevented by 10 microM serosal bumetanide. Then, initial filling of the Ussing-chamber was imitated during the course of the experiment by removal and immediate re-addition of the bathing fluid. This procedure caused ISC-changes of similar appearance as the initial ISC-transient. To verify that indeed mechanical stretch is the sensory stimulus triggering the initial ISC-transient, the effect of small pressure oscillations was studied. This also produced an ISC-transient which was TTX-sensitive and was abolished after removal of the submucosal plexus Meissner by total stripping.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Carbetimer, a new synthetic low molecular weight polyelectrolyte with a novel structure displayed antitumor activity in a number of animal tumor model systems and in vitro investigations. Based on these findings it was brought to a phase I clinical trial in patients with advanced malignant disease after failure of conventional treatment or with no conventional treatment available. Forty-eight patients received 98 courses. The schedule was a one hour i.v. infusion every four weeks. The starting dose was 180 mg/m2 and dose escalation was performed according to a modified Fibonacci formula up to 16,690 mg/m2. At least three patients were treated at each dose level and each patient was eligible to receive repeat courses at the same dose, until progressive disease or dose-limiting toxicity intervened. No hematological toxicity was encountered. Some adverse effects such as reversible proteinuria, hypercalcaemia, pain at infusion site, nausea and vomiting and fatigue were seen partly in a dose-related manner but did not represent the maximum tolerated dose (MTD). The limiting toxicity at the highest dose level of 16,690 mg/m2 consisted of ocular symptoms ('light flashes') accompanied by a modest decrease of blood pressure and nausea or vomiting during a one hour infusion. 16,690 mg/m2/1 hour was considered the MTD. There were four deaths on study, all considered disease-related. Fourteen patients had stable disease for more than two courses, which, however, could also be explained by the natural course of disease. No clear-cut antitumor responses were noted in our study center. The recommended dose for phase II trials derived from our results is 12,550 mg/m2/2 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Self-filling blind loops of rat jejunum exhibit hyperregenerative transformation of the mucosa. We used this experimental model to characterise mechanisms, which may occur under similar conditions in man (stagnant loop syndrome). Epithelial and subepithelial resistance were measured in the Ussing-chamber by voltage divider ratio measurements after positioning a microelectrode between epithelium and subepithelial tissue layers. In the blind loop, epithelial resistance increased from 8 +/- 1 to 23 +/- 1 omega cm2 and subepithelial resistance from 39 +/- 4 to 86 +/- 8 omega cm2 as compared with control jejunum. The increase in the subepithelial resistance was paralleled anatomically by an increase in the thickness of the subepithelial tissue layers from 63 +/- 4 microns to 177 +/- 19 microns. Ultrastructural analysis of the tight junction area by freeze fracture electron microscopy revealed an increase in the total junctional 'depth' in the crypts from 243 +/- 9 nm in control jejunum to 396 +/- 17 nm in the blind loop, while the number of horizontally oriented 'strands' remained unchanged. Villus tight junctions did not differ between blind loop and control. We interpret the alterations in the self-filling blind loop as an adaptive response of the epithelium which reduces backleakage of already absorbed electrolytes across the tight junction into the intestinal lumen. This mechanism is suitable to support the intestine in maintaining body electrolyte and water contents during cellular electrolyte malabsorption.

