Abstract
We previously showed that pancreatic beta-cells express neuronal nitric oxide synthase (nNOS) that controls insulin secretion through two catalytic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now provide evidence that the endogenous protein inhibitor of nNOS (PIN) is expressed in rat pancreatic islets and INS-1 cells. Double-immunofluorescence studies showed a colocalization of PIN with both nNOS and myosin Va in insulin-secreting beta-cells. Electron microscopy studies confirmed that PIN is mainly associated with insulin secretory granules and colocated with nNOS in the latter. In addition, PIN overexpression in INS-1 cells enhanced glucose-induced insulin secretion, which is only partly reversed by addition of an NO donor, sodium nitroprusside (SNP), and unaffected by the inhibitor of cytochrome c reductase activity, miconazole. In contrast, the pharmacological inhibitor of nNOS, Nomega-nitro-l-arginine methyl ester, amplified glucose-induced insulin secretion, an effect insensitive to SNP but completely normalized by the addition of miconazole. Thus, PIN insulinotropic effect could be related to its colocalization with the actin-based molecular motor myosin Va and as such be implicated in the physiological regulation of glucose-induced insulin secretion at the level of the exocytotic machinery.
Highlights
We previously showed that pancreatic -cells express neuronal nitric oxide synthase that controls insulin secretion through two catalytic activities: nitric oxide (NO) production and cytochrome c reductase activity
Our data clearly show that protein inhibitor of nNOS (PIN) is expressed in rat pancreatic -cells, as previously shown for neuronal nitric oxide synthase (nNOS) [29], and positively modulates insulin secretion
Coincidence of nNOS and PIN occurs in other tissues and has been reported in brain [18], kidney [20], and ventilatory muscles [19]. nNOS is always coexpressed with its endogenous inhibitor, but PIN, which is moderately expressed in all rat and human tissues [16,17], does not necessarily occur with nNOS
Summary
Protein Inhibitor of Neuronal Nitric Oxide Synthase (PIN) Is a New Regulator of Glucose-Induced Insulin Secretion. We previously showed that pancreatic -cells express neuronal nitric oxide synthase (nNOS) that controls insulin secretion through two catalytic activities: nitric oxide (NO) production and cytochrome c reductase activity. Stitutive NOSs produce small amounts of NO, implicated in physiological functions such as synaptic transmission [2] and vascular tone [3], whereas iNOS produces high amounts of NO, acting as a cytotoxic mediator in immune response [4] In addition to these beneficial roles, NO exerts deleterious effects as a result of its overproduction, leading to neuronal death during brain ischemia and certain neurodegenerative pathologies [5] and to autoimmune diseases [6]. This prompted us, first, to investigate if PIN is expressed in rat pancreatic -cells, second, to study its subcellular localization, and to determine, by using the insulin-secreting cell line INS-1, whether PIN could play a role in the regulation of glucose-induced insulin secretion
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