The Rho guanine nucleotide exchange factors Intersectin 1L and ß-Pix control calcium-regulated exocytosis in neuroendocrine PC12 cells.

GTPases of the Rho family are molecular switches that play an important role in a wide range of membrane-trafficking processes including neurotransmission and hormone release. We have previously demonstrated that RhoA and Cdc42 regulate calcium-dependent exocytosis in chromaffin cells by controlling actin dynamics, whereas Rac1 regulates lipid organisation. These findings raised the question of the upstream mechanism activating these GTPases during exocytosis. The guanine nucleotide exchange factors (GEFs) that catalyse the exchange of GDP for GTP are crucial elements regulating Rho signalling. Using an RNA interference approach, we have recently demonstrated that the GEFs Intersectin-1L and ß-Pix, play essential roles in neuroendocrine exocytosis by controlling the activity of Cdc42 and Rac1, respectively. This review summarizes these results and discusses the functional importance of Rho GEFs in the exocytotic machinery in neuroendocrine cells.

The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C.

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.

Secretory cell actin-binding proteins: identification of a gelsolin-like protein in chromaffin cells.

Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory process. Actin and Ca++-sensitive actin-binding proteins were purified from bovine adrenal medulla on affinity columns using DNase-I as a ligand. Buffers that contained decreasing Ca++ concentrations were used to elute three major proteins of 93, 91, and 85 kD. The bulk of the actin was eluted with guanidine-HCl buffer plus some 93- and 91-kD proteins. These Ca++-sensitive regulatory proteins were shown to inhibit the gelation of actin using the low-shear falling ball viscometer and by electron microscopy. Actin filaments were found to be shortened by fragmentation. Using antibody raised against rabbit lung macrophage gelsolin, proteolytic digestion with Staphylococcus V8 protease and two-dimensional gel electrophoresis, the 91-kD actin-binding protein was shown to be a gelsolin-like protein. The 93-kD actin-binding protein also showed cross-reactivity with anti-gelsolin antibody, similar peptide maps, and a basic-shift in pHi indicating that this 93-kD protein is a brevin-like protein, derived from blood present abundantly in adrenal medulla. Purification from isolated chromaffin cells demonstrated the presence of 91- and 85-kD proteins, whereas the 93-kD protein was hardly detectable. The 85-kD protein is not a breakdown product of brevin-like or gelsolin-like proteins. It did not cross-react with anti-gelsolin antibody and showed a very different peptide map after mild digestion with V8 protease. Antibodies were raised against the 93- and 91-kD actin-binding proteins and the 85-kD actin-binding protein. Antibody against the 85-kD protein did not cross-react with 93- and 91-kD proteins and vice versa. In vivo, the cytoskeleton organization of chromaffin secretory cells is not known, but appears to be under the control of the intracellular concentration of free calcium. The ability of calcium to activate the gelsolin-like protein, and as shown elsewhere to alter fodrin localization, provides a mechanism for gel-sol transition that might be essential for granule movement and membrane-membrane interactions involved in the secretory process.

Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells.

Annexin II is a Ca(2+)-dependent membrane-binding protein present in a wide variety of cells and tissues. Within cells, annexin II is found either as a 36-kD monomer (p36) or as a heterotetrameric complex (p90) coupled with the S-100-related protein, p11. Annexin II has been suggested to be involved in exocytosis as it can restore the secretory responsiveness of permeabilized chromaffin cells. By quantitative confocal immunofluorescence, immunoreplica analysis and immunoprecipitation, we show here the translocation of p36 from the cytosol to a subplasmalemmal Triton X-100 insoluble fraction in chromaffin cells following nicotinic stimulation. A synthetic peptide corresponding to the NH2-terminal domain of p36 which contains the phosphorylation sites was microinjected into individual chromaffin cells and catecholamine secretion was monitored by amperometry. This peptide blocked completely the nicotine-induced recruitment of p36 to the cell periphery and strongly inhibited exocytosis evoked by either nicotine or high K+. The light chain of annexin II, p11, was selectively expressed by adrenergic chromaffin cells, and was only present in the subplasmalemmal Triton X-100 insoluble protein fraction of both resting and stimulated cells. p11 can modify the Ca(2+)- and/or the phospholipid-binding properties of p36. We found that loss Ca2+ was required to stimulate the translocation of p36 and to trigger exocytosis in adrenergic chromaffin cells. Our findings suggest that the translocation of p36 to the subplasmalemmal region is an essential event in regulated exocytosis and support the idea that the presence of p11 in adrenergic cells may confer a higher Ca2+ affinity to the exocytotic pathway in these cells.

