Abstract: Weibel-Palade bodies (WPBs) are the storage organelles for von Willebrand factor (VWF) in endothelial cells. VWF forms multimers that assemble into tubular structures in WPBs. Upon demand, VWF is secreted into the blood circulation, where it unfolds into strings that capture platelets during the onset of primary hemostasis. Numerous mutations affecting VWF lead to the bleeding disorder von Willebrand disease. This review reports the recent findings on the effects of VWF mutations on the biosynthetic pathway of VWF and its storage in WPBs. These new findings have deepened our understanding of VWF synthesis, storage, secretion, and function.
Abstract: Patients with von Willebrand disease (VWD) are often heterozygous for a missense mutation in the von Willebrand factor (VWF) gene. Investigating the pathogenic features of VWF mutations in cells directly derived from patients has been challenging. Here, we have used blood outgrowth endothelial cells (BOECs) isolated from human peripheral blood to analyze the storage and secretion of VWF. BOECs showed full endothelial characteristics and responded to Weibel-Palade body (WPB) secretagogues except desmopressin. We examined BOECs derived from a single subject heterozygous for a type 2N mutation (p.Arg854Gln) and from 4 patients with type 1 VWD who were, respectively, heterozygous for p.Ser1285Pro, p.Leu1307Pro, p.Tyr1584Cys, and p.Cys2693Tyr. Compared with normal BOECs, BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr showed morphologically abnormal WPB and retention of VWF in the endoplasmic reticulum, whereas BOECs heterozygous for p.Arg854Gln or p.Tyr1584Cys showed normal WPB. The agonist-induced exocytosis of WPB from BOECs and formation of VWF strings on BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr, but not for p.Arg854Gln or p.Tyr1584Cys, were reduced. In conclusion, VWD phenotype can be recapitulated in BOECs, and thus BOECs provide a feasible bona fide cell model to study the pathogenic effects of VWF mutations.
Abstract: Vascular endothelial cells contain unique storage organelles, designated Weibel-Palade bodies (WPBs), that deliver inflammatory and hemostatic mediators to the vascular lumen in response to agonists like thrombin and vasopressin. The main component of WPBs is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation. In addition to VWF, several other components are known to be stored in WPBs, like osteoprotegerin, monocyte chemoattractant protein-1 and angiopoetin-2 (Ang-2). Here, we used an unbiased proteomics approach to identify additional residents of WPBs. Mass spectrometry analysis of purified WPBs revealed the presence of several known components such as VWF, Ang-2, and P-selectin. Thirty-five novel candidate WPB residents were identified that included insulin-like growth factor binding protein-7 (IGFBP7), which has been proposed to regulate angiogenesis. Immunocytochemistry revealed that IGFBP7 is a bona fide WPB component. Cotransfection studies showed that IGFBP7 trafficked to pseudo-WPB in HEK293 cells. Using a series of deletion variants of VWF, we showed that targeting of IGFBP7 to pseudo-WPBs was dependent on the carboxy-terminal D4-C1-C2-C3-CK domains of VWF. IGFBP7 remained attached to ultralarge VWF strings released upon exocytosis of WPBs under flow. The presence of IGFBP7 in WPBs highlights the role of this subcellular compartment in regulation of angiogenesis.
Abstract: Background Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand's disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies. DESIGN AND METHODS: The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate. RESULTS: p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser. Conclusions We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulum.
Abstract: Weibel-Palade bodies (WPBs) are elongated secretory organelles specific to endothelial cells that contain von Willebrand factor (VWF) and a variety of other proteins that contribute to inflammation, angiogenesis, and tissue repair. The remarkable architecture of WPBs is because of the unique properties of their major constituent VWF. VWF is stored inside WPBs as tubules, but on its release, forms strikingly long strings that arrest bleeding by recruiting blood platelets to sites of vascular injury. In recent years considerable progress has been made regarding the molecular events that underlie the packaging of VWF multimers into tubules and the processes leading to the formation of elongated WPBs. Mechanisms directing the conversion of tightly packaged VWF tubules into VWF strings on the surface of endothelial cells are starting to be unraveled. Several modes of exocytosis have now been described for WPBs, emphasizing the plasticity of these organelles. WPB exocytosis plays a role in the pathophysiology and treatment of von Willebrand disease and may have impact on common hematologic and cardiovascular disorders. This review summarizes the major advances made on the biogenesis and exocytosis of WPBs and places these recent discoveries in the context of von Willebrand disease.
