Yoshinori Marunaka

Abstract: Anxiety disorders are the most popular psychiatric disease in any human societies irrespective of nation, culture, religion, economics or politics. Anxiety expression mediated by the amygdala may be suppressed by signals transmitted from the prefrontal cortex and hippocampus. KF-1 is an endoplasmic reticulum (ER)-based E3-ubiquitin (Ub) ligase with a RING-H2 finger motif at the C-terminus. The kf-1 gene expression is up-regulated in the frontal cortex and hippocampus in rats after anti-depressant treatments. The kf-1 null mice show no apparent abnormalities, but exhibit selectively pronounced anxiety-like behaviors or increased timidity-like responses. The kf-1 orthologous genes had been generated after the Poriferan emergence, and are found widely in all animals except insects, arachnids and threadworms such as Drosophila, Ixodes and Caenorhabditis, respectively. This suggests that the kf-1 gene may be relevant to some biological functions characteristic to animals. Based on these observations, the Anxiety Suppressor Model has been proposed, which assumes that KF-1 Ub ligase may suppress the amygdala-mediated anxiety by degrading some anxiety promoting protein(s), such as a neurotransmitter receptor, through the ER-associated degradation pathway in the frontal cortex and hippocampus. According to this model, the emotional sensitivity to environmental stresses may be regulated by the cellular protein level of KF-1 relative to that of the putative anxiety promoter. The kf-1 null mice should be useful in elucidating the molecular mechanisms of the anxiety regulation and for screening novel anxiolytic compounds, which may block the putative anxiety promoter.

Abstract: Limb salvage involving wide resection and reconstruction is now well established for managing musculoskeletal sarcomas. However, involvement of major nerves and vessels with a large volume of muscle and skin may result in a useless limb, contributing to depression and a low quality of life. We have been studying alternative treatments for musculoskeletal sarcoma since 1990, and have recently established a regime using photodynamic surgery with cells labelled with acridine orange, photodynamic therapy with cells treated similarly and radiodynamic treatment using the effect of X-rays on such cells. These techniques have been used after marginal or intralesional resection of tumours since 1999 and have enabled maintenance of excellent limb function in patients with sarcomas.

Abstract: In the present study, we investigated if the intracellular Cl(-) affects cell growth and cell cycle progression of androgen-independent prostate cancer PC3 cells. PC3 cells cultured in a medium containing 113 mM Cl(-) for 96 h grew up 9-fold in cell number, while PC3 cells cultured in an 8 mM-Cl(-)-containing culture medium showed complete arrest of cell growth even after culture for 96 h. Exposure of cells to the 8 mM-Cl(-) culture medium diminished phosphorylation levels of Rb and cdc2, which are respectively key accelerators of transition from G(1) to S phase and G(2) to M phase in cell cycle progression. Culturing cells in the 8 mM-Cl(-)-containing culture medium upregulated the protein expression level of p21 (a CDK inhibitor) inhibiting transition of G(1) to S phase, and diminished the incorporation of 5-ethynyl-2'-deoxyuridine (EdU; a thymidine analogue) into DNA. These results suggest that cells cultured in the low Cl(-) medium prolonged the duration of all phases of the cell cycle (G(1), S, and G(2)/M), thereby abolishing overall cell cycle progression. Effects of culturing cells in the low Cl(-) culture medium on cell cycle progression would be mediated via a change in the intracellular Cl(-) concentration ([Cl(-)](i)), since [Cl(-)](i) was decreased under a low Cl(-) culture medium. To clarify this possibility, we studied effects of furosemide and bumetanide, Na+/K+/2Cl(-) cotransporter (NKCC) inhibitors, on proliferation of PC3 cells. Furosemide and bumetanide decreased [Cl(-)](i) and cell growth of PC3 cells. These results suggest that a change in [Cl(-)](i) would play a critical role in this growth mechanism.

Abstract: In multicellular organisms, epithelia separate and divide the internal environment maintaining appropriate conditions in each compartment. To maintain homeostasis in these compartments, claudins, major cell adhesion molecules in tight junctions (TJs), regulate movements of several substances through the paracellular pathway (barrier function). In this study, we investigated effects of the flux of several substances between apical and basolateral side on paracellular transport and TJ protein localization. NaCl flux from apical to basolateral side increased paracellular conductance (Gp) and recruited claudin-1 from lateral cell membrane to the apical end with the colocalization with occludin, one of the TJ proteins concentrated at TJ strands. Oppositely-directed flux of sucrose against NaCl flux inhibited these reactions and same directional flux of sucrose with NaCl enhanced the increase of Gp, whereas 10-kDa dextran inhibited these reactions regardless of the side of administration. Our present findings indicated that TJ protein localization and barrier function are regulated depending on the environmental differences between apical and basolateral side.

Abstract: Indomethacin (IDM, 10 microm), not aspirin (ASA; 10 microm), enhanced the Ca(2+)-regulated exocytosis stimulated by 1 microm acetylcholine (ACh) in guinea-pig antral mucous cells. Indomethacin inhibits prostaglandin G/H (PGG/H) and 15R-hydroperoxy-eicosatetraenoic acid (15R-HPETE) production from arachidonic acid (AA), while ASA inhibits PGG/H production but accelerates 15R-HPETE production. This suggests that IDM accumulates AA. Arachidonic acid (2 microm) enhanced Ca(2+)-regulated exocytosis in antral mucous cells to a similar extent to IDM. Moreover, a stable analogue of AA, arachidonyltrifluoromethyl ketone (AACOCF(3)), also enhanced Ca(2+)-regulated exocytosis, indicating that AA, not products from AA, enhances Ca(2+)-regulated exocytosis. We hypothesized that AA activates peroxisome proliferation activation receptor alpha (PPARalpha), because AA is a natural ligand for PPARalpha. A PPARalpha agonist (WY14643; 1 microm) enhanced Ca(2+)-regulated exocytosis, and a PPARalpha blocker (MK886; 50 microm) abolished the enhancement of Ca(2+)-regulated exocytosis induced by AA, IDM, AACOCF(3) and WY14643. Western blotting and immunohistochemical examinations demonstrated that PPARalpha exists in antral mucous cells. Moreover, MK886 decreased the frequency of Ca(2+)-regulated exocytosis activated by 1 microm ACh or 2 microm thapsigargin alone by 25-30%. Thus, ACh stimulates AA accumulation via an [Ca(2+)](i) increase, which activates PPARalpha, leading to enhancement of Ca(2+)-regulated exocytosis in antral mucous cells. A novel autocrine mechanism mediated via PPARalpha enhances Ca(2+)-regulated exocytosis in guinea-pig antral mucous cells.

Abstract: Recently, we reported that reduction of intracellular Cl(-) concentration ([Cl(-)](i)) inhibited proliferation of MKN28 gastric cancer cells by diminishing the transition rate from G(1) to S cell-cycle phase through upregulation of p21, cyclin-dependent kinase inhibitor, in a p53-independent manner. However, it is still unknown how intracellular Cl(-) regulates p21 expression level. In this study, we demonstrate that mitogen-activated protein kinases (MAPKs) are involved in the p21 upregulation and cell-cycle arrest induced by reduction of [Cl(-)](i). Culture of MKN28 cells in a low Cl(-) medium significantly induced phosphorylation (activation) of MAPKs (ERK, p38, and JNK) and G(1)/S cell-cycle arrest. To clarify the involvement of MAPKs in p21 upregulation and cell growth inhibition in the low Cl(-) medium, we studied effects of specific MAPKs inhibitors on p21 upregulation and G(1)/S cell-cycle arrest in MKN28 cells. Treatment with an inhibitor of p38 or JNK significantly suppressed p21 upregulation caused by culture in a low Cl(-) medium and rescued MKN28 cells from the low Cl(-)-induced G(1) cell-cycle arrest, whereas treatment with an ERK inhibitor had no significant effect on p21 expression or the growth of MKN28 cells in the low Cl(-) medium. These results strongly suggest that the intracellular Cl(-) affects the cell proliferation via activation of p38 and/or JNK cascades through upregulation of the cyclin-dependent kinase inhibitor (p21) in a p53-independent manner in MKN28 cells.

Abstract: Trafficking is one of the primary mechanisms of epithelial Na(+) channel (ENaC) regulation. Although it is known that ENaCs are recycled between the apical membrane and the intracellular channel pool, it has been difficult to investigate the recycling of ENaCs; especially endogenously expressed ENaCs. The aim of the present study is to investigate if the recycling rates of ENaCs depend on the total amount of recycled ENaCs. To accomplish this point, we established a novel method to estimate the total amount of recycled ENaCs and the ENaC recycling rates by using a specific blocker (benzamil) of ENaC with a high-affinity for functional label of the channels in recycling. Applying this method, we studied if a decrease in the total amount of ENaCs caused by brefeldin A (5 μg/mL, 1 h) affects respectively the rates of insertion and endocytosis of ENaCs to and from the apical membrane in monolayers of renal epithelial A6 cells. Our observations indicate that: 1) both insertion and endocytosis rates of ENaC increase when the total amount of ENaCs decreases, 2) the increase in the insertion rate is larger than that in the endocytosis rate, and 3) this larger increase in the insertion rate than the endocytosis rate caused by the decrease in the total amount of ENaCs plays an important role in preventing Na(+) transport from drastically diminishing due to a decrease in the total amount of ENaCs. The newly established analysis of blocker-labeled ENaCs in the present study provides a useful tool to investigate the recycling of endogenously expressed ENaCs.

Abstract: Continuous positive airway pressure (CPAP) therapy of obstructive sleep apnea syndrome (OSAS) is widely accepted. Recently it is reported that central type apnea increases in some patients with OSAS with the application of CPAP, and this type of sleep-disordered breathing is called complex sleep apnea syndrome (comp. SAS). However, until now its concept, mechanism and therapy have not been fully established. We treated 2 cases of comp. SAS with CPAP therapy. When we performed a polysomnography (PSG) examination in case 1 one year later, the symptoms had diminished and the central apnea had decreased, indicating the effectiveness CPAP therapy in case 1. In case 2, the symptoms had not diminished one year later. We therefore performed Adaptive Servo-Ventilation (ASV) therapy, resulting in improvement of symptoms and decrease of the central apnea. CPAP is not always effective in comp. SAS, and ASV can be suitable in such cases.

Abstract: We investigated regulatory mechanisms of Cl- secretion playing an essential role in maintenance of surface fluid in human airway epithelial Calu-3 cells. The present study reports that quercetin (a flavonoid) stimulated bumetanide-sensitive Cl- secretion with reduction of apical Cl- conductance, suggesting that quercetin stimulates Cl- secretion by activating an entry step of Cl- across the basolateral membrane through Na(+)/K(+)/2Cl(-) cotransporter (NKCC1). To clarify the stimulatory mechanism of NKCC1 by quercetin, we verified involvement of PKA, PKC, PTK and cytosolic Ca2+-dependent pathways. A PKA inhibitor (PKI-14-22 amide), a PKC inhibitor (Gö 6983) or with a Ca2+ chelating agent did not affect the quercetin-stimulated Cl- secretion. On the other hand, a PTK inhibitor (AG18) significantly diminished the stimulatory action of quercetin on Cl- secretion without inhibitory effects on apical Cl- conductance, suggesting that a PTK-mediated pathway is involved in the stimulatory action of quercetin. The quercetin action on Cl- secretion was suppressed with brefeldin A (BFA, an inhibitor of vesicular transport from ER to Golgi), and the BFA-sensitive Cl- secretion was not observed in the presence of an EGFR kinase inhibitor (AG1478), suggesting that quercetin stimulates Cl- secretion by causing the EGFR kinase-mediated translocation of NKCC1 or an NKCC1-activating factor to the basolateral membrane in human airway epithelial Calu-3 cells. However, the surface density of NKCC1 was not increased by quercetin, but quercetin elevated the activity of NKCC1. These observations indicate that quercetin stimulates Cl- secretion by activating NKCC1 via translocation of an NKCC1-activating factor via an EGFR kinase-dependent pathway.

Abstract: Cisplatin is widely used for the treatment of non-small-cell lung cancer. However, it can cause unpleasant side effects and also requires prolonged hydration. We conducted a Phase II study of weekly gemcitabine and split-dose cisplatin in patients with advanced non-small-cell lung cancer (NSCLC) in order to reduce toxicity and shorten the time taken by administration. Our aims were to determine the response rate, toxicity and survival time with this regimen in patients with Stage IIIB/IV disease.

Abstract: In the epithelia and endothelia, tight junctions regulate the movement of several substances through the paracellular pathway, maintaining several gradients between apical and basal compartments including osmolality and hydrostatic pressure. In this study, we show that the change of hydrostatic pressure gradient affected tight junctions as well as actin cytoskeleton, cell height and transcellular ion transport. Hydrostatic pressure gradient from basolateral to apical side increased transepithelial conductance and altered claudin-1 localization within several tens of minutes. These changes were promptly restored by the elimination of hydrostatic pressure gradient. Hydrostatic pressure gradient also induced dynamic changes in the actin structure and cell height. We further found that hydrostatic pressure gradient from basolateral to apical side stimulates transcellular Cl(-) transport. Our present findings indicate that the epithelial cell structures and functions are regulated by the hydrostatic pressure gradient which is generated and maintained by the epithelia themselves.

Abstract: There is considerable evidence that schizophrenia is associated with immune system dysregulation. For example, blood and cerebrospinal fluid (CSF) levels of proinflammatory cytokines are significantly increased in schizophrenic patients, and their normalization correlates with improvement in psychotic symptoms. In fact, typical and atypical antipsychotics are reported to modulate immune function in in vitro and in vivo studies. In the present study, we examined the anti-inflammatory effect of antipsychotics, clozapine, olanzapine, risperidone and haloperidol, on serum cytokine levels in lipopolysaccharide (LPS)-treated mice. Atypical antipsychotics, such as clozapine, olanzapine and risperidone, but not haloperidol, suppressed tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and up-regulated IL-10. Moreover, only clozapine, robustly increased the serum levels of IL-10. Clozapine reproduced its anti-inflammatory feature in polyinsinic-polycytidylic acid sodium salt (Poly[I:C])-induced inflammation. Thus, the anti-inflammatory effect of clozapine would adapt to inflammation induced by some varieties of antigens. Several receptor ligands, such as 8-OH-DPAT, ketanserin, prazosin and scopolamine, were also examined as to their anti-inflammatory effects on serum cytokine levels in LPS-treated mice. Ketanserin and prazosin, but not 8-OH-DPAT nor scopolamine, behaved similarly to atypical antipsychotics. However, the remarkable increase of serum IL-10 level observed in clozapine was not detected in ketanserin and prazosin. These results suggest the unique efficacy of atypical antipsychotics in the suppression of proinflammatory cytokines, and the increase of anti-inflammatory cytokine, IL-10.

Abstract: We report the effects of new N-acylated ambroxol derivatives (TEI-588a, TEI-588b, TEI-589a, TEI-589b, TEI-602a and TEI-602b: a, aromatic amine-acylated derivative; b, aliphatic amine-acylated derivative) induced from ambroxol (a mucolytic agent to treat human lung diseases) on Cl(-) secretion in human submucosal serous Calu-3 cells under a Na(+)/K(+)/2Cl(-) cotransporter-1 (NKCC1)-mediated hyper-secreting condition. TEI-589a, TEI-589b and TEI-602a diminished hyper-secretion of Cl(-) by diminishing the activity of NKCC1 without blockade of apical Cl(-) channel (TEI-589a>TEI-602a>TEI-589b), while any other tested compounds including ambroxol had no effects on Cl(-) secretion. These indicate that the inhibitory action of an aromatic amine-acylated derivative on Cl(-) secretion is stronger that that of an aliphatic amine-acylated derivative, and that 3-(2,5-dimethyl)furoyl group has a strong action in inhibition of Cl(-) secretion than cyclopropanoyl group. We here indicate that TEI-589a, TEI-589b and TEI-602a reduce hyper-secretion to an appropriate level in the airway, providing a possibility that the compound can be an effective drug in airway obstructive diseases including COPD by reducing the airway resistance under a hyper-secreting condition.

Abstract: We evaluated the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57BL/6N mice fed a magnesium (Mg(2+))-deficient diet. On the 3rd week, Mg(2+)-deficient mice displayed increased anxiety- and depression-like behavior. In the Mg(2+)-deficient mice, a low does (10mg/kg) of MPTP treatment decreased dopamine (DA) and its metabolites contents in the striatum, but not in control mice. The same dose of MPTP did not influence these neurochemical markers in the mice fed Mg(2+)-deficient diet for 1 week which did not exhibit the altered emotional behavior. These results indicate that Mg(2+)-deficient mice with altered emotional behavior appear to increase the susceptibility to MPTP neurotoxicity in C57BL/6N mice.

Abstract: The aim of the present study is to investigate whether the chloride affects cell growth and cell-cycle progression of cancer cells. In human gastric cancer MKN28 cells, the culture in the Cl(-)-replaced medium (replacement of Cl(-) by NO(3)(-)) decreased the intracellular chloride concentration ([Cl(-)](i)) and inhibited cell growth. The inhibition of cell growth was due to cell-cycle arrest at the G(0)/G(1) phase caused by diminution of CDK2 and phosphorylated Rb. The culture of cells in the Cl(-)-replaced medium significantly increased expressions of p21 mRNA and protein without any effects on p53. These observations indicate that chloride ions play important roles in cell-cycle progression by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells, leading to a novel, unique therapeutic strategy for gastric cancer treatment via control of [Cl(-)](i).

Abstract: Excessive nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) aggravates acute lung injury (ALI) by producing peroxinitrite. We previously showed that the expression of iNOS and lung injury were suppressed by inhalation of a novel iNOS inhibitor, ONO-1714, in mice with Candida-induced ALI, and that nitric oxide produced by iNOS and apoptosis of epithelial cells were found to have a crucial role in Candida-induced ALI. In the present study, we investigated the effect of NO on the apoptosis of alveolar epithelial cells in Candida-induced ALI. Mice were pretreated by inhalation of ONO-1714 or saline (vehicle control of ONO-1714), and were given an intravenous injection of Candida albicans to induce ALI. After 24 h from injection of Candida albicans, we performed bronchoalveolar lavage and removed lung tissues. We assessed apoptosis on the basis of TUNEL staining and caspase 3 activity. Our results showed that apoptosis was suppressed by inhibition of iNOS-derived NO production by ONO-1714 inhalation. The augmented production of NO increased FasL, TNF-alpha, and mRNA production of Bax of lung that induced apoptosis of alveolar epithelial cells. Inhibition of iNOS-derived NO production by ONO-1714 inhalation ameliorated Candida-induced ALI and improved survival by suppressing apoptosis of alveolar epithelial cells.

