MASTOPARAN STIMULATES EXOCYTOSIS AT A CA2-INDEPENDENT LATE SITE IN STIMULUS-SECRETION COUPLING - STUDIES WITH THE RINM5F BETA-CELL LINE()

Mastoparan, a tetradecapeptide from wasp venom, stimulated exocytosis in a concentration-dependent manner, which was enhanced by pertussis t oxin pretreatment, in the insulin secreting beta-cell line RINm5F. Mas toparan (3-20 muM) also elevated cytosolic free calcium concentration ([Ca2+]i), a rise that was not attenuated by nitrendipine. Divalent ca tion-free Krebs-Ringer bicarbonate (KRB) medium with 0.1 mm EGTA nulli fied the mastoparan-induced increase in [Ca2+]i, suggesting that the p eptide increased Ca2+ influx but not through the L-type voltage-depend ent Ca2+ channel. Depletion of the intracellular Ca2+ pool did not aff ect the mastoparan-induced elevation of [Ca2+]i. Remarkably, in divale nt cation-free KRB medium with 0.1 mM EGTA and 2 muM thapsigargin in w hich mastoparan reduced [Ca2+]i, the mastoparan-stimulated insulin rel ease was similar to that in normal Ca2+-containing KRB medium. Inhibit ors of protein kinase C, such as bisindolylmaleimide, staurosporine, a nd 1-O-hexadecyl-2-O-methyl-rac-glycerol did not suppress the mastopar an-stimulated insulin release. Mastoparan at 10-20 muM did not increas e cellular cAMP levels, nor did mastoparan at 5-10 muM affect [H-3]ara chidonic acid release. In conclusion, although mastoparan increased [C a2+]i, this increase was not involved in the stimulation of insulin re lease. Rather, the data suggest that mastoparan directly stimulates ex ocytosis in a Ca2+-independent manner. As GTP-binding proteins (G prot eins) are thought to be involved in the process of exocytosis and as m astoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimu latory G(e) (exocytosis) protein is likely.