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: 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: We have investigated the role for diacylglycerol (DAG) in membrane bud formation in the Golgi apparatus. Addition of propranolol to specifically inhibit phosphatidate phosphohydrolase (PAP), an enzyme responsible for converting phosphatidic acid into DAG, effectively prevents formation of membrane buds. The effect of PAP inhibition on Golgi membranes is rapid and occurs within 3 min. Removal of the PAP inhibitor then results in a rapid burst of buds, vesicles, and tubules that peaks within 2 min. The inability to form buds in the presence of propranolol does not appear to be correlated with a loss of ARFGAP1 from Golgi membranes, as knockdown of ARFGAP1 by RNA interference has little or no effect on actual bud formation. Rather, knockdown of ARFGAP1 results in an increase in membrane buds and a decrease of vesicles and tubules suggesting it functions in the late stages of scission. How DAG promotes bud formation is discussed.
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: Locating areas of interest by electron microscopy can be laborious. This is particularly true for electron tomography, where the use of thicker sections may obscure relevant details in the projection images. We evaluated the applicability of fluorescent probes to thin plastic sections, in combination with fluorescence microscopy, as an aid in selecting areas for subsequent electron microscopic analysis. We show that pre-embedding labeling of DNA and RNA with acridine orange yielded a predominant nuclear stain. The stain greatly reduced the time needed to scan sections for mitotic cells, or cells with characteristic nuclei such as neutrophils. Post-embedding labeling with SYTOX green yielded a nuclear stain comparable to acridine orange, and wheat germ agglutinin (WGA) conjugated to Alexa Fluor 488 labeled mucous granules and the Golgi area in intestinal goblet cells. The fluorescent labels were visualized directly on sections on electron microscope grids. It was therefore possible to establish a coordinate system based on the position of the grid bars, allowing for easy retrieval of selected areas. Because the fluorescent probes were incompatible with osmium tetroxide treatment, contrast in the sections was faint. We propose a simplified electron tomography procedure for the generation of 2D views with enhanced contrast and resolution.
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: Correlative microscopy is a powerful technique that combines the strengths of fluorescence microscopy and electron microscopy. The first enables rapid searching for regions of interest in large fields of view while the latter exhibits superior resolution over a narrow field of view. Routine use of correlative microscopy is seriously hampered by the cumbersome and elaborate experimental procedures. This is partly due to the use of two separate microscopes for fluorescence and electron microscopy. Here, an integrated approach to correlative microscopy is presented based on a laser scanning fluorescence microscope integrated in a transmission electron microscope. Using this approach the search for features in the specimen is greatly simplified and the time to carry out the experiment is strongly reduced. The potential of the integrated approach is demonstrated at room temperature on specimens of rat intestine cells labeled with AlexaFluor488 conjugated to wheat germ agglutinin and on rat liver peroxisomes immunolabeled with anti-catalase antibodies and secondary AlexaFluor488 antibodies and 10nm protein A-gold.
Abstract: Rab3D is a small GTP-binding protein that associates with secretory granules of endocrine and exocrine cells. The physiological role of Rab3D remains unclear. While it has initially been implicated in the control of regulated exocytosis, recent deletion-mutation studies have suggested that Rab3D is involved in the biogenesis of secretory granules. Here, we report the unexpected finding that Rab3D also associates with early Golgi compartments in intestinal goblet cells and in Brunner's gland acinar cells. Expression of Rab3D in the intestine was demonstrated by SDS-PAGE and Western blot analysis of homogenates prepared from the rat duodenum and colon. Confocal laser scanning microscopy revealed Rab3D immunofluorescence in the Golgi area of goblet cells of the duodenum and colon and in Brunner's gland acinar cells. There was no colocalization between Rab3D and a trans-Golgi network marker, TGN-38. In contrast, Rab3D colocalized partially with a cis-Golgi marker, GM-130, and with a marker of cis-Golgi and coat protein complex I vesicles, beta-COP. Strong colocalization was observed between Rab3D and the lectins Griffonia simplicifolia agglutinin II and soybean agglutinin, which have been described as markers of the medial and cis-Golgi, respectively. Rabphilin, a putative effector of Rab3D, displayed an identical pattern of Golgi localization. Incubation of colon tissue with carbamylcholine or deoxycholate to stimulate exocytosis by goblet cells caused a partial redistribution of Rab3D to the cytoplasm and mucous granule field and a concomitant transformation of the Golgi architecture. Taken together, the present data suggest that Rab3D and rabphilin may regulate the secretory pathway at a much earlier stage than what has hitherto been assumed.