Abstract: Thiobutabarbital anaesthetized and abdominally operated control rats develop high endogenous plasma levels of both aldosterone and corticosterone during the course of a 12 h experiment. This effect was used as a model for examining 'acute' steroid action (i) on net ion and water fluxes and (ii) on zero flux luminal limiting concentrations in rat upper colon (proximal 50% of large intestine) and rectum (distal 40%). Experiments of both kinds consisted of 8 independent 90 min measuring periods. (i) In rectum net fluxes of Na, K, osmolytes (sum of all solutes) and water started at low levels around zero, began to rise about 2 h after plasma levels of aldosterone had increased, and reached plateau values around the 6th hour of anaesthesia. In upper colon, fluxes of Na, K, Cl, and osmolytes were high from the beginning and did not vary significantly with time. (ii) At zero flux conditions limiting concentrations of Na in the hormonally unstimulated phase of the experiment were 20 +/- 3 mM in upper colon and 22 +/- 3 mM in rectum. After maximal endogenous aldosterone liberation zero flux concentrations were 5.2 mM in upper colon and 2.2 mM in rectum, corresponding to luminal fluid to plasma ratios (LF/P) of 0.040 and 0.016, respectively. Amiloride reduced the maximal Na gradient in rectum to a LF/P of 0.3 but was not effective in upper colon and did not prevent the stimulating effect of aldosterone in this segment. Under all experimental conditions zero flow concentrations of K were higher than consistent with a solely passive distribution, indicating simultaneous passive and active secretion in both segments. In contrast to the findings of others, the luminal fluid remained isoosmolar with plasma in all zero flux experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Self-filling blind loops of rat jejunum were used as a model for the blind loop syndrome in humans. Electrical resistance, short circuit current, and unidirectional sodium and chloride fluxes were measured using the Ussing technique. Whereas net fluxes for sodium and chloride did not differ significantly from zero in the blind loop or in the control, unidirectional fluxes of either direction were decreased and electrical resistance was increased, indicating an increase in the tightness of the intestinal wall. Measurements of alternating current impedance and micropuncture experiments revealed that this was due to an increase in epithelial resistance from 9 +/- 1 omega X cm2 (n = 15, results of both methods) to 27 +/- 4 omega X cm2 (n = 15) and in subepithelial resistance from 40 +/- 2 omega X cm2 (n = 15) to 76 +/- 7 omega X cm2 (n = 15). As the ratio of epithelial to subepithelial resistance was similar in the blind loop and in the control, lower transport rates in the blind loop are indicative of impaired epithelial transport function. Subsequently, two different transport systems were characterized. First, the 3-o-methyl-glucose-induced, phlorizin-reversible increase in short circuit current, representing glucose-coupled sodium absorption, showed a 77% decrease in maximum velocity in the blind loop and no change in Km. Second, the chloride-induced, bumetanide-reversible increase in short circuit current in tissues stimulated simultaneously by prostaglandin E1 and theophylline, representing rheogenic chloride secretion, also showed a decrease in maximum velocity (of 83%) and no change in Km. A morphometric analysis revealed that the crypt surface area increased by 100% in the blind loop, whereas the villous surface area was not significantly different between blind loops and controls. We conclude that the jejunal self-filling blind loop is characterized by impaired active ion transport processes and an increase in epithelial and subepithelial resistance.

Abstract: Protamine is a naturally occurring basic protein (pI; 9.7 to 12.0). We have recently reported that protamine dissolved in the mucosal bath (2 to 20 microM), induces about a twofold increase in transepithelial resistance in Necturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity. In this leaky epithelium, where greater than 90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular action a priori. To confirm this, ionic conductance across the apical cell membrane was studied with microelectrodes. Protamine increased transepithelial resistance without changing apical cell membrane voltage or fractional membrane resistance. Variation in extracellular K concentration (6 to 50 mM) caused changes in apical membrane voltage not different from control. To determine if protamine-induced resistance changes were associated with structural alteration of tight junctions, gallbladders were fixed in situ at peak response and analyzed by freeze-fracture electron microscopy. According to a morphometrical analysis, the tight junctional intramembranous domain expands vertically due to incorporation of new strands (fibrils) into the main compact fibrillar meshwork. Since morphologic changes are complete within 10 min, strands are probably recycled into and out of the tight junctional membrane domain possibly by the cytoskeleton either from cytoplasmic vesicles or from intramembranous precursors. Regulation of tight junctional permeability by protamine and other perturbations may constitute a common mechanism by which leaky epithelia regulate transport, and protamine, in concentrations employed in this study, seems reasonably specific for the tight junction.

Abstract: Epithelial and subepithelial electrical resistances of rat large intestine were measured by means of a 4-electrode AC impedance technique in three segments, colon ascendens, colon descendens and rectum. Epithelial resistance of colon ascendens and colon descendens was about 35 omega X cm2 and not different between these two segments. It was, however, about 3 times higher in rectum (99 omega X cm2). This finding is in accord with our previous observation of about 3-fold higher net fluxes of ions and water in colon ascendens and colon descendens than in rectum. It confirms the concept of a main functional difference between the terminal part of the large intestine (rectum) and the more proximal segments (colon). The acutely (within hours) varied level of aldosterone by keeping the rats for 7 h in anaesthesia caused in the rectum a more than 10-fold increase in short circuit current (Isc) and transepithelial voltage but no significant decrease in resistance. Similarly, the decline in Isc, as regularly observed in the early phase of in vitro measurements on partially stripped large intestine, was paralleled by voltage changes but not by changes in resistance. We conclude that the wide range of resistance values published so far was caused to a great extent by including various portions of colon or rectum. By comparing intact (not stripped) and partially stripped preparations (muscularis propria removed) of the rectum it was shown that partial stripping did not alter the epithelial resistance but reduced the subepithelial resistance in this segment from 26 to 8 omega X cm2, or by 68%.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract:

Abstract: Twenty children with soft tissue sarcomas of the head and neck, treated at the Children's Hospital of Philadelphia and the Hospital of the University of Pennsylvania from 1972 to 1981, were evaluated for the late deleterious effects of treatment. All patients received radiation therapy and combination chemotherapy with vincristine, dactinomycin, and cyclophosphamide; certain patients also received Adriamycin (doxorubicin). All had ophthalmologic, otologic, growth, and cosmetic evaluations; 15 also had dental and maxillofacial examinations. The median age at diagnosis was 6 years (range, 7 months-13 years). Median follow-up from time of diagnosis was 5.5 years with a minimum of 3 years in all but four patients. The major problems encountered were related to the eyes (xerophthalmia and cataracts), ears (hearing loss), teeth (maleruption and caries), glandular structures (xerostomia, hypopituitarism), and development (craniofacial deformity). It is concluded that children treated for soft tissue sarcomas of the head and neck with combined modality therapy, including radiation enhancers, may show a variety of late treatment-related adversities. These children require close multidisciplinary follow-up for detection of late effects in order that appropriate prophylactic or symptomatic treatment can be instituted to minimize their consequences.

Abstract: Epithelial and subepithelial resistance of rat jejunum was measured in vitro by two independent methods. (i) Transepithelial AC impedance data were interpreted in terms of a simple parallel RpCp element (representing the epithelial cell layer) in series with an ohmic resistor RS (representing the subepithelial layers). (ii) In separate experiments, the tip of a microelectrode was positioned between epithelium and subepithelial layers and the respective resistances were obtained from DC-pulse voltage divider ratios between both structures. The total tissue resistance as measured in conventional Ussing-chamber experiments (49 +/- 4 Ohm X cm2, mean of both methods) was formed to 81 +/- 6% (40 +/- 3 Ohm X cm2) by subepithelial layers and to only 19 +/- 3% (9 +/- 1 Ohm X cm2) by the epithelial cell line. We conclude that rat jejunum is more conductive than assumed so far. In in vitro flux studies on intact jejunal sheets a pronounced back-diffusion of absorbed substances will lead to an underestimation of the true net transport capacity of this structure. This error averages about fivefold and will be found likewise in conventional short-circuit measurements.

Abstract: Protamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested in Necturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 micrograms/ml = 20 microM), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage (psi ms) turned from slightly mucosa-positive values to mucosa-negative values of approximately +1 to -5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 micrograms/ml but a maximum response was achieved between 10 and 20 micrograms/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5 X 10(-4) M) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability.

Abstract: We have developed a general method for electrically introducing DNA into plant cells. Gene transfer occurs when a high-voltage electric pulse is applied to a solution containing protoplasts and DNA. Carrot protoplasts were used as a model system to optimize gene-transfer efficiency, which was measured 24-48 hr after electroporation by the amount of chloramphenicol acetyltransferase activity resulting from the expression of the introduced chimeric plasmids. Gene-transfer efficiency increased with the DNA concentration and was affected by the amplitude and duration of the electric pulse as well as by the composition of the electroporation medium. Our optimized gene-transfer conditions were effective when applied to tobacco and maize protoplasts, demonstrating that the method is applicable to both monocot and dicot protoplasts.