Influence of hypertonic solutions on catecholamine release from intact and permeabilized cultured chromaffin cells.

Chromaffin cells purified from bovine adrenal medulla and maintained in primary culture were used to study the effects of hyperosmolarity on the nicotine- and high potassium-induced secretory response. A similar study was also performed on cells permeabilized with digitonin and with alpha-toxin from Staphylococcus aureus. Hyperosmolarity does not affect the spontaneous release of catecholamines from either intact cells or permeabilized cells. The nicotine-induced secretion and high potassium-induced secretion from intact cells are inhibited by hypertonic solutions; a 100% inhibition of net release was observed at 660 mOsm (sucrose as osmotic agent). Veratridine- and the cation ionophore X537-A-induced release were both depressed under hyperosmotic conditions. Hyperosmolarity was shown to have reversible effects on the secretory response of intact cells. Finally, hyperosmolarity has intracellular effects on catecholamine release evoked by calcium from both detergent- and alpha-toxin-permeabilized cells. Our data show that hyperosmolarity has multiple effects on the cell membrane and the protein constituents associated with it, but has also a significant effect on intracellular reactions concerned with exocytosis.

Proteolytic processing of chromogranin A in cultured chromaffin cells.

The prohormone chromogranin A is the major soluble component of secretory granules in chromaffin cells of adrenal medulla and in many other different endocrine cell types. The proteolytic processing of chromogranin A was studied in cultured bovine chromaffin cells using [35S]methionine to label proteins and a specific antibody to immunoprecipitate the native protein and its breakdown products. In resting cells, it was found that the degradation of chromogranin A is a slow process, since no degradation was observed after a 40 h incubation with radiolabelled methionine. Stimulation of cells with a single pulse or with successive pulses of nicotine did not significantly enhance the degree of proteolytic processing of chromogranin A. As it has recently been shown (Simon, J.P., Bader, M.F. and Aunis, D. Biochem. J. (1989) 260, 915-922) that protein kinase C may be involved in the regulation of chromogranin A synthesis, the possibility that prohormone processing may also be controlled by protein kinase C was examined using the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA). However, incubation of cells with TPA did not significantly modify chromogranin A processing, indicating that biosynthesis and proteolytic processing of chromogranin A are two distinctly regulated mechanisms. Glucocorticoids are known to exert regulatory control of chromaffin cell metabolism; however, incubation of cells with dexamethasone did not alter slow chromogranin A processing. Stimulation of labelled cells rapidly released newly synthesized chromogranin A into external medium. In addition, released chromogranin A was found to be actively processed into its 60 kDa and 43 kDa breakdown products. This extracellular proteolytic degradation mechanism may be of importance with regard to the function of chromogranin A as a prohormone.

Structural analysis of junctions formed between lipid membranes and several annexins by cryo-electron microscopy.

The (annexin II-p11)2 tetramer has been proposed to participate in exocytosis and several other members of the annexin superfamily have been reported to aggregate liposomes in vitro. In this context, the Ca2+-dependent binding of several annexins to chromaffin granules and liposomes was investigated by cryo-electron microscopy. The Ca2+-dependent aggregation of lipid membranes by (annexin II-p11)2 results from the spontaneous self-organization of the protein into two-dimensional plaques, which are visualized in projection as characteristic junctions. The junctions have a constant thickness of 210(+/-10) A and present a symmetrical distribution of electron-dense material arranged into seven stripes. They were observed over a wide range of Ca2+ concentrations, down to 2 microM. The molecular components corresponding to the seven electron-dense stripes were assigned as follows: the two associated membranes give rise to two outer stripes each and the three central stripes correspond to the (annexin II-p11)2 tetramer. Each annexin II molecule interacts with the outer lipid leaflet of one membrane, giving rise to one stripe, while the central stripe is due to the (p11)2 dimer with which both annexin II molecules interact. Both annexin II and annexin I also induced the Ca2+-dependent aggregation of liposomes via junctions that lack the central (p11)2 moiety and present only six high-density stripes. As expected, both annexin V and annexin III bind to liposomes without inducing their aggregation.