Abstract: Rab3D is a small GTP-binding protein associated with secretory vesicles in various exocrine and endocrine cells, where it has been implicated in regulated exocytosis. Data obtained previously in pancreas have suggested that rab3D is involved in the coating of secretory granules with filamentous actin. In the present study we employed Western blot analysis, immunofluorescence, and immunoelectron microscopy to examine the distribution of rab3D in rat lung. Rab3D immunoreactivity was detected in bronchiolar Clara cells and alveolar epithelial type II (AET-II) cells. In both cell types, rab3D displayed preferential localization to secretory vesicles that were identified using specific antibodies against Clara Cell Secretory Protein and p180 lamellar body protein, respectively. Interestingly, rab3D was associated with only 24% of the lamellar bodies in AET-II cells. Rab3D-positive lamellar bodies were typically in close proximity of the apical plasma membrane, where exocytosis occurs. Another subpopulation of lamellar bodies, constituting only 2%, was not only rab3D-positive but could also be labeled with the filamentous-actin probe phalloidin. A third subpopulation, constituting 9%, displayed actin coating without rab3D staining. We propose that these three lamellar body subpopulations represent consecutive intermediates along the regulated exocytotic pathway, implying that rab3D release and actin coating are intimately linked processes.
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: Rab3D is a small GTPase implicated in regulated exocytosis, and is a marker of secretory granules in exocrine cells. We have previously shown that rab3D undergoes reversible carboxyl-methylation in adult rat pancreatic acinar cells, and that carboxyl-methylation of rab3D is developmentally regulated concomitantly with the maturation of the regulated secretory apparatus in rat pancreas. We also observed that dexamethasone treatment of the rat pancreatic acinar tumor cell line, AR42J, led to a significant increase in the size of the unmethylated pool of a rab3-like protein. The current study was designed to further characterize this rab3-like protein. Here we show that AR42J cells express rab3D, and that the protein focuses on 2D gels as two spots with pI values of 4.9 and 5.0. Treatment of AR42J cells with N-acetyl-S-geranylgeranyl-l-cysteine, an inhibitor of carboxyl-methylation, led to a decrease in the basic form of rab3D and a proportional increase in the acidic form. In contrast, N-acetyl-S-farnesyl-l-cysteine, which inhibits carboxyl-methylation of farnesylated proteins, had no effect. Lovastatin, an inhibitor of geranylgeranylation, also induced an accumulation of the acidic form of rab3D. Taken together, these data indicate that rab3D can undergo reversible carboxyl-methylation in AR42J cells by a geranylgeranyl-specific methyltransferase. The 2D gel and immunoblotting analyses indicated that dexamethasone treatment of AR42J cells led to an increase in the proportion of the unmethylated form of rab3D concurrent to inducing a regulated secretory pathway, similar to the rab3D profile change in developing rat pancreas. Our data, along with previous studies done on developing rat pancreas, indicate that the tumor cell line AR42J represents a good model system for studying the regulated secretory pathway, and that carboxyl-methylation of rab3D may play a role in the acquisition of stimulus-secretion coupling.
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: 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: Several GTPases of the rab family, including rab3A, are methylesterifled on their carboxy-terminal prenylcysteine residue. The significance of this reversible posttranslational modification for the function of rab proteins is unknown, although it has been postulated that carboxyl methylation facilitates the membrane association of prenylated proteins through a hydrophobic mechanism. We here demonstrate, that pancreatic rab3D undergoes developmentally regulated carboxyl methylation concurrently with the maturation of the regulated secretory apparatus in pancreatic acinar cells: in fetal glands, which are refractive to hormone stimulation, the majority of the rab3D protein was methylated, whereas in neonatal and adult glands, which are secretory competent, only 50% was methylated. The methylated form of rab3D was also predominant in a transplantable acinar cell tumor which displays impaired secretory responsiveness and morphological characteristics reminiscent of the fetal pancreas. In addition, treatment of AR42J pancreatic acinar tumor cells with dexamethasone to induce a regulated secretory pathway, led to a significant increase in the size of the unmethylated pool of a rab3-like protein. Strikingly, membrane preparations from adult pancreata and parotid glands contained both methylated and unmethylated forms of rab3D indiscriminately. These results suggest that the acquisition of stimulus-secretion coupling by the exocrine pancreas correlates with the methylation state of rab3D, and that carboxyl methylation plays no significant role in enhancing the membrane association or determining the subcellular distribution of rab3D.
