Influence of nitric oxide synthase inhibition, nitric oxide and hydroperoxide on insulin release induced by various secretagogues

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Abstract
Recent studies have suggested that the generation of nitric oxide (NO) and hydrogen peroxide (H2O2) by islet NO synthase and monoamine oxidase, respectively, may have a regulatory influence on insulin secretory processes. We have investigated the pattern of insulin release from isolated islets of Langerhans in the presence of various pharmacological agents known to perturb the intracellular levels of NO and the oxidation state of SH‐groups. The NO synthase inhibitor, NG‐nitro‐l‐arginine methyl ester (l‐NAME) dose‐dependently increased l‐arginine‐induced insulin release. d‐Arginine did not influence l‐arginine‐induced insulin secretion. However, d‐NAME which reportedly has no inhibitory action on NO synthase, modestly increased l‐arginine‐induced insulin release, but was less effective than l‐NAME. High concentrations (10 mm) of d‐arginine as well as l‐NAME and d‐NAME could enhance basal insulin release. The intracellular NO donor, hydroxylamine, dose‐dependently inhibited insulin secretion induced by l‐arginine and l‐arginine + l‐NAME. Glucose‐induced insulin release was increased by NO synthase inhibition (l‐NAME) and inhibited by the intracellular NO donor, hydroxylamine. Sydnonimine‐1 (SIN‐1), an extracellular donor of NO and superoxide, induced a modest suppression of glucose‐stimulated insulin release. SIN‐1 did not influence insulin secretion induced by l‐arginine or the adenylate cyclase activator, forskolin. The intracellular ‘hydroperoxide donor’ tert‐butylhydroperoxide in the concentration range of 0.03–3mm inhibited insulin release stimulated by the nutrient secretagogues glucose and l‐arginine. Low concentrations (0.03–30 μm) of tert‐butylhydroperoxide, however enhanced insulin secretion induced by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). Islet guanosine 3’:5′‐cyclic monophosphate (cyclic GMP) content was not influenced by 10 mm l‐arginine or tert‐butylhydroperoxide at 3 or 300 μm but was markedly increased (14 fold) by a high hydroxylamine concentration (300 μm). In contrast, islet adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) content was increased (3 fold) by l‐arginine (10 mm) and (2 fold) by tert‐butylhydroperoxide (300 μm). Our results strongly suggest that NO is a negative modulator of insulin release induced by the nutrient secretagogues l‐arginine and glucose. This effect is probably not mediated to any major extent by the guanylate cyclase‐cyclic GMP system but may rather be exerted by the S‐nitrosylation of critical thiol groups involved in the secretory process. Similarly the inhibitory effect of tert‐butylhydroperoxide is likely to be elicited through affecting critical thiol groups. The mechanism underlying the secretion‐promoting action of tert‐butylhydroperoxide on IBMX‐induced insulin release is probably linked to intracellular Ca2+‐perturbations affecting exocytosis. Taken together with previous data the present results suggest that islet production of low physiological levels of free radicals such as NO and H2O2 may serve as important modulators of insulin secretory processes.