Peripheral actin filaments control calcium-mediated catecholamine release from streptolysin-O-permeabilized chromaffin cells.

Adrenal medullary chromaffin cells were permeabilized by treatment with a streptococcal cytotoxin streptolysin O (SLO) which generates pores of macromolecular dimensions in the plasma membrane. SLO did not provoke spontaneous release of catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However, the addition of micromolar free calcium concentration induced the corelease of noradrenaline and chromogranin A, indicating that secretory products are liberated by exocytosis. Calcium-dependent exocytosis from SLO-permeabilized cells required Mg-ATP and could not occur in the presence of other nucleotides. The pores generated by the toxin were large enough to introduce proteins, e.g., immunoglobulins, but also caused efflux of the cytosolic marker lactate dehydrogenase. Despite this, the cells remained responsive to calcium for up to 30 min after permeabilization, indicating that they retained their secretory machinery. In the search for a functional role of cytoskeletal proteins in the secretory process, we used SLO-permeabilized cells to examine the localization of filamentous actin, using rhodamine-phalloidin, and that of the actin-severing protein, gelsolin, using specific antibodies. It was found that both F-actin and gelsolin were exclusively localized in the subplasmalemmal region of the cell. We examined the relationship between actin disassembly, the elevation of intracellular calcium and secretion in SLO-treated cells. F-Actin destabilizing agents such as cytochalasin D or DNase I were found to potentiate calcium-stimulated release. The maximal effect was observed at low calcium concentrations (1-4 microM) and at the later stages of the secretory response (after 10 min stimulation). In addition, using rhodamine-phalloidin, we observed that calcium provoked simultaneously both cortical actin disassembly and catecholamine release in SLO-permeabilized cells. These results demonstrate that a close relationship exists between the secretory response and actin disassembly and provide further evidence that intracellular calcium controls the subplasmalemmal cytoskeletal actin organization and thereby the access of secretory granules to exocytotic sites.

Regulation of exocytosis in chromaffin cells by phosducin-like protein, a protein interacting with G protein betagamma subunits.

Phosducin and related proteins have been identified as ubiquitous regulators of signalling mediated by betagamma subunits of trimeric G proteins. To explore a role for phosducin in regulated exocytosis, we have examined the distribution and putative function of phosducin-like protein (PhLP) in adrenal medullary chromaffin cells. The full-length cDNA encoding the short splice variant of PhLP (PhLPs) was cloned from cultured chromaffin cells. Native PhLPs was found associated with plasma membranes and detected in the subplasmalemmal area of resting chromaffin cells by confocal immunofluorescence analysis. Stimulation with secretagogues triggered a massive redistribution of PhLPs into the cytoplasm. When microinjected into individual chromaffin cells, recombinant PhLPs inhibited catecholamine secretion evoked by a depolarizing concentration of K+ without affecting calcium mobilization. Thus, PhLPs may participate directly in the regulation of calcium-evoked exocytosis.

Insight in the exocytotic process in chromaffin cells: regulation by trimeric and monomeric G proteins.

Catecholamine secretion from chromaffin cells has been used for a long time as a general model to study exocytosis of large dense core secretory granules. Permeabilization and microinjection techniques have brought the possibility to dissect at the molecular level the multi-protein machinery involved in this complex physiological process. Regulated exocytosis comprises distinct and sequential steps including the priming of secretory granules, the formation of a docking complex between granules and the plasma membrane and the subsequent fusion of the granule with the plasma membrane. Key proteins involved in the exocytotic machinery have been identified. For instance, SNAREs which participate in the docking events in most intracellular transport steps along the secretory pathway, play a role in exocytosis in both neuronal and endocrine cells. However, in contrast to intracellular transport processes for which the highest fusion efficiency is required after correct targeting of the vesicles, the number of exocytotic events in activated secretory cells needs to be tightly controlled. We describe here the multistep control exerted by heterotrimeric and monomeric G proteins on the progression of secretory granules from docking to fusion and the molecular nature of some of their downstream effectors in neuroendocrine chromaffin cells.

Morphology and secretory activity of digitonin- and alpha-toxin-permeabilized chromaffin cells.

