DISTINCT HETEROTRIMERIC GTP-BINDING-PROTEINS ACT IN SERIES TO CONTROLTHE EXOCYTOTIC MACHINERY IN CHROMAFFIN CELLS

Regulated exocytosis requires both calcium and MgATP. Although the bio chemical events responsible for ATP-dependent calcium-activated secret ion have not been elucidated yet, some progress has been made in deter mining the relative order of the ATP- and calcium-dependent steps. Stu dies on permeabilized secretory cells have shown that MgATP acts befor e calcium and maintains the secretory apparatus in a ''primed'' state. In this paper, we examine the possible role of heterotrimeric G-prote ins in these two steps of exocytosis in permeabilized chromaffin cells . We show that mastoparan and other activators of heterotrimeric G-pro teins inhibit the MgATP-dependent reaction, but stimulate the late cal cium-dependent step of exocytosis. Nonhydrolyzable GTP analogues (GTP- gamma-S and GMP-PNP) mimick the dual effects of mastoparan on secretio n, but with different potencies, suggesting the involvement of two dis tinct heterotrimeric G-proteins in regulated exocytosis. GPAnt-2, a su bstance P related peptide known to inhibit the stimulation of Gi and G o by mastoparan, reverses, in a dose-dependent manner, both the inhibi tory and stimulatory effects of mastoparan on secretion. These results indicate that two distinct heterotrimeric G-proteins from the Gi/o fa mily may act in series in the exocytotic pathway in chromaffin cells: one controls the ATP-dependent priming step, whereas the second is inv olved in the late calcium-dependent fusion step which does not require ATP.