Possible involvement of phosphatidylinositol 3-kinase in regulated exocytosis: studies in chromaffin cells with inhibitor LY294002.

Several lines of evidence suggest that phosphorylated products of phosphatidylinositol play critical functions in the regulation of membrane trafficking along the secretory pathway. To probe the possible involvement of phosphatidylinositol 3-kinase (PI 3-kinase) in regulated exocytosis, we have examined its subcellular distribution in cultured chromaffin cells by immunoreplica analysis and confocal immunofluorescence. We found that the PI 3-kinase heterodimer consisting of the regulatory and catalytic subunits was associated essentially with the subplasmalemmal cytoskeleton in both resting and nicotine-stimulated chromaffin cells. Attempts to immunoprecipitate PI 3-kinase with anti-phosphotyrosine antibodies failed, suggesting that the activity of PI 3-kinase was not modulated by tyrosine phosphorylation and/or physical interaction with SH2-containing proteins in stimulated chromaffin cells. LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a potent inhibitor of PI 3-kinase, produced a dose-dependent inhibition of catecholamine secretion evoked by various secretagogues. Furthermore, cytochemical experiments with rhodamine-labeled phalloidin revealed that LY294002 blocked the disassembly of cortical actin in chromaffin cells stimulated by a depolarizing concentration of potassium. Our results suggest that PI 3-kinase may be one of the important regulatory exocytotic components involved in the signaling cascade controlling actin rearrangements required for catecholamine secretion.

Exocytosis in single chromaffin cells: regulation by a secretory granule-associated Go protein.

1. Besides having a role in signal transduction, trimeric G proteins may also be involved in membrane trafficking events. In chromaffin cells, G alpha o has been found associated with the membrane of secretory granules. Here we examined the role of Go in regulated exocytosis using pressure microinjection combined with amperometric measurement of catecholamine secretion from individual chromaffin cells. 2. Microinjection of GTP gamma S and mastoparan strongly inhibits the amperometric response to either nicotine or high K+. 3. The presence of mastoparan in the cell incubation medium had no effect on K(+)-evoked secretion, suggesting that mastoparan blocks the exocytotic machinery through an intracellular target protein not located just beneath the plasma membrane. 4. Microinjection of anti-G alpha o antibodies potentiates by more than 50% the K(+)-evoked secretion, whereas anti-G alpha i1/2 antibodies have no effect. 5. Thus an inhibitory Go protein, probably associated with secretory granules, controls exocytosis in chromaffin cells. The intracellular proteins controlling organelle-associated G proteins are currently unknown. The neuronal cytosolic protein GAP-43 stimulates G alpha o in purified chromaffin granule membranes and inhibits exocytosis in permeabilized cells. We show here that microinjection of a synthetic peptide corresponding to the domain of GAP-43 that interacts with Go inhibits secretion. We suggest that GAP-43 or a related cytosolic protein controls the exocytotic priming step in chromaffin, cells by stimulating a granule-associated Go protein.

Regulated exocytosis in chromaffin cells. A potential role for a secretory granule-associated ARF6 protein.

The ADP-ribosylation factor (ARF) GTP-binding proteins are believed to function as regulators of vesicular budding and fusion along the secretory pathway. To investigate the role of ARF in regulated exocytosis, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation and immunoreplica analysis. We found that ARF6 is specifically associated with the membrane of purified secretory chromaffin granules. Chemical cross-linking and immunoprecipitation experiments suggested that ARF6 may be part of a complex with betagamma subunits of trimeric G proteins. Stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid dissociation of ARF6 from secretory granules. This effect could be inhibited by AlF4- which selectively activates trimeric G proteins. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 strongly inhibited calcium-evoked secretion in streptolysin-O-permeabilized chromaffin cells. The possibility that ARF6 plays a role in the effector pathway by which trimeric G proteins control exocytosis in chromaffin cells is discussed.

GAP-43 controls the availability of secretory chromaffin granules for regulated exocytosis by stimulating a granule-associated G0.

Besides having a role in signal transduction, heterotrimeric G proteins may also be involved in membrane trafficking events as suggested by their presence in specific intracellular compartments. In chromaffin cells, G alpha 0 is associated with secretory organelles, and its activation inhibits exocytosis. Although plasma membrane-bound G proteins are activated by cell-surface receptors, the intracellular proteins controlling organelle-associated G proteins are currently unknown. GAP-43, a neuronal protein enriched in axonal growth cones and presynaptic terminals, is one possible candidate since it can directly stimulate purified G0. We have investigated the interaction of adrenal medullary GAP-43 with chromaffin granule-associated G0 and its effect on catecholamine secretion. Cytosolic and depalmitoylated membrane-extracted GAP-43 were found to stimulate guanine nucleotide binding and exchange activity in chromaffin granule membranes. In permeabilized chromaffin cells, both forms of GAP-43 blocked calcium-dependent exocytosis, and this effect was inhibited by specific antibodies against G alpha 0. A synthetic peptide corresponding to the GAP-43 domain that interacts with G0 inhibited catecholamine secretion. This effect could be selectively reversed by the COOH-terminal peptide of G alpha 0. These results indicate that GAP-43 may be an endogenous pseudoreceptor for the secretory granule-bound form of G0 and can thereby control calcium-regulated exocytosis in chromaffin cells.

