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 tetradeca peptide that activates heterotrimeric G-proteins, inhibits Ca2+-induce d MgATP-dependent secretion from streptolysin-O-permeabilized chromaff in cells [Vitale, Mukai, Rouot, Thierse, Aunis and Bader (1993) J. Bio l. Chem. 268, 14715-14723]. Our observations suggest the involvement o f an inhibitory G(o)-protein, possibly located on the membrane of secr etory granules, in the final stages of the exocytotic pathway in chrom affin cells. Here, we demonstrate that mastoparan is also able to stim ulate the Ca2+-dependent secretion of catecholamines in the absence of MgATP in the medium. This MgATP-independent secretion is totally bloc ked by tetanus toxin, a potent inhibitor of exocytosis in all neurosec retory cells so far investigated, suggesting that the mastoparan targe t is a component of the exocytotic machinery. Mas17, a mastoparan anal ogue inactive on G-proteins, had no effect on catecholamine secretion whereas both Mas7, a highly active analogue of mastoparan, and AIF(4)- , which selectively activates trimeric G-proteins, triggered MgATP-ind ependent secretion. Nonhydrolysable 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 su ggest the involvement of two distinct G-proteins. Accordingly, the mas toparan-induced MgATP-independent secretion is highly sensitive to per tussis toxin (PTX) whereas the inhibition by mastoparan of secretion i n the presence of MgATP is resistant to PTX treatment. When permeabili zed cells were incubated with mastoparan, the release of arachidonic a cid increased in a PTX-sensitive manner. 7,7-Dimethyl-5,8-eicosadienoi c acid, a potent inhibitor of intracellular phospholipase A(2), inhibi ted both the arachidonate release and the MgATP-independent catecholam ine secretion evoked by mastoparan. In contrast, neomycin, an inhibito r of phospholipase C, had no significant effect on either the release of arachidonic acid or the secretion of catecholamines provoked by mas toparan. We conclude that two distinct heterotrimeric G-proteins act i n series in the exocytotic pathway in chromaffin cells: one controls a n ATP-dependent priming step through an effector pathway that remains to be determined, and the second is involved in a late Ca2+-dependent step which does not require MgATP but possibly involves the generation of arachidonic acid.