Abstract: In the present study, we investigated the effect of osmolality on the paracellular ion conductance (Gp) composed of the Na(+) conductance (G(Na)) and the Cl(-) conductance (G(Cl)). An osmotic gradient generated by NaCl with relatively apical hypertonicity (NaCl-absorption-direction) induced a large increase in the G(Na) associated with a small increase in the G(Cl), whereas an osmotic gradient generated by NaCl with relatively basolateral hypertonicity (NaCl-secretion-direction) induced small increases in the G(Na) and the G(Cl). These increases in the Gp caused by NaCl-generated osmotic gradients were diminished by the application of sucrose canceling the NaCl-generated osmotic gradient. The osmotic gradient generated by basolateral application of sucrose without any NaCl gradients had little effects on the Gp. However, this basolateral application of sucrose produced a precondition drastically quickening the time course of the action of the NaCl-generated osmotic gradient on the Gp. Further, we found that application of the basolateral hypotonicity generated by reduction of NaCl concentration shifted the localization of claudin-1 to the apical from the basolateral side. These results indicate that the osmotic gradient regulates the paracellular ion conductive pathway of tight junctions via a mechanism dependent on the direction of NaCl gradients associated with a shift of claudin-1 localization to the apical side in renal A6 epithelial cells.

Abstract: In A6 cells, a renal cell line derived from Xenopus laevis, hypotonic stress stimulates the amiloride-sensitive Na(+) transport. Hypotonic action on Na(+) transport consists of two phases, a nongenomic early phase and a genomic delayed phase. Although it has been reported that, during the genomic phase, hypotonic stress stimulates transcription of Na(+) transport-related genes, such as serum- and glucocorticoid-inducible kinase 1 (SGK1) and subunits of the epithelial Na(+) channel (ENaC), increasing Na(+) transport, the mechanism remains unknown. We focused the present study on the role of intracellular Ca(2+) in hypotonicity-induced SGK1 and ENaC subunit transcription. Since hypotonic stress raises intracellular Ca(2+) concentration in A6 cells, we hypothesized that Ca(2+)-dependent signals participate in the genomic action. Using real-time quantitative RT-PCR and Western blot techniques and measuring short-circuit currents, we observed that 1) BAPTA-AM and W7 blunted the hypotonicity-induced expression of SGK1 mRNA and protein, 2) ionomycin dose dependently stimulated expression of SGK1 mRNA and protein under an isotonic condition and the time course of the stimulatory effect of ionomycin on SGK1 mRNA was remarkably similar to that of hypotonic action on SGK1 mRNA, 3) hypotonic stress stimulated transcription of three ENaC subunits in an intracellular Ca(2+)-dependent manner, and 4) BAPTA-AM retarded the delayed phase of hypotonic stress-induced Na(+) transport but had no effect on the early phase. These observations indicate for the first time that intracellular Ca(2+) plays a role as the second messenger in hypotonic stress-induced Na(+) transport by stimulating transcription of SGK1 and ENaC subunits.

Abstract: BACKGROUND: The mechanisms of hyperoxia-induced lung injury remain poorly defined. Thioredoxin-1 (TRX-1) is a small ubiquitous protein that acts as an important radical scavenger. We investigated the effect of TRX-1 on apoptosis in hyperoxia-induced lung injury. METHODS: Mice were exposed to 98% O(2) to produce a model of hyperoxia-induced lung injury. Using transgenic mice overexpressing human TRX-1 (hTRX-1), we assessed lung structure (n=4 per group), immunohistochemical staining for 8-hydroxy-deoxyguanosine (n=4 per group), TUNEL staining (n=5 per group), cytokine (n=5 per group) of IL-1beta and IL-6, and protein (n=6 per group) and m-RNA levels (n=4 per group) (or both) of cytochrome c, Bcl-2, Bax, p21, and p53 in the lungs. RESULTS: After exposure to hyperoxia, hTRX-1 transgenic mice had significantly decreased alveolar damage. The apoptotic index was significantly lower in hTRX-1 transgenic mice than in wild-type (WT) mice after exposure to hyperoxia. Protein expression of cytochrome c in the lung was significantly lower in hTRX-1 transgenic mice than in WT mice after exposure to hyperoxia. Protein expression and m-RNA levels of Bcl-2 in the lung were significantly higher in hTRX-1 transgenic mice than in WT mice after exposure to hyperoxia. TRX-1 had no effect on the protein and m-RNA levels of Bax and p21. The protein and m-RNA levels of p53 was unaffected by hyperoxia in hTRX-1 transgenic mice. The cytokine level of IL-6 was significantly higher in hTRX-1 transgenic mice than in WT mice after exposure to hyperoxia. TRX-1 had no effect on cytokine level of IL-1beta. CONCLUSIONS: These findings suggest that overexpression of hTRX-1 protects against hyperoxia-induced apoptosis in cells of the alveolar walls. The up-regulating Bcl-2 protein is considered to be one of antiapoptotic effects of TRX-1 in hyperoxia-induced lung injury.

Abstract: BACKGROUND: Exposure of animals to hyperoxia causes lung injury, characterized by diffuse alveolar damage and exudation of plasma into the alveolar space. Reactive oxygen species (ROS) play an important role in the development of hyperoxic lung injury. Mitochondrial oxidative phosphorylation is one of the major sources of ROS. N-acetylcysteine (NAC) is a precursor of glutathione (GSH), which functions as an antioxidant by reducing hydrogen peroxide to water and alcohols. NAC has been shown to diminish lung injury in a large variety of animal models. AIM: We elucidated the mechanism underlying the protective effects of NAC in hyperoxia-induced lung injury. METHODS: Male BALB/c mice were exposed to 98% oxygen for 72 h. The mice inhaled NAC or saline twice a day from 72 h before oxygen exposure to the end of experiment. RESULTS: Inhaled NAC increased the GSH level in lung homogenate. NAC also attenuated cellular infiltrations in both bronchoalveolar lavage fluid (BALF) and lung tissue. The total protein level in BALF and the level of 8-isoprostane, a marker of lipid peroxidation, in lung homogenate were decreased by inhalation of NAC. Inhaled NAC induced the overexpression of Mn superoxide dismutase (MnSOD) mRNA and protein, but did not alter the expressions of other antioxidant enzymes, including CuZnSOD, extracellular SOD, and glutathione peroxydase 1. CONCLUSION: These findings suggest that the antioxidant properties of NAC in hyperoxic lung injury involve a decrease in mitochondrial ROS in association with the induction of MnSOD, in addition to its role as a precursor of GSH.

Abstract: STUDY OBJECTIVES: High-concentration oxygen therapy is used to treat tissue hypoxia, but hyperoxia causes lung injury. Overproduction of nitric oxide by nitric oxide synthase (NOS) is thought to promote hyperoxic lung injury. The present study was conducted to examine the role of inducible nitric oxide synthase (iNOS) in hyperoxic lung injury in mice. MEASUREMENTS AND RESULTS: Mice were exposed to >98% oxygen for 72 h, and ONO-1714 (0.05 mg/kg) (ONO) was subcutaneously administered to block iNOS. Hyperoxia significantly increased total cell count, protein concentration, and nitrites/nitrates in the bronchoalveolar lavage fluid and proinflammatory cytokines in the lung tissue. ONO significantly prevented the increases in all of these variables. ONO suppressed histologic evidence of lung injury. ONO markedly inhibited iNOS protein expression and nitrotyrosine production in lung homogenates. After exposure to hyperoxia, alveolar epithelial cells stained positively for 8-hydroxy-2'-deoxyguanosine, a proper marker of oxidative DNA damage by reactive oxygen species. ONO attenuated this finding. CONCLUSIONS: NOS play important roles in the pathogenesis of hyperoxic lung injury. Selective iNOS inhibitors may be useful for the treatment of hyperoxic lung injury.

Abstract: Epithelial sodium channel (ENaC) plays a crucial role in controlling sodium reabsorption in the kidney keeping the normal blood pressure. We previously reported that the expression of ENaC mRNA in the kidney of Dahl salt-sensitive (DS) rats was abnormally regulated by aldosterone, however it is unknown if dietary sodium affects the expression of ENaC and serum and glucocorticoid-regulated kinase 1 (SGK1), which plays an important role in ENaC activation, in DS rats. In the present study, we investigated whether dietary sodium abnormally affects the expression of ENaC and SGK1 mRNA in DS rats. DS and Dahl salt-resistant (DR) rats (8 weeks old) were divided into three different groups, respectively: (1) low sodium diet (0.005% NaCl), (2) normal sodium diet (0.3% NaCl), and (3) high sodium diet (8% NaCl). The high sodium diet for 4 weeks in DS rats elevated the systolic blood pressure, but did not in any other groups. The expression of alpha-ENaC mRNA in DS rats was abnormally increased by high sodium diet in contrast to DR rats, while it was normally increased by low sodium diet in DS rats similar to DR rats. The expression of beta- and gamma-ENaC mRNA in DS rats was also abnormally increased by high sodium diet unlike DR rats. The expression of SGK1 mRNA was elevated by high sodium diet in DS rats, but it was decreased in DR rats. These observations indicate that the expression of ENaC and SGK1 mRNA is abnormally regulated by dietary sodium in salt-sensitively hypertensive rats, and that this abnormal expression would be one of the factors causing salt-sensitive hypertension.

Abstract: Aldosterone and osmotic stress are well known to regulate the epithelial Na(+) channel (ENaC)-mediated Na(+) transport in renal epithelial cells. However, we have no information on how aldosterone and osmotic stress interact on stimulation of ENaC-mediated Na(+) transport in renal epithelium. In the present report, we studied how application of aldosterone (1 microM for 1 day) modifies the action of hypotonic stress on the ENaC-mediated Na(+) transport in renal A6 epithelial cells by measuring the benzamil (a specific inhibitor for ENaC)-sensitive short-circuit current. The present study suggests that: (1) most ENaCs in cells without aldosterone treatment are translocated to Golgi apparatus, (2) major parts of aldosterone-generated ENaCs are located at the endoplasmic reticulum, (3) aldosterone diminishes the endocytosis rate of ENaCs from the apical membrane without any significant changes in the insertion rate of ENaCs into the apical membrane, and (4) application of sucrose after hypotonic stress stimulates the endocytosis of ENaCs, and elongates the functional life time of ENaCs by enhancing recycle of ENaCs into the endoplasmic reticulum in a retrograde manner.

Abstract: Intracellular pH (pHi) after the NH+4 pulse addition and its removal were measured in isolated alveolar type II cells (ATII cells) using BCECF fluorescence. In the absence of HCO(-3), the NH+4 pulse addition increased pHi (alkali jump) and its removal decreased pH(i) (acid jump) to the control level (no overacidification). This pHi change was induced by reaction 1 (NH3 + H+ <--> NH+4). However, in the presence of HCO(-3), the NH+4 pulse removal decreased pHi (acid jump) with overacidification. The extent of overacidification was decreased by acetazolamide (a carbonic anhydrase inhibitor), bumetanide (an inhibitor of Na+/K+/2Cl(-) cotransporter [NKCC]), and NPPB (an inhibitor of Cl(-) channel). The NH+4 pulse addition led to the accumulation of NH+4 in ATII cells via reaction 1 and NKCC, and the NH+4 pulse removal induced reaction 2 (NH+4 + HCO(-3) --> NH3 + H+ HCO(-3)) in addition to the reversal of reaction 1. Thus, NH+4 that entered via NKCC reacts with HCO(-3) (reaction 2) to produce H+, which induces overacidification in the acid jump. After the overacidification, the pH(i) recovery consisted of a rapid recovery (first phase) followed by a slow recovery (second phase). The first phase was inhibited by NPPB, glybenclamide, amiloride, and an Na+-free solution, and the second phase was inhibited by DIDS, MIA, and an Na+-free solution. Both phases were accelerated by a high extracellular HCO(-3) concentration. These observations indicate that the first phase was induced by HCO(-3) entry via Cl(-) channels coupled with Na+ channels activities, and that the second phase was induced by H+ extrusion via Na+/H+ exchanger and by HCO(-3) entry via HCO(-3) cotransporter. Thus, in ATII cells, HCO(-3) entry via Cl(-) channels is essential for recovering pHi after overacidification during the acid jump and for removing NH+4 that entered via NKCC from ATII cells, suggesting HCO(-3)-dependent NH3 excretion from lungs.

Abstract: In the present study, we investigated if Cl(-) regulates the proliferation of the MC3T3-E1 osteoblastic cells. The proliferation of MC3T3-E1 osteoblastic cells was diminished by lowering the extracellular Cl(-) concentration ([Cl(-)](o)) in the culture medium. The lowered in [Cl(-)](o) increased the periods of the G(0)/G(1) and the G(2)/M phases in cell cycle. We further studied the effects of [Cl(-)](o) on the key enzymes, Rb and cdc2, playing key roles in checking points of the G(0)/G(1) and the G(2)/M phases in cell cycle. The lowered in [Cl(-)](o) diminished the active forms of enzymes, Rb and cdc2. We further found that the action of lowered [Cl(-)](o) on the cell proliferation, the cell cycle, Rb and cdc2 was abolished by the presence of 2mM glutamine, but not by that of pyruvate as another Krebs cycle substrate. Taken together, these observations indicate here for the first time that Cl(-) modulates Rb and cdc2, enhancing the proliferation of the MC3T3-E1 osteoblastic cells.

Abstract: The purpose of the present study is to characterize the ENaC-mediated Na+ absorption in human upper airway epithelia, nasal cavity, and paranasal sinus. To perform the purpose, we obtained epithelial cells from human nasal polyp (NP) and paranasal sinus mucosa (PSM) by endoscopic surgery. We measured the short-circuit current (I(sc)) sensitive to benzamil (a specific ENaC blocker). The benzamil-sensitive I(sc) (Na+ absorption) in NP was larger than that in PSM. The mRNA expression of three subunits of ENaC was as follows: alpha>beta>gamma in both tissue, NP and MS. The mRNA expression of gamma subunit of ENaC in NP was larger than that in PSM, but no difference of mRNA expression of alpha or beta ENaC subunit between NP and PSM was detected. We found correlation of the Na+ absorption to mRNA expression of gamma ENaC in NP and PSM. Forskolin diminished the Na+ absorption associated with an increase in Cl- secretion. These observations suggest that: (1) human NP absorbs more ENaC-mediated Na+ than PSM, (2) expression of gamma ENaC in plays a key role in the ENaC-mediated Na+ absorption in NP and PSM, and (3) cAMP diminishes the ENaC-mediated Na+ absorption by stimulating Cl- secretion (diminution of driving force for Na+ absorption) in NP and PSM.

Abstract: In the present study, we clarified how the NaCl gradient across the epithelial cells regulates the paracellular ion conductance. Under isotonic conditions, the absorption-directed NaCl gradient elevated the paracellular conductances of Na(+) (G(Na)) and Cl(-) (G(Cl)), while the secretion-directed NaCl gradient diminished the G(Na) and G(Cl). We further investigated the paracellular ionic conductances of NMDG (G(NMDG)) and gluconate (G(gluconate)) by replacing Na(+) with NMDG or Cl(-) with gluconate. The G(NMDG) was lower than the G(Na) and the replacement of Na(+) with NMDG decreased the G(Cl). The G(gluconate) was lower than the G(Cl) and the replacement of Cl(-) with gluconate also decreased the G(Na). These observations suggest the interaction of cations and anions on paracellular ionic conductances; i.e., cations affect paracellular anion conductances and anions affect paracellular cation conductances.

Abstract: The Na(+)/K(+)/2Cl(-) cotransporter (NKCC) mediates electroneutral transport of 2Cl(-) coupled with Na(+) and K(+) across the plasma membrane, and plays crucial roles in Cl(-) uptake into the cells, homeostasis of cellular Cl(-), and cell volume regulation. However, we have very limited information on the roles of ion transporters in neurite outgrowth in neuronal cells. In the present study, we report the role of NKCC1 (an isoform of NKCC) in NGF-induced neurite outgrowth of rat pheochromocytoma PC12D cells. The expression level of NKCC1 protein was increased by NGF treatment. Knock-down of NKCC1 by RNA interference (RNAi) drastically diminished the NGF-induced neurite outgrowth. Transfection of enhanced green fluorescent protein (EGFP)-tagged rat NKCC1 into cells for clarification of intracellular localization of NKCC1 revealed that the EGFP-rNKCC1 was mainly localized in the plasma membrane at growth cone during neurite outgrowth. These observations suggest that NKCC1 plays a fundamental role in NGF-induced neurite outgrowth of PC12D cells.

Abstract: Excessive nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) aggravates acute lung injury (ALI) by producing peroxynitrite. We previously showed by immunostaining that the expression of iNOS was suppressed by inhalation of N(G)-nitro-L-arginine methyl ester in mice with Candida-induced ALI. This study tested the hypothesis that a novel iNOS inhibitor suppresses not only iNOS expression, but also iNOS messenger RNA (mRNA) production by interrupting a positive feedback loop at the time of NO production in Candida-induced ALI. Mice were pretreated by inhalation of saline or ONO-1714, a selective iNOS inhibitor, and were given an intravenous injection of Candida albicans to induce ALI. After inhalation of 1 mM aerosolized ONO-1714, the nitrite-nitrate concentration in bronchoalveolar lavage fluid (BALF) at 24 h was significantly lower than that after inhalation of saline. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) levels and neutrophils in BALF were decreased by inhalation of ONO-1714. Inhalation of ONO-1714 markedly suppressed nitrotyrosine production and inhibited the expression of iNOS mRNA as well as proteins in the lung. Survival was prolonged by inhalation of ONO-1714. We conclude that pretreatment with inhaled ONO-1714 suppresses the production of peroxinitrite and decreases oxidative stress associated with peroxinitrite in Candida-induced ALI.