Abstract: The transepithelial voltage (psi ms) of rat rectum in vivo increases for several hours in experiments under general anaesthesia. So far this was attributed by indirect evidence to increasing aldosterone plasma levels during the course of the experiment. We performed direct measurements of aldosterone and corticosterone plasma concentrations during intestinal perfusion experiments on barbiturate anaesthetized rats. Experiments were terminated for blood sampling at 10, 75, 300, 400, 800, or 1,800 min, respectively. (i) After 75 min of anaesthesia, surgical preparation was finished and plasma levels of aldosterone and of corticosterone were found increased by the factors 5 and 3, respectively, as compared to conscious controls. (ii) During the following 12 h, aldosterone further increased to levels 10 times as high as those of controls. In contrast, during the same period corticosterone slowly decreased but still remained elevated as compared to controls. (iii) The increase of both hormones was attenuated when abdominal surgery was omitted. (iv) The use of pentobarbital (Nembutal) instead of thiobarbital (Inactin) did not influence the adrenal response. (v) In adrenalectomized rats a continuous substitution with 65 ng X h-1 X kg-1 BWT aldosterone resulted in plasma levels as high as in conscious intact animals. (vi) Rectal psi ms started to move to higher lumen-negative values with a time delay of 1-1 1/2 h as compared with the increase of hormone levels. psi ms then stayed elevated until to the end of the experiments. We conclude that in vivo experiments of several hours duration in thio- or pentobarbital anaesthetized rats take place under conditions of aldosterone and corticosterone plasma levels which are high as compared to those of conscious unstressed animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The site of adenosine 3',5'-monophosphate-mediated fluid and electrolyte secretion across mammalian large intestine was found to be the crypts of Lieberkühn by means of two techniques. First, the formation of fluid droplets was visualized on the oil-covered mucosal surface directly over crypt duct openings when secretion was stimulated. Second, microelectrode impalement of individual surface and crypt cells revealed that only crypts cells produced a pattern of secretagogue induced alterations in membrane potential and resistance that was characteristic of secretory epithelia.

Abstract: The SV40 early region promoter, previously localized to the DNA segment bounded by the HpaII and HindIII restriction sites (nucleotides 346 and 5171), was further defined by construction of an extensive set of deletions within this region and measurement of their effects on (a) viral DNA replication, (b) virus multiplication and the ability to complement early and late mutations, (c) transformation of rat cells, (d) large T antigen formation, and (e) the location of the 5' ends of early mRNAs. One set of mutations is represented by deletions that begin at the HpaII site and extend unidirectionally for varying lengths toward the BglI site at ori. A second set of mutants contains deletions that start at ori and extend unidirectionally for varying lengths towards the HpaII site. A third set of mutants, with deletions or duplications of various lengths and boundaries, lie between the HpaII and BglI sites. Our studies indicate the following. (a) Ori, the sequence needed for initiating SV40 DNA replication, extends from the sequences needed for initiating SV40 DNA replication, extends from the sequences needed to bind T antigen to the palindrome in site II to nucleotide 34, the late region edge of the AT block. Flanking sequences adjacent to the AT block facilitate DNA replication. (b) The SV40 early region promoter comprises two functionally distinct nucleotide sequence elements. One is flanked by nucleotides 5231 and 107, and contains the RNA initiation sites at nucleotides 5231-5237, a positioning element resembling the TATAAATA consensus sequence about 20-25 nucleotides upstream, and an RNA polymerase II recognition sequence contributed by short GC-rich sequences clustered between nucleotides 35 and 107; we refer to this as the RNA polymerase II interaction site. The second distinct sequence element is contained within each of two 72-bp segments located between nucleotides 107 and 250; the behavior of this element suggests that it may influence the accessibility of RNA polymerase II for the interaction site or the efficiency of RNA chain initiation. Large T antigen binding sites I, II, and III overlap with the putative RNA polymerase II interaction site; since large T antigen does not prevent elongation of RNA transcripts initiated upstream, T antigen probably represses early region expression by preventing RNA polymerase II binding to the promoter.

Abstract: The results of previous studies indicate that the bidirectional fluxes of K across short-circuited rabbit descending colon are attributable to passive diffusion through paracellular pathways and that this route is ten times more permeable to K than to Na and Cl. However, transepithelial diffusion potentials in the presence of large transepithelial Na and K concentration differences are much lower than those predicted by the "constant field equation" and appear to be inconsistent with this high K selectivity. The results of the present studies, designed to resolve this apparent contradiction, indicate that: (a) The ratios of the bidirectional transepithelial fluxes of K determined over a wide range of combined chemical and electrical potential differences conform reasonably well with those predicted by the Ussing flux-ratio equation. (b) The permeability coefficient of K (PK), determined from the net fluxes in the presence of concentration differences and from unidirectional fluxes under short-circuit conditions, decreases with increasing K concentration; in the presence of low K concentrations, PK is approximately ten-times PNa, but it approaches PNa in the presence of high K concentrations. PNa is not affected under these conditions. These results provide an explanation for the failure to observe large transepithelial diffusion potentials in the presence of large transepithelial Na and K concentration differences. In addition, these results are consistent with the notion that K diffuses across this preparation through two parallel pathways, one that does not discriminate among K, Na and Cl (a "free-solution" shunt) and another that is highly K selective and involves an interaction with one, or at most two, sites along the route.