An ultrastructural examination of cultured bovine chromaffin cells permeabilized with Staphylococcus aureus alpha-toxin or digitonin revealed differences in the preservation of cell morphology. The toxin-treated cells closely resembled control cultured cells whereas digitonin-treated cells showed gradations in cytoplasmic densities suggesting extraction, some swelling of the endoplasmic reticulum and, occasionally, discontinuities in the plasma membrane and free granules in the extracellular medium. In both cell models, there was a swelling of the mitochondria. Horseradish peroxidase labelling of permeabilized cells marked the cytoplasm of digitonin-treated cells but only the surface of toxin-treated cells, demonstrating that larger lesions were caused by digitonin. In stimulated cells, the decrease in volumetric density of chromaffin granules correlated well with catecholamine release. The sites of secretory activity could be demonstrated in toxin-treated cells using horseradish peroxidase as a surface marker. Although both cell systems secrete catecholamines in response to calcium stimulation, their calcium requirements and the kinetics of release were different. In alpha-toxin-treated cells, 100 microM free calcium induced maximal catecholamine release. In digitonin-treated cells, 20 microM evoked maximal release but secretion was blocked at 100 microM. Catecholamine release terminated in digitonin-treated cells within 10 min but continued in alpha-toxin-treated cells for at least 60 min. In addition, the maximal release observed in toxin-treated cells (50%) was always greater than that observed in digitonin-permeabilized cells (20%). The results suggest that both exocytosis and granule translocation are operational in alpha-toxin-treated cells, but that the translocation step or the docking of granules at the plasma membrane may be impaired in digitonin-treated cells.

Chromogranin A triggers a phenotypic transformation and the generation of nitric oxide in brain microglial cells.

Chromogranin A is an ubiquitous 48,000 mol. wt secretory protein stored and released from many neuroendocrine cells and neurons. In human brain, chromogranin A is a common feature of regions that are known to be affected by various neurodegenerative pathologies such as Alzheimer's, Parkinson's and Pick's diseases. Brain degenerative areas are generally infiltrated by activated microglial cells, the resident macrophage cell population within the central nervous system. Here, we report that both recombinant human chromogranin A and chromogranin A purified from bovine chromaffin granules trigger drastic morphological changes in rat microglial cells maintained in culture. Microglial cells exposed to chromogranin A adopted a flattened amoeboid shape and, this change was associated with an accumulation of actin in the subplasmalemmal region, as observed by immunocytochemistry and confocal laser microscopy. In single microglial cells loaded with indo-1, chromogranin A elicited a rapid and transient increase in [Ca2+]i which preceded the reorganization of actin cytoskeleton. The activity of nitric oxide synthase was estimated by measuring the accumulation of nitrite in the culture medium. Both recombinant human chromogranin A and bovine chromogranin A triggered an important accumulation of nitrite comparable to that induced by lipopolysaccharide, a well-known activator of microglia. The effect of chromogranin A was dose dependent, inhibited by N omega-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase, and by cycloheximide, an inhibitor of protein synthesis. These findings suggest that chromogranin A induces an activated phenotype of microglia, and thus may have a role in the pathogenesis of neuronal degeneration in the brain.

Chromogranin A in the pancreatic islet: cellular and subcellular distribution.

Chromogranin A (CGA) is the major soluble protein within secretory vesicles of chromaffin cells. A polyclonal antiserum was raised against bovine CGA and characterized in two-dimensional immunoblots. Cellular and subcellular distribution of CGA in bovine pancreatic islet was investigated by immunocytochemistry. At the light microscopic level, CGA-like immunoreactivity was found in the same cells that react with antibodies against insulin, glucagon, and somatostatin. A minority of cells containing pancreatic polypeptide also showed faint immunostaining. At the ultrastructural level (protein A-gold technique), CGA-like immunoreactivity was confined exclusively to the secretory vesicles. Whereas the hormones were localized mainly in the central part of the secretory vesicles, CGA was present predominantly in the periphery. These findings indicate that a CGA-like protein is a regular constituent of the matrix of secretory vesicles in pancreatic endocrine cells.

Regulation of chromogranin a and chromogranin B (secretogranin I) synthesis in bovine cultured chromaffin cells.