Abstract: Several missense mutations in the von Willebrand Factor (VWF) gene of von Willebrand disease (VWD) patients have been shown to cause impaired constitutive secretion and intracellular retention of VWF. However, the effects of those mutations on the intracellular storage in Weibel-Palade bodies (WPBs) of endothelial cells and regulated secretion of VWF remain unknown. We demonstrate, by expression of quantitative VWF mutants in HEK293 cells, that four missense mutations in the D3 and CK-domain of VWF diminished the storage in pseudo-WPBs, and led to retention of VWF within the endoplasmic reticulum (ER). Immunofluorescence and electron microscopy data showed that the pseudo-WPBs formed by missense mutant C1060Y are indistinguishable from those formed by normal VWF. C1149R, C2739Y, and C2754W formed relatively few pseudo-WPBs, which were often short and sometimes round rather than cigar-shaped. The regulated secretion of VWF was impaired slightly for C1060Y but severely for C1149R, C2739Y, and C2754W. Upon co-transfection with wild-type VWF, both intracellular storage and regulated secretion of all mutants were (partly) corrected. In conclusion, defects in the intracellular storage and regulated secretion of VWF following ER retention may be a common mechanism underlying VWD with a quantitative deficiency of VWF.
Abstract: In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative light-electron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultra-large VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIII-covered VWF strings, and impairs recruitment of platelets. Apparently, intra-cellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings.
Abstract: Regulated exocytosis of Weibel-Palade bodies (WPBs) is a pivotal mechanism via which vascular endothelial cells initiate repair in response to injury and inflammation. Several pathways have been proposed to enable differential release of bioactive molecules from WPBs under different pathophysiologic conditions. Due to the complexity, many aspects of WPB biogenesis and exocytosis are still poorly understood. Herein, we have investigated the regulated exocytosis of the major WPB constituent, von Willebrand Factor (VWF), which upon its release forms strings of up to several millimeters long that capture circulating platelets and thereby initiate the formation of a haemostatic plug. Using correlative, fluorescence, and electron microscopic imaging techniques, we provide evidence that multigranular exocytosis is an important pathway for VWF release in secretagogue-challenged human umbilical vein endothelial cells. A novel membrane-delimited structure (secretory pod) was identified as the site of WPB coalescence and VWF exocytosis. Clathrin-coated profiles present on the secretory pods suggested remodeling via compensatory membrane retrieval. Small, 30- to 40-nm cytoplasmic vesicles (nanovesicles) mediated the fusion of WPBs with secretory pods. Multigranular exocytosis may facilitate VWF string formation by pooling the content of multiple WPBs. In addition, it may provide a novel mechanism for the differential release of WPB cargo.
Abstract: The insect lipophorin receptor (LpR), an LDL receptor (LDLR) homologue that is expressed during restricted periods of insect development, binds and endocytoses high-density lipophorin (HDLp). However, in contrast to LDL, HDLp is not lysosomally degraded, but recycled in a transferrin-like manner, leaving a function of receptor-mediated uptake of HDLp to be uncovered. Since a hallmark of circulatory HDLp is its ability to function as a reusable shuttle that selectively loads and unloads lipids at target tissues without being endocytosed or degraded, circulatory HDLp can exist in several forms with respect to lipid loading. To investigate whether lipid content of the lipoprotein affects binding and subsequent endocytosis by LpR, HDLp was partially delipidated in vitro by incubation with alpha-cyclodextrin, yielding a particle of buoyant density 1.17g/mL (HDLp-1.17). Binding experiments demonstrated that LpR bound HDLp-1.17 with a substantially higher affinity than HDLp both in LpR-transfected Chinese hamster ovary (CHO) cells and isolated insect fat body tissue endogenously expressing LpR. Similar to HDLp, HDLp-1.17 was targeted to the endocytic recycling compartment after endocytosis in CHO(LpR) cells. The complex of HDLp-1.17 and LpR appeared to be resistant to endosomal pH, as was recently demonstrated for the LpR-HDLp complex, corroborating that HDLp-1.17 is recycled similar to HDLp. This conclusion was further supported by the observation of a significant decrease with time of HDLp-1.17-containing vesicles after endocytosis of HDLp-1.17 in LpR-expressing insect fat body tissue. Collectively, our results indicate that LpR favors the binding and subsequent endocytosis of HDLp-1.17 over HDLp, suggesting a physiological role for LpR in selective endocytosis of relatively lipid-unloaded HDLp particles, while lipid reloading during their intracellular itinerary might result in decreased affinity for LpR and thus allows recycling.
Abstract: Cryo-electron tomography (cryo-ET) allows for the visualization of biological material in a close-to-native state, in three dimensions and with nanometer scale resolution. However, due to the low signal-to-noise ratio inherent to imaging of the radiation-sensitive frozen-hydrated samples, it appears often times impossible to localize structures within heterogeneous samples. Because a major potential for cryo-ET is thereby left unused, we set out to combine cryo-ET with cryo-fluorescence microscopy (cryo-FM), in order to facilitate the search for structures of interest. We describe a cryo-FM setup and workflow for correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) that can be easily implemented. Cells are grown on finder grids, vitally labeled with one or two fluorescent dyes, and vitrified. After a structure is located by cryo-FM (with 0.4microm resolution), its image coordinates are translated to cryo-ET stage coordinates via a home-built software routine. We tested our workflow on whole mount primary human umbilical vein endothelial cells. The correlative routine enabled us to investigate mitochondrial ultrastructure for the first time on intact human mitochondria, and led us to find mitochondrial cristae that were connected to the intermembrane space via large slits, which challenges the current view that such connections are established exclusively via small circular pores. Taken together, this study emphasizes that cryo-CLEM can be a routinely used technique that opens up exciting new possibilities for cryo-ET.
Abstract: Multimers of von Willebrand Factor (vWF), a protein mediating blood clotting in response to vascular injury, are stored as tubular structures by endothelial cells in specific organelles, the Weibel-Palade Bodies (WPBs). To date very little is known about the 3D structure of WPBs in relation to the organization of the tubules. Therefore, we have initiated a thorough electron microscopic study in human umbilical vein endothelial cells (HUVECs) using electron tomography to gain further understanding of the ultrastructure of WPBs. We found that in addition to the well-documented cigar-shape, WPBs adopt irregular forms, which appeared to result from homotypic fusion. In transverse views of WPBs the tubular striations appear evenly spaced, which indicates a high level of organization that is likely to involve an underlying scaffold of structural proteins. Additionally, we found that the tubular striations twisted in an orderly fashion, suggesting that they are stored within the WPBs by a spring-loading mechanism. Altogether these data suggest that WPBs undergo a relatively complex maturation process involving homotypic fusion. Although the mechanism of assembly of vWF multimers into tubules is still unknown, the curled arrangement of the tubules within WPBs suggests a high degree of folding of the protein inside the organelle.
Abstract: Rab3D, a member of the Rab3 subfamily of the Rab/ypt GTPases, is expressed on zymogen granules in the pancreas as well as on secretory vesicles in mast cells and in the parotid gland. To shed light on the function of Rab3D, we have generated Rab3D-deficient mice. These mice are viable and have no obvious phenotypic changes. Secretion of mast cells is normal as revealed by capacitance patch clamping. Furthermore, enzyme content and overall morphology are unchanged in pancreatic and parotid acinar cells of knockout mice. Both the exocrine pancreas and the parotid gland show normal release kinetics in response to secretagogue stimulation, suggesting that Rab3D is not involved in exocytosis. However, the size of secretory granules in both the exocrine pancreas and the parotid gland is significantly increased, with the volume being doubled. We conclude that Rab3D exerts its function during granule maturation, possibly by preventing homotypic fusion of secretory granules.
Abstract: The p41 splice variant of major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains a 65 aa segment that binds to the active site of cathepsin L (CatL), a lysosomal cysteine protease involved in MHC class II-restricted antigen presentation. This segment is absent from the predominant form of Ii, p31. Here we document the in vivo significance of the p41-CatL interaction. By biochemical means and electron microscopy, we demonstrate that the levels of active CatL are strongly reduced in bone marrow-derived antigen-presenting cells that lack p41. This defect mainly concerns the mature two-chain forms of CatL, which depend on p41 to be expressed at wild-type levels. Indeed, pulse-chase analysis suggests that these mature forms of CatL are degraded by endocytic proteases when p41 is absent. We conclude that p41 is required for activity of CatL by stabilizing the mature forms of the enzyme. This suggests that p41 is not merely an inhibitor of CatL enzymatic activity, but serves as a chaperone to help maintain a pool of mature enzyme in late-endocytic compartments of antigen-presenting cells.
Abstract: The present study describes a novel phenomenon in pancreatic acinar cells undergoing regulated exocytosis. When acinar cell preparations were challenged with the secretagogue carbamylcholine, a subpopulation of zymogen granules became coated with filamentous actin. These zymogen granules were always in proximity of the acinar cell apical membrane (the site of exocytosis) but did not appear to have fused yet. They were distinct from regular zymogen granules not only because of their association with filamentous actin, but also because the majority of them lacked the zymogen granule marker rab3D, a small GTPase implicated in regulated exocytosis. The apparent loss of rab3D, presumed to result from the release of rab3D from the granule membranes, could be prevented by agents that modulate the actomyosin system as well as by GTP[gammaS]. These data suggest that zymogen granules engaging in exocytosis become coated with actin before fusion and that this actin coating is tightly coupled to the release of rab3D. We propose that rab3D is involved in the regulation of actin polymerization around secretory granules and that actin coating might facilitate the movement of granules across the subapical actin network and toward their fusion site.
Abstract: The small GTP-binding protein, rab4, is involved in recycling of transferrin receptors and translocation of GLUT4. Recent studies suggest that rab4 controls regulated exocytosis in the exocrine pancreas. We conducted the present study to further investigate the role of rab4 in the exocrine pancreas. We found that the exocrine pancreas expresses two rab4 immunoanalogs, one of approximately 28 kDa identified previously in neonatal glands, and one of approximately 24 kDa which is similar to rab4 characterized in other systems. The latter species was mostly membrane-anchored and localized to endosome-like structures in a supranuclear region that was immunopositive for the transferrin receptor. The approximately 24-kDa rab4 form also localized to the apical plasmamembrane, and this immunofluorescence increased greatly in tissue challenged with a secretagogue. We propose that the approximately 24-kDa rab4 species is involved in compensatory membrane retrieval following regulated exocytosis, and that rab4-positive endocytic vesicles move through a supranuclear recycling compartment.
Abstract: The neuropeptide calcitonin gene-related peptide (CGRP) is concentrated in fine sensory nerve endings innervating all tissues, including bone. CGRP inhibits osteoclasts, stimulates insulin-like growth factor I and inhibits tumor necrosis factor alpha production by osteoblasts in vitro. To investigate the role of CGRP in bone in vivo, mice were engineered to express CGRP in osteoblasts by placing the human CGRP gene under the control of the rat osteocalcin promoter (Ost-CGRP tg+ mice). Calvaria cultures from transgene positive (tg+), but not tg- mice, produced bioactive CGRP. Trabecular bone density and bone volume, determined by peripheral quantitative computed tomography and bone histomorphometry, respectively, were higher in tg+ than tg- littermates. This increase in bone volume was associated with an increased bone formation rate. Trabecular bone density decreased in tg+ mice as a result of ovariectomy, but remained higher than in sham tg- mice. Targeting CGRP to osteoblasts appears to favor the establishment of a higher trabecular bone mass in mice.