Abstract: This study was undertaken in order to determine directly the rates of K leakage (JK) out of the tips of microelectrodes into a solution of 100 mM KCl (approximating the K concentration of the cell interior) and to relate these rates to the concentration of the filling solution and the tip resistance. The values of JK for electrodes filled with 3 M KCl having resistances of 16 and 30 M omega (when measured in 3 M KCl) were 10 and 5.5 fmol/sec, respectively. When the same electrodes were filled with 0.5 M KCl, the resistances (measured in 0.5 M KCl) increased to 62 and 115 M omega, respectively, and JK fell to 1.8 and 1.0 fmol/sec, respectively. These values are in reasonable agreement with what would be expected from theoretical considerations if leakage of KCl were the result of diffusion plus convective flow due to the hydrostatic pressure of the filling solution. We conclude that K leakage out of microelectrodes filled with 3 M KCl is unnecessarily high; leakage can be reduced fivefold by filling electrodes with 0.5 M KCl without incurring significant increases in tip or diffusion potentials or unmanageable tip resistances. Finally, the lowest rate of K leakage observed (1 fmol/sec) is still very considerable for the case of animal cells with an intracellular volume of approximately 1 pl and a K content of approximately 100 fmol. The finding of stable intracellular potentials, often for many minutes, in some tissues suggests that K which enters the cell rapidly diffuses into neighboring cells via high conductance intercellular communications.

Abstract: A simple and versatile tool facilitating micropuncture of small cells is described which utilizes a commercial piezoelectric element made from a stacked column of monomorph ceramic discs. The device is able to advance complete input stage-electrode-assemblies with high speed and can be used in combination with conventional micromanipulators. Advancing characteristics as recorded optically at high magnification demonstrated less axial vibration, although faster action, than two other modern micropositioners driven by step motors. In biological experiments on selected tissues (Necturus gallbladder epithelium, Amphiuma renal distal tubule cells, rabbit and human corneal endothelium) the combined use of micromanipulator and piezo-stepper was, in all cases, superior to the use of a micromanipulator alone: the percentage of successful cell penetrations increased, cell potentials were stable for a longer time, and the durability of electrode-tips improved.

Abstract: Functionally isolated segments of rat colon and rectum were perfused in situ in a closed loop system. Rectum was defined as the lower 25--35% of the length of large intestine (cecum excluded). Perfusion conditions were optimized at 0.5 ml.min-1 and 3 cm H2O luminal pressure. Variation of perfusion rate between 0.2 and 2 ml.min-1 did not influence net volume transport (JNV). Luminal distension following elevation of hydrostatic pressure to 18 cm H2O reversibly increased Jnv. Under control conditions Jnv and Na+-transport rates (JnNa) of colon were 2--3 times higher than those of rectum. In colon transepithelial electrical potential difference (psims) was time independent --12 mV (lumen negative) whereas rectal psims increased with time from --6 mV, reaching a plateau of --67 mV within 6 h. Amiloride 10(-4) mol.l-1 had no effect on psims, Jnv, and JnNa in colon but did slightly depress K+-secretion in colon descendens. In contrast, psims in rectum was dose-dependently depressed, being reversed to +7 mV at 10(-4) mol.l-1. Jnv and JnNa were decreased by half. Acetazolamide in addition to amiloride lowered the positive post-amiloride rectal psims by half. Adrenalectomy had no effect on colonic psims, but abolished psims of the rectum. A single dose of 40 microgram.kg-1 b.w. aldosterone during the experiment restored the typical time course of rectal psims, but did not affect psims in colon. It is concluded that aldosterone induces an amiloride-sensitive Na+-pathway only in rectum, but not in colon, and that colon and rectum differ basically in their transport properties, quantitatively as well as qualitatively, as do the kidney distal convoluted tubule and the cortical collecting duct.