Abstract: Regulatory volume decrease (RVD) occurs after hypotonicity-caused cell swelling. RVD is caused by activation of ion channels and transporters, which cause effluxes of K(+), Cl(-), and H(2)O, leading to cell shrinkage. Recently, we showed that hypotonicity stimulated transepithelial Na(+) reabsorption via elevation of epithelial Na(+) channel (alpha-ENaC) expression in renal epithelia A6 cells in an RVD-dependent manner and that reduction of intracellular Cl(-) concentration ([Cl(-)](i)) stimulated the Na(+) reabsorption. These suggest that RVD would reveal its stimulatory action on the Na(+) reabsorption by reducing [Cl(-)](i). However, the reduction of [Cl(-)](i) during RVD has not been definitely analyzed due to technical difficulties involved in halide-sensitive fluorescent dyes. In the present study, we developed a new method for the measurement of [Cl(-)](i) change during RVD by using a high-resolution flow cytometer with a halide-specific fluorescent dye, N-(6-methoxyquinolyl) acetoethyl ester. The [Cl(-)](i) in A6 cells in an isotonic medium was 43.6 +/- 3.1 mM. After hypotonic shock (268 to 134 mosmol/kgH(2)O), a rapid increase of cell volume followed by RVD occurred. The RVD caused drastic diminution of [Cl(-)](i) from 43.6 to 10.8 mM. Under an RVD-blocked condition with NPPB (Cl(-) channel blocker) or quinine (K(+) channel blocker), we did not detect the reduction of [Cl(-)](i). Based on these observations, we conclude that one of the physiological significances of RVD is the reduction of [Cl(-)](i) and that RVD shows its action via reduction of [Cl(-)](i) acting as an intracellular signal regulating cellular physiological functions.

Abstract: We previously reported that hypotonic stress stimulated transepithelial Na(+) transport via a pathway dependent on protein tyrosine kinase (PTK; Niisato N, Van Driessche W, Liu M, Marunaka Y. J Membr Biol 175: 63-77, 2000). However, it is still unknown what type of PTK mediates this stimulation. In the present study, we investigated the role of receptor tyrosine kinase (RTK) in the hypotonic stimulation of Na(+) transport. In renal epithelial A6 cells, we observed inhibitory effects of AG1478 [an inhibitor of the EGF receptor (EGFR)] and AG1296 [an inhibitor of the PDGF receptor (PDGFR)] on both the hypotonic stress-induced stimulation of Na(+) transport and the hypotonic stress-induced ligand-independent activation of EGFR. We further studied whether hypotonic stress activates members of the MAP kinase family, ERK1/2, p38 MAPK, and JNK/SAPK, via an RTK-dependent pathway. The present study indicates that hypotonic stress induced phosphorylation of ERK1/2 and JNK/SAPK, but not p38 MAPK, that the hypotonic stress-induced phosphorylation of ERK1/2 and JNK/SAPK was diminished by coapplication of AG1478 and AG1296, and that only JNK/SAPK was involved in the hypotonic stimulation of Na(+) transport. A further study using cyclohexamide (a protein synthesis inhibitor) suggests that both RTK and JNK/SAPK contributed to the protein synthesis-independent early phase in hypotonic stress-induced Na(+) transport, but not to the protein synthesis-dependent late phase. The present study also suggests involvement of phosphatidylinositol 3-kinase (PI3-kinase) in RTK-JNK/SAPK cascade-mediated Na(+) transport. These observations indicate that 1) hypotonic stress activates JNK/SAPK via RTKs in a ligand-independent pathway, 2) the RTK-JNK/SAPK cascade acts as a mediator of hypotonic stress for stimulation of Na(+) transport, and 3) PI3-kinase is involved in the RTK-JNK/SAPK cascade for the hypotonic stress-induced stimulation of Na(+) transport.

Abstract: Neltenexine has been applied to human lung diseases such as chronic obstructive pulmonary disease (COPD) as a mucolytic agent. However, we have no information on the neltenexine action in bronchial epithelial cells. We studied the neltenexine action on the ion transport in human submucosal serous Calu-3 cells. Under a hyper-secreting condition caused by terbutaline (a beta2-adrenergic agonist), neltenexine diminished anion secretion by inhibiting the Cl- and HCO3- uptake via Na+/K+/2Cl- cotransporter and Na+/HCO3- cotransporter without blockade of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, and also diminished anion secretion via stimulation of Cl-/HCO3- exchanger, which facilitates the extrusion of more CFTR-permeant anion, Cl-, with the uptake of less CFTR-permeant anion, HCO3-. Thus, neltenexine reduced the hyper-secretion to keep an appropriate fluid level in the airway, providing a possibility that neltenexine can be an effective drug in airway obstructive diseases by decreasing the airway resistance under a hyper-secreting condition.

Abstract: We investigated a role of p38 MAPK in the regulation of transepithelial Na(+) reabsorption by chronic application (20-24h) of hypotonicity (hypotonic stress) in renal epithelial A6 cells. Pretreatment with a specific p38 MAPK inhibitor (SB202190) significantly reduced the chronic hypotonicity-stimulated transepithelial Na(+) reabsorption by diminishing the Na(+) entry through epithelial Na(+) channel (ENaC) in the apical membrane and the Na(+) extrusion via the Na(+)/K(+) ATPase (pump), although the rate limiting step was still the Na(+) entry step. We further examined whether the inhibitory effects of SB202190 on the transepithelial Na(+) reabsorption is caused through suppression of mRNA expression of ENaC participating in the transepithelial Na(+) reabsorption as the Na(+) entry pathway. The chronic hypotonicity increased the mRNA expression of alpha-, beta-, and gamma-subunits of ENaC. Moreover, we found that inhibition of p38 MAPK by SB202190 diminished the mRNA expression of beta- and gamma-ENaC but not alpha-ENaC. Based on these observations, it is suggested that the chronic hypotonicity stimulates the renal transepithelial Na(+) reabsorption by upregulating the mRNA expression of beta- and gamma-ENaC via a p38 MAPK-dependent pathway.

Abstract: Nasal cavity and paranasal sinus have various functions. However, little information is available on ion transport in these upper airway epithelia. In the present study, we measured the anion secretion and the anion channel activity to characterize the ion transport in epithelial cells prepared from human paranasal sinus mucosa (PSM) and nasal polyp (NP). To estimate the anion secretion and the anion channel activity, we measured the short-circuit current (Isc) and the transepithelial conductance (Gt) sensitive to NPPB (a Cl(-) channel blocker). The NPPB-sensitive Isc in PSM was larger than that in NP, correlating to the NPPB-sensitive Gt (Cl(-) channel activity). Forskolin stably elevated the NPPB-sensitive Isc associated with an increase in the NPPB-sensitive Gt in PSM and NP. UTP transiently stimulated the Isc associated with an elevation of Gt in PSM and NP. The stimulatory action of UTP on Isc and Gt was diminished by application of NPPB but not benzamil in PSM and NP, suggesting that UTP induced the NPPB-sensitive Isc (Cl(-) secretion) and Gt (Cl(-) channel activity). These observations suggest that in human PSM and NP, cAMP stably stimulates anion secretion by activating the Cl(-) (anion) channels, and that UTP just transiently elevates anion secretion via activation of some Cl(-) (anion) channels.

Abstract: Since our experimental results suggest that UT-B1 functions as active water transporter against osmotic gradient in C6 glial cells, we report here for the first time the evidence for the active water transport. Exposure of C6 cells to a hyperosmotic solution containing glycerol or sucrose produced cell shrinkage due to water efflux according to osmotic gradient for water movement. On the other hand, C6 cells show cell swelling against osmotic gradient for water movement just after exposure to a hyperosmotic solution containing urea, indicating that water influx against osmotic gradient for water movement is accelerated by urea; i.e., urea performs active water transport. A specific inhibitor of UT-B, pCMBS, blocked the urea-induced swelling. The urea-induced cell swelling was significantly suppressed in the siRNA-induced UT-B1-knockdown C6 cells. Taken together, these observations indicate that UT-B1 acts as an active water transporter, providing a new model on active water transport.

Abstract: OBJECTIVES: We conducted phase I and II studies of biweekly docetaxel and cisplatin with concurrent radiotherapy, followed by consolidation chemotherapy with the same drugs in patients with locally advanced, unresectable non-small-cell lung cancer (NSCLC). Our objectives were to define the maximum-tolerated dose and dose-limiting toxicity (DLT) in the phase I study, and to determine the response rate, toxicity, and survival rate at the recommended dose (RD) in the phase II study. METHODS: Patients with unresectable stage IIIA and IIIB NSCLC were studied. Six to eight cycles of docetaxel and cisplatin were administered at 2-week intervals. In the phase I study, patients received four dose levels: level 1, docetaxel/cisplatin=30/40 mg/m2; level 2, 35/40; level 3, 40/40; and level 4, 45/40. Radiotherapy was delivered at a rate of 2 Gy per fraction/day up to a total dose of 60 Gy over the course of 6 weeks, during the first three cycles of chemotherapy. RESULTS: DLT comprised neutropenia at level 4 in the phase I study (n=15), and level 3 was considered the RD. In the phase II study (n=46), two patients had a complete response (4.3%) and 34 had a partial response (73.9%), for an overall response rate of 78.2% [95% CI (66.3-90.2%)]. The survival rate was 69.1% at 1 year and 39.6% at 2 years, with a median survival time of 19.1 months. Leukopenia, neutropenia, anemia, and radiation esophagitis were the most common toxic reactions, with Grade > or = 3 reactions occurring at rates of 77, 70, 17, and 8%, respectively. CONCLUSION: Biweekly docetaxel and cisplatin with concurrent RT was active and well tolerated in patients with unresectable stage III NSCLC.

Abstract: Furosemide, a blocker of Na(+)/K(+)/2Cl(-) cotransporter (NKCC), is often used as a diuretic to improve edema, ascites, and pleural effusion of patients with cancers. The aim of the present study was to investigate whether an NKCC blocker affects cancer cell growth. If so, we would clarify the mechanism of this action. We found that poorly differentiated gastric adenocarcinoma cells (MKN45) expressed the mRNA of NKCC1 three times higher than moderately differentiated ones (MKN28) and that the NKCC in MKN45 showed higher activity than that in MKN28. A cell proliferation assay indicates that furosemide significantly inhibited cell growth in MKN45 cells, but not in MKN28 cells. Using flow cytometrical analysis, we found that the exposure to furosemide brought MKN45 cells to spend more time at the G(0)/G(1) phase, but not MKN28 cells. Based on these observations, we indicate that furosemide diminishes cell growth by delaying the G(1)-S phase progression in poorly differentiated gastric adenocarcinoma cells, which show high expression and activity of NKCC, but not in moderately differentiated gastric adenocarcinoma cells with low expression and NKCC activity.

Abstract: Ambroxol is often used as a mucolytic agent in various lung diseases. However, it is unclear how ambroxol acts on bronchial epithelial cells. To clarify the action of ambroxol, we studied the effects of ambroxol on the ion transport in human Calu-3 cells, a human submucosal serous cell line, measuring the transepithelial short-circuit current and conductance across monolayers of Calu-3 cells. Ambroxol of 100 microM diminished the terbutaline (a beta2-adrenergic agonist)-stimulated Cl-/HCO3(-)-dependent secretion without any decreases in the conductance of cystic fibrosis transmembrane conductance regulator (CFTR) channel locating on the apical membrane. On the other hand, under the basal (unstimulated) condition ambroxol increased the Cl(-)-dependent secretion with no significant change in the apical CFTR channel conductance and decreased the HCO3- secretion associated with a decrease in the apical CFTR channel conductance. Ambroxol had no major action on the epithelial Na+ channel (ENaC) or the ENaC-mediated Na+ absorption. These results indicate that in Calu-3 cells: (1) under the basal (unstimulated) condition ambroxol increases Cl- secretion by stimulating the entry step of Cl- and decreases HCO3- secretion by diminishing the activity of the CFTR channel and/or the Na+/HCO3(-)-dependent cotransporter, (2) under the adrenergic agonist-stimulated condition, ambroxol decreases Cl- secretion by acting on the Cl-/HCO3- exchanger, and (3) ambroxol has a more powerful action than the adrenergic agonist on the Cl-/HCO3- exchanger, leading fluid secretion to a moderately stimulated level from a hyper-stimulated level.

Abstract:

Abstract: Aldosterone plays a crucial role in controlling mineral balance in our body. The mechanism of aldosterone has been reported to elevate renal Na+ reabsorption by stimulating expression of epithelial Na+ channel (ENaC) and also activate an ENaC-regulating protein kinase, serum and glucocorticoid-regulated kinase 1 (SGK1). However, it is unknown whether aldosterone shows its stimulatory action on ENaC and SGK1 under an abnormal, salt-sensitive hypertensive condition. To clarify this point, we studied how aldosterone regulates expression of ENaC and SGK1 in Dahl salt-sensitive (DS) rat that shows hypertension with high salt diet. RNA and protein were extracted from the kidney 6 h after application of aldosterone (1.5 mg/kg body weight) subcutaneously injected into adrenalectomized DS and Dahl salt-resistant (DR) rats. Aldosterone decreased mRNA expression of beta- and gamma-ENaC in DS rat unlike DR rat, while aldosterone increased alpha-ENaC mRNA expression in DS rat similar to DR rat. Further, we found that aldosterone elevated SGK1 expression in DR rat, but not in DS rat. These observations indicate that ENaC and SGK1 are abnormally regulated by aldosterone in salt-sensitive hypertensive rats, suggesting that disturbance of the aldosterone regulation would be one of factors causing salt-sensitive hypertension.

Abstract: In guinea pig antral mucous cells, ACh stimulates the Ca(2+)-regulated exocytosis, which has a characteristics feature: an initial transient phase followed by a sustained phase. The effects of cGMP on ACh-stimulated exocytosis were studied in guinea pig antral mucous cells using video microscopy. cGMP enhanced the frequency of ACh-stimulated exocytotic events, whereas cGMP alone did not induce any exocytotic events under the ACh-unstimulated condition. cGMP did not stimulate either Ca(2+) mobilization or cAMP accumulation. The Ca(2+) dose-response studies demonstrated that cGMP shifted the dose-response curve upward with no shift to the lower concentration. This indicates that cGMP increased maximal responsiveness of the Ca(2+)-regulated exocytosis, but not the Ca(2+) sensitivity. Moreover, under a condition of ATP depletion by dinitrophenol (DNP) or anoxia (N(2) bubbling), ACh evoked only a sustained phase in exocytotic events with no initial transient phase. However, ACh evoked an initial transient phase followed by a sustained phase with addition of cGMP before ATP depletion, whereas only a sustained phase was evoked in a case of cGMP addition after ATP depletion. Thus cGMP-induced enhancement in ACh-stimulated exocytotic events requires ATP, suggesting that cGMP modulates ATP-dependent priming of Ca(2+)-regulated exocytosis in antral mucous cells. In conclusion, cGMP increases the number of primed granules via acceleration of the ATP-dependent priming, which enhances the Ca(2+)-regulated exocytosis stimulated by ACh.

Abstract: The epithelial Na(+) transport via an epithelial Na(+) channel (ENaC) expressed in the lung epithelium would play a key role in recovery from lung edema at acute lung injury by removing the fluid in lung luminal space. The lung edema causes dysfunction of gas exchange, decreasing oxygen pressure level of artery [P(aO(2))]. To study if ENaC plays a key role in recovering P(aO(2)) from a decreased level to a normal one in acute lung injury, we applied benzamil (20microM, a specific blocker of ENaC) to the lung luminal space in acute lung injury treated with high frequency oscillation ventilation (HFOV) that is a lung-protective ventilation with a lower tidal volume and a smaller pressure swing than conventional mechanical ventilation (CMV). Benzamil facilitated the recovery of P(aO(2)) in acutely injured lung with HFOV but not CMV. The observation suggests that in acutely injured lung treated with HFOV an ENaC blocker, benzamil, can be applied as a therapeutic drug for acute lung injury combing with HFOV.

Abstract: The present study provides a new concept of the spare receptor. Model [A]: 1) Several receptors connect with an effector; 2) if an agonist occupies one of the receptors connecting with one effector, the effector fully functions. When the number of receptors connecting with one effector is "m", the relationship between the functional effectors (E) and the concentration of agonists ([a]) is as follows: [formula: see text] where Rt is the total number of receptors and Kd is the agonist dissociation constant from the receptor. Model [B]: 1) Several receptors connect with an effector; 2) only when agonists occupy all of the receptors connecting with one effector, the effector functions. The relationship between E and [a] is as follows: [formula: see text] If m=1, equations (I) and (II) are exactly the same as the Michaelis-Menten equation. If m is larger than 1, the apparent saturation in the effector efficiency becomes larger in Model [A], and smaller in Model [B], respectively. The dissociation of the fractional efficiency of effectors from the fractional binding of agonists to receptors becomes larger as m becomes larger in both models. Further, we propose a variable model, including the concept of agonist-occupancy-dependent stability in the functional conformation change of the effector; only when more than j pieces of receptors connecting with one effector are occupied by agonists, the effector functions (Model [M]). The relationship between E and [a] is as follows: [formula: see text]

Abstract: Transepithelial Cl(-) secretion in polarized renal A6 cells is composed of two steps: (1) Cl(-) entry step across the basolateral membrane mediated by Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and (2) Cl(-) releasing step across the apical membrane via cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. We estimated CFTR Cl(-) channel activity and transcellular Cl(-) secretion by measuring 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB, a blocker of CFTR Cl(-) channel)-sensitive transepithelial conductance (Gt) and short-circuit current (Isc), respectively. Pretreatment with 1 microM insulin for 24 h had no effects on NPPB-sensitive Gt or Isc. On the other hand, in A6 cells treated with carbobenzoxy-L-leucyl-leucyl-L-leucinal (MG132; 100 microM for 2 h) that inhibits endocytosis of proteins at the plasma membrane into the cytosolic space, insulin pretreatment increased the NPPB-sensitive Isc with no effects on NPPB-sensitive Gt. Genistein (100 microM) induced sustained increases in NPPB-sensitive Gt and Isc, which were diminished by brefeldin A (a blocker of protein translocation to Golgi apparatus from endoplasmic reticulum). Co-application of insulin and genistein synergically stimulated the NPPB-sensitive Isc without any effects on NPPB-sensitive Gt. These observations suggest that: (1) insertion and endocytosis of NKCC are stimulated by insulin, (2) the insulin-induced stimulation of NKCC insertion into the basolateral membrane is offset by the stimulatory action on NKCC endocytosis from the basolateral membrane, (3) genistein stimulates insertion of both CFTR Cl(-) channel into the apical membrane and NKCC into the basolateral membrane, and (4) insulin and genistein synergically stimulated NKCC insertion into the basolateral membrane.

