Microelectrodes were used to record the effects of glucose on the membrane potential of single mouse B cells. In most cells, the slow waves of depolarization and the intervals of repolarization produced by a constant concentration of glucose displayed a great regularity. However, cyclic variations in the duration of these slow waves and/or intervals were observed in a certain number of B cells. These oscillations were more clearly visible and more frequent (47%) in the presence of 15 mM glucose, than in the presence of 10 mM glucose (19%). They sometimes disappeared with time, but sometimes persisted for over 90 min and were not affected by atropine, propanolol and phentolamine. Their mean period was 203 s at 10 mM glucose and 235 s at 15 mM glucose. The membrane potential and the degree of electrical activity were not different in B cells exhibiting these cyclic variations or not. These oscillations in the duration of slow waves and intervals induced by glucose could be due to fluctuations in metabolic events and in cytoplasmic K+ activity in B cells.

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.

HB 699 is a benzoic acid derivative similar to the non-sulphonylurea moiety of glibenclamide. The mechanisms whereby it affects B-cell function have been studied in vitro with mouse islets. In the presence of 3 mmol/l glucose, HB 699 decreased 86Rb+ efflux and accelerated 45Ca2+ efflux from islet cells, depolarized the B-cell membrane and induced an electrical activity similar to that triggered by stimulatory concentrations of glucose, and increased insulin release. The changes in 45Ca2+ efflux and insulin release, but not the inhibition of 86Rb+ efflux, were abolished in the absence of Ca2+. In the presence of 10 mmol/l glucose, HB 699 increased 86Rb+ and 45Ca2+ efflux from the islets, caused a persistent depolarization of the B-cell membrane with continuous electrical activity and markedly potentiated insulin release. All these changes were suppressed by omission of extracellular Ca2+. In the presence of 15 mmol/l glucose, diazoxide increased 86Rb+ efflux, hyperpolarized the B-cell membrane, suppressed electrical activity and inhibited insulin release. HB 699 reversed these effects of diazoxide. It is suggested that HB 699 decreases K+ permeability of the B-cell membrane, thereby causing a depolarization which leads to activation of voltage-dependent Ca channels and Ca2+ influx, and eventually increases insulin release. A sulphonylurea group is thus not a prerequisite to trigger the sequence of events that is also thought to underlie the releasing effects of tolbutamide and glibenclamide.

1 The mechanisms whereby extracellular adenine nucleotides modulate pancreatic #-cell function were studied with mouse islets stimulated by 15 mm glucose.2 Adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP) (100pM) inhibited insulin release, 45Ca efflux and 86Rb efflux from islet cells, and decreased electrical activity in fl-cells. These changes were rapid but small and transient. 3 x,#-Methylene ADP caused a rapid and sustained inhibition of insulin release, 45Ca efflux and 86Rb efflux from islet cells. It also produced a slight hyperpolarization of the #-cell membrane, with sustained modification of the pattern but only transient decrease of the intensity of the electrical activity. In the absence of extracellular Ca2, a,#-methylene ADP increased 45Ca and 86Rb efflux without changing insulin release. Most effects of a,#-methylene ATP were qualitatively similar but quantitatively smaller than those of the ADP-analogue. 4 Adenylylimido-diphosphate (AMP-PNP) slightly increased 45Ca and 86Rb efflux and potentiated insulin release in the presence of extracellular Ca22. However, its effects on electrical activity in fl-cells were qualitatively similar to those of the a,#-methylene analogues. 5 The small effects of ATP and ADP could result from their degradation into adenosine. a,#-Methylene ADP appears to increase K+ permeability of the fl-cell membrane and to produce a second, intracellular, effect which largely contributes to the inhibition of insulin release. Another recognition site, with higher affinity for triphosphate derivatives, could mediate the small stimulatory effects of AMP-PNP.

The mechanisms whereby activation of the cyclic AMP-dependent protein kinase A or the Ca2+-phospholipid-dependent protein kinase C amplifies insulin release were studied with mouse islets. Forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) were used to stimulate adenylate cyclase and protein kinase C respectively. The sulphonylurea tolbutamide was used to initiate insulin release in the presence of 3 mM-glucose. Tolbutamide alone inhibited 86Rb+ efflux, depolarized ,-cell membrane, triggered electrical activity, accelerated 45Ca2+ influx and efflux and stimulated insulin release. Forskolin alone only slightly inhibited 86Rb+ efflux, but markedly increased the effects of tolbutamide on electrical activity, 45Ca2+ influx and effilux, and insulin release. In the absence of Ca2+, only the inhibition of 86Rb+ efflux persisted. TPA (100 nM) alone slightly accelerated 45Ca2+ efflux and insulin release without affecting 45Ca2+ influx or fl-cell membrane potential. It increased the effects of tolbutamide on 45Ca2+ efflux and insulin release without changing 86Rb+ efflux, 45Ca2+ influx or electrical activity. Omission of extracellular Ca2+ suppressed all effects due to the combination of TPA and tolbutamide, but not those of TPA alone. Though ineffective alone, 10 nM-TPA amplified the releasing action of tolbutamide without affecting its ionic and electrical effects. In conclusion, the two amplification systems of insulin release involve at least partially distinct mechanisms. The cyclic AMP but not the protein kinase C system increases the initiating signal (Ca2+ influx) triggered by the primary secretagogue.

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