Exogenous Nitric Oxide and Endogenous Glucose-Stimulated β-Cell Nitric Oxide Augment Insulin Release

Abstract

The role nitric oxide (NO) plays in physiological insulin secretion has been controversial. Here we present evidence that exogenous NO stimulates insulin secretion, and that endogenous NO production occurs and is involved in the regulation of insulin release. Radioimmunoassay measurement of insulin release and a dynamic assay of exocytosis using the dye FM1-43 demonstrated that three different NO donors-hydroxylamine (HA), sodium nitroprusside, and 3-morpholinosydnonimine (SIN-1)-each stimulated a marked increase in insulin secretion from INS-1 cells. Pharmacological manipulation of the guanylate cyclase/guanosine 3,5-cyclic monophosphate pathway indicated that this pathway was involved in mediating the effect of the intracellular NO donor, HA, which was used to simulate endogenous NO production. This effect was further characterized as involving membrane depolarization and intracellular T he endogenous synthesis of nitric oxide (NO) from L-arginine (L-arg) has been shown to play a critical role in a wide variety of physiological functions including neurotransmission, vascular tone, platelet aggregation, immunological reactions, penile erection, and endocrine and exocrine function (1). Cellular NO is synthesized by a family of NO synthase (NOS) enzymes, comprised of constitutively expressed, Ca 2ϩ / calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS), and a Ca 2ϩ /calmodulin-independent inducibly expressed NOS (iNOS) (2).Pancreatic ␤-cells are able to express the iNOS enzyme in response to inflammatory stimuli, leading to high cytotoxic levels of NO production, which appear to be involved in ␤-cell damage, dysfunction, and death and the pathogenesis of type 1 diabetes (3,4). The presence of a constitutive NOS (cNOS) enzyme in ␤-cells is substantiated by considerable evidence, including biochemical, histochemical, immunohistochemical, immunofluorescence, RT-PCR, and protein immunoblot analyses (5-11). Given that the amino acid NO precursor L-arg has long been known to stimulate insulin release (12), it has been postulated that a low level of NO produced from the ␤-cell cNOS isoform functions in the regulation of insulin release. However, several reports in which cNOS activity was manipulated or exogenous NO was applied have yielded seemingly conflicting results. NOS inhibition has been reported to produce an inhibitory effect (5,13-15), a stimulatory effect (6,9,16 -18), and no effect at all (19,20) on insulin release. Similarly, exogenously applied NO has been reported to exert a stimulatory (5,13,14,21,22) and an inhibitory effect (6,18,23-27) on insulin release. These discrepant data may be the result of variations in the specifics of the experimental conditions, including differences in the agents used (e.g., NOS substrate, NOS inhibitors, NO donors), the concentration of these agents, whether other stimulatory pathways were activated concurrently (e.g., with glucose), the experimental model used (e.g., ␤-cell line, islets, pancreas), and the species. These critical differences may result in d...

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