Abstract: In renal epithelial A6 cells, aldosterone applied for 24 h increased the transepithelial Cl- secretion over 30-fold due to activation of the Na+/K+/2Cl- cotransporter and stimulated the transepithelial Na+ absorption, activity of epithelial Na+ channel (ENaC), and alpha-ENaC mRNA expression. The stimulatory action of aldosterone on the transepithelial Na+ absorption, ENaC activity, and alpha-ENaC mRNA expression was diminished by 24h-pretreatment with quercetin (an activator of Na+/K+/2Cl- cotransporter participating in Cl- entry into the cytosolic space) or 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB) (a blocker of Cl- channel participating in Cl- release from the cytosolic space), while 24h-pretreatment with bumetanide (a blocker of Na+/K+/2Cl- cotransporter) enhanced the stimulatory action of aldosterone on transepithelial Na+ absorption. On the other hand, under the basal (aldosterone-unstimulated) condition, quercetin, NPPB or bumetanide had no effect on transepithelial Na+ absorption, activity of ENaC or alpha-ENaC mRNA expression. These observations suggest that although aldosterone shows overall its stimulatory action on ENaC (transepithelial Na+ transport), aldosterone has an inhibitory action on ENaC (transepithelial Na+ transport) via activation of the Na+/K+/2Cl- cotransporter, and that modification of activity of Cl- transporter/channel participating in the transepithelial Cl- secretion influences the aldosterone-stimulated ENaC (transepithelial Na+ transport).

Abstract: Hypotonicity stimulates transepithelial Na(+) reabsorption in renal A6 cells, but the mechanism for this stimulation is not fully understood. In the present study, we found that hypotonicity stimulated Na(+) reabsorption through increases in mRNA expression of the alpha-subunit of the epithelial Na(+) channel (alpha-ENaC). Hypotonicity decreases cytosolic Cl(-) concentration; therefore, we hypothesized that hypotonicity-induced decreases in cytosolic Cl(-) concentration could act as a signal to regulate Na(+) reabsorption through changes in alpha-ENaC mRNA expression. Treatment with the flavone apigenin, which activates the Na(+)-K(+)-2Cl(-) cotransporter and increases cytosolic Cl(-) concentration, markedly suppressed the hypotonicity-induced increase in alpha-ENaC mRNA expression. On the other hand, blockade of the Na(+)-K(+)-2Cl(-) cotransporter decreases cytosolic Cl(-) concentration and increased alpha-ENaC mRNA expression and Na(+) reabsorption. Blocking Cl(-) channels with 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the hypotonicity-induced decrease in cytosolic Cl(-) concentration and suppressed the hypotonicity-induced increase in alpha-ENaC mRNA expression. Coapplication of NPPB and apigenin synergistically suppressed alpha-ENaC mRNA expression. Thus, in every case, changes in cytosolic Cl(-) concentration were associated with changes in alpha-ENaC mRNA expression and changes in Na(+) reabsorption: decreases in cytosolic Cl(-) concentration increased alpha-ENaC mRNA and increased Na(+) reabsorption, whereas increases in cytosolic Cl(-) concentration decreased alpha-ENaC mRNA and decreased Na(+) reabsorption. These findings support the hypothesis that changes in cytosolic Cl(-) concentration are an important and novel signal in hypotonicity-induced regulation of alpha-ENaC expression and Na(+) reabsorption.

Abstract: Flavonoid, a plant extract, exhibits various biological actions. Dietary flavonoid intake is reported to reduce an elevated blood pressure, however the mechanism is unknown. The epithelial Na+ channel (ENaC) in the kidney plays a key role in the regulation of blood pressure by contributing to the Na+ reabsorption in renal tubules. Thus, we investigated the effect of quercetin, a flavonoid, on ENaC mRNA expression in the kidney of hypertensive Dahl salt-sensitive rats. Dahl salt-sensitive rats of 8 weeks were acclimated for 1 week in a metabolic cage and were subsequently kept for 4 weeks under four different conditions: (1) normal salt diet (0.3% NaCl), (2) normal salt diet with quercetin (10 mg/kg/day), (3) high-salt diet (8% NaCl), and (4) high-salt diet with quercetin. Quercetin diminished the alphaENaC mRNA expression in the kidney associated with reduction of the systolic blood pressure elevated by high-salt diet, suggesting that one of the mechanisms of the flavonoid's antihypertensive effect on salt-sensitive hypertension would be mediated through downregulation of ENaC expression in the kidney.

Abstract: We have reported that in renal epithelial A6 cells flavones stimulate the transepithelial Cl- secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel and/or the Na+/K+/2Cl- cotransporter. On the other hand, it has been established that cAMP activates the CFTR Cl- channel and the Na+/K+/2Cl- cotransporter. However, no information is available on the interaction between cAMP and flavones on stimulation of the CFTR Cl- channel and the Na+/K+/2Cl- cotransporter. To clarify the interaction between cAMP and flavones, we studied the regulatory mechanism of the CFTR Cl- channel and the Na+/K+/2Cl- cotransporter by flavones (apigenin, luteolin, kaempherol, and quercetin) under the basal and cAMP-stimulated conditions in renal epithelial A6 cells. Under the basal (cAMP-unstimulated) condition, these flavones stimulated the Cl- secretion by activating the Na+/K+/2Cl- cotransporter without any significant effects on the CFTR Cl- channel activity. On the other hand, these flavones diminished the activity of the cAMP-stimulated Na+/K+/2Cl- cotransporter without any significant effects on the CFTR Cl- channel activity. Interestingly, the level of the flavone-induced Cl- secretion under the basal condition was identical to that under the cAMP-stimulated condition. Based on these results, it is suggested that although both cAMP and flavones activate the Na+/K+/2Cl- cotransporter, these flavones have more powerful effects than cAMP on the Na+/K+/2Cl- cotransporter.

Abstract: Maintaining the extracellular K(+) concentration ([K(+)](o)) between 15 and 60 mM induced oscillations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat submandibular acinar cells during stimulation with acetylcholine (ACh, 1 micro M). These [Ca(2+)](i) oscillations were also induced by 1 micro M thapsigargin and were inhibited by 50 micro M La(3+), 1 micro M Gd(3+), or the removal of extracellular Ca(2+), indicating that the [Ca(2+)](i) oscillations were generated by store-operated Ca(2+) entry (SOC). The frequency of the ACh-evoked [Ca(2+)](i) oscillations increased from 0.8 to 2.3 mHz as [K(+)](o) was increased from 15 to 50 mM. TEA (an inhibitor of K(+) channels) also induced [Ca(2+)](i) oscillations at [K(+)](o) of 4.5 or 7.5 mM in ACh-stimulated cells. These data suggest that depolarization causes [Ca(2+)](i) to oscillate in ACh-stimulated submandibular acinar cells. Pertussis toxin (PTX, an inhibitor of G proteins) caused [Ca(2+)](i) to be sustained at a high level in ACh-stimulated cells at 25 mM or 60 mM [K(+)](o). This suggests that the [Ca(2+)](i) oscillations are generated by a periodic inactivation of the SOC channels via PTX-sensitive G proteins, which are stimulated by depolarization. Moreover, in the presence of DBcAMP or forskolin which accumulated cAMP the frequency of the [Ca(2+)](i) oscillations remained constant (approximately 1.2 mHz) when [K(+)](o) was maintained in the range 25-60 mM. Based on these observations in ACh-stimulated submandibular acinar cells, we conclude that depolarization stimulates the PTX-sensitive G proteins, which inactivate the SOC channels periodically ([Ca(2+)](i) oscillation), while hyperpolarization or PTX inhibits the G proteins, maintaining the activation of the SOC channels. Accumulation of cAMP is likely to modulate the PTX-sensitive G proteins.

Abstract: The present study was performed to clarify the effect of H89, an inhibitor of cAMP-activated protein kinase (protein kinase A; PKA), on Na(+) absorption in fetal rat alveolar type II epithelium. H89 stimulated the Na(+) absorption by increasing the open probability (Po) and number of a nonselective cation (NSC) channel composed of four alpha subunits of epithelial Na(+) channel (ENaC). Brefeldin A (BFA), an inhibitor of intracellular protein translocation, blocked the stimulatory action of H89 on the Na(+) absorption by interrupting the action of H89 on the Po and number of the NSC channel. H85, an inactive form of H89, showed an effect similar to H89, suggesting that H89 does not show its effect by inhibiting PKA, but acts on the channel depending the structure. These observations indicate that: (1) the H89 induced increase in number of the channel at the apical membrane is due to translocation of alpha subunit of ENaC to the apical membrane, (2) the elevation of Po of the channel is mediated through translocation of a protein activating alpha subunit of ENaC, and (3) the effect of H89 is dependent on its structure without any relation to PKA.

Abstract: The epithelial Na+ channel (ENaC), composed of three subunits (alphabetagamma), is expressed in various Na(+)-absorbing epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. By using patch clamp techniques, we have examined the effect of cytosolic ATP on the activity of the rat alphabetagammaENaC (rENaC) stably expressed in NIH-3T3 cells and in Madin-Darby canine kidney epithelial cells. The inward whole-cell current attributable to rENaC activity ran down when these cells were dialyzed with an ATP-free pipette solution in the conventional whole-cell voltage-clamping technique. This run down was prevented by 2 mM ATP (but not by AMP or ADP) in the pipette solution or by the poorly or non-hydrolyzable analogues of ATP (adenosine 5'-O-(thiotriphosphate) and adenosine 5'-(beta,gamma-imino)triphosphate) in both cell lines, suggesting that protection from run down was mediated through non-hydrolytic nucleotide binding. Accordingly, we demonstrate binding of ATP (but not AMP) to alpharENaC expressed in Madin-Darby canine kidney cells, which was inhibited upon mutation of the two putative nucleotide-binding motifs of alpharENaC. Single channel analyses indicated that the run down of currents observed in the whole-cell recording was attributable to run down of channel activity, defined as NPo (the product of the number of channels and open probability). We propose that this novel ATP regulation of ENaC may be, at least in part, involved in the fine-tuning of ENaC activity under physiologic and pathophysiologic conditions.

Abstract: We studied effects of tyrphostin A23 (an inhibitor of protein tyrosine kinase; PTK) and tyrphostin A63 (an inactive analog of tyrphostin A23) on forskolin-activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and Cl(-) secretion in renal epithelial A6 cells. Tyrphostin A23 and A63 had no effects on the basal CFTR Cl(-) channel and Cl(-) secretion. However, under the forskolin-stimulated condition, tyrphostin A23 and A63 stimulated Cl(-) secretion by activating CFTR Cl(-) channels. These observations suggest that: 1) tyrphostin A23 and A63 stimulate the cAMP-activated CFTR Cl(-) channel via a PTK-independent, structure-dependent mechanism, and 2) tyrphostin A23 and A63 do not stimulate the basal CFTR Cl(-) channel. These lead us to an idea that: 1) cAMP might cause a conformational change of CFTR Cl(-) channel which is accessible by tyrphostins, and 2) tyrphostins would stimulate translocation of the cAMP-modified channel to the apical membrane by binding to the channel.

Abstract: Hypotonicity stimulates translocation of epithelial Na(+) channel (ENaC) to the apical membrane from the intracellular store site of ENaC by activating protein tyrosine kinase (PTK) in renal epithelial A6 cells. Based upon the fact that calmodulin shows its action on other enzymes through PTK caused phosphorylation of tyrosine residues of calmodulin itself, we studied whether a calmodulin-dependent pathway is involved in the action of hypotonicity on ENaC. W7, an antagonist of calmodulin, diminished the stimulatory action of hypotonicity on ENaC, irrespective of W7 treatment before or after application of hypotonicity. Calmodulin is known to regulate three pathways: (1) protein phosphatase 2B (PP2B), (2) Ca(2+)/calmodulin-dependent protein kinase II (CaMK II), and (3) myosin light chain kinase (MLCK). Pretreatment with cyclosporin A, an inhibitor of PP2B, did not influence the hypotonicity action on ENaC. The hypotonicity action on ENaC was partially inhibited by pretreatment with KN93, an inhibitor of CaMK II, but not by addition of KN93 after hypotonic stimulation had been applied. ML-7, an inhibitor of MLCK, showed the action similar to KN93. These observations indicate that: (1) the hypotonicity-induced translocation of ENaC depends on CaMK II and MLCK and (2) ENaC translocated to the apical membrane by hypotonicity is maintained in its activity and/or stability at the apical membrane through a calmodulin-dependent pathway.

Abstract: A beta-adrenergic agonist (beta-agonist), terbutaline, stimulated amiloride-sensitive Na(+) absorption in fetal rat alveolar type II epithelium, contributing to the clearance of lung fluid. Cytosolic Ca(2+) plays an important role in terbutaline-stimulated Na(+) absorption, since Ca(2+)-activated, amiloride-sensitive Na(+)-permeable channels are involved in transcellular Na(+) absorption and terbutaline stably elevates the cytosolic Ca(2+) concentration by stimulating Ca(2+) influx. Therefore, we studied whether Ca(2+) channel blockers (Ni(2+), verapamil, and nifedipine) affect terbutaline-stimulated transcellular Na(+) absorption. Ni(2+) partially blocked the channel responsible for the terbutaline-stimulated Na(+) absorption at the Na(+) entry pathway across the apical membrane of the epithelium, but did not diminish the terbutaline-stimulated transcellular Na(+) absorption. By measuring the capacity of the Na(+),K(+)-pump activity, we determined that the rate-limiting step of the terbutaline-stimulated transcellular Na(+) absorption was the extrusion step across the basolateral membrane by the Na(+),K(+)-pump. The other Ca(2+) channel blockers, verapamil and nifedipine, had effects identical to those of Ni(2+). Based upon these observations, we conclude that, in the beta-agonist-stimulated fetal rat alveolar type II epithelium, Ca(2+) channel blockers diminish amiloride-sensitive channels, but do not affect transcellular Na(+) absorption, since under the beta-agonist-stimulated condition the Na(+),K(+)-pump is the rate-limiting step in Na(+) transport.

Abstract: Lignocaine suppresses insulin-stimulated glucose transport into the cells and insulin-stimulated glycogenesis at doses equivalent to that used in the treatment of muscle pain disorder. We evaluated the direct effect of lignocaine on insulin receptor (IR) kinase activity. After lignocaine (40 mM, approximately equivalent to 1%) or an equal volume (100 microl) saline had been injected into the tibialis anterior muscle of rat, insulin (50 mM g-1 body weight) was administered into the portal vein in vivo. Immunoprecipitation and immunoblotting were used to detect insulin-mediated tyrosine phosphorylation of both IR-beta and insulin receptor substrate (IRS)-1, and insulin-stimulated binding of IRS-1 to p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K) in the extracted muscle. In the in vitro study, purified IR from rat liver and/or recombinant IRS-1 protein with adenosine triphosphate were incubated with lignocaine (4 or 40 mM). Lignocaine reduced insulin-stimulated tyrosine phosphorylation of IR-beta to 12.6+/-5.7% (P<0.001), and IRS-1 to 32.1+/-18.8% (P<0.01), and also reduced insulin-stimulated binding of IRS-1 to p85 to 27.4+/-12.7% (P<0.001) relative to control (100%) in muscle in vivo. The in vitro study revealed that lignocaine directly inhibited both basal and insulin-stimulated tyrosine phosphorylation of IR. These results indicate that clinically used doses of lignocaine inhibit insulin signalling in skeletal muscle. The inhibitory effect of lignocaine on tyrosine kinase activity of the IR underlies the suppression of insulin signalling with lignocaine.

Abstract: An amiloride-sensitive, Ca(2+)-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na+ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca2+ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (Po) of the NSC channel to 0.62 +/- 0.07 from 0.03 +/- 0.01 (mean +/- SE; n = 8) 30 min after application of terbutaline in a solution containing 1 mM Ca2+. The Po of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca2+ to 0.26 +/- 0.05 (n = 8). The cytosolic Ca2+ concentration ([Ca2+]c) in the presence and absence of extracellular Ca2+ was, respectively, 100 +/- 6 and 20 +/- 2 nM (n = 7) 30 min after application of terbutaline. The cytosolic Cl- concentration ([Cl-]c) in the presence and absence of extracellular Ca2+ was, respectively, 20 +/- 1 and 40 +/- 2 mM (n = 7) 30 min after application of terbutaline. The diminution of [Ca2+]c from 100 to 20 nM itself had no significant effects on the Po if the [Cl-]c was reduced to 20 mM; the Po was 0.58 +/- 0.10 at 100 nM [Ca2+]c and 0.55 +/- 0.09 at 20 nM [Ca2+]c (n = 8) with 20 mM [Cl-]c in inside-out patches. On the other hand, the Po (0.28 +/- 0.10) at 20 nM [Ca2+]c with 40 mM [Cl-]c was significantly lower than that (0.58 +/- 0.10; P < 0.01; n = 8) at 100 nM [Ca2+]c with 20 mM [Cl-]c' suggesting that reduction of [Cl-]c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca2+ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl-]c.

Abstract: Forskolin induced the transepithelial Cl- transport (secretion) by activating the apical Cl- channel and basolateral Na+/K+/2Cl- cotransporter in renal epithelial A6 cells via an increase in cytosolic cAMP concentration. The cAMP activation of apical Cl- channel and Na+/K+/2Cl- cotransporter was partially mediated through a protein kinase A (PKA)-dependent pathway, but a PKA-independent pathway was also suggested to be involved in the cAMP activation. Therefore, we assessed a possibility of involvement of protein tyrosine kinase (PTK)-dependent pathway as a PKA-independent pathway in the cAMP activation by applying a PTK inhibitor, tyrphostin A23 (AG18). Tyrphostin A23 abolished the forskolin-induced transepithelial Cl- secretion by partially diminishing the activity of the Cl- channel and completely inhibiting the Na+/K+/2Cl- cotransporter. Further, forskolin increased phosphorylation of protein tyrosine, suggesting that cAMP activates PTK. These observations suggest that cAMP activates the Cl- channel and the Na+/K+/2Cl- cotransporter by activating PTK.

Abstract: We have shown that the apical membrane of renal epithelial A6 cells has a 29-pS stretch-activated nonselective cation (NSC) channel, which is activated by cytosolic cyclic AMP (cAMP) (J Gen Physiol 1997;110:327-36). In general, downstream signalings of cAMP are mediated through a cAMP-activated protein kinase (protein kinase A, PKA)-dependent pathway. Therefore, to study if the channel is activated by a PKA-dependent pathway, we applied a PKA catalytic subunit directly to the channel from the cytosolic surface in cytosol-free excised inside-out patches, using the single channel recording (patch clamp) technique. Application of PKA catalytic subunit with 2 mM ATP increased the open probability (P(o)) of the channel from 0.11 +/- 0.04 to 0.58 +/- 0.10 (mean +/- SD, N = 11, P < 0.001). The channel has a gating kinetics "C(L) <--> C(S) <--> O, " where C(L,) C(S,) and O are the long closed state, the short closed state, and the open state, respectively. PKA influenced the communication of the channel between C(L) and C(S) without affecting the communication between C(S) and O, leading the channel to only stay in C(S) and O. The PKA-induced increase in P(o) was attributable to the interruption of communication between C(L) and C(S) or to the reduction of time the channel stays in C(L.) Pretreatment with cytochalasin D (Cyt-D), an inhibitor of the polymerization of actin filaments, blocked the stimulatory effect of PKA on the channel. These observations suggest that phosphorylation of polymerized actin filaments regulates the gating kinetics of a stretch-activated channel in renal epithelium.

Abstract: Renal A6 cells have been reported in which hyposmolality stimulates Na(+) transport by increasing the number of conducting amiloride-sensitive 4-pS Na(+) channels at the apical membrane. To study a possible role of protein tyrosine kinase (PTK) in the hyposmolality-induced signaling, we investigated effects of PTK inhibitors on the hyposmolality-induced Na(+) transport in A6 cells. Tyrphostin A23 (a PTK inhibitor) blocked the stimulatory action of hyposmolality on a number of the conducting Na(+) channels. Tyrphostin A23 also abolished macroscopic Na(+) currents (amiloride-sensitive short-circuit current, I(Na)) by decreasing the elevating rate of the hyposmolality-increased I(Na). Genistein (another type of PTK inhibitor) also showed an effect similar to tyrphostin A23. Brefeldin A (BFA), which is an inhibitor of intracellular translocation of protein, blocked the action of hyposmolality on I(Na) by diminishing the elevating rate of the hyposmolality-increased I(Na), mimicking the inhibitory action of PTK inhibitor. Further, hyposmolality increased the activity of PTK. These observations suggest that hyposmolality would stimulate Na(+) transport by translocating the Na(+) channel protein (or regulatory protein) to the apical membrane via a PTK-dependent pathway. Further, hyposmolality also caused an increase in the plasma (apical) membrane capacitance, which was remarkably blocked by treatment with tyrphostin A23 or BFA. These observations also suggest that a PTK-dependent pathway would be involved in the hyposmolality-stimulated membrane fusion in A6 cells.

Abstract: 1) A beta agonist stimulated Na+ transport and decreased the intracellular Cl concentration ([Cl]c) associated with cell shrinkage via an increase in cytosolic cAMP level by activating adenylate cyclase in rat fetal distal lung epithelial (FDLE) cells. 2) Lowering [Cl-]c activated a 28-pS nonselective cation (NSC) channel by elongating the open time of the channel. 3) cAMP signals were converted to a protein tyrosine kinase (PTK)-mediated signal. 4) The PTK-mediated signal was involved in the cAMP-stimulated Na+ transport in rat FDLE cells.

Abstract: The aim of the present study was to investigate the roles of Ca2+ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca2+ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca2+ (<1 nm). This insulin action in a Ca2+-containing solution was completely blocked by co-application of bumetanide (50 microm, an inhibitor of Na+/K+/2Cl- cotransporter) and amiloride (10 microm, an inhibitor of epithelial Na+ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca2+-free solution was completely blocked by quinine (1 mm, a blocker of Ca2+-activated K+ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl- cotransporter in the presence of 1 mm extracellular Ca2+, that the stimulatory action of insulin on an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl- cotransporter requires Ca2+, and that in a Ca2+-free solution insulin activates a quinine-sensitive K+ channel but not in the presence of 1 mm Ca2+. The insulin action on cell volume in a Ca2+-free solution was almost completely blocked by treatment with BAPTA (10 microm) or thapsigargin (1 microM, an inhibitor of Ca2+-ATPase which depletes the intracellular Ca2+ pool). Further, lavendustin A (10 microm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca2+-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K+ channel is mediated through PTK activity in a cytosolic Ca2+-dependent manner. Lavendustin A, further, completely blocked the activity of the Na+/K+/2Cl- cotransporter in a Ca2+-free solution, but only partially blocked the activity of the Na+/K+/2Cl- cotransporter in the presence of 1 mm Ca2+. This observation suggests that the activity of the Na+/K+/2Cl- cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca2+-independent pathway and the other is a PTK-independent, Ca2+-dependent pathway. Further, we observed that removal of extracellular Ca2+ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na+ channel and the bumetanide-sensitive Na+/K+/2Cl- cotransporter, and that removal of extracellular Ca2+ abolished the activity of the quinine-sensitive K+ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca2+ results from diverse effects on the cotransporter and Na+ and K+ channels.

Abstract: The Na+ transport in alveolar type II epithelial cells of rat fetal lung was stimulated by cAMP, which is generally thought to act through activation of protein kinase A (PKA). PKA inhibitors (H8, H89 and H7) stimulated amiloride-sensitive Na+ transport in the alveolar type II epithelial cells. H85, an inactive form of H89 as a PKA inhibitor, had also mimicked the stimulatory action of H89 on the Na+ transport. On the other hand, another type of PKA inhibitor, KT5720 or myristoylated PKA inhibitory peptide [14-22] amide, did not stimulate the Na+ transport, but inhibited the Na+ transport unlike H-compounds. These observations suggest that H-compounds act on the Na+ transport depending on the structure.

Abstract: To study a cAMP-mediated signaling pathway in the regulation of amiloride-sensitive Na(+) transport in rat fetal distal lung epithelial cells, we measured an amiloride-sensitive short-circuit current (Na(+) transport). Forskolin, which increases the cytosolic cAMP concentration, stimulated the Na(+) transport. Forskolin also activated cAMP-dependent protein kinase (PKA). A beta-adrenergic agonist and cAMP mimicked the forskolin action. PKA inhibitors KT-5720, H-8, and myristoylated PKA-inhibitory peptide amide-(14-22) did not influence the forskolin action. These results suggest that forskolin stimulates Na(+) transport through a PKA-independent pathway. Furthermore, forskolin increased tyrosine phosphorylation of approximately 70- to 80-, approximately 97-, and approximately 110- to 120-kDa proteins. Protein tyrosine kinase (PTK) inhibitors (tyrphostin A23 and genistein) abolished the forskolin action. Moreover, 5-nitro-2-(3-phenylpropylamino)benzoate (a Cl(-)-channel blocker) prevented the stimulatory action of forskolin on Na(+) transport via abolishment of the forskolin-induced cell shrinkage and tyrosine phosphorylation. Based on these results, we conclude that forskolin (and cAMP) stimulates Na(+) transport in a PTK-dependent but not a PKA-dependent pathway by causing cell shrinkage, which activates PTK in rat fetal distal lung epithelial cells.

Abstract: Osmotic shock is well recognized as one of the factors activating stress-activated protein kinases (SAPKs), p38 MAP kinase and c-Jun N-terminal kinases (JNKs). In renal epithelial A6 cells, hypo-osmotic shock transiently activated SAPKs with maximal activation at 5 min. A6 cells showed a regulatory volume decrease (RVD) after swelling when the cells were exposed to a hypo-osmotic solution. In contrast, activation of SAPKs was maintained over 90 min after hypo-osmotic shock in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl(-) channel blocker), which completely blocked the RVD and kept the cells continuously swelling. Exposure of the cells to a high K(+) iso-osmotic solution containing nystatin, which induces continuous cell swelling, also continuously activated SAPKs. Furthermore, membrane deformation induced by chlorpromazine activated SAPKs. These results suggest that changes in membrane tension by cell swelling or chlorpromazine, but not osmolality, are important steps for activation of SAPKs in A6 cells.

Abstract: 1. We studied the regulatory mechanism of Na+ transport by hyposmolality in renal epithelial A6 cells. 2. Hyposmolality increased (1) Na+ absorption, which was detected as an amiloride-sensitive short-circuit current (INa), (2) Na+-K+ pump activity, (3) basolateral Cl- conductance (Gb,Cl), and (4) phosphorylation of tyrosine, suggesting an increase in activity of protein tyrosine kinase (PTK). 3. A Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which abolished Gb, Cl, blocked the INa by inhibiting the Na+-K+ pump without any direct effect on amiloride-sensitive Na+ channels. Diminution of Gb,Cl by Cl- replacement with a less permeable anion, gluconate, also decreased the hyposmolality-increased Na+-K+ pump activity. 4. The PTK inhibitors tyrphostin A23 and genistein induced diminution of the hyposmolality-stimulated Gb,Cl, which was associated with attenuation of the hyposmolality-increased Na+-K+ pump activity. 5. Taken together, these observations suggest that: (1) hyposmolality activates PTK; (2) the activated PTK increases Gb,Cl; and (3) the PTK-increased Gb,Cl stimulates the Na+-K+ pump. 6. This PTK-activated Gb,Cl-mediated signalling of hyposmolality is a novel pathway for stimulation of the Na+-K+ pump.

Abstract: 1. In cell-attached patches formed on the apical membrane of fetal alveolar epithelium, terbutaline (a specific beta2-adrenergic agonist) increased the open probability (Po) of an amiloride-sensitive Na+-permeable non-selective cation (NSC) channel (control, 0.03 +/- 0.04; terbutaline, 0.62 +/- 0.18; n = 8, P < 0. 00001) by increasing the mean open time 100-fold without any significant change in the mean closed time and without any change in the single channel conductance (control, 27.8 +/- 2.3 pS; terbutaline, 28.2 +/- 2.1 pS; n = 8). 2. The Po of the unstimulated channel increased when the apical membrane was depolarized due to a decrease in the closing rate and an increase in the opening rate, while the Po of the terbutaline-stimulated channel did not depend on the membrane potential. 3. Increased cytosolic [Ca2+] also increased the Po of the channel in a manner consistent with one Ca2+-binding site on the cytosolic surface of the channel. Terbutaline increased the sensitivity of the channel to cytosolic Ca2+ by shifting the concentration of cytosolic Ca2+ ([Ca2+]c) required for half-maximal activation to a lower [Ca2+]c value, leading to an increase in Po. 4. An increase in the cytosolic Cl- concentration ([Cl-]c) decreased the Po of the channel consistent with two Cl--binding sites by increasing the closing rate without any significant change in the opening rate. Terbutaline increased Po by reducing the effect of cytosolic Cl- to promote channel closing. 5. Taken together, these observations indicate that terbutaline activates a Ca2+-activated, Cl--inhibitable, amiloride-sensitive, Na+-permeable NSC channel in fetal rat alveolar epithelium in two ways: first, through an increase in Ca2+ sensitivity, and second, through a reduction in the effect of cytosolic Cl- to promote channel closing.

Abstract: The present study investigates regulation of Cl- channels and Na+/K+/2Cl- cotransporter in a renal epithelial cell line, A6, by flavones: genistein [an inhibitor of protein tyrosine kinases (PTK)], daidzein (an inactive compound of genistein), and apigenin [an inhibitor of mitogen-activated protein (MAP) kinase]. Genistein and daidzein activated Cl- channels. Genistein and apigenin had a stimulatory effect on the bumetanide-sensitive Na+/K+/2Cl- cotransporter. Other PTK inhibitors, tyrphostin A23, lavendustin A, and herbimycin A, which do not contain a structure flavone, had no stimulatory action on Cl- channels or the Na+/K+/2Cl- cotransporter. These observations conclude that (i) genistein activates a Cl- channel and the Na+/K+/2Cl- cotransporter; and (ii) the stimulatory action is not mediated through its inhibitory action on protein tyrosine kinase, but rather the structure of flavone itself plays a crucial role in stimulatory regulation of Cl- channels and Na+/K+/2Cl- cotransporter.

Abstract: Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current (Isc) without affecting total Isc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the Isc. The effect was reversed by the addition of N-monomethyl-L-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.

Abstract: The epithelial Na+ channel (ENaC), composed of three subunits (alpha, beta, and gamma), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat alpha beta gamma ENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the alpha beta gamma rENaC-expressing MDCK cells exhibited greater whole cell Na+ current at -143 mV (-1,466.2 +/- 297.5 pA) than did untransfected cells (-47.6 +/- 10.7 pA). This conductance was completely and reversibly inhibited by 10 microM amiloride, with a Ki of 20 nM at a membrane potential of -103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing alpha beta or alpha gamma subunits alone was -115.2 +/- 41.4 pA and -52.1 +/- 24.5 pA at -143 mV, respectively, similar to the whole-cell Na+ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na+ conductance was Li+ > Na+ >> K+ = N-methyl-D-glucamine+ (NMDG+). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na+ channel current, was found to be approximately 5 and 8 pS when Na+ and Li+ were used as a charge carrier, respectively. K+ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (Po), was increased by membrane hyperpolarization. Both whole-cell Na+ current and conductance were saturated with increased extracellular Na+ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nPo) was significantly decreased when cytosolic Na+ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na+ conductance (with Li+ as a charge carrier) was inhibited by the addition of ionomycin (microM) and Ca2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 microM Ca2+ caused a biphasic inhibition, with time constants of 1.7 +/- 0.3 min (n = 3) and 128.4 +/- 33.4 min (n = 3). An increase in cytosolic Ca2+ concentration from <1 nM to 1 microM was accompanied by a decrease in channel activity. Increasing cytosolic Ca2+ to 10 microM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca2+ concentrations from <1 nM to 10 microM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na+ and Ca2+.

Abstract: The apical membrane of distal nephron epithelium (A6) has a Ca(2+)-dependent outwardly rectifying Cl- channel with single channel conductances of 3 pS for outward current and 1 pS for inward current under the basal condition. The single channel conductance for inward currents increased as cytosolic Ca2+ concentration ([Ca2+]c) was elevated, while the single channel conductance for outward currents did not change at the range of [Ca2+]c from 10 nM to 1 mM. Insulin (100 nM) increased the single channel conductance for the inward current by increasing the sensitivity to cytosolic Ca2+ by 400-fold, but did not affect the single channel conductance for the outward current. Further, insulin increased the open probability of the channel. These effects of insulin were completely blocked by cyclosporin-A, an inhibitor of protein phosphatase type 2B (PP2B) which dephosphorylates phospho-tyrosine in addition to phosphoserine/threonine, but not by okadaic acid, an inhibitor of protein phosphatase type 1 and 2A. Further, these effects of insulin were also completely blocked by W7, an antagonist of calmodulin which is required for activation of PP2B. Lavendustin A, an inhibitor of protein tyrosine kinase (PTK), mimicked these effects of insulin; this action of lavendustin A required 1 hr after its application, while within 30 min after its application lavendustin A had no significant effects on the single channel conductance. On the other hand, lavendustin A blocked the insulin action for a relatively short time period (i.e., within 30 min after their application). However, H89 (an inhibitor of protein kinase A) or H7 (an inhibitor of protein kinases A, C and G) did not mimic the insulin action. Application of PP2B or protein tyrosine phosphatase to the cytosolic surface of the inside-out patch membrane increased the single channel conductance and the open probability as did insulin in cell-attached patches. The insulin-induced increases in single channel conductance and open probability were reversibly decreased by application of PTK catalytic subunit in the presence of ATP through a decrease in the sensitivity to cytosolic Ca2+, but not by protein kinase A. These observations suggest that as intracellular signalling of insulin action, PP2B-mediated dephosphorylation of phospho-tyrosine of the channel protein (or channel-associated protein) is a novel mechanism for regulation of single channel conductance, and that at least two different types of PTKs regulate the channel characteristics.

Abstract: It is currently believed that a nonselective cation (NSC) channel, which responds to arginine vasotocin (an antidiuretic hormone) and stretch, regulates Na+ absorption in the distal nephron. However, the mechanisms of regulation of this channel remain incompletely characterized. To study the mechanisms of regulation of this channel, we used renal epithelial cells (A6) cultured on permeable supports. The apical membrane of confluent monolayers of A6 cells expressed a 29-pS channel, which was activated by stretch or by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase. This channel had an identical selectivity for Na+, K+, Li+, and Cs+, but little selectivity for Ca2+ (PCa/PNa < 0.005) or Cl- (PCl/PNa < 0.01), identifying it as an NSC channel. Stretch had no additional effects on the open probability (Po) of the IBMX-activated channel. This channel had one open ("O") and two closed (short "CS" and long "CL") states under basal, stretch-, or IBMX-stimulated conditions. Both stretch and IBMX increased the Po of the channel without any detectable changes in the mean open or closed times. These observations led us to the conclusion that a kinetic model "CL <--> CS <--> O" was the most suitable among three possible linear models. According to this model, IBMX or stretch would decrease the leaving rate of the channel for CL from CS, resulting in an increase in Po. Cytochalasin D pretreatment abolished the response to stretch or IBMX without altering the basal activity. H89 (an inhibitor of cAMP-dependent protein kinase) completely abolished the response to both stretch and IBMX, but, unlike cytochalasin D, also diminished the basal activity. We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.

Abstract: The effects of terbutaline (a selective beta 2-adrenoceptor agonist) on cell volume and ion transport in rat fetal distal lung epithelial (FDLE) cells were studied. In FDLE cells, benzamil (1 microM) induced cell shrinkage, while cell volume was increased by 1 mM quinine or 2 mM Ba2+ but was not affected by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 20 microM). Terbutaline (10 nM) induced transient cell swelling, which was suppressed by benzamil, while 10 microM terbutaline induced initial rapid cell shrinkage followed by delayed slow cell shrinkage, which was suppressed by 1 mM quinine or 20 microM NPPB but not by 2 mM Ba2+. Application of benzamil enhanced the cell shrinkage induced by 10 microM terbutaline. These observations suggest that: (1) benzamil-blockable Na(+)-permeable channels and quinine- and Ba(2+)-blockable K+ channels contribute to maintenance of cell volume of FDLE cells; (2) terbutaline at both 10 nM and 10 microM activates benzamil-blockable Na(+)-permeable channels; (3) quinine-blockable K+ channels are activated by 10 microM terbutaline; and (4) NPPB-blockable Cl- channels are responsible for Cl- movement in cell volume changes induced by terbutaline. The present study demonstrates that the cellular mechanisms of volume change induced by terbutaline are closely related to activation of Na+ absorption and KCl release.

