The mechanisms by which cationic amino acids influence pancreatic B-cell function have been studied by monitoring simultaneously (86)Rb(+) efflux and insulin release from perifused rat islets. The effects of two reference amino acids arginine and lysine were compared with those of closely related substances to define the structural requirements for recognition of these molecules as secretagogues. Arginine accelerated (86)Rb(+) efflux and increased insulin release in the absence or in the presence of 7mm-glucose. Its effects on efflux did not require the presence of extracellular Ca(2+) or Na(+), but its insulinotropic effects were suppressed in a Ca(2+)-free medium and inhibited in an Na(+)-free medium. Among arginine derivatives, only 2-amino-3-guanidinopropionic acid mimicked its effects on (86)Rb(+) efflux and insulin release; citrulline, guanidinoacetic acid, 3-guanidinopropionic acid and guanidine were inactive. Norvaline and valine also increased (86)Rb(+) efflux, but their effect required the presence of extracellular Na(+); they did not stimulate insulin release. Lysine as well as the shorter-chain cationic amino acids ornithine and 2,4-diaminobutyric acid accelerated (86)Rb(+) efflux in a Ca(2+)- and Na(+)-independent manner. Their stimulation of insulin release was suppressed by Ca(2+) omission, but only partially inhibited in an Na(+)-free medium. The uncharged glutamine and norleucine increased the rate of (86)Rb(+) efflux in the presence of glucose, only if extracellular Na(+) was present. Norleucine slightly increased release in a Ca(2+)- and Na(+)-dependent manner. The effects of lysine on efflux and release were not mimicked by other related substances such as 1,5-diaminopentane and 6-aminohexanoic acid. The results suggest that the depolarizing effect of cationic amino acids is due to accumulation of these positively charged molecules in B-cells. This causes acceleration of the efflux of K(+) ((86)Rb(+)) and activation of the influx of Ca(2+) (which triggers insulin release). The prerequisite for the stimulation of B-cells by this mechanism appears to be the presence of a positive charge on the side chain of the amino acid, rather than a specific group.

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 /xM) 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 86 Rb + 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 a-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. DIABETES 37:776-783, September 1982. D iazoxide remains the drug of choice for medical treatment of chronic hypoglycemia due to hyperinsulinemia. 1 ' 2 A stimulation of epinephrine release and mainly an inhibition of insulin release account for its hyperglycemic action. 3 -4 The direct inhibitory effect of the drug on the pancreatic B-cell has been demonstrated in vitro, 5 " 7 but its mechanisms have long remained elusive. The latest experimental evidence 8 has ascribed the inhibition of glucose-stimulated release to the ability of diazoxide to hyperpolarize the B-cell membrane by increasing its potassium permeability. Nevertheless, certain aspects of its mode of action are still unclear. It has been suggested that diazoxide directly activates a-adrenergic receptors in B-ceils, 9~12 but such a mechanism appears difficult to reconcile with the clinically useful relaxation of vascular smooth muscles that the drug, also produces. 13 Furthermore, diazoxide inhibition of insulin release shows a certain stimulus selectivity in vitro 14 and in vivo. 15 Iri particular, the int...

The effects of three types of amino acids on 45Ca2+ fluxes in rat pancreatic islets have been compared. Alanine, a non-insulinotropic neutral amino acid, transported with Na+, increased 45Ca2+ efflux in the presence or in the absence of extracellular Ca2+, but not in the absence of Na+. Its effects in Na+-solutions were practically abolished by 7 mM-glucose. Alanine slightly stimulated 45Ca2+ influx (5 min uptake) only when Na+ was present. Two insulinotropic cationic amino acids (arginine and lysine) triggered similar changes in 45Ca2+ efflux. They accelerated the efflux in the presence of Ca2+ and inhibited the efflux in a Ca2+-free medium, whether glucose was present or not. In an Na+-free Ca2+-medium, arginine and lysine markedly accelerated 45Ca2+ efflux, but this effect was suppressed by 7 mM-glucose. Arginine stimulated 45Ca2+ influx irrespective of the presence or absence of glucose and Na+. Leucine, a neutral insulinotropic amino acid well metabolized by islet cells, inhibited 45Ca2+ efflux from the islets in a Ca2+-free medium; this effect was potentiated by glutamine. In the presence of Ca2+ and Na+, leucine was ineffective alone, but triggered a marked increase in 45Ca2+ efflux when combined with glutamine. In an Na+-free Ca2+-medium, leucine accelerated 45Ca2+ efflux to the same extent with or without glutamine. Leucine also stimulated 45Ca2+ influx in the presence or in the absence of Na+, but its effects were potentiated by glutamine only in the presence of Na+. The results show that amino acids of various types cause distinct changes in 45Ca2+ fluxes in pancreatic islets. Certain of these changes involve an Na+-mediated mobilization of cellular Ca2+ from sequestering sites where glucose appears to exert an opposite effect.

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