Abstract: The epithelial sodium channel (ENaC) provides the rate-limiting step in the reabsorption of sodium by many epithelia. The number of channels at the cell surface is tightly regulated; most cells express only a few channels. We have examined the biosynthesis and cell surface expression of ENaC in Xenopus oocytes. The subunits of ENaC are readily synthesized in the endoplasmic reticulum, but most of them remain as immature proteins in pre-Golgi compartments, where they are degraded by the proteasomal pathway without apparent ubiquitination. Even when the three subunits, alpha, beta, and gamma, are expressed in the same cell, only a very small fraction of the total channel population leave the endoplasmic reticulum, acquire complex oligosaccharides, and reach the plasma membrane. Overexpression of subunits does not increase the number of channels in the plasma membrane but results in the appearance of cytoplasmic subunits in a form not membrane bound. The data indicate that maturation and assembly of the subunits are slow and inefficient processes, and constitute limiting steps for the expression of functional ENaC channels in the plasma membrane.
Abstract: Rab4 is a small GTP-binding protein that has been implicated in the regulation of membrane traffic and recycling of transferrin receptors and GLUT4 transporters along the endocytic pathway. Here we present data that suggest a novel and very different role for rab4 during development in the rat exocrine pancreas. On immunoblots of pancreatic homogenates, a dramatic increase in rab4 expression occurred over the first 40 h after birth, concomitant with the time of acquisition of stimulus-secretion coupling. Following high-speed centrifugation of postnuclear supernatants prepared from 1-day neonatal pancreatic homogenates, rab4 partitioned into a Triton X-100 insoluble particulate fraction and was partially solubilized upon extraction with 0.1 M Na2CO3, pH 11.5, or 1 M KCl, suggesting that rab4 was not an integral membrane protein. This was confirmed by Triton X-114 extractions of post-nuclear supernatants showing that rab4 partitioned into the aqueous phase of Triton X-114, which is indicative of a lack of isoprenylation. Confocal and electron microscopic immunocytochemistry revealed that rab4 colocalized with the actin terminal web and microvilli in the apical region of the exocrine acinar cells. In view of these findings, we propose that rab4 is involved in the maturation of the regulated secretory pathway in pancreatic acinar cells through an interaction with the apical actin cytoskeleton.
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: This study reports of presence of rab3D, a low M(r) GTP-binding protein, in rat pancreatic acinar cells and islets using a combination of Western blot analysis, two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis/isoelectric focusing, and light and electron microscopic immunocytochemistry. For these purposes, we used an affinity-purified rabbit polyclonal antibody generated against the exclusive amino terminus of rab3D. Failure to detect rab3A, B or C in pancreatic acinar cells with their respective antisera indicated that the rab3D immunoreactivity was not due to cross-reaction with rab3A, B or C. A monoclonal antiserum which recognized rab3A, B, C and D (clone 42.1) detected a second lower M, band in gradient gels. This protein may be an additional member of the rab family. Double label electron microscopic immunogold localizations for rab3D, and the monoclonal antibody that recognizes all members of the rab3 family, showed a preferential localization of rab3D to zymogen granules. In contrast, clone 42.1 detected both zymogen granules and elements of the Golgi complex. Rab3D also localized to the secretory granule field in pancreatic islet cells which additionally expressed rab3A. The majority of rab3D in acinar cells was tightly associated with membrane fractions as indicated by its resistance to alkaline pH extraction. It is likely associated with membranes via isoprenyl groups as suggested by its partitioning into the detergent phase in Triton X-114 extractions. In contrast, bacterially expressed rab3D partitioned solely into the aqueous phase in Triton X-114 extractions. Because of its exclusive location on zymogen granules, rab3D may play a role in regulated exocytosis from pancreatic acinar cells.
