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.
OBJECTIVE
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.


CONCLUSIONS
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.
Trovati
M
M
Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, Orbassano, Italy.
Massucco
P
P
Mattiello
L
L
Costamagna
C
C
Aldieri
E
E
Cavalot
F
F
Anfossi
G
G
Bosia
A
A
Ghigo
D
D
D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured / Q000187:drug effects; D002956:Citrulline / Q000378:metabolