Changes in the Dimeric State of Neuronal Nitric Oxide Synthase Affect the Kinetics of Secretagogue-Induced Insulin Response

Changes in the Dimeric State of Neuronal Nitric Oxide Synthase Affect the Kinetics of Secretagogue-Induced Insulin Response Anne-Dominique Lajoix 1 2 , Martine Pugnière 2 , Françoise Roquet 2 , Jean-Claude Mani 2 , Samuel Dietz 1 2 , Nathalie Linck 2 , Fleur Faurie 2 , Gérard Ribes 2 , Pierre Petit 2 and René Gross 2 1 Innodia SAS, Montpellier, France 2 UMR 5160 CNRS, Montpellier, France Address correspondence and reprint requests to René Gross, CNRS UMR 5160, Institut de Biologie, 4 Boulevard Henri IV, 34960 Montpellier Cedex 2, France. E-mail: rene.gross{at}univ-montp1.fr Abstract We previously showed that pancreatic β-cells express a neuronal isoform of nitric oxide synthase (nNOS) that controls insulin secretion by exerting two enzymatic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now bring evidence that two inhibitors of nNOS, N -ω-nitro- l -arginine methyl ester ( l -NAME) and 7-nitroindazole (7-NI), increase glucose-induced insulin secretion but affect β-cell function differently. In the presence of l -NAME, insulin response is monophasic, whereas 7-NI preserves the normal biphasic secretory pattern. In addition, the alterations of β-cell functional response induced by the inhibitors also differ by their sensitivity to a substitutive treatment with sodium nitroprusside, a chemical NO donor. These differences are probably related to the nature of the two inhibitors. Indeed, using low-temperature SDS-PAGE and real-time analysis of nNOS dimerization by surface plasmon resonance, we could show that 7-NI, which competes with arginine and tetrahydrobiopterin (BH 4 ), an essential cofactor for nNOS dimer formation, inhibits dimerization of the enzyme, whereas the substrate-based inhibitor l -NAME stabilizes the homodimeric state of nNOS. The latter effect could be reproduced by the two endogenous inhibitors of NOS, N -ω-methyl- l -arginine and asymmetric dimethylarginine, and resulted interestingly in a reduced ability of the protein inhibitor of nNOS (PIN) to dissociate nNOS dimers. We conclude that intracellular factors able to induce abnormalities in the nNOS monomer/dimer equilibrium could lead to pancreatic β-cell dysfunction. 7-NI, 7-nitroindazole ADMA, asymmetric dimethyl-arginine BH4, tetrahydrobiopterin eNOS, endothelial NOS iNOS, inducible NOS l-NAME, N-ω-nitro-l-arginine methyl ester l-NNA, N-ω-nitro-l-arginine l-MMA, N-ω-methyl-l-arginine NOS, nitric oxide synthase nNOS, neuronal NOS PIN, protein inhibitor o