Abstract: Although the secretory apparatus of rat pancreatic acinar cells already has a mature appearance in late stage fetuses, the regulated exocytotic pathway becomes functional only after birth. In this study we tested the hypothesis that the acquisition of stimulus-secretion coupling in the acinar cells depends on the developmental expression of rab3D, a small GTP-binding protein which we have previously shown to be associated with zymogen granule membranes in the adult pancreatic acinar cell. On immunoblots of pancreatic homogenates, rab3D became detectable on gestational day 18. This developmental pattern was consistent with that observed by confocal immunocytochemistry. In 20- and 21-day-old fetal pancreata, rab3D was mostly associated with the cytosolic fraction, whereas a redistribution to the membrane fraction occurred after birth, concomitant with a decrease in rab-GDI expression. Overlays of Western blots with 32P-alpha-GTP revealed that rab3D immunoprecipitated from fetal pancreatic homogenates possessed very little GTP-binding capacity as compared to adult homogenates. Triton X-114 extractions showed that the fetal rab3D partitioned into the detergent phase, suggesting that it was posttranslationally isoprenylated. Taken together, the present data indicate that the expression and localization of rab3D is developmentally regulated and strongly suggest that the maturation of the regulated exocytotic pathway in the exocrine pancreas depends on the membrane association of rab3D.
Abstract: A patch-clamp study was conducted on cultured frog pituitary melanotrophs, in order to investigate the effects of adrenaline on the electrical activity of these cells. In the whole-cell configuration, adrenaline (1 microM) caused hyperpolarization that was accompanied by a fall in membrane input resistance and a blockage of spontaneous action potentials. Under voltage clamp, adrenaline elicited a net-outward current. The hyperpolarization became undetectable at a command voltage of -100 mV which corresponded to the equilibrium potential of potassium ions. The effect of adrenaline on membrane potential and spontaneous activity was blocked by the alpha 2-adrenergic receptor antagonist yohimbine (1-10 microM) but could not be mimicked by the alpha 2-adrenergic agonist clonidine (1-10 microM). In the cell-attached configuration, exposure of the extra-patch membrane to adrenaline increased the occurrence of single-channel currents with a slope conductance of 100 pS. The deduced reversal potential of these currents corresponded to the equilibrium potential of potassium ions. These results suggest that frog melanotrophs display an alpha 2-adrenergic receptor subtype coupled to potassium channels involved in hyperpolarization.
Abstract: The molecular mechanisms regulating GABAA receptor activity in cultured frog melanotrophs were studied using the patch-clamp technique. In the whole-cell configuration, application of GABA evoked a dose-related increase of inward chloride currents. The ED50 value, estimated from the sigmoidal dose-response curve was 2 x 10(-6) M and the Hill coefficient was 1.55. The amplitude of the GABA-induced current decayed with time. Kinetics analysis of the desensitization revealed that the time-course of the current decrement was fitted by one exponential. Graded doses of GABA or association of GABA with the benzodiazepine receptor agonist flunitrazepam accelerated the desensitization process. In contrast, the time-course of the current did not significantly vary at different holding potentials. In the outside-out configuration, GABA was found to activate channels which displayed three unitary conductance levels (8, 15 and 30 pS). The channel openings of the more frequent conductance level (30 pS) exhibited short and long lasting open states (1.2 and 28.3 ms at -60 mV). Altogether these data reveal that frog melanotrophs possess a single population of GABAA receptors which interconvert into a higher affinity state in the presence of benzodiazepine receptor agonists. Two GABA molecules must bind to the receptor to trigger long lasting channel openings. In addition, the activity of the GABAA receptor appears to be independent of the accumulation of intracellular chloride ions.