Exocytosis in chromaffin cells: evidence for a MgATP-independent step that requires a pertussis toxin-sensitive GTP-binding protein.

We have previously described that mastoparan, an amphiphilic tetradecapeptide that activates heterotrimeric G-proteins, inhibits Ca(2+)-induced MgATP-dependent secretion from streptolysin-O-permeabilized chromaffin cells [Vitale, Mukai, Rouot, Thiersé, Aunis and Bader (1993) J. Biol. Chem. 268, 14715-14723]. Our observations suggest the involvement of an inhibitory G(o)-protein, possibly located on the membrane of secretory granules, in the final stages of the exocytotic pathway in chromaffin cells. Here, we demonstrate that mastoparan is also able to stimulate the Ca(2+)-dependent secretion of catecholamines in the absence of MgATP in the medium. This MgATP-independent secretion is totally blocked by tetanus toxin, a potent inhibitor of exocytosis in all neurosecretory cells so far investigated, suggesting that the mastoparan target is a component of the exocytotic machinery. Mas17, a mastoparan analogue inactive on G-proteins, had no effect on catecholamine secretion whereas both Mas7, a highly active analogue of mastoparan, and AlF4-, which selectively activates trimeric G-proteins, triggered MgATP-independent secretion. Non-hydrolysable GTP analogues (GTP[S] and p[NH]ppG) mimicked the dual effects of mastoparan on secretion: they inhibited exocytosis in the presence of MgATP and stimulated MgATP-independent secretion. The different potencies displayed by these two analogues suggest the involvement of two distinct G-proteins. Accordingly, the mastoparan-induced MgATP-independent secretion is highly sensitive to pertussis toxin (PTX) whereas the inhibition by mastoparan of secretion in the presence of MgATP is resistant to PTX treatment. When permeabilized cells were incubated with mastoparan, the release of arachidonic acid increased in a PTX-sensitive manner. 7,7-Dimethyl-5,8-eicosadienoic acid, a potent inhibitor of intracellular phospholipase A2, inhibited both the arachidonate release and the MgATP-independent catecholamine secretion evoked by mastoparan. In contrast, neomycin, an inhibitor of phospholipase C, had no significant effect on either the release of arachidonic acid or the secretion of catecholamines provoked by mastoparan. We conclude that two distinct heterotrimeric G-proteins act in series in the exocytotic pathway in chromaffin cells: one controls an ATP-dependent priming step through an effector pathway that remains to be determined, and the second is involved in a late Ca(2+)-dependent step which does not require MgATP but possibly involves the generation of arachidonic acid.

Exocytosis in chromaffin cells. Possible involvement of the heterotrimeric GTP-binding protein G(o).

The use of non-hydrolyzable analogues of GTP in permeabilized secretory cells suggests that guanine nucleotide-binding regulatory proteins (G proteins) may be involved in regulated exocytosis. Because GTP analogues are known to modulate both monomeric low molecular mass G proteins and heterotrimeric G proteins, we have examined the effect of mastoparan, an activator of heterotrimeric G proteins, on secretion from intact and permeabilized chromaffin cells. In intact cells, mastoparan inhibited catecholamine secretion evoked by nicotine but had no effect on release induced by other secretagogues. In permeabilized cells, mastoparan inhibited calcium-dependent secretion providing that the pores created in the plasma membrane allow the penetration of the peptide into the cytoplasm. These results indicate that mastoparan blocks the exocytotic machinery through an intracellular target protein that may not be located just beneath the plasma membrane. Accordingly, mastoparan was able to stimulate G proteins associated with purified chromaffin granule membranes, in a range of concentration and Mg2+ requirement that was similar to its inhibitory effect on secretion. Mas 17, a mastoparan analogue inactive on purified G proteins, neither modified catecholamine secretion nor stimulated chromaffin granule G proteins. The substance P-related peptide, GPAnt-2, known to antagonize the effects of mastoparan on G(o), blocked both the inhibitory effect of mastoparan on secretion and the mastoparan-stimulated GTPase activity in chromaffin granule membranes. Moreover, specific antibodies raised against the carboxyl terminus of G(o) alpha reversed in a dose-dependent manner the inhibition by mastoparan on catecholamine release and the stimulation by mastoparan of chromaffin granule-associated G proteins. These results suggest that the secretory machinery in chromaffin cells can be blocked by activating a G(o) protein. Consistent with this finding, two other known activators of heterotrimeric G proteins, aluminum fluoride and benzalkonium chloride, inhibited calcium-evoked catecholamine secretion in streptolysin O-permeabilized chromaffin cells. We conclude that an inhibitory G(o) protein, possibly located on the membrane of secretory granules, is involved in the final stages of exocytosis in chromaffin cells.

