RAPID ATP-DEPENDENT PRIMING OF SECRETORY GRANULES PRECEDES CA2-INDUCED EXOCYTOSIS IN MOUSE PANCREATIC B-CELLS()

1. The glucose and ATP dependence of exocytosis were investigated in s ingle mouse pancreatic B-cells by monitoring changes in cell capacitan ce evoked by voltage-clamp depolarizations, infusion of high-[Ca2+](i) buffers or photorelease of caged Ca2+ or ATP. 2. In intact B-cells, u sing the perforated patch whole-cell technique, glucose (5 mM) increas ed exocytotic responses evoked by membrane depolarization 5-fold over that observed in the absence of the sugar. Increasing the glucose conc entration to 20 mM produced a further doubling of exocytosis. The stim ulatory action of glucose was attributable to glucose metabolism and a bolished by mannoheptulose, an inhibitor of glucose phosphorylation. 3 . Exocytosis triggered by infusion of high [Ca2+](i) and ATP was reduc ed by 80% when ATP was replaced by its non-hydrolysable analogue adeno sine 5'-[beta,gamma-methylene]triphosphate (AMP-PCP) in standard whole -cell experiments. Exocytosis elicited by GTP gamma S was similarly af fected by replacement of ATP with the stable analogue. 4. Photoreleasi ng ATP in the presence of 170 nM [Ca2+](i), following the complete was h-out of endogenous ATP, produced a prompt (latency, <400 ms) and biph asic stimulation of exocytosis. 5. Elevation of [Ca2+](i) to exocytoti c levels by photorelease from Ca2+-nitrophenyl EGTA preloaded into the cell evoked a biphasic stimulation in the presence of Mg-ATP. The res ponse consisted of an initial rapid (completed in < 200 ms) phase foll owed by a slower (lasting greater than or equal to 10 s) sustained com ponent. Replacement of ATP with AMP-PCP abolished the late component b ut did not affect the initial phase. The latency between elevation of [Ca2+](i) and exocytosis was determined as < 45 ms. Inclusion of N-eth ylmaleimide (NEM; 0.5 mM for 3 min) in the intracellular solution exer ted effects similar to those obtained by substituting AMP-PCP for ATP. 6. We conclude that the B-cell contains a small pool (40 granules) of primed granules which are immediately available for release and which are capable of undergoing exocytosis in an ATP-independent fashion, W e propose that this pool of granules is preferentially released during first phase glucose-stimulated insulin secretion. The short latency b etween the application of ATP and the onset of exocytosis finally sugg ests that priming takes place with sufficient speed to participate in the rapid adjustment of the secretory capacity of the B-cell.