Abstract In the present study we investigated the regulation of Chromogranin A (CGA) and Chromogranin B (CGB) biosynthesis in bovine chromaffin cells maintained in primary culture. Cellular proteins were labelled with [(35)S]methionine and the incorporated radioactivity was used as an index of the synthesis rate. The radioactivity incorporated into CGA was determined by immunoprecipitation, and that into CGB was quantified by a dot immunobinding assay using specific antibodies. Incubation of cells with carbamylcholine, nigh K(+) or histamine, three potent stimulators of catecholamine secretion in chromaffin cells, increased the rate of CGA and CGB synthesis. On the other hand bradykinin, angiotensin II and prostaglandin E(2), which cause little secretion, also produced an increase in both CGA and CGB synthesis. These results suggest that in chromaffin cells, the biosynthesis of chromogranins is not closely linked to the secretory activity. Inhibition of protein kinase C by sphingosine or by long-term treatment with phorbol esters, completely abolished the synthesis of CGA and CGB induced by carbamylcholine, bradykinin and prostaglandin E(2) but decreased only partially the stimulating effect of histamine. Thus, protein kinase C may not be the sole effector involved in the secretagogue-induced modulation of Chromogranin synthesis. Forskolin, an activator of adenylate cyclase had no effect on CGA synthesis, but significantly enhanced the incorporation of radioactivity into CGB. The effect of forskolin was not modified by protein kinase C inhibitors and was additive to that induced by phorbol esters indicating that cyclic AMP did not stimulate CGB synthesis through a protein kinase C-dependent pathway. These observations suggest that the biosynthesis of CGA and CGB in chromaffin granules is independently regulated.

Secretogranin II: regulation of synthesis and post-translational proteolysis in bovine adrenal chromaffin cells.

Secretogranin II (SgII), also called chromogranin C, is an acidic tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurones. Although less abundant than chromogranin A (CGA) and chromogranin B (CGB), SgII is found in adrenal medullary chromaffin granules. In the present study we investigated the regulation of SgII biosynthesis in bovine chromaffin cells maintained in primary culture. Cellular proteins were labelled with [35S]methionine and the heat stable chromogranin enriched fraction was isolated. Following electrophoretic separation, the 86 kDa SgII band was identified by sequence analysis using the Edman degradation procedure. The radioactivity incorporated in the 86 kDa SgII band was used as an index of the SgII synthesis rate. We found that stimulation of chromaffin cells with nicotine and histamine and to a smaller extent with angiotensin II and bradykinin significantly enhanced the rate of SgII synthesis. In contrast direct depolarization with K+ may not be sufficient to induce modifications in SgII synthesis suggesting that the raise of cytosolic calcium evoked by high K+ may not be sufficient to induce modifications in SgII synthesis . The possible second messenger pathways involved in the control of SgII biosynthesis were investigated by using protein kinase C and adenylate cyclase activators. We observed that 12-O-tetradecanoylphorbol 13-acetate (TPA) and forskolin increased the basal rate of SgII synthesis. Incubation with both TPA and forskolin was required to obtain an effect comparable to that produced by nicotine or histamine suggesting that these secretagogues recruit both protein kinase C- and cyclic AMP-dependent mechanisms to stimulate SgII synthesis.

Regulation of enkephalin, VIP, and chromogranin biosynthesis in actively secreting chromaffin cells. Multiple strategies for multiple peptides.