Voltage-gated Ca entry in isolated bovine capillary endothelial cells: evidence of a new type of BAY K 8644-sensitive channel.

Isolated bovine capillary endothelial cells have been examined for voltage-dependent Ca entry. All cells displayed a low threshold activity, with the main characteristics of a T-type transient current, when examined using whole-cell recording for activation and inactivation and cell-attached conditions or inside-out patches for the elementary conductance (8 pS). 25% of the cells displayed an additional sustained current in 5 mM CaCl2 above -40 mV, which was enhanced by application of BAY K 8644, but almost insensitive to superfusion with nicardipine. Two types of channels (2.8 and 21 pS, in 110 mM BaCl2) were shown to have a BAY K 8644 sensitivity. The large conductance channels were L-type channels. The smaller events were elicited at more hyperpolarized potentials (by some 30 mV). Their mean open time was 16 ms in control conditions. In presence of BAY K 8644, additional long open times were observed (up to 100 ms as compared to 7.8 ms for the time constants of the slow mode of the L-type channel). We refer to these channels as SB channels: of small conductance and sensitive to BAY K 8644. In the presence of nicardipine, SB channels are not noticeably modified, in contrast to the L-type openings which are abolished. Also, SB open times are close to control values when nicardipine is added after a BAY K 8644 application. We suggest that, at physiological concentrations of divalent ions, an SB-type activity is elicited above -40 mV which generates the low threshold sustained current.

A pertussis-toxin-sensitive protein controls exocytosis in chromaffin cells at a step distal to the generation of second messengers.

The role of GTP-binding proteins (G-proteins) in the secretory process in chromaffin cells was investigated by studying the effects of pertussis toxin (PTX) on catecholamine release and generation of various second messengers. PTX was found to stimulate the catecholamine secretion induced by nicotine, 59 mM-K+ or veratridine. PTX also potentiated Ca2(+)-evoked catecholamine release from permeabilized chromaffin cells, suggesting that PTX substrate(s) regulate the exocytotic machinery at a step distal to the rise in intracellular Ca2+. We have investigated the possible intracellular pathways involved in the stimulation of secretion by PTX. PTX did not modify the translocation of protein kinase C (PKC) to membranes in intact or permeabilized cells; in addition, neither inhibitors nor activators of PKC had any effect on catecholamine release induced by PTX. Thus it seems unlikely that the effect of PTX on secretion is mediated by activation of PKC. The effect of PTX is also cyclic AMP-independent, as PTX did not change cytoplasmic cyclic AMP levels. The relationship between PTX treatment and arachidonic acid release was also examined. We found that an increase in cytoplasmic arachidonic acid concentration enhanced Ca2(+)-evoked catecholamine release in permeabilized cells, but arachidonic acid did not mimic the effect of PTX on the Ca2(+)-dose-response curve for secretion. Furthermore, PTX did not significantly modify the release of arachidonic acid measured in resting or stimulated chromaffin cells, suggesting that the stimulatory effect of PTX on secretion is not mediated by an activation of phospholipase A2. Taken together, these results suggest that PTX may modulate the intracellular machinery of secretion at a step distal to the generation of second messengers. In alpha-toxin-permeabilized cells, full retention of the PTX-induced activation of secretion was observed even 30 min after permeabilization. In contrast, when chromaffin cells were permeabilized with streptolysin-O (SLO), there was a marked progressive loss of the PTX effect. We found that SLO caused the rapid leakage of three G-protein alpha-subunits which are specifically ADP-ribosylated by PTX. We propose that a PTX-sensitive G-protein may play an inhibitory role in the final stages of the Ca2(+)-evoked secretory process in chromaffin cells.

A reassessment of guanine nucleotide effects on catecholamine secretion from permeabilized adrenal chromaffin cells.

