T. Tamagawa

Diazoxide and D600 Inhibition of Insulin Release: Distinct Mechanisms Explain the Specificity for Different Stimuli

The mechanisms by which diazoxide and D600 affect insulin release have been compared in experiments using isolated rat islets. Diazoxide (20–400 (JLM) and D600 (1–50 μM) produced a dose-dependent inhibition of glucose-stimulated release. Diazoxide also inhibited the insulinotropic effect of leucine and related substances (ketoisocaproate and BCH), but not that of potassium or of arginine and other cationic amino acids. Diazoxide suppressed glucose and leucine stimulation of Ca uptake in islet cells, but had no effect on the stimulation by potassium and arginine. By contrast, D600 suppressed the effect of all these agents on both Ca uptake and insulin release. Theophylline partially antagonized the inhibitory effect of D600 on release, in the presence of diazoxide, theophylline was much less effective, except when combined with cationic amino acids. Diazoxide inhibition of glucose-induced release was prevented by phentolamine, but hot by dihydroergotamine and yohimbine, two other blpckers of a-adrenergic receptors. Epinephrine abolished the insulinotropic effect of arginine alone or with theophylline. Diazoxide increased 86Rb+ efflux from islet cells, whereas D600 and epinephrine decreased it. The acceleration of efflux by diazoxide was inhibited by D600 and phentolamine, but not by epinephrine or dihydroergotamine. It thus appears that the effects of diazoxide on B-cells are not due to activation of α-adrenergic receptors. The results suggest that, in contrast to the direct blockade of Ca channels by D600, the blockade of these channels by diazoxide is secondary to the hyperpolarization of the B-cell membrane. Since the latter results from an increase in K permeability, the inhibitory effects of diazoxide are restricted to stimulators that depolarize the B-cell membrane by decreasing its K permeability (glucose, leucine, and related substances) and do not affect the stimulation by K and cationic amino acids, which depolarize by other mechanisms.

Effects of acute sodium omission on insulin release, ionic flux and membrane potential in mouse pancreatic B-cells

The effects of acute omission of extracellular Na+ on pancreatic B-cell function were studied in mouse islets, using choline and lithium salts as impermeant and permeant substitutes, respectively. In the absence of glucose, choline substitution for Na+ hyperpolarized the B-cell membrane, inhibited 86Rb+ and 45Ca2+ efflux, but did not affect insulin release. In contrast, Li+ substitution for Na+ depolarized the B-cell membrane and caused a Ca2+-independent, transient acceleration of 45Ca2+ efflux and insulin release. Na+ replacement by choline in the presence of 10 mM glucose and 2.5 mM Ca2+ again rapidly hyperpolarized the B-cell membrane. This hyperpolarization was then followed by a phase of depolarization with continuous spike activity, before long slow waves of the membrane potential resumed. Under these conditions, 86Rb+ efflux first decreased before accelerating, concomitantly with marked and parallel increases in 45Ca2+ efflux and insulin release. In the absence of Ca2+, 45Ca2+ and 86Rb+ efflux were inhibited and insulin release was unaffected by choline substitution for Na+. Na+ replacement by Li+ in the presence of 10 mM glucose rapidly depolarized the B-cell membrane, caused an intense continuous spike activity, and accelerated 45Ca2+ efflux, 86Rb+ efflux and insulin release. In the absence of extracellular Ca2+, Li+ still caused a rapid but transient increase in 45Ca2+ and 86Rb+ efflux and in insulin release. Although not indispensable for insulin release, Na+ plays an important regulatory role in stimulus-secretion coupling by modulating, among others, membrane potential and ionic fluxes in B-cells.

Chloride Modulation of Insulin Release, 86Rb+ Efflux, and 45Ca2+ Fluxes in Rat Islets Stimulated by Various Secretagogues

Substitution of extracellular CI− by impermeant isethionate (5 mM residual CI−) caused a monophasic inhibition of glucose-stimulated insulin release, accompanied by an initial transient increase and a secondary lasting decrease in 86Rb+ efflux from perifused islets. CI− reintroduction restored insulin release with an overshoot above control values and successively produced a small decrease and a large increase in efflux. Theophylline potentiated the insulinotropic effect of glucose more markedly at low CI− than at normal CI−, but did not restore a normal rate of 86Rb+ efflux. Lowering the concentration of CI− did not alter the effect of glucose, tolbutamide, or arginine on 86Rb+ efflux, but simply shifted the efflux rates to lower values. The first phase of glucose-stimulated insulin release was not modified, but the second phase was inhibited. The insulinotropic effect of tolbutamide was augmented at low CI− and that of arginine (at 7 mM glucose) was not affected. In incubated islets, the stimulation of insulin release by glyceraldehyde was barely inhibited when CI− was substituted by isethionate and the marked decrease of the effect of glucose could be prevented by glutamine. In a glucose-free, low CI− medium, the insulinotropic effect of leucine, arginine, and lysine was inhibited; this inhibition was reversed by glutamine, but not by theophylline. Lowering the concentration of CI− had no effect on 45Ca2+ influx or efflux in the absence of glucose, did not alter the increase in influx and efflux during the first 5 min of glucose stimulation, but impaired both influx and efflux during the second phase. Leucine-induced 45Ca2+ uptake was inhibited at low CI− and this inhibition was prevented by glutamine. In conclusion, islet cells possess a CI−-activated modality of K efflux, which does not seem to play a role in the stimulus-secretion coupling. Since CI− substitution by an impermeant anion does not inhibit the stimulation of insulin release by all agents, the role of CI− ions does not appear to be restricted to a chemiosmotic mechanism of exocytosis. No single mechanism explains the multiple changes in B-cell function resulting from the decrease in CI− concentration, but it is proposed that some of them could result from modifications of intracellular pH.