Abstract: We examined the effects of disruption of the actin cytoskeleton by cytochalasin D (cytoD) on basal and carbamylcholine-stimulated exocytosis and on compensatory membrane retrieval in pancreatic acinar cells. Although the involvement of actin in exocytosis is reasonably well established, its role in these coupled processes is not understood. Our findings suggested that cytoD inhibited stimulated secretion of amylase. However, morphometry revealed that exocytosis had occurred: the number of zymogen granules decreased, the size of the lumen increased, and large vacuolar structures continuous with the lumen formed into which amylase accumulated. Large amounts of amylase were released to the medium on removal of secretagogue and cytoD, suggesting that the subapical actin network provides contractile forces that expel the lumenal contents. Strikingly, we observed that at the apical pole of the cells where exocytosis occurred, cytoD induced an accumulation of membrane invaginations into a vastly enlarged apical membrane. These pits were often surrounded by a clathrin-like coat. Concomitantly, AP-2-, clathrin-, dynamin- and caveolin-like immunoreactivity concentrated around the enlarged lumina, suggesting that incorporation of zymogen granule membrane into the apical plasma membrane triggered the recruitment of these proteins. After wash out of cytoD and carbamylcholine and reformation of the subapical actin cytoskeleton, the coated invaginations largely disappeared in association with a reduction in lumenal size, and relocation of clathrin, AP-2, dynamin and caveolin into the cell. We suggest that the actin terminal web also controls compensatory membrane retrieval following exocytosis.
Abstract: This review summarizes new insights into the role of the actin cytoskeleton in exocytosis and compensatory membrane retrieval from mammalian regulated secretory cells. Data from our lab and others now indicate that the actin cytoskeleton is involved in exocytosis both as a negative regulator of membrane fusion under resting conditions and as a facilitator of movement of secretory granules to their site of fusion with the apical plasmalemma. Coating of docked secretory granules with actin filaments correlates with the dissociation of secretory-granule-associated rab3D, pointing out a novel role for rab proteins in modulating the actin cytoskeleton during regulated exocytosis. Compensatory membrane retrieval following regulated exocytosis is also critically dependent on the actin cytoskeleton both in initiating the formation of clathrin-coated retrieval vesicles and subsequent trafficking back into the cell. We propose that insertion of secretory granule membrane into the plasmalemma initiates a trigger for membrane retrieval, possibly by exposing sites where proteins involved in compensatory membrane retrieval are assembled. The results summarized in this review were derived primarily from investigations on the pancreatic acinar cell, an old friend who is providing modern wisdom not attainable in other simpler systems.
Abstract: The rat TRH precursor contains five copies of TRH separated by connecting peptides. Previous studies have shown that the decapeptide prepro-TRH (160-169; Ps4) potentiates the effect of TRH on TSH secretion. In the present study, we have characterized Ps4 receptors in the rat pituitary by in vitro autoradiography using [125I-Tyr0]Ps4 as a radioligand, and we have investigated the evolution of receptor density during ontogenesis. Incubation of rat pituitary slices with [125I-Tyr0]Ps4 revealed intense binding in the anterior lobe and virtually no binding in the neurointermediate lobe. Biochemical characterization of the Ps4-binding sites suggested the existence of a single class of sites exhibiting high affinity for [Tyr0]Ps4 (IC50 = 8.3 +/- 1.2 nM) and a much lower affinity for Ps4 (IC50 = 9.3 +/- 1.2 microM). Emulsion-coated cytoautoradiography performed on cultured anterior pituitary cells showed that only 26% of the cells possessed [125I-Tyr0]Ps4-binding sites. Immunocytochemical analysis using antibodies against the different anterior pituitary hormones indicated that the cells possessing [125I-Tyr0]Ps4-binding sites did not correspond to TSH-, PRL-, GH-, ACTH-, or LH-secreting cells. In contrast, cells expressing Ps4 receptors were immunoreactive for the S-100 protein, a marker of folliculo-stellate cells. During postnatal development, a 4-fold increase in the concentration of [125I-Tyr0]Ps4-binding sites occurred from birth to weaning in the pituitary, with a marked and transient increase at the time of weaning. Thereafter, the density of sites declined gradually until day 60. In conclusion, the present study shows that folliculo-stellate cells express [125I-Tyr0]Ps4-binding sites in the anterior pituitary, and that these sites are developmentally regulated. The present data suggest that the potentiating effect of Ps4 on TRH-induced TSH secretion is mediated by folliculo-stellate cells.
