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

Abstract

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.

Role of heterotrimeric GTP binding proteins in vesicular protein transport: indications for both classical and alternative G protein cycles

“…interaction with G proteins via the C-terminal region of G proteins) is reversed both by synthetic peptides that correspond to the C-terminus of G~_3 and by antibodies directed against this region [46]. In addition, the activation of a G protein in the calcium-dependent stage (presumably G~_3) with mastoparan is reversed by pertussis toxin treatment [47]. Finally, receptor-mimicking peptides, pre-activated Gi/Go proteins, as well as transient expression of active mutants of Gi/Go inhibited the calcium-dependent step of exocytosis [18].…”

“…In contrast, the G protein involved in the ATPdependent priming step is likely subject to a non-classical mechanism of activation. Activation of this G protein by mastoparan results in inhibition of secretion which can not be reversed by PTX [47]. The inhibition is reversed, however, by antibodies directed against the C-terminus of Go~ [48].…”

Identification of a Potential Effector Pathway for the Trimeric Go Protein Associated with Secretory Granules

“…By using permeabilized cell models, it has been possible to establish that exocytosis consists of sequential ATP-dependent and ATP-independent phases of release (7,43,44). The ATP-independent phase has been attributed to the fusion of a small number of docked granules (45,46), whereas the ATP-dependent component has been assumed to include docking of granules and priming of the exocytotic machinery (43).…”

Trimeric G Proteins Control Exocytosis in Chromaffin Cells

“…Thus, the inactivation of Rho by C3 transferase prevented mastoparan from stabilizing the actin cytoskeleton and inhibiting secretion, indicating that Rho might be involved in the pathway by which the granule-associated G o protein controls the organization of the cortical cytoskeleton in chromaffin cells. DISCUSSION We previously investigated the function(s) of trimeric G proteins in regulated exocytosis in chromaffin cells using mastoparan (6,9,10,40). Mastoparan is a peptide from wasp venom that stimulates the GTPase of G i and G o proteins by a mechanism that is virtually identical with that of agonist-bound receptors (34,38).…”

“…Accordingly, the participation of a plasma membrane-bound G i3 protein in the late stages of exocytosis in mast cells has been demonstrated (7). Direct control of exocytosis by G i and G o proteins has also been described in insulin-secreting cells (8) and in chromaffin cells (6,9,10). Thus regulated exocytosis may represent a possible effector pathway for trimeric G proteins, although the mechanism by which this class of G proteins relates to the exocytotic machinery remains to be elucidated.…”