Abstract: 1. Melanostatin, a thirty-six amino acid peptide recently isolated from the frog brain due to its ability to inhibit alpha-melanocyte-stimulating hormone (alpha-MSH) release, is the amphibian counterpart of mammalian neuropeptide Y (NPY). The effect of synthetic melanostatin on the bioelectrical activity of cultured frog melanotrophs was studied in 124 cells by using the whole-cell patch-clamp technique. 2. In current-clamp experiments, melanostatin (1 microM) provoked a reversible hyperpolarization and a suppression of spontaneous action potentials. In some cells the hyperpolarizing response was absent, but an arrest of spike firing still occurred. 3. Melanostatin-induced hyperpolarization was associated with a decrease in membrane resistance. In voltage-clamp experiments, melanostatin induced an outward current at a constant command potential. This hyperpolarizing outward current appeared to be carried by potassium ions. 4. Cell dialysis with the non-hydrolysable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) sustained the outward current produced by melanostatin. Dopamine (1 microM), which generates a similar hyperpolarizing outward current in frog melanotrophs, was not capable of increasing the current provoked by melanostatin and sustained by GTP gamma S. 5. Melanostatin also modulated voltage-operated currents. The amplitude of voltage-activated potassium current was increased by 30%. 6. Melanostatin reduced the fast sodium current. This inhibitory effect was rather persistent compared to the other modulated currents. 7. Melanostatin markedly scaled down high voltage-activated N- and L-like calcium currents. The activation kinetics of these two calcium currents were not altered by the peptide. 8. Pretreatment of melanotrophs with pertussis toxin (1 microgram ml-1) blocked melanostatin-induced inhibition of N- and L-like calcium currents. 9. It is concluded that the NPY-related peptide melanostatin generates a very complex pattern of electrical responses in frog melanotrophs, including hyperpolarization and modulation of voltage-activated currents underlying action potentials. G proteins appear to mediate at least part of these effects.
Abstract: Dopamine (1 microM) reversibly scaled down barium current through high-voltage activated (HVA) calcium channels but had little effect on the time course of current activation in cultured frog melanotrophs. Intracellular perfusion with guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM) sustained the effect of dopamine. Moreover, GTP gamma S drastically slowed down the current activation kinetics. The latter effect was in part reversed by dopamine. A conditioning prepulse to +70 mV facilitated the current in GTP gamma S-dialyzed cells but not in cells exposed to dopamine. These results suggest the existence of a dual G protein-mediated mechanism for reducing HVA calcium current.
Abstract: The effects of gamma-aminobutyric acid (GABA) and benzodiazepines on the electrical activity of cultured frog melanotrophs were studied using the patch-clamp technique. In the cell-attached configuration, the exposure to GABA caused a blockage of the spontaneous firing. In the whole-cell configuration, with physiological chloride concentrations, GABA evoked a hyperpolarization associated with a decrease of membrane resistance, generating an inward chloride current. Clonazepam, a central-type benzodiazepine agonist, potentiated the GABA-induced current and the resulting hyperpolarization. In addition, the benzodiazepine inverse agonist Ro 19-4603 totally abolished GABA-induced hyperpolarizing chloride current. Since the pars intermedia of the frog pituitary is composed of a 'pure' population of endocrine cells enriched with GABAA receptors, our results indicate that these cells represent a valuable model in which to investigate the electrophysiological effects of ligands for the GABAA benzodiazepine receptor complex.
Abstract: A patch-clamp study was conducted in order to investigate the effects of dopamine on the ionic currents in cultured frog melanotrophs. Brief applications of dopamine (1 microM) hyperpolarized the cell and inhibited the spontaneous action potentials. The hyperpolarization was accompanied by an increase in membrane conductance. Under voltage clamp, dopamine evoked a net outward current. The dopamine-induced outward current was negligible at the equilibrium potential for potassium ions. It was also observed that dopamine increased the intensity of a voltage-dependent outward potassium current monitored by constant depolarizing pulses. In addition, voltage-dependent L- and N-like calcium currents and sodium current were reduced. In the cell-attached configuration, two distinct channel types were activated and one channel type was blocked by dopamine exposure to the extrapatch membrane, which indicates the involvement of an intracellular factor in the signal transduction pathway. A higher conductance channel (100 pS) was characterized by a very low basal activity which rapidly increased upon dopamine application. A lower conductance channel (30 pS) displayed a basal activity with frequent opening events, and a delayed (30-40 s) increase of activity in response to dopamine. Both currents reversed at a deduced potential corresponding to the equilibrium potential for potassium ions. The channel type inhibited by dopamine had a low conductance of 15 pS. The inhibition of the electrical activity induced by dopamine was totally blocked by the D2 receptor antagonist S(-)-sulpiride (1 microM) but was not affected by the D1 receptor antagonist SKF-83566 (1 microM). It is concluded that dopamine activates potassium channels and inhibits calcium and sodium channels in frog melanotrophs. The results also indicate that stimulus-response coupling is mediated by intracellular messenger system(s).