Abstract: To investigate whether the immunosuppressive neuropeptide calcitonin gene-related peptide (CGRP) was a potential candidate for tissue-specific gene therapy, we engineered nonobese diabetic (NOD) mice to produce CGRP in beta cells by placing the modified calcitonin gene under the control of the rat insulin promoter. CGRP inhibits CD4 T cell production of the cytokines that have been implicated in the pathogeny of type I diabetes. Three transgene-positive mouse lines were obtained, two of which expressed immunoreactive CGRP in beta cells (NOD-CGRP mice). Isolated islets from one of these two transgene-positive founders produced active CGRP, whereas islets from transgene-negative littermates did not. The production of CGRP by beta cells prevented insulin-dependent diabetes mellitus in male and reduced its incidence by 63% in female mice. This prevention was due to a local immunosuppressive effect of CGRP as no difference was detected between NOD-CGRP and NOD littermate lymph node, spleen, and thymus cells by either FACS analysis or proliferative response to stimulation by Ag, alloantigen or anti-CD3. These data suggest that CGRP is a potential therapeutic molecule to prevent or treat diabetes and possibly other diseases and conditions in which immune cells are involved. These data also suggest that endogenous CGRP concentrated in sensory nerve endings may regulate locally the immune response, further strengthening the importance of the functional neuroimmune link.
Abstract: Studies with the Na+ channel blocker tetrodotoxin (TTx) on Ca(2+)-dependent hormone release by mammalian and amphibian pituitary melanotrophs have suggested that the Na+ spikes these cells generate are not responsible for triggering Ca2+ influx and consequently secretion. In contrast, we found in Xenopus laevis melanotrophs that the spontaneously occurring elevations in cytosolic free Ca2+ concentration ("Ca2+ pulses') were dependent on the presence of extracellular Na+ and sensitive to TTx and the Na+ channel activator, veratridine. However, an inhibitory effect of TTx could only be demonstrated when the extracellular Na+ concentration was lowered to near-threshold levels. In voltage-clamp experiments, two distinct Na+ currents were recorded, one sensitive to TTx and the other insensitive to TTx but blocked by micromolar concentrations of Cd2+. Together they appeared to control action potential activity and spontaneous Ca2+ pulsing. These data strongly suggest that Na+ action potentials do regulate cytosolic free Ca2+ concentration in melanotrophs.
Abstract: Calcitonin gene-related peptide (CGRP) is a neuropeptide abundantly concentrated in sensory nerve endings innervating bone metaphysis and periosteum, which indicates that it plays a local role in bone metabolism. CGRP-alpha and -beta share structural and functional homology with calcitonin (CT) and have been shown to inhibit bone resorption in vitro and to induce hypocalcemia in vivo. We recently reported that CGRP stimulates the production of the growth factor insulin-like growth factor-I and inhibits that of the cytokine tumor necrosis factor-alpha by osteoblasts, suggesting that CGRP may control bone cell activity. To investigate this possibility, we used ovariectomized (ovx) rats as a high bone turnover model and compared the effects of CGRP to those of CT. ovx young female rats were injected daily starting the day after surgery with either phosphate-buffered saline, CGRP-alpha (1.15 mg/kg per day), or CT (3 micrograms/kg per day) for 28 days. Ovariectomy induced an increase in bone turnover associated with a 60% loss in trabecular bone volume of the proximal tibia. CGRP inhibited bone resorption but not bone formation, and was nevertheless less efficient than CT in preventing bone loss, since CGRP-treated rats had a loss of 46% of cancellous bone, whereas CT-treated rats had a loss of 21%. This suggests that CGRP is either less potent than CT at inhibiting bone resorption or else very rapidly degraded. These data indicate that CGRP can control bone cells through a mechanism that is in part different from that of CT, and further suggest that CGRP may play a local role in bone metabolism.