Abstract: We studied regulation of Cl- transport by cAMP and Ca2+ in renal epithelial A6 cells. Stimulation of A6 cells by 1 mM 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase), which increased cytosolic cAMP, elicited biphasic increases in short-circuit current (Isc), i.e., a transient phase followed by a sustained one. Apical application of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, a Cl- channel blocker) markedly and dose-dependently inhibited the IBMX-induced Isc. Pretreatment with nifedipine (100 microM, a Ca2+ channel blocker) or 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA/AM, 10 microM, a Ca2+ chelator) partially but markedly inhibited the Isc. On the other hand, a cAMP-dependent protein kinase inhibitor, H89 (0.5 microM for 1 h), also reduced the IBMX-induced Isc to a level similar to that following nifedipine or BAPTA pretreatment. Nifedipine had no synergistic effects on the IBMX-induced Isc in cells treated with H89. Ionomycin (a Ca2+ ionophore) could mimic the transient increase dose dependently, and H89 did not block the ionomycin-induced Isc. Taken together, our observations suggest that: (1) part of the IBMX-stimulated Cl- release is regulated by an increased cytosolic Ca2+ through nifedipine-sensitive Ca2+ influx; (2) cAMP-dependent phosphorylation may be required for elevation of the cytosolic Ca2+ concentration but not for activation of Cl- channels, which are directly activated by cytosolic Ca2+; and (3) the IBMX-induced sustained Cl- release requires cAMP elevation in addition to an increase in the cytosolic Ca2+ concentration.

Abstract: One of the most important factors controlling blood pressure is the total body Na+ content, which depends upon Na+ intake and excretion. The kidney influences body Na+ content by regulating the tubular absorption of the Na+ filtered through the glomeruli. Thus, the regulation of Na+ absorption in the tubules of the kidney plays an important role in controlling blood pressure. More than 99% of the Na+ passing through the glomerulus is reabsorbed in the kidney. About 90% of the filtered Na+ through the glomerulus is reabsorbed in the proximal tubule and the ascending limb of the loop of Henle. The remainder of the Na+ absorption occurs in the distal nephron. This process is regulated by hormones such as aldosterone and antidiuretic hormone (ADH), and also by the osmolality of the plasma. These observations suggest that the regulation of Na+ transport in the distal nephron by hormones and osmolality plays an important role in the control of blood pressure. The distal nephron is composed of two different types of epithelial cells: the principal cell and the intercalated cell. The latter is also composed of two types of cells: alpha and beta intercalated cells. In addition to Na+ absorption, the distal-nephron epithelial cells also participate in K+ and H+ secretion. Na+ absorption is mediated through the principal cell, which also contributes to K+ secretion, whereas H+ is secreted through both types of intercalated cells, alpha and beta, in different ways. There are, in general, two steps in the transepithelial ion movement across the epithelium, including the distal-nephron epithelium. For example, in the case of Na+ absorption, one is the entry step of Na+ across the apical membrane and the other is the extrusion step of Na+ across the basolateral membrane. This means that there are two major regulatory sites of transepithelial Na+ absorption: namely, regulation of the entry and extrusion steps of Na+. It is generally thought that the entry step of Na+ across the apical membrane is the rate-limiting step in the transepithelial Na+ transport and that Na+ channels in the apical membrane play an important role as an entry step of Na+ and are regulated by hormones and plasma osmolality. In this review, we describe the regulatory mechanisms of Na+ absorption in renal distal-nephron epithelium by aldosterone, ADH and osmolality. Further, we will review the regulatory mechanisms of Cl- transport, which also plays an important role in Na+ transport as a major counter ion, and discuss other roles of Cl- in the active regulation of Na+ transport.

Abstract:

Abstract: To assess the action of antidiuretic hormone (ADH) and the osmolality of bathing solution on amiloride-sensitive Na+ transport, we measured the amiloride-sensitive short-circuit current (Isc) and single-channel currents in renal epithelial A6 cells. The A6 cells were cultured on permeable supports for 9-13 d without aldosterone treatment. The basal amiloride-sensitive Isc and the density of the amiloride-sensitive Na+ channel at the apical membrane increased as the osmolality of the bathing solution decreased. ADH stimulated the amiloride-sensitive Isc. The stimulatory action of ADH was enhanced by low osmolality. The stimulatory action of hyposmolality on the amiloride-sensitive Isc was significantly diminished by pretreatment with brefeldin A (BFA, a blocker of protein translocation), while BFA had no significant effect on the ratio of ADH-stimulated amiloride-sensitive Isc to basal amiloride-sensitive Isc. These results suggest that hyposmolality stimulates the translocation of amiloride-sensitive Na+ channels to the apical membrane from the cytosolic store sites of the channel, and that ADH may activate amiloride-sensitive Na+ channels pre-existing in the apical membrane or translocate the channel via BFA-insensitive pathways in a manner dependent on the osmolality of the bathing solution in aldosterone-untreated A6 cells, differently from aldosterone-treated A6 cells in which ADH stimulates the translocation of amiloride-sensitive Na+ channels via BFA-sensitive pathways.

Abstract: We studied cAMP-dependent regulation of ion transport in aldosterone-untreated renal epithelial A6 cells by measuring short-circuit current (Isc). Biphasic increases in Isc, a transient phase followed by a sustained one, were elicited in response to 1 mm 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase) which increased cytosolic cAMP concentration. IBMX increased the apical Cl- conductance. The sustained phase of Isc induced by IBMX was reduced by 50 microM bumetanide (Na+/K+/2 Cl- cotransporter inhibitor) or 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, an inhibitor of Cl-/HCO3- exchanger). Under the normal condition, the inhibitory effect of bumetanide was much larger than that of DIDS. On the other hand, under a low Cl- condition, the effect of DIDS was more effective than that of bumetanide. Further, under a Cl--free condition Na+/HCO3- symporter contributed to the IBMX-generated Isc. Taken together, our observations suggest that in A6 cells (i) IBMX stimulates Cl- secretion associated with an increase in apical Cl- conductances, (ii) the ionic components to generate the IBMX-induced Isc are mainly maintained by bumetanide-sensitive Na+/K+/2 Cl- cotransporter and DIDS-sensitive Cl-/HCO3- exchanger, (iii) Cl-/HCO3- exchanger coupled to Na+/HCO3- symporter under a low-Cl- condition or Na+/HCO3- symporter under a Cl--free condition contributes to the IBMX-induced Isc, compensating for diminishment of the Na+/K+/2Cl- cotransporter-mediated Cl- secretion, (iv) IBMX increases Cl- and HCO3- conductances in the apical membrane.

Abstract: We studied the effect of brefeldin A, which inhibits the intracellular trafficking of membrane proteins from the cytosolic pool to the cell surface, on terbutaline (a beta2-specific adrenergic agonist)-induced alterations in ion transport by primary monolayer cultures of fetal rat distal lung epithelium. The amiloride-sensitive short circuit current (Isc) increased 2.5-fold 50 min after application of terbutaline (10 microM) from basolateral side; this response was abolished by pretreatment with brefeldin A (1 microg/ml). Brefeldin A did not suppress the Na+/K+ pump capacity. Single channel patch clamp experiments demonstrated that terbutaline increased the density of amiloride-sensitive Na+-permeable nonselective cation channels on the apical cell membrane and this action was blocked by brefeldin A. These observations suggest that beta2-specific adrenergic agonists promote the trafficking of amiloride sensitive Na+-permeable nonselective cation channels to the apical cell surface.

Abstract: The effects of beta 2-adrenoceptor agonist (beta 2 agonist) and cAMP on cytosolic Ca2+ concentration ([Ca2+]c) and cell volume were studied in fetal distal lung epithelial cells. Both terbutaline (a specific beta 2 agonist, 10 microM) and dibutyryl cAMP (DBcAMP, 1 mM) increased [Ca2+]c in the presence of extracellular Ca2+. Even in the absence of extracellular Ca2+, the terbutaline-induced increase in [Ca2+]c was still observed, although the increase was transient. However, DBcAMP caused no significant change in [Ca2+]c. In the presence of 1 mM extracellular Ca2+, terbutaline and DBcAMP induced quinine (a blocker of K+ channel) sensitive cell shrinkage. However, in a Ca2(+)-free solution, terbutaline induced rapid cell shrinkage, followed by benzamil (a specific blocker of Na+ channel, an analogue of amiloride) sensitive transient cell swelling. In a Ca2(+)-free solution, DBcAMP induced benzamil-sensitive transient cell swelling without cell shrinkage. Taken together, our observations indicate that the beta 2 agonist induced an elevation of [Ca2+]c by increasing both a Ca2+ influx from the extracellular space and a Ca2+ release from intracellular Ca2+ stores, whereas DBcAMP only stimulated Ca2+ influx from the extracellular space. Furthermore, it is suggested that terbutaline and DBcAMP activated benzamil-sensitive channels independently of an increase in [Ca2+]c.

Abstract: Cell-volume changes induced by terbutaline (a specific beta2-agonist) were studied morphometrically in rat fetal distal lung epithelium (FDLE) cells. Cell-volume changes qualitatively differed with the concentration of terbutaline. Terbutaline of 10(-10)-10(-8) M induced transient cell swelling. Terbutaline of 10(-7) M induced transient cell swelling followed by slow cell shrinkage. Terbutaline of 10(-6)-10(-5) M induced rapid cell shrinkage followed by slow cell shrinkage. Terbutaline of 10(-3) M induced transient cell shrinkage; then cell volume oscillated during stimulation. Benzamil of 10(-6) M suppressed the cell swelling induced by 10(-10)-10(-8) M terbutaline and quinine of 10(-3) M inhibited the cell shrinkage induced by 10(-6)-10(-5) M terbutaline. These results suggest that cell swelling would be induced by NaCl influx and the cell shrinkage is by KCl efflux. Dibutyryl cyclic AMP (DBcAMP) also induced similar cell-volume changes over a wide range of concentrations (10(-9)-10(-3) M): a low concentration induced transient cell swelling; a high concentration, rapid and slow cell shrinkage. Forskolin (10(-4) M), like terbutaline (10(-5) M), induced rapid cell shrinkage followed by slow cell shrinkage, and this decrease in the cell volume was enhanced by the presence of benzamil. On the other hand, cell shrinkage was induced by ionomycin (even low concentration; 3 x 10(-10) M ionomycin), and after that cell volume remained at a plateau level. Removal of extracellular Ca2+ abolished the cell swelling caused by terbutaline of 10(-10)-10(-8) M. With removal of extracellular Ca2+, the initial, rapid cell shrinkage induced by 10(-5) M terbutaline became transient, but we still detected slow cell shrinkage similar to that in the presence of extracellular Ca2+. Overall, at low concentrations (10(-10)-10(-8) M), terbutaline induced benzamil-sensitive cell swelling in FDLE cells, which was cAMP- and Ca2+-dependent; high concentrations (> or =10(-6)) induced quinine-sensitive rapid cell shrinkage, which was Ca2+-dependent; high concentrations (> or = 10(-7)) induced slow cell shrinkage, which was cAMP-dependent. These findings suggest that terbutaline regulates cell volume in FDLE cells by cytosolic cAMP and Ca2+ through activation of Na+ and K+ channels.

Abstract: The apical membrane of renal epithelial A6 cells has 3 and 8 pS Cl- channels. Both types of the channels were blocked by NPPB (5-Nitro-2-(3-phenylpropylamino)-benzoic acid; 100 microM). 8-bromo-cAMP (Br-cAMP) increased the open probability of 3 pS Cl- channel and this action was inhibited by pretreatment with H89, an inhibitor of cAMP-dependent protein kinase (PKA). On the other hand, the number of 8 pS Cl- channel was increased by Br-cAMP. The increase in number of 8 pS Cl- channel by Br-cAMP was inhibited by brefeldin A (a blocker of movement of membrane protein from an intracellular pool to the cell surface) but not by H89. These observations indicate that cAMP could activate the 3 pS Cl- channel through PKA-dependent phosphorylation, and that cAMP could stimulate translocation of the 8 pS Cl- channel through PKA-independent pathways. We conclude that PKA-independent pathways are involved in cAMP signaling mechanisms in addition to PKA-dependent pathways.

Abstract: We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca2+ concentrations ([Ca2+]c, 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 approximately 14 days in media with supplemental aldosterone (1 microM). At 30 nm [Ca2+]c, basal conductances mainly consisted of Cl- conductances, which were sensitive to 5-nitro-2-(3-phenyl-propylamino)-benzoate. Reduction of [Ca2+]c to 2 nm abolished the basal Cl- conductance. AVT evoked Cl- conductances at 2 as well as 30 nm [Ca2+]c. In addition to Cl- conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca2+]c but not at 2 nm [Ca2+]c. Thus, cytosolic Ca2+ regulates NSC and Cl- conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca2+]c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells.

Abstract: The Na+ transport function of alveolar epithelium represents an important mechanism for clearance of fluid in air space at birth. I observed the activity of two types of amiloride-blockable Na+-permeant cation channels in the apical membrane of fetal distal lung epithelium cultured on permeable filters for 2 days after harvesting of the cells from Wistar rats of 20 days gestation (term = 22 days). One type was a nonselective cation (NSC) channel and had a linear current/voltage (I/V) relationship with a single-channel conductance of 26.9 +/- 0.8 pS (n = 5). The other type was highly Na+ selective (i.e. Na+ channel) and had an inwardly rectifying I/V relationship with a single-channel conductance of 11.8 +/- 0.2 pS (n = 5) around resting membrane potential. The NSC channel was more frequently observed (1.37 +/- 0.15 per patch membrane; n = 73) than the Na+ channel (0.15 +/- 0.40 per patch membrane; n = 73). However, the open probability of the NSC channel was smaller than that of the Na+ channel. Both types of the channels were activated by cytosolic Ca2+, however the sensitivity to cytosolic Ca2+ was much higher in the Na+ channel than in the NSC channel. Furthermore, both types of the channels were blocked by amiloride or benzamil. The half-maximal inhibitory concentration (IC50) of amiloride or benzamil of the Na+ channel was 1-2 microM, while that of NSC channel was less than 1 microM. Both channels were activated by insulin.

Abstract: The apical membrane of distal nephron epithelium (A6) had a Ca(2+)-activated outwardly rectifying Cl- channel with single-channel conductances of 3 pS for outward current and 0.8 pS for inward current. The single-channel conductance for inward current was dependent on cytosolic Ca2+ concentration. Insulin increased the single-channel conductance for the inward current by increasing Ca2+ sensitivity about 300-fold. The insulin action was diminished by vanadyl hydroperoxide (vanadate, an inhibitor of protein tyrosine phosphatase (PTP)). Application of protein tyrosine kinase, p60c-src, reversibly diminished the insulin-induced increase in single-channel conductance. Further, the application of PTP to the cytosolic surface of the inside-out patch membrane, like insulin, increased single-channel conductance. PTP-mediated dephosphorylation of the phospho-tyrosine of the channel protein, as a mechanism of intracellular signaling of insulin action, is a novel mechanism for regulating single-channel conductance by modulating Ca2+ sensitivity.

Abstract: The aim of the present study was to investigate the roles of tyrosine kinase (TK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocyte. Insulin (100 nmol/l) increased cell volume, and this insulin (100 nmol/l) action was completely blocked by 50 micromol/l bumetanide (BMT) and 10 micromol/l amiloride (AML). This observation indicates that 100 nmol/l insulin activates BMT-sensitive Na+/K+/2Cl- cotransporter and AML-sensitive pathways. The stimulatory action of 100 nmol/l insulin on BMT-sensitive Na+/K+/2Cl- cotransporter was completely abolished by 10 micromol/l lavendustin A (LAV-A, an inhibitor of TK), however 100 nmol/l insulin could stimulate AML-sensitive pathways even in LAV-A (10 micromol/l)-treated cells. These observations indicate that the insulin (100 nmol/l) action on the BMT-sensitive Na+/K+/2Cl- cotransporter is mediated through TK-dependent pathways, while 100 nmol/l insulin requires a TK-independent pathway to show the stimulatory action on the AML-sensitive pathways. From these observations we conclude that TK-dependent and -independent pathways are involved in the insulin (100 nmol/l) signaling in fetal rat type II pneumocyte.

Abstract: Short-circuit current measurement in renal epithelium (A6) indicated that the amount of antidiuretic hormone-stimulated Na+ entry in aldosterone-treated cells exceeded the ability of the Na+/K(+)-pump to extrude Na+ in aldosterone-untreated cells. This suggests that, if aldosterone has no stimulatory action on Na+/K(+)-pump activity, the activity of Na+/K(+)-pump is not large enough to extrude Na(+) entering through antidiuretic hormone-activated Na(+)-permeant channels in the apical membrane. The present study concludes that the stimulatory action of aldosterone on the Na+/K+ pump is a physiological requirement in antidiuretic hormone-stimulated Na+ absorption.

Abstract: Insulin activated a 28 pS amiloride-blockable nonselective cation (NSC) channel in rat fetal distal lung epithelium. Benzamil, an analogue of amiloride, decreased the open probability (Po) of the insulin-unstimulated channel from 0.06 +/- 0.02 to 0.0013 +/- 0.0006 (mean +/- SEM, n = 5-7; 100 microM benzamil application to extracellular surface), but increased the Po of the insulin-stimulated channel from 0.10 +/- 0.02 to 0.69 +/- 0.02 (mean +/- SEM, n = 5-7; 100 microM benzamil application to extracellular surface). The effects of benzamil could be observed in either case that it was applied to the extracellular or cytosolic surface. Unlike benzamil, amiloride decreased the Po of both insulin-unstimulated and -stimulated channels. These observations suggest that benzamil acts as a blocker on the insulin-unstimulated NSC channel but as an opener on the insulin-stimulated NSC channel.

Abstract: 1. To clarify the effect of arginine vasotocin (AVT) on intracellular pH (pHi) of A6 cells (an amphibian renal cell line), we measured pHi with a single-cell fluorescent imaging system in an HCO3(-)-nominally-free medium. 2. AVT (40 mU/ml) significantly decreased pHi from 7.44 +/- 0.11 to 7.19 +/- 0.22 (mean +/- SE) by inhibiting the Na+/H+ exchanger. 3. A membrane-permeable analogue of cAMP, dibutyryl cAMP (1 mM), significantly decreased pHi from 7.40 +/- 0.02 to 7.17 +/- 0.02, a response similar to that of AVT. 4. These data indicate that, in A6 cells, AVT suppresses the Na+/H+ exchanger via a cAMP-dependent pathway and the Na+/H+ exchanger maintains pHi under HCO3(-)-nominally-free conditions.