The role of guanine nucleotides in catecholamine secretion was investigated in alpha-toxin-permeabilized chromaffin cells. The stable GTP analogues, GTP-gamma-S (guanosine 5'-(gamma-thio)triphosphate) and GMP-PNP (guanosine 5'-(beta,gamma-imido)triphosphate), potentiated calcium-evoked catecholamine release in a dose-dependent manner. This effect was reversed by GDP-beta-S (guanosine 5'-(beta-thio)diphosphate) indicating that a GTP-binding protein plays a modulatory role in the calcium-dependent secretory process in chromaffin cells. Calcium and the phosphorylating nucleotide ATP were both necessary for secretion, even in the presence of GTP analogues, suggesting that the activation of a GTP-regulatory protein alone does not trigger exocytosis in these cells. TPA (12-O-tetradecanoylphorbol-13-acetate), a direct activator of protein kinase C, was found to mimic the effects of the GTP analogues, inducing a dose-dependent potentiation of the calcium-evoked release in alpha-toxin-permeabilized cells. Treatment of the permeabilized cells with sphingosine, a potent inhibitor of protein kinase C, completely abolished the stimulatory effects of both TPA and GTP-gamma-S. Moreover, long term incubation of chromaffin cells with TPA, a treatment which depletes cells of protein kinase C activity, suppressed the stimulatory effects of GTP-gamma-S. Protein kinase C is activated when it becomes membrane-bound in the presence of calcium and diacylglycerol; here, GTP-gamma-S was found to enhance the calcium-induced translocation of protein kinase C to membranes in alpha-toxin-permeabilized cells. These results suggest that guanine nucleotides modulate secretion by activating protein kinase C-linked events in chromaffin cells. Furthermore, the potentiation of calcium-induced secretion in alpha-toxin-permeabilized cells following activation of protein kinase C either directly with TPA or indirectly with GTP analogues provides additional support for the concept that protein kinase C may exert a positive control directly on the intracellular exocytotic machinery.

Characterization of hormone and protein release from alpha-toxin-permeabilized chromaffin cells in primary culture.

Addition of Staphylococcus aureus alpha-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular 86Rb+. The alpha-toxin does not provoke a spontaneous release of either 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 Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In alpha-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+-dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from alpha-toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus alpha-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact.

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.

Evidence for a spectrin-like protein as a major component of the synaptosomal membrane cytoskeleton.

After treatment of synaptosomes with Nonidet-containing buffers, a proportion of the proteins remained insoluble. The major component (50%) of the residue was identified as a spectrin-like protein by immunodetection after mono- and bi-dimensional gel electrophoresis and transfer to nitrocellulose paper. Actin was also present.

Immunocytochemical study of microtubules in chromaffin cells in culture and evidence that tubulin is not an integral protein of the chromaffin granule membrane.

Bovine adrenal chromaffin cells were maintained in culture in Dulbecco's modified Eagle's medium containing 20% foetal calf serum and 10 units per ml of Nerve Growth Factor. Under these conditions, chromaffin cells developed up to five neurites per cell. The neurites showed lateral branches and varicosities along their trunk which ended with thick growth cone-like structures. Cultures of chromaffin cells were stained by indirect immunofluorescence with antibodies against (a) chromogranin A to follow the distribution of chromaffin granules, the catecholamine-storing organelles, and (b) tubulin, to study the microtubular system during outgrowth of neurites. Chromogranin A antibodies showed a very intensely staining punctate pattern, not randomly distributed but localized in neurites. Chromaffin granules were found to migrate from the cell body to reach neurite endings where they were densely packed. Intense staining was also observed in varicosities; a linear arrangement of granules was evident along neurite trunks. Tubulin antibodies decorated a complex network, clearly visible at the cell periphery and also in the growth cone-like structures, in the palm region of the growth cone. Colchicine treatment effected retraction of neurites and disappearance of organized microtubule networks; chromaffin granules were found in the perinuclear region of the cell. Some tubulin (0.2% of total membrane proteins) was found in the purified chromaffin granule membrane preparation; however, this tubulin is probably associated with contaminating plasma membranes. By the criteria of morphology and staining with antitubulin antibodies, adult bovine chromaffin cells in culture display characteristics similar to those of sympathetic neurones. In addition, they showed an exaggerated transport of granules. Adult bovine chromaffin cells in culture offer an excellent model for studying the role of microtubules and the contractile apparatus in relation to cell morphological changes and neurosecretion.

Immunofluorescent localization of microfilaments in neuronal and glial primary cell cultures with antibody against adrenal medullary myosin.

The localization of myosin was studied in rat neuronal and glial cell maintained in primary culture, using the double antibody immunofluorescent method. Antibodies were raised against myosin purified from bovine adrenal medulla. Myosin-specific immunoreactivity was found in the cell body and neurites of neuronal cells and in the cytoplasm of glial cells. In the former no typical substructure was observable, whilst in the latter myosin-rich filaments were found forming either a cage entrapping the nucleus or as long cables in cellular morphogenic expansions.