Insulin increases NO-stimulated guanylate cyclase activity in cultured VSMC while raising redox potential

Insulin acutely stimulates cyclic guanosine monophosphate (cGMP) production in primary confluent cultured vascular smooth muscle cells (VSMC) from can ine femoral artery, but the mechanism is not known. These cells contain the inducible isoform of nitric oxide (NO) synthase (iNOS), and insulin-stimul ated cGMP production in confluent cultured cells is blocked by the NOS inhi bitor, N-G-monomethyl-L-arginine (L-NMMA). In the present study, it is show n that iNOS is also present in freshly dispersed VSMC from this artery, ind icating that iNOS expression in cultured VSMC is not an artifact of the cul ture process. Insulin did not stimulate NOS activity in primary confluent c ultured cells because it did not affect citrulline or combined NO3-/NO2- pr oduction. To see whether insulin required the permissive presence of NO to stimulate cGMP production, iNOS and basal cGMP production were inhibited wi th L-NMMA, and the cells were incubated with or without 1 nM insulin and/or the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at a concentrati on (0.1 mu M) that restored cGMP production to the basal value. In the pres ence of L-NMMA, insulin no longer affected cGMP production but when insulin was added to L-NMMA plus SNAP, cGMP production was increased by 69% (P < 0 .05 vs. L-NMMA plus SNAP). Insulin, which increases glucose uptake by these cells, increased the cell lactate content and the lactate-to-pyruvate rati o (LPR) by 81 and 97%, respectively (both P < 0.05), indicating that the ho rmone increased aerobic glycolysis and the redox potential. The effects of insulin on LPR and cGMP production were blocked by removing glucose or by a dding 2-deoxyglucose to the incubation media and were duplicated by the red ucing substrate, beta-hydroxybutyrate. We conclude that insulin does not ac utely affect iNOS activity in these VSMC but it does augment cGMP productio n induced by the NO already present in the cell while increasing aerobic gl ycolysis and the cell redox potential.