Abstract: 1. We studied the effects of bumetanide (a Na+/K+/2Cl- cotransport inhibitor) and HCO3(-) on beta-adrenergic-agonist-stimulated short-circuit current (beta-Isc) in rat fetal distal lung epithelium (FDLE). 2. Bumetanide significantly increased beta-Isc in the absence of amiloride but decreased it in its presence. The amiloride- and bumetanide-insensitive beta-Isc was diminished by a removal of HCO3(-) in the bathing solution. 3. Our results suggest that bumetanide stimulates the amiloride-sensitive beta-Isc in FDLE and that the amiloride-insensitive beta-Isc is composed of two different pathways: bumetanide-sensitive and HCO3(-)-dependent Cl- secretion in FDLE.

Abstract: The whole cell patch-clamp technique was used to study ionic conductances in fetal distal lung epithelial (FDLE) cells. In unstimulated FDLE cells, K+ conductances were detected in lowered intracellular Cl- concentration ([Cl-]i, < or = 50 mM). The whole cell currents of FDLE cells were increased by elevation of intracellular Ca2+ concentration ([Ca2+]i) or intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration ([cAMP]i). The elevation of [Ca2+]i activated the K+ currents. The amiloride-blockable whole cell currents were activated by [cAMP]i of 1 mM with [Cl-]i of 20 mM and were more frequently detected in the pipette solution without ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) than with it (0.5 mM). When the [Cl-]i was fixed at 50 or 145 mM, however, the increase in these currents was not detected even with cAMP and without EGTA. The amiloride-blockable currents were detected in both the Na+ and K+ pipette solutions. Thus the increase in amiloride-blockable whole cell currents was due to the activation of nonselective cation channels. In FDLE cells treated with terbutaline, which is a beta 2-adrenergic receptor agonist, or forskolin, these currents were detected in the pipette solution containing 20 mM Cl- but were suppressed with time when the pipette solution contained 50 or 145 mM Cl-. It seems likely that maintenance of [Cl-]i at the lowered level is an important requirement for the FDLE cells to activate the amiloride-blockable whole cell currents. It is proposed that cellular mechanisms, such as cell shrinkage, exist to reduce the [Cl-]i in response to cAMP.

Abstract: 1. The apical membrane of fetal distal lung epithelium had two types of amiloride-blockable Na(+)-permeant cation channels; (1) nonselective cation (NSC) channel with a single channel conductance of 27 pS and (2) Na+ channel with a single channel conductance of 12 pS around resting membrane potential. 2. The IC50 of amiloride to the Na+ channel was 1-2 microM, while the IC50 of amiloride to the NSC channel was less 1 microM. The open probability of the Na+ channel was about 10-fold larger than that of the NSC channel. 3. Insulin (100 nM) increased the open probability of both channels.

Abstract: We studied the effects of aldosterone (Aldo) and arginine vasotocin (AVT) on ion transport of renal epithelial cell line (A6) by measuring short-circuit current (Isc). AVT induced a rapid, transient increase in Isc, followed by a decrease toward the baseline in cells untreated with Aldo. In cells treated with Aldo, Isc showed a biphasic response to AVT, i.e., both transient and sustained increases over 40 min after addition of AVT. The transient increase was composed only of amiloride-insensitive Isc regardless of Aldo treatment, whereas the sustained increase contained both amiloride-sensitive and amiloride-insensitive components. The main part of the amiloride-insensitive, sustained Isc depended on HCO3(-). In cells treated with Aldo for 1 day, removal of HCO3(-) in the bathing solution enhanced the amiloride-sensitive component and decreased the amiloride-insensitive one. These data suggest that 1) Aldo treatment is necessary for an AVT-induced sustained increase of Isc and 2) a HCO3(-)-dependent Isc mainly contributes to the sustained increase in amiloride-insensitive Isc.

Abstract: The effect of antidiuretic hormone (ADH) on a distal nephron cell line (A6) was studied using the whole cell patch-clamp technique. A6 cells were cultured on a permeable support filter for 10-14 days in media containing 10% fetal bovine serum without supplemental aldosterone. In the unstimulated condition A6 cells had very small conductances of Na+,K+, and Cl-. Arginine vasotocin (AVT, 140 mU/ml, 280 nM) evoked a "transient" increase in whole cell currents as did dibutyryl-adenosine 3',5'-cyclic monophosphate (5 mM). These transients consisted of two components; one was the nonselective cation conductance, and the other was the Cl- conductance. Activation of these conductances was dependent on intracellular Cl- concentration ([Cl-]i). At low [Cl-]i (< or = 50 mM) both conductances were activated, whereas when [Cl-]i was 80 mM, only the Cl- conductance was activated. At high [Cl-]i (125 mM), both conductances were inhibited. It seems likely that the [Cl-]i maintained at a low level (< or = 50 mM) is an important requirement for A6 cells to respond to AVT.

Abstract: A6 cells cultured on permeable supports for 10 to 14 days have two types of Cl- channels in the apical membrane that have single channel conductances of 3 and 8 pS. In cells without arginine vasopressin (AVP) pretreatment, the 3 pS Cl- channel, which was Ca(2+)-activated and had an outward rectification, was more frequently observed than the 8 pS Cl- channel, which had a linear current-voltage relationship. AVP increased the 3 pS channel's single channel conductance through an increase in the Ca(2+)-sensitivity about 100-fold and the open probability (Po) without significantly changing the Po or conductance of the 8 pS Cl- channel. On the other hand, AVP did not affect the number of the 3 pS Cl- channel, but increased the number of 8 pS Cl- channels. These observations suggest that AVP has two different pathways to increase apical membrane chloride conductance in distal nephron A6 cells; i.e., 1) increases the Po and single channel conductance of 3 pS Cl- channels and 2) increases the number of 8 pS Cl- channels.

Abstract: We studied the regulatory mechanism of Na+ absorption in fetal distal lung epithelium (FDLE), using patch clamp and single cell imaging techniques. A beta 2-agonist activated a 28 pS non-selective cation channel (NSCC) by: 1) producing a dependency of the NSCC activity on the cytosolic [Cl-] ([Cl-]c); 2) inducing a reduction in the [Cl-]c from 45 to 25 mM which directly activated the beta 2-treated NSCC. The beta 2-agonist-induced decrease in [Cl-]c was caused by activation of Ca(2+)-activated K+ channel and cAMP-activated Cl- channel. A development of the [Cl-]c-dependency and a reduction in [Cl-]c act as a second messenger of the beta 2 agonist signal transduction pathway in this Na+ transporting epithelium.

Abstract: The effect of antidiuretic hormone (ADH) on a distal nephron cell line (A6) was studied using the whole cell patch clamp technique. Arginine vasotocin (AVT, 140mU/ml) evoked a "transient" increase in whole cell currents as did dibutyryl cyclic AMP (5 mM). These transients consisted of two components: one was the non-selective cation conductance and the other was the Cl- conductance. The transient was evoked by AVT in the lower cytosolic Cl- concentration ([Cl-]i) (< approximately 50 mM), and was suppressed by higher concentrations of [Cl-]i (125 mM) in A6 cells. It seems likely that the [Cl-]i maintained at a lower level is an important requirement for A6 cells to respond to AVT.

Abstract: The Na+ transport function of alveolar epithelium represents an important mechanism for air space fluid clearance after acute lung injury. We studied the effect of endotoxin-stimulated rat alveolar macrophages on lung epithelial ion transport and permeability in vitro. Cultured rat distal lung (alveolar) epithelial monolayers incubated with both endotoxin and macrophages demonstrated a 75% decline in transepithelial resistance and a selective 60% reduction in amiloride-sensitive short-circuit current (Isc). Single-channel patch-clamp analysis demonstrated a 60% decrease in the density of 25-pS nonselective cation (NSC) channels on the apical membrane of epithelium exposed to both endotoxin and macrophages. A concurrent reduction in epithelial F-actin content suggested a role for actin depolymerization in mediating this effect. Incubation of cocultures with the methylated L-arginine (Arg) derivative NG-monomethyl-L-arginine prevented the reduction in epithelial Isc, as did substitution of L-Arg with D-Arg or incubation in L-Arg-free medium. Furthermore, the stable and products of Arg metabolism were found to have no effect on epithelial ion transport. These studies show that endotoxin-stimulated alveolar macrophages impair distal lung epithelial ion transport by an L-Arg-dependent mechanism by inactivating amiloride-sensitive 25-pS NSC channels. This may represent a novel mechanism whereby local inflammatory cells regulate lung epithelial ion transport. This could affect the ability of the lung to clear fluid from the air space.

Abstract: Arginine vasotocin (AVT, 70 mU/ml) added from the basolateral side transiently activated a nonselective cation (NSC) channel with a single-channel conductance of 28.5 pS and almost identical selectivity for Na+ and K+ in the apical membrane of distal nephron cells (A6) cultured on permeable supports for 10-12 days in media containing 10% fetal bovine serum without supplemental aldosterone. The open probability (Po) of the NSC channel at the apical resting membrane potential in cell-attached patches was approximately 0.09 and increased when the apical membrane depolarized. The Po of the NSC channel was decreased by a rise in cytosolic Ca2+ concentration within a range of 30 nM-1 microM but not affected by cytosolic pH within a range of 6-8. The channel was activated by the application of negative pressure (10-60 cmH2O) into the patch pipette. Gadolinium (2 microM), an inhibitor of stretch-activated channels, decreased the Po by 40%. This blocking action of gadolinium was more effective after the channel was activated by stretch, i.e., 2 microM gadolinium decreased the Po by 70% when a negative pressure (60 cmH2O) was applied into the patch pipette. Amiloride (10 and 100 microM) showed a blocking action on the channel only when the NSC channel was activated by stretch.

Abstract: Nonselective cation (NSC) channels have been identified in the apical membrane of fetal distal lung epithelium (FDLE). However, their physiological role in Na+ transport is uncertain. Because terbutaline, a beta 2-agonist, increases Na+ transport by FDLE, we studied its effect and selected signal transduction mechanisms on NSC channel activity. Using patch-clamp and single-cell imaging techniques, we found that terbutaline activated the NSC channel by 1) increasing its sensitivity to cytosolic Ca2+ concentration ([Ca2+]c) by 100- to 1,000-fold, 2) increasing [Ca2+]c from 35 nM to 3.3 microM, 3) producing a dependency of the NSC channel activity on the cytosolic Cl- concentration ([Cl-]c) at a physiological [Ca2+]c, and 4) inducing a reduction in the [Cl-]c from 45 to 25 mM, which directly activates the beta 2-treated NSC channel. These observations indicate that a beta 2-agonist physiologically activates an amiloride-blockable NSC channel in FDLE through an increase in its sensitivity to [Ca2+]c, resulting in the development of a [Cl-]c dependency at a physiological [Ca2+]c associated with both an increase in [Ca2+]c and a reduction in [Cl-]c. A development of the [Cl-]c dependency and a reduction in [Cl-]c act as a second messenger of the beta-agonist signal transduction pathway in this Na(+)-transporting epithelium.

Abstract: We have investigated how two types of Cl- channels found in sodium transporting epithelium are regulated by arginine vasopressin (AVP). A6 cells cultured on permeable supports for 10 to 14 days have two types of Cl- channels in the apical membrane that have single channel conductances of 3 and 8 pS. In cells without AVP pretreatment, the 3 pS Cl- channel was more frequently observed than the 8 pS Cl- channel. AVP increased the open probability (Po) and single channel conductance of the 3 pS Cl- channel without significantly changing the Po or conductance of the 8 pS Cl- channels. On the other hand, AVP did not affect the number of the 3 pS Cl- channel, but increased the number of 8 pS Cl- channels. These observations suggest that AVP has two different pathways to increase apical membrane chloride conductance in distal nephron A6 cells; i.e., (1) increases the Po and single channel conductance of 3 pS Cl- channels and (2) increases the number of 8 pS Cl- channels.

Abstract: To determine whether primary cultures of rat fetal distal lung epithelium (FDLE) possessed L-type Na+ channels on their plasma membrane we performed experiments with 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and other amiloride analogs. Short-circuit current (Isc) was decreased by the apical application of amiloride and benzamil, but was unaffected by 10 microM dimethylamiloride (DMA). EIPA decreased Isc when added to either the apical or basal sides. Greatest effects were seen with bilateral EIPA, where half-maximal effects occurred in the micromolar range. Measurements of intracellular pH with the fluorescent dye BCECF demonstrated that DMA impaired (IC50 = 71 nM) the ability of FDLE to recover from intracellular acidification. Nystatin perforated patch clamp techniques showed that FDLE had nonrectifying Na+ currents but no detectable Cl- currents. The whole-cell currents were reversibly decreased by 20 microM concentrations of EIPA, benzamil, and amiloride but were unaffected by 20 microM DMA. These studies indicate that there are EIPA-sensitive Na+ conductances in intact FDLE and suggest the presence of L-type Na+ conductances on their apical membrane and EIPA-sensitive K+ channels on the basolateral membrane.

Abstract: The Cl- conductance of a mouse fibroblast cell line (LTK- cells) that was stably transfected with the human CFTR (cystic fibrosis transmembrane conductance regulator) complementary DNA was studied. Single Cl- channel activity was observed only after treatment of the cells with forskolin, the single-channel conductance being 6.2 +/- 0.2 pS with a linear current-voltage relationship. In CFTR+ cells, the whole-cell current at +90 mV increased from 7.3 +/- 2.7 pA/pF (n = 12) to 46.1 +/- 11.2 pA/pF (n = 5) after addition of dibutyryl-cyclic AMP (10(-4) M) to the bath. Increasing the intracellular Cl- concentration to 150 mM activated linear Cl- currents in the absence of cyclic AMP in CFTR+ (n = 42) but not in CFTR- cells (n = 4). Similar Cl- current was also activated by high intracellular I- concentration. These results indicate that the CFTR-induced Cl- conductance in LTK- cells can be activated by either cyclic AMP or high intracellular halide concentrations.

Abstract: We have used the whole-cell patch-clamp technique to identify and characterize Cl- currents in a cell line derived from human peripheral airway epithelium (NCI-H-441-4). The permeability sequence and relative selectivity for different anions was Br- (1.4) approximately I- (1.3) > Cl- (1.0) > F- (0.6) > gluconate (0.4) > glutamate (0.2). The current-voltage relationship displayed rectification in the outward direction. Diphenylamine-2-carboxylate (10(-4) M) applied intracellularly blocked the outward-rectified current, while extracellularly applied diphenylamine-2-carboxylate had no effect on Cl- current. This current was also blocked by extracellularly applied 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), with an estimated IC50 of 15.2 microM. Dibutyryl-cyclic AMP (10(-4) M) increased outward current, whereas pretreatment with 100 ng/mL pertussis toxin almost completely abolished the Cl- current. Pertussis toxin inhibition of this current could be partially reversed by dialysis of the cell interior with the activated alpha i-2 subunit of Gi protein. This cell line provides an opportunity to study directly the regulation of Cl- channels in cells derived from the peripheral human lung airways.

Abstract: The apical membrane of renal epithelium (A6) had a Ca(2+)-activated, outwardly rectified chloride channel with the single channel conductance of 3 pS for outward current and 0.5 pS for inward current. The outward rectification was dependent on the cytosolic Ca2+ concentration. Vasopressin (arginine vasopressin, AVP) and cholera toxin (CTX) increased the single channel conductance for inward current through an increase in the Ca(2+)-sensitivity about 100-fold.

Abstract: Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

Abstract: The cytosolic Ca2+ concentration ([Ca2+]i) affects many cell functions, including the modulation of ion channel activity. In this study patch clamp experiments using primary cultures of fetal distal lung epithelium (FDLE) demonstrated that the elevation of [Ca2+]i modulated a 25pS amiloride-blockable non selective cation (NSC) channel's ion selectivity and sensitivity to amiloride. An elevation of [Ca2+]i from 0.1 microM to 1mM both increased open probability (Po) and decreased the ratio of the permeability to Na to the permeability to K (PNa/PK) from 1.96 +/- 0.11 (SEM, n = 6) to 0.88 +/- 0.04 (n = 6). Within the range of [Ca2+]i from 0.1 microM to 100 microM amiloride (0.5 microM) decreased Po, however amiloride (0.5 microM) no longer affected Po of the NSC channel when [Ca2+]i was increased to 1mM under physiologic membrane potentials. A beta adrenergic agonist (terbutaline, 10 microM) increased Po in cell-attached patches from almost 0 (Po less than 0.01; n = 9) to 0.39 +/- 0.09 (n = 9) and [Ca2+]i from 40 +/- 6nM (n = 9) to more than 1 microM. This suggested that amiloride-blockable NSC channel activity and ion permeability are modulated by changes in [Ca2+]i near physiologic membrane potentials and a beta adrenergic agonist increases [Ca2+]i to more than 1 microM (unlike other epithelial including adult alveolar cells) which is associated with activation the NSC channel.

Abstract: We studied the effect of insulin and lavendustin-A (a tyrosine kinase inhibitor) on the short-circuit current (ISC) of primary cultures of fetal distal rat lung epithelium (FDLE). Insulin (2 microM) on the basolateral side of the monolayer increased ISC from 5.76 +/- 0.83 microA/cm2 (SEM, n = 7) to 7.23 +/- 1.00 microA/cm2 (p less than 0.01) under control conditions, and from 1.00 +/- 0.31 microA/cm1 to 1.53 +/- 0.34 microA/cm2 (p less than 0.05, n = 4) when amiloride (10 microM) was present on the apical side of the monolayer. Thus insulin increased both the amiloride-sensitive and insensitive ISC with the insulin-induced increase in ISC in the absence of amiloride (1.47 +/- 0.22 microA/cm2, n = 7) being significantly larger than that in the presence of 10 microM amiloride (0.53 +/- 0.14 microA/cm2, n = 4; p less than 0.025). Insulin's effect reached steady state in 1 hr. Lavendustin-A (10 microM), a tyrosine kinase inhibitor, applied to the apical side of the monolayer attenuated but did not completely block insulin's ability to increase in ISC; i.e., insulin increased ISC in lavendustin-A treated monolayers (0.63 +/- 0.09 microA/cm2, n = 5; p less than 0.0025) but the increase was significantly smaller than that without the pretreatment of lavendustin-A (p less than 0.05). In the presence of amiloride (10 microM) and lavendustin-A (10 microM) insulin was no longer able to increase ISC (change in ISC = 0.04 +/- 0.03 microA/cm2, n = 6), suggesting that lavendustin-A had blocked the insulin's effect on the amiloride-insensitive ISC. Lavendustin-A (10 microM) had no significant effect on the basal ISC in control and amiloride treated monolayers. Our studies demonstrate that insulin increases amiloride-insensitive ISC in FDLE via lavendustin-A sensitive tyrosine kinase and that insulin's action on the amiloride-sensitive ISC of FDLE is mediated through a lavendustin-A insensitive (and presumably tyrosine kinase-independent) pathway.