Enkephalins, vasoactive intestinal polypeptide, and chromogranin A are all contained in the secretory vesicles of chromaffin cells in culture, and are all released from this compartment by secretagogues in a calcium-dependent way. The biosynthesis of each of these peptides, however, is under quite independent regulation. The synthesis and secretion of enkephalin is tightly coupled to acetylcholine and elevated potassium stimulation by calcium influx. Once calcium enters the cell, calcium acts at pharmacologically distinct sites to elicit secretion and enhanced biosynthesis of Metenkephalin. This is demonstrated by the calcium-independent stimulation of enkephalin secretion by 1 mM barium, in contrast to the dependence on extracellular calcium of barium-stimulated biosynthesis of this peptide. The synthesis and secretion of VIP is also coupled to acetylcholine and elevated potassium stimulation by calcium influx. Treatment with barium demonstrates that calcium acts at distinct sites to stimulate secretion and biosynthesis of this peptide; however induction of VIP by barium and veratridine shows greater sensitivity to the calcium channel blocker methoxyverapamil (D600) than does the induction of Met-enkephalin by these agents. These differences in D600 sensitivity may be due to differences in calcium metabolism or voltage-dependent calcium channels in enkephalin-producing and VIP-inducible subpopulations of chromaffin cells. Chromogranin A levels are essentially unaffected by any of the agents which increase enkephalin and VIP levels, although it is secreted in parallel with enkephalins and catecholamines from chromaffin cells in response to secretagogues. We suggest that peptide hormones such as VIP and enkephalins are regulated by calcium-dependent stimulus-secretion-synthesis coupling in the chromaffin cell. Cyclic AMP is a positive regulator of enkephalin and VIP biosynthesis, but does not affect acute release of these peptides. The cAMP/protein kinase A system may be a distal mediator of peptide biosynthesis stimulated by secretagogues. Alternatively, cAMP may be involved in early developmental establishment of phenotype or long-term regulation of peptide biosynthesis by other hormones or neurotransmitters. Chromogranin A may represent a class of intravesicular, soluble proteins that are expressed constitutively by the chromaffin cell in the presence or absence of positive regulators of other systems. The biosynthesis of chromogranin A may be coupled to the production or assembly of the secretory vesicle itself.

Recombinant human chromogranin A: expression, purification and characterization of the N-terminal derived peptides.

Chromogranin A (CGA) is an ubiquitous 48 kDa secretory protein stored and released from most endocrine cells and is present in nanomolar concentration in the human vascular system. Recent data suggest that CGA may be the precursor of several peptides with a defined biological activity. The present report describes the expression of human CGA in Escherichia coli using the pET3a vector system, the purification and characterization of the recombinant protein and the production of antibody against the expressed protein. The expressed CGA was purified by a multi-step protocol including heat treatment, gel filtration and high performance-anion exchange chromatography and two-dimensional gel electrophoresis. Two major forms of recombinant human CGA (rhCGA) were purified from the bacterial cytosol: a 70 kDa form which corresponded to the native full-length CGA and a major proteolytic 63 kDa product recognized by antibodies raised against the 70 kDa rhCGA or to synthetic peptides localized in the N-terminal part of the bovine CGA sequence. This E. coli expression system provides a method for producing a suitable protein which will permit the identification of CGA-derived peptides with defined biological function in human. Fragments containing the N-terminal domain were generated by acidic cleavage of the two forms of rhCGA. A two-step purification using high-performance reverse-phase chromatography yielded 6 peptide bands ranging in apparent molecular mass from 7 to 18 kDa. Four components (molecular mass range 12-18 kDa) were immunostained with antibodies directed against synthetic sequences of bovine vasostatin II (bCGA1-113) while the two others (molecular mass range 7-8 kDa) were immunostained only with antibodies directed against vasostatin I (bCGA1-76). From protein staining the ratio vasostatins II/I was 10:1. The vasoinhibitory activity of this preparation was examined on isolated human saphenous vein segments. An inhibitory effect was obtained in paired vessel segments from 7 patients undergoing surgery for coronary artery bypass, however with low potency for supression of the endothelin-1 evoked sustained tension in these vessels.

Quantitative evaluation of the properties of a pyridazinyl GABA derivative (SR 95531) as a GABAA competitive antagonist. An electrophysiological approach.

We have investigated the effects of an aryl-aminopyridazine derivative of GABA (SR 95531) on dose-response curves of GABA-induced depolarizations from dorsal root ganglion neurones recorded intracellularly. The reversible shift to the right of the dose-response curves in a parallel fashion and the dissociation constant (KB) value of 0.13 +/- 0.02 microM (n = 15) indicate that this compound is a potent competitive GABAA antagonist. The competitive nature of SR 95531-induced antagonism was confirmed by single channel analysis. In excised membrane patches from bovine chromaffin cells (outside out configuration), 0.2-0.5 microM SR 95531 did not alter the mean open time of GABA-activated channels and did not introduce further short closing gaps within bursts. Whole cell recordings from cultured nodose ganglion neurones indicated that SR 95531 (10 microM) did not modify significantly any of the 3 types of calcium currents already reported in sensory neurones. This result might be of importance for further studies of presynaptic GABA actions on transmitter release.