Human vascular smooth muscle cells express a constitutive nitric oxide synthase that insulin rapidly activates, thus increasing guanosine 3':5'-cyclic monophosphate and adenosine 3':5'-cyclic monophosphate concentrations

Diabetologia. 1999 Jul;42(7):831-9. doi: 10.1007/s001250051234.

Abstract

Aims/hypothesis: Insulin incubation of human vascular smooth muscle cells (hVSMC) for 120 min increases both guanosine 3':5'-cyclic monophosphate (cGMP) and adenosine 3':5'-cyclic monophosphate (cAMP) and these effects are blocked by inhibiting nitric oxide synthase (NOS). These data suggest that insulin activates a constitutive Ca2+-dependent NOS (cNOS), not described at yet in hVSMC. To test this hypothesis, we evaluated in hVSMC: i) the kinetics of the insulin-induced enhancement of the two cyclic nucleotides; ii) the ability of nitric oxide (NO) to increase both cyclic nucleotides; iii) NO involvement in the short-term influence of insulin on both cyclic nucleotides; iv) the ability of insulin to increase NO production in a few minutes; v) the presence of a cNOS activity; vi) the expression of mRNA for cNOS.

Methods: In hVSMC incubated with insulin, NO donors and the Ca2+ ionophore ionomycin, we measured cAMP and cGMP (RIA); in hVSMC incubated with insulin and ionomycin we measured NO, evaluated as L-(3H)-citrulline production from L-(3H)-arginine; by northern blot hybridization, we measured the expression of cNOS mRNA.

Results: i) By incubating hVSMC with 2 nmol/l insulin for 0-240 min, we observed an increase of both cGMP and cAMP (ANOVA: p = 0.0001). Cyclic GMP rose from 0.74 +/- 0.01 to 2.62 +/- 0.10 pmol/10(6) cells at 30 min (p = 0.0001); cAMP rose from 0.9 +/- 0.09 to 11.65 +/- 0.74 pmol/10(6) cells at 15 min (p=0.0001). ii) Sodium nitroprusside (100 mol/l) and glyceryltrinitrate (100 micromol/l) increased both cGMP and cAMP (p = 0.0001). iii) The effects of insulin on cyclic nucleotides were blocked by NOS inhibition. iv) An increase of NO was observed by incubating hVSMC for 5 min with 2 nmol/l insulin (p = 0.0001). v) Ionomycin (1 micromol/l) enhanced NO production (p = 0.0001) and increased both cyclic nucleotides (p = 0.0001). vi) hVSMC expressed mRNA of cNOS.

Conclusion/interpretation: Human VSMC express cNOS, which is rapidly activated by insulin with a consequent increase of both cGMP and cAMP, suggesting that insulin-induced vasodilation in vivo is not entirely endothelium-mediated.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / pharmacology
  • Cells, Cultured / drug effects
  • Citrulline / metabolism
  • Cyclic AMP / analysis*
  • Cyclic GMP / analysis*
  • Gene Expression*
  • Humans
  • Insulin / pharmacology*
  • Ionomycin / pharmacology
  • Muscle, Smooth, Vascular / cytology*
  • Muscle, Smooth, Vascular / enzymology
  • Nitric Oxide / biosynthesis
  • Nitric Oxide / physiology
  • Nitric Oxide Synthase / chemistry*
  • Nitric Oxide Synthase / drug effects
  • Nitric Oxide Synthase / genetics
  • Nitric Oxide Synthase / metabolism*
  • Nitric Oxide Synthase Type III
  • Nitroprusside / pharmacology
  • RNA, Messenger / analysis
  • Transcription, Genetic

Substances

  • Insulin
  • RNA, Messenger
  • Nitroprusside
  • Citrulline
  • Nitric Oxide
  • Ionomycin
  • Cyclic AMP
  • NOS3 protein, human
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type III
  • Cyclic GMP
  • Calcium