Mouse islets were used to test the hypothesis that the B cell membrane must be depolarized for acetylcholine to increase insulin release. The resting membrane potential of B cells (at 3 mM glucose) was slightly decreased (5 mV) by acetylcholine, but no electrical activity appeared. This depolarization was accompanied by a Ca-independent acceleration of 86Rb and 45Ca efflux but no insulin release. When the B cell membrane was depolarized by a stimulatory concentration of glucose (10 mM), acetylcholine potentiated electrical activity, accelerated 86Rb and 45Ca efflux, and increased insulin release. This latter effect, but not the acceleration of 45Ca efflux, was totally dependent on extracellular Ca. If glucose-induced depolarization of the B cell membrane was prevented by diazoxide, acetylcholine lost all effects but those produced at low glucose. In contrast, when the B cell membrane was depolarized by leucine or tolbutamide (at 3 mM glucose), acetylcholine triggered a further depolarization with appearance of electrical activity, accelerated 86Rb and 45Ca efflux, and stimulated insulin release. Acetylcholine produced similar effects (except for electrical activity) in the presence of high K or arginine which, unlike the above test agents, depolarize the B cell membrane by a mechanism other than a decrease in K+ permeability. Omission of extracellular Ca abolished the releasing effect of acetylcholine under all conditions but only partially decreased the stimulation of 45Ca efflux. The results show thus that acetylcholine stimulation of insulin release does not result from mobilization of cellular Ca but requires that the B cell membrane be sufficiently depolarized to reach the threshold potential where Ca channels are activated. This may explain why acetylcholine alone does not initiate release but becomes active in the presence of a variety of agents.
The possible role of an osmotic lysis of insulin granules during exocytosis has been studied in perifused mouse pancreatic islets. Raising the osmolarity of the extracellular medium by addition of 400 mM sucrose reversibly inhibited glucose-stimulated insulin release. This inhibition was accompanied by a decrease in the rates of 86Rb+ or 45Ca2+ efflux from the islets. Increasing the osmolarity and restoring a normotonic medium in the presence of a nonstimulatory concentration of glucose accelerated 86Rb+ and 45Ca2+ efflux and augmented basal insulin release in both the presence and absence of Ca2+. Hyperosmolarity did not prevent a rise in glucose concentration from decreasing 86Rb+ efflux from islet cells or from inhibiting 45Ca2+ efflux in Ca2+-free medium. However, the stimulation of 45Ca2+ efflux otherwise produced by glucose in the presence of Ca2+ was abolished, and the stimulation of insulin release was almost suppressed. Hyperosmolarity also strongly impaired the release of insulin during stimulation by eight experimental conditions known to act through at least partially different mechanisms. The changes in 45Ca2+ efflux brought about by these different agents were also altered by hyperosmolarity, whether they resulted from direct mobilization of intracellular Ca2+ or were secondary to increased Ca2+ influx. The blockade of insulin release by hyperosmolarity, whatever the mode of action of the stimulus, is compatible with the participation of osmotic events in exocytosis. However, the marked alterations in Ca2+ handling that occur concomitantly and might account for the inhibition of release make it impossible to demonstrate their exact role in intact islet cells.
The mechanism whereby nutrient insulin secretagogues decrease 4sCa 2+ efllux from islet cells is controversial. It was studied with mouse islets perifused with Caz+-free solutions. In the presence of Na+, glucose and ketoisocaproate inhibited 4sCa2+ efllux by about 50%. Substitution of choline+ salts for Na+ salts decreased the efllux rate by 45x, but did not prevent glucose from decreasing it further. Ketoisocaproate also inhibited 45Ca2+ etllux, but less markedly than in an Na+ medium. Omission of Na+ decreased the efllux rate even when it was already lowered by glucose or ketoisocaproate. It is thus clear that nutrient insulin secretagogues decrease 45CaZ+ efflux from islet cells by a mechanism other than the inhibition of the Na+-Caz+ countertransport, possibly by increasing sequestration of the ion in cellular organelles.
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