Abstract: 1. The sensitivity of Na efflux to ouabain in frog skeletal muscle cells was studied in the presence and absence of insulin. 2. Insulin increased the ouabain-sensitive Na efflux, about two-fold, without any significant effect on the ouabain-insensitive Na efflux; i.e. all components of the Na efflux increased by insulin can be blocked by ouabain. 3. There was no significant difference between the time course of the inhibitory action of ouabain on Na efflux in the presence and absence of insulin: i.e. the binding affinity of the insulin-stimulated Na/K pump to ouabain is same as that of the control.

Abstract: To determine the mechanism by which vasopressin increases sodium transport in sodium-transporting, tight epithelia, we examined single amiloride-blockable Na channels in membrane patches from cultured distal nephron cells (A6) either before or after treatment with arginine vasopressin. Pretreatment of cells with vasopressin (40 mU/ml) for 40-50 min increases NPo (N, the number of Na channels; Po, the open probability of an individual Na channel). The increase in NPo is due to an increase in the number of conductive Na channels with little or no change in the open probability of individual Na channels. Pretreatment of cells for 1 h with 1 mM N6,2'-O-dibutyryladenosine 3', 5'-cyclic monophosphate (DBcAMP) also increased NPo. The increase in NPo caused by DBcAMP pretreatment is also due to the increase in the number of conductive Na channels with no change in the open probability of individual Na channels. Cells pretreated with cholera toxin (CTX; 250 ng/ml) for 4 h appeared similar to cells that had been treated with vasopressin or DBcAMP; that is, the number of Na channels per patch increased with little or no effect on the open probability of individual Na channels. For patches from many untreated cells, when the frequency of occurrence is plotted against the number of channels in an individual patch, the histogram consists of a single peak with a number of channels per patch of 2.0 +/- 1.5 (+/- SD, 126 patches). After pretreatment of cells with vasopressin, DBcAMP, or CTX, the same histogram contains two peaks after vasopressin of 1.8 +/- 1.2 and 9.2 +/- 1.5 (+/- SD, 38 and 53 patches, respectively). These observations suggest that pretreatment of cells with vasopressin, DBcAMP, or CTX may act by promoting insertion of clusters of new sodium channels.

Abstract: We studied the effects of guanosine 3',5'-cyclic monophosphate (cGMP) and nitroprusside on ion channels in the apical membrane of confluent A6 cells (a distal nephron cell line) cultured on permeable supports for 10-14 days using patch clamp techniques. In cell-attached patches without any detectable channel activity, activity of a non-selective cation channel with a single-channel conductance of 1 pS was observed after adding nitroprusside. After adding cGMP to the cytosolic surface of inside-out patches with no detectable channel activity, we observed single channel activity similar to the channel observed after adding nitroprusside. These observations imply that nitroprusside activates a non-selective cation channel with small single channel conductance (1 pS) via an increase in cGMP which activates the channel.

Abstract: In this report, single-channel recording methods were used to determine whether there are Cl- conductive pathways in the apical membrane of cultured renal distal nephron cells (A6). Two different types of single Cl- channels were observed. In cell-attached patches, one had a unit conductance of 3 pS, whereas the unit conductance of the other was 8 pS. In cell-attached patches, the currents associated with the 3-pS Cl- channel outwardly rectified, whereas the current voltage relationship for the 8-pS Cl-channel was linear. The 3-pS Cl- channel has one open and one closed state; the 8-pS Cl- channel has one open and two closed states. The open probability of the 3-pS Cl- channel was voltage dependent (increasing with depolarization of the membrane) but even at very depolarized potentials (+140 mV) remained small (always less than 0.1). On the other hand, the open probability of the 8-pS Cl- channel was large (approximately 0.8) and voltage independent. The closing rate of the 3-pS Cl- channel was decreased when the patch membrane was depolarized, whereas the opening rate was increased. In contrast, the closing rate of the 8-pS Cl- channel decreased with depolarization, but the opening rates were voltage independent. The outward rectification of the 3-pS channel was markedly reduced in inside-out patches when high calcium concentrations (10-800 microM) were present on the intracellular surface. The open probability of the 3-pS Cl- channel is increased by membrane permeable analogues of adenosine 3',5'-cyclic monophosphate primarily by decreasing the mean closed time.

Abstract: A Cl- channel with a small single-channel conductance (3 pS) was observed in cell-attached patches formed on the apical membrane of cells from the distal nephron cell line (A6) cultured on permeable supports. The current-voltage (I-V) relationship from cell-attached patches or inside-out patches with 1 microM cytosolic Ca2+ strongly rectified with no inward current at potentials more negative than ECl. However, the rectification decreased (i.e., inward current increased) when the cytosolic Ca2+ concentration ([Ca2+]i) was increased above 1 microM. If [Ca2+]i is increased to 800 microM, the I-V relationship became linear. Besides the change in the I-V relationship, an increase in [Ca2+]i also increases the open probability of the channel. Regardless of the recording condition, the channel has one open and one closed state. Both closing and opening rates were dependent on [Ca2+]i; an increase of [Ca2+]i decreased the closing rate and increased the opening rate. The Ca2+ dependence of transition rates at positive membrane potentials (cell interior with respect to external surface) were much larger than the dependence at negative intracellular potentials. The I-V relationship of chloride channels in inside-out patches from cells pretreated with insulin was linear even with 1 microM [Ca2+]i, while channel currents from cells under similar conditions but without insulin still strongly rectified. Alkaline phosphatase applied to the intracellular surface of inside-out patches altered the outward rectification of single channels in a manner qualitatively similar to that of insulin pretreatment. These observations suggest that phosphorylation/dephosphorylation of the channel modulates the sensitivity of the Cl- channel to cytosolic Ca2+ and that insulin produces its effect by promoting dephosphorylation of the channel.

Abstract: The first step in net active transepithelial transport of sodium in tight epithelia is mediated by the amiloride-blockable sodium channel in the apical membrane. This sodium channel is the primary site for discretionary control of total body sodium and, therefore, investigating its regulatory mechanisms is important to our understanding of the physiology of fluid and electrolyte balance. Because essentially all of the regulatory sites on the channel are on the intracellular surface, patch clamp methods have proven extremely useful in the electrophysiological characterization of the sodium channel by isolating it from other channel proteins in the epithelial membrane and by allowing access to the intracellular surface of the protein. We have examined three different regulatory mechanisms. (1) Inhibition of channel activity by activation of protein kinase C; (2) activation of the channel by agents which activate G-proteins; and (3) modulation of channel kinetics and channel number by mineralocorticoids. Activation of protein kinase C by phorbol esters or synthetic diacylglycerols reduces the open probability of sodium channels. Protein kinase C can be activated in a physiological context by enhancing apical sodium entry. Actions which reduce sodium entry (low luminal sodium concentrations or the apical application of amiloride) increase channel open probability. The link between sodium entry and activation of protein kinase C appears to be mediated by intracellular calcium activity linked to sodium via a sodium/calcium exchange system. Thus, the intracellular sodium concentration is coupled to sodium entry in a negative feedback loop which promotes constant total entry of sodium. Activation of G-proteins by pertussis toxin greatly increases the open probability of sodium channels. Since channels can also be activated by pertussis toxin or GTP gamma S in excised patches, the G-protein appears to be closely linked in the apical membrane to the sodium channel protein itself. The mechanism for activation of this apical G-protein, when most hormonal and transmitter receptors are physically located on the basolateral membrane, is unclear. Mineralocorticoids such as aldosterone have at least two distinct effects. First, as expected, increasing levels of aldosterone increase the density of functional channels detectable in the apical membrane. Second, contrary to expectations, application of aldosterone increases the open probability of sodium channels. Thus aldosterone promotes the functional appearance of new sodium channels and promotes increased sodium entry through both new and pre-existant channels.

Abstract: Resting membrane potentials (Em) increased in the negative direction during first cleavage in Cynops pyrrhogaster eggs whose new membranes formed at first cleavage were exposed to bathing solutions by removing the vitelline envelopes. Em was -11.4 and -87.2 mV at the one- and two-cell stages, respectively. Na/K pump activity contributed to Em at the two-cell stage by about -30 mV. The distribution of Na/K ATPase activity was cytochemically studied by Ernst's method (S. A. Ernst, 1972, J. Histochem. Cytochem. 20, 23-38). The new membrane of the eggs at the two-cell stage showed the pump activity. But the activity was detected neither in the preexisting outer membrane of the eggs at the two-cell stage nor in the membrane at the one-cell stage.

Abstract: To clarify the dependency of the Na/K coupling of the Na,K-pump on internal Na and external K concentrations in skeletal muscle, the ouabain-induced change in membrane potential, the ouabain-induced change in Na efflux and the membrane resistance were measured at various internal Na and external K concentrations in bullfrog sartorius muscle. Upon raising the internal Na concentration from 6 mmol/kg muscle water to 20 mmol/kg muscle water, the magnitude of the ouabain-induced change in membrane potential increased about eightfold and the magnitude of the ouabain-induced change in Na efflux increased about fivefold while the membrane resistance was not significantly changed. As the external K concentration increased from 1 to 10 mM, the magnitude of the ouabain-induced change in membrane potential decreased (1/5.5 fold), while the magnitude of the ouabain-induced change in Na efflux increased (about 1.5-fold). The membrane resistance decreased upon raising the external K concentration from 1 to 10 mM (1/2-fold). These observations imply that the values of the Na/K coupling of the Na,K-pump increases upon raising the internal Na concentration and decreases upon raising the external K concentration.

Abstract: 1. It is well known that insulin has various effects on glucose transport and the Na,K-pump in muscles. It is also known to have some effects on the membrane potential--in general, insulin induces a hyperpolarization of the membrane in muscles. Furthermore, it is suggested that the actions of insulin are modified by changes in ionic surroundings. 2. In this review article, the actions of ionic surroundings and insulin on glucose transport in muscles are discussed; in particular, the effects of changes in extracellular and/or intracellular concentrations of Na, K, Ca and H ions will be mentioned. 3. The actions of ionic surroundings and insulin on the Na,K-pump in muscles are discussed; in particular, the effects of changes in extracellular an/or intracellular concentrations of Na, K, Ca and H ions will be examined. 4. The relationship between the actions of ionic surroundings and insulin are discussed. 5. In particular, the effects of changes in ionic surroundings on the insulin-induced hyperpolarization of the membrane are discussed by relating it to the Na,K-pump function. The relationship between the insulin-induced change in membrane potential and glucose transport will be also mentioned.

Abstract: Insulin induced a hyperpolarization of the membrane by stimulating the Na,K-pump in frog skeletal muscles. The Na,K-pump activity was dependent on the internal Na concentration. As the internal Na concentration was raised from 5 mmol/kg muscle water to 18 mmol/kg muscle water, the magnitude of the insulin-induced increase in the ouabain-sensitive Na efflux (an index of the Na,K-pump activity) rose by 5-fold and the magnitude of the insulin-induced hyperpolarization rose by 8.5-fold. On the other hand, the specific membrane resistance was not significantly changed by a rise in the internal Na concentration. The Na/K coupling of the Na,K-pump was calculated at low, normal or high internal Na concentration by using the values of the insulin-induced changes in the ouabain-sensitive Na efflux and the membrane potential. As a result of the calculation, it was suggested that in frog skeletal muscles the Na/K coupling would increase with a rise of the internal Na concentration.

Abstract: Insulin induced a hyperpolarization of the membrane and stimulated the 3-O-methylglucose (3-O-MG) uptake in frog skeletal muscle. In the present study, the relationship between the insulin-induced changes in the membrane potential and the 3-O-MG uptake was investigated. The stimulatory action of insulin on the 3-O-MG uptake was mediated by two different mechanisms. One of them was dependent on the change in the membrane potential and the other was independent of the change in the membrane potential. Both values of the insulin-induced changes in the membrane potential and the 3-O-MG uptake were diminished by increasing the external K concentration. One of the causes for the diminution of the 3-O-MG uptake with a rise of the external K concentration would be the decrease in the magnitude of the insulin-induced hyperpolarization.

Abstract: It has been suggested that the insulin-induced hyperpolarization might be a mediator of the stimulatory action of insulin on glucose transport. The purpose of the present study was to investigate the relationship between the insulin-induced hyperpolarization and the stimulatory action of insulin on glucose transport in skeletal muscle. Satorius muscles dissected from bullfrogs (Rana catesbeiana) were used. Insulin induced a hyperpolarization of the membrane and an increase in the 3-O-Methyl-D-glucose (3-O-MG) uptake and extrusion. In the presence of valinomycin, insulin had no significant effect on the membrane potential. Insulin still had the stimulatory action on both the 3-O-MG uptake and extrusion even in the presence of valinomycin, under whose condition insulin had no significant effect on the membrane potential. The magnitude of the stimulatory action of insulin on the 3-O-MG uptake in the presence of valinomycin was smaller than that in the absence of valinomycin. The magnitude of the stimulatory action of insulin on the 3-O-MG extrusion was, on the contrary, larger than that in the absence of valinomycin. The abolishment of the insulin-induced hyperpolarization decreased the 3-O-MG uptake and increased the 3-O-MG extrusion. The observation in the present study concludes that insulin has two different actions on glucose transport. One of them is developed through the insulin-induced hyperpolarization, which increases the 3-O-MG uptake and decreases the 3-O-MG extrusion. The other action is irrelevant of the insulin-induced hyperpolarization and stimulates both the 3-O-MG uptake and extrusion.

Abstract: Insulin hyperpolarized the membrane of frog skeletal muscle by stimulating the electrogenic Na,K-pump. At external K concentrations of 1,2,5 and 10 mM, both the insulin-induced hyperpolarization and the insulin-stimulated ouabain-sensitive Na efflux (an index of Na,K-pump activity) were observed. By increasing the external K concentration, the insulin-stimulated Na efflux increased, but the magnitude of the insulin-induced hyperpolarization decreased; i.e., although the activity of the insulin-stimulated Na,K-pump increased, on the contrary, the magnitude of the hyperpolarization decreased. To clarify the causes of this phenomenon, the specific membrane resistance was measured and found to decrease upon increasing the external K concentration. One of the reasons for the decrease in magnitude of the hyperpolarization is the decrease in the specific membrane resistance. However, the decrease in magnitude of the hyperpolarization with a rise of the external K concentration, which increased the insulin-stimulated Na,K-pump activity, cannot be explained only by the decrease in the specific membrane resistance. It is suggested that the decrease in magnitude of the hyperpolarization is mainly caused by a decrease in the electrogenicity of the insulin-stimulated Na,K-pump upon an increase in the external K concentration. The conclusion of the present study is that the electrogenicity of the insulin-stimulated Na,K-pump in muscles is variable and decreases with increasing the external K concentration.

Abstract: A Na+-sensitive uptake of 3-O-methylglucose (3-O-MG), a nonmetabolized sugar, was characterized in frog skeletal muscle. A removal of Na+ from the bathing solution reduced 3-O-MG uptake, depending on the amount of Na+ removed. At a 3-O-MG concentration of 2 mM, the Na+-sensitive component of uptake in Ringer's solution was estimated to be about 26% of the total uptake. The magnitude of Na+-sensitive component sigmoidally increased with an increase of 3-O-MG in bathing solution, whereas in Na+-free Ringer's solution the uptake was proportional to the concentration. The half saturation of the Na+-sensitive component was at a 3-O-MG concentration of about 13 mM, and the Hill coefficient was 1.4 to 1.6. Phlorizin (5 mM), a potent inhibitor specific for Na+-coupled glucose transport, reduced the uptake in a solution containing Na+ to the level in Na+-free Ringer's solution. Glucose of concentrations higher than 20 mM suppressed 3-O-MG uptake to a level slightly lower than that in Na+-free Ringer's solution. These observations indicate that there are Na+-coupled sugar transport systems in frog skeletal muscle which are shared by both glucose and 3-O-MG.

Abstract: To gain an insight into the mechanism of stimulation by insulin of Na efflux, the effect of high concentrations of internal Na on the inhibition by ouabain of Na efflux was compared to the inhibition by ouabain of insulin-stimulated Na efflux. The rate coefficient of 22Na efflux from "high-Na" muscle exposed to ouabain was lower than that from "low-Na" muscle exposed to ouabain. Similarly, the rate of net Na loss from the "high-Na" muscle which had been exposed to ouabain was lower than that frm the "low-Na" muscle. These findings indicate that Na transport units whose internal Na-binding sites have been occupied by Na is susceptible to ouabain. The insulin-stimulated Na efflux was inhibited by ouabain to a larger extent than was the Na efflux from the control muscle, although the final level of the rate coefficient of 22Na efflux from the muscle exposed to both insulin and ouabain was not always lower than that from the muscle exposed only to ouabain. The similarity between the effects of ouabain in "high-Na" muscle and in insulin-preincubated muscle suggests that insulin increases the fraction of Na transport units whose internal Na-binding sites have been occupied by Na.

Abstract: The dependency of Na efflux on intracellular Na concentration was examined on sartorius muscles of Rana catesbeiana. In normal Ringer, the rate coefficient of 22Na efflux is nearly proportional to the internal Na concentration, i.e., Hill's coefficient in the dependency of Na efflux on internal Na concentration was around two. As long as insulin is present in the bathing solution, the rate coefficient of 22Na efflux retains the stimulated level. Insulin caused a leftward shift of the relationship between Na efflux and the logarithm of internal Na concentration, and concomitantly decreased Hill's coefficient. A model of the coupling of Na transport and hydrolysis of ATP was proposed. The theoretical relation derived from this model accounted for these findings quite satisfactorily. According to this model, insulin shifts as a cofactor the equilibrium between dephosphorylated and phosphorylated states of carriers, whose Na binding sites are not occupied by Na, toward the phosphorylated state.