Abstract: The purpose of this study was to investigate the mechanisms involved in the reduced thyroid function in starved, young female rats. Food deprivation for 3 days reduced the hypothalamic content of prothyrotrophin-releasing hormone (proTRH) mRNA, the amount of proTRH-derived peptides (TRH and proTRH160-169) in the paraventricular nucleus, the release of proTRH-derived peptides into hypophysial portal blood and the pituitary levels of TSH beta mRNA. Plasma TSH was either not affected or slightly reduced by starvation, but food deprivation induced marked increases in plasma corticosterone and decreases in plasma thyroid hormones. Refeeding after starvation normalized these parameters. Since the molar ratio of TRH and proTRH160-169 in hypophysial portal blood was not affected by food deprivation, it seems unlikely that proTRH processing is altered by starvation. The median eminence content of pGlu-His-Pro-Gly (TRH-Gly, a presumed immediate precursor of TRH), proTRH160-169 or TRH were not affected by food deprivation. Since median eminence TRH-Gly levels were very low compared with other proTRH-derived peptides it is unlikely that alpha-amidation is a rate-limiting step in hypothalamic TRH synthesis. Possible negative effects of the increased corticosterone levels during starvation on proTRH and TSH synthesis were studied in adrenalectomized rats which were treated with corticosterone in their drinking water (0.2 mg/ml). In this way, the starvation-induced increase in plasma corticosterone could be prevented. Although plasma levels of thyroid hormones remained reduced, food deprivation no longer had negative effects on hypothalamic proTRH mRNA, pituitary TSH beta mRNA and plasma TSH in starved adrenalectomized rats. Thus, high levels of corticosteroids seem to exert negative effects on the synthesis and release of proTRH and TSH. This conclusion is corroborated by the observation that TRH release into hypophysial portal blood became reduced after administration of the synthetic glucocorticosteroid dexamethasone. On the basis of these results, it is suggested that the reduced thyroid function during starvation is due to a reduced synthesis and release of TRH and TSH. Furthermore, the reduced TRH and TSH synthesis during food deprivation are probably caused by the starvation-induced enhanced adrenal secretion of corticosterone.
Abstract: The effects of GABAa receptor activation on the secretion of alpha-melanocyte-stimulating hormone (alpha-MSH) from the superfused pars intermedia of the amphibian Xenopus laevis were examined. The GABAa receptor agonist isoguvacine inhibited secretion of alpha-MSH from the pars intermedia, an action completely antagonized by the chloride channel blocker picrotoxin. Isoguvacine stimulated secretion from picrotoxin-treated tissue. Both effects were blocked by the GABAA receptor antagonist bicuculline, indicating that it is a specific action via the GABAA receptor. We conclude that, besides the inhibitory chloride channel, there is a stimulatory signaling property associated with the GABAA receptor. Isoguvacine stimulated the production of c-AMP, an action that was not blocked by picrotoxin. This suggests that the stimulatory mechanism of the GABAA receptor involves, directly or indirectly, the generation of c-AMP.
