Constitutive nitric oxide synthases in rat pancreatic islets: direct imaging of glucose-induced nitric oxide production in beta-cells.

The production of nitric oxide (NO) by constitutive nitric oxide synthase (NOS) was investigated in isolated rat pancreatic islets and dispersed beta-cells. Double-immunocytochemical analyses with a confocal microscope demonstrated the presence of NOS1 in alpha-, beta-, delta-, and PP-cells and that of NOS3 in beta-, delta-, and PP-cells, but not alpha-cells, in the isolated rat islets. Image analyses with the NO-reactive fluorescence dye DAF-2 clearly showed that an elevation in glucose concentrations from 0 to 11.1 mM increased intracellular NO in most cells of the isolated islets. The glucose-induced elevation of intracellular NO in the islet cells was abolished in the presence of the Ca2+ channel blocker nicardipine and after treatment with the NOS inhibitor NG-nitro-L-arginine. Similarly, the ATP-sensitive K+ channel blocker tolbutamide, which elevates intracellular Ca2+ concentrations, increased DAF-2 fluorescence in most cells of the isolated islets. In isolated beta-cells, 11.1 mM glucose increased DAF-2 fluorescence, which was suppressed by NG-nitro-L-arginine and by reducing the glucose concentration to 0 mM. DAF-2 fluorescence in beta-cells was also increased by 50 mM K+, which was suppressed by NG-nitro-L-arginine. These results suggest that NOS1 and NOS3 are present in rat pancreatic beta-cells, and that glucose produces NO by Ca(2+)-dependent activation of the constitutive NOS isoforms.