Abstract: Recently we have demonstrated that dopamine inhibits action potentials in cultured frog melanotrophs through D2 receptor-mediated activation of hyperpolarizing potassium current and reduction of calcium and sodium currents. Herein, the respective roles of G proteins, guanosine-5'-triphosphate and adenosine-3':5'-cyclic-monophosphate in dopamine-induced electrical responses were investigated using the whole-cell patch-clamp technique. Pretreatment of melanotrophs with pertussis toxin (1 microgram/ml) abolished the hyperpolarization and arrest of action potentials evoked by dopamine (1 microM) in 77% of the cells studied. Addition of guanosine-5'-O-(2-thiodiphosphate) (500 microM) to the intracellular solution did not alter the effects of a first exposure to dopamine, but completely blocked the response of cultured melanotrophs to subsequent pulses of dopamine. In cells which were dialysed with guanosine-5'-O-(3-thiotriphosphate) (100 microM) dopamine caused a sustained hyperpolarization and an irreversible inhibition of spikes. Voltage-clamp recordings with electrodes containing guanosine-5'-O-(3-thiotriphosphate), showed that the increase of potassium current and decrease of calcium and sodium currents caused by dopamine were irreversible. These effects were not modified when the pipette contained, in addition to guanosine-5'-O-(3-thiotriphosphate), a high concentration of adenosine-3':5'-cyclic-monophosphate (100 microM) together with the inhibitor of phosphodiesterases 3-isobutyl-1-methylxanthine (100 microM). It is concluded that, in cultured frog melanotrophs, a pertussis toxin-sensitive G protein is implicated in the coupling of dopamine D2 receptors to activation of potassium channels and inhibition of calcium and sodium channels. Our results also indicate that the G protein-mediated signal transduction does not involve the adenylate cyclase system.
Abstract: The firing patterns of cultured frog melanotrophs were studied using the patch-clamp technique. In the cell-attached mode, unitary currents were frequently observed as well as biphasic waveforms which were attributed to action potentials 'leaking' through the patch membrane. An inwardly rectifying single-unit current was observed with pipette solutions containing either 100 mM K+ or 100 mM Na+. Under both conditions, these channels displayed an identical I/V relationship, yielding a unitary conductance of 110 pS. The channel opening time was extremely long (50-3000 ms) and single-channel currents showed typical relaxations, which triggered bursts of action currents. In the whole-cell configuration large (2-12 mV) fluctuations in the membrane voltage of current-clamped cells frequently occurred. The deflections appeared to result from single-channel currents. Depolarizing 'events' often led to the discharge of action potentials. Taken together, our data provide evidence for the existence of high-conductance cationic channels in frog pars intermedia cells. These channels may, at least in some cases, be responsible for the generation of pacemaker depolarizations, thereby regulating firing behaviour. It is concluded, that the current traversing a single channel can seriously affect the membrane potential and excitability of frog melanotrophs.
Abstract: Due to the high complexity of the mammalian central nervous system, sampling of immunohistochemically processed brain tissues for electronmicroscopy requires an accurate and reliable technique. For this reason, the flat-embedding method, which allows light microscopical examination of tissue before sampling, is generally employed. Because of the osmification process, however, the tissue is blackish and opaque which hampers light microscopical selection of tissue areas of interest. We have found that tissue translucency is highly improved by an osmification process using an osmium tetroxide-ferrocyanide mixture. We describe a transilluminated chuck that enables visualization of immunostaining in specimens mounted on a trimming instrument, thus allowing for extremely precise sampling of the tissue.