Abstract: The rat thyrotropin-releasing hormone (TRH) precursor (prepro-TRH) contains five copies of the TRH progenitor sequence linked together by intervening sequences. Recently, we have shown that the connecting peptides prepro-TRH-(160-169) (Ps4) and prepro-TRH-(178-199) (Ps5) are released from rat hypothalamic neurones in response to elevated potassium concentrations, in a calcium-dependent manner. In the present study, the role of voltage-operated calcium channels in potassium-induced release of Ps4 and Ps5 was investigated, using a perifusion system for rat hypothalamic slices. The release of Ps4 and Ps5 stimulated by potassium (70 mM) was blocked by the inorganic ions Co2+ (2.6 mM) and Ni2+ (5 mM). In contrast, the stimulatory effect of KCl was insensitive to Cd2+ (100 microM). The dihydropyridine antagonist nifedipine (10 microM) had no effect on K(+)-evoked release of Ps4 and Ps5. Furthermore, the response to KCl was not affected by nifedipine (10 microM) in combination with diltiazem (1 microM), a benzothiazepine which increases the affinity of dihydropyridine antagonists for their receptor. The dihydropyridine agonist BAY K 8644, at concentrations as high as 1 mM, did not stimulate the basal secretion of Ps4 and Ps5. In addition, BAY K 8644 had no potentiating effect on K(+)-induced release of Ps4 and Ps5. The marine cone snail toxin omega-conotoxin, a blocker of both L- and N-type calcium channels had no effect on the release of Ps4 and Ps5 stimulated by potassium. Similarly, the omega-conopeptide SNX-111, a selective blocker of N-type calcium channels, did not inhibit the stimulatory effect of potassium. The release of Ps4 and Ps5 evoked by high K+ was insensitive to the non-selective calcium channel blocker verapamil (20 microM). Amiloride (1 microM), a putative blocker of T-type calcium channels, did not affect KCl-induced secretion of the two connecting peptides. Taken together, these results indicate that two connecting peptides derived from the pro-TRH, Ps4 and Ps5, are released by K(+)-induced depolarization through activation of voltage-sensitive calcium channels. The calcium channels appear to have a pharmacological profile different from that of L- and N-type channels. Although, their insensitivity to low Cd2+ concentrations and sensitivity to Ni2+ ions would support the involvement of T-type calcium channels, the lack of effect of amiloride suggests that they belong to a yet undefined class of calcium channels.
Abstract: Thyrotropin-releasing hormone prohormone contains five copies of the thyrotropin-releasing hormone progenitor sequence Gln-His-Pro-Gly, each flanked by pairs of basic amino acids and separated by intervening sequences (connecting peptides). Using a perifusion system for rat hypothalamic slices, we have studied the ionic mechanisms underlying the release of two connecting peptides originating from the thyrotropin-releasing hormone precursor: prepro-thyrotropin-releasing hormone-(160-169) (Ps4) and prepro-thyrotropin-releasing hormone-(178-199) (Ps5). Quantification of these two peptides in the effluent fluid was performed using sensitive and highly specific radioimmunoassay procedures. Reverse phase high performance liquid chromatography analysis of the effluent perifusate showed that released peptides co-eluted with synthetic Ps4 and Ps5. The secretion of Ps4 and Ps5 was stimulated by depolarizing agents such as (i) high potassium concentrations, (ii) ouabain, an Na+/K(+)-ATPase inhibitor, and (iii) veratridine, a stimulator of voltage-operated Na+ channels. The response to potassium (70 mM) was not affected by the specific Na+ channel blocker tetrodotoxin. The K+ channel blocker tetraethylammonium did not modify K(+)-evoked release of Ps4 and Ps5. These data suggest that voltage-operated Na+ channels are not involved in the stimulatory effect of high K+ on the release of Ps4 and Ps5. The lack of effect of picrotoxin, a Cl- channel blocker, on the secretion of the connecting peptides indicates that chloride ions play a minor role in the release process. In contrast, deprivation of Ca2+ in the perifusion medium suppressed K(+)-evoked release of the two peptides, indicating that voltage-operated Ca2+ channels are implicated in the release process. Taken together, the present results show that non-thyrotropin-releasing hormone peptides originating from the thyrotropin-releasing hormone precursor are secreted by mediobasal hypothalamic fragments. The release of these peptides is stimulated by depolarization through a calcium-dependent process. These data indicate that Ps4 and Ps5 may be released at the level of the median eminence into the portal circulation, suggesting that these peptides may play a role in the control of anterior pituitary cells.
