A Neuronal Isoform of Nitric Oxide Synthase Expressed in Pancreatic β-Cells Controls Insulin Secretion

Abstract

Evidence is presented showing that a neuronal isoform of nitric oxide synthase (NOS) is expressed in rat pancreatic islets and INS-1 cells. Sequencing of the coding region indicated a 99.8% homology with rat neuronal NOS (nNOS) with four mutations, three of them resulting in modifications of the amino acid sequence. Doubleimmunofluorescence studies demonstrated the presence of nNOS in insulin-secreting ␤-cells. Electron microscopy studies showed that nNOS was mainly localized in insulin secretory granules and to a lesser extent in the mitochondria and the nucleus. We also studied the mechanism involved in the dysfunction of the ␤-cell response to arginine and glucose after nNOS blockade with N Gnitro-L-arginine methyl ester. Our data show that miconazole, an inhibitor of nNOS cytochrome c reductase activity, either alone for the experiments with arginine or combined with sodium nitroprusside for glucose, is able to restore normal secretory patterns in response to the two secretagogues. Furthermore, these results were corroborated by the demonstration of a direct enzymesubstrate interaction between nNOS and cytochrome c, which is strongly reinforced in the presence of the NOS inhibitor. Thus, we provide immunochemical and pharmacological evidence that ␤-cell nNOS exerts, like brain nNOS, two catalytic activities: a nitric oxide production and an NOS nonoxidating reductase activity, both of which are essential for normal ␤-cell function. In conclusion, we suggest that an imbalance between these activities might be implicated in ␤-cell dysregulation involved in certain pathological hyperinsulinic states. T he short-lived free radical gas nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as NO synthases (NOSs). Three NOS isoenzymes, encoded by three separate genes, have been described, including the Ca 2ϩ /calmodulin-dependent and constitutively expressed neuronal NOS (nNOS) and endothelial NOS (eNOS) enzymes and a calmodulin-independent cytokine-inducible NOS (iNOS) enzyme found in various cell types (rev. in 1). The small amounts of NO, produced by the constitutive forms in response to increases in intracellular calcium, play a crucial role in a number of physiological functions, including neurotransmission (2), vascular tone (3) and platelet aggregation (4), whereas the large amounts, produced by iNOS in a calcium-independent manner over prolonged periods of time, are implicated in pathological functions, such as cytotoxicity of activated macrophages (5).Even though the inducible isoform has been cloned in insulin-producing cells after induction by cytokines (6), it is unclear whether pancreatic ␤-cells express a constitutive NOS. Both NADPH-diaphorase (NADPH-d) histochemical staining previously shown to be specific for NOS (7) and immunohistochemical studies using various nNOS antisera yielded apparently conflicting data. Positive NADPH-d and immunoreactive nNOS stainings have been found to be colocalized in most pancreatic endocrine cells (8), a finding not confirmed in other studies...

Protein Inhibitor of Neuronal Nitric Oxide Synthase (PIN) Is a New Regulator of Glucose-Induced Insulin Secretion

“…2C, F, I, and L). This strong colocalization suggests that PIN is associated with insulin secretory granules, as previously demonstrated for nNOS (29).…”

“…Pancreatic ␤-cell nNOS is for a great part localized in insulin secretory granules but also in mitochondria and nucleus. The enzyme is implicated in the control of insulin secretion and, as previously shown for brain NOS, is able to exert two catalytic activities: NO production and a cytochrome c reductase activity (29,30). Furthermore, we could show that a normal balance between the two catalytic activities is essential for ␤-cell function, but the mechanisms that could regulate nNOS catalytic activities in pancreatic ␤-cells remain to be investigated.…”

“…A possible decrease in NO production was tested using sodium nitroprusside (SNP), an exogenous NO donor, and an increased cytochrome c reductase activity was challenged with a specific inhibitor, miconazole (29). Data from PIN-overexpressing cells were also compared with those obtained in nontransfected cells in the presence of L-NAME, a nonmetabolizable analog of arginine, an inhibitor of nNOS activity.…”

“…In previous studies, we showed that pancreatic ␤-cells express nNOS that displays three mutations when compared with cerebellar nNOS (29). Pancreatic ␤-cell nNOS is for a great part localized in insulin secretory granules but also in mitochondria and nucleus.…”

Expression of islet inducible nitric oxide synthase and inhibition of glucose-stimulated insulin release after long-term lipid infusion in the rat is counteracted by PACAP27

“…In contrast with the consistent experimental support of a critical role of iNOS for degenerative and apoptotic processes in ␤-cells, significant controversy exists with regard to the role played by ncNOS in the regulation of insulin secretion (16,22,34,44). A clear indication that ncNOS-derived NO deserves consideration as a negative modulator of glucose-stimulated insulin release is coming from the findings that inhibition of ncNOS by different NOS inhibitors positively affects the insulin response to glucose (1, 14 -16, 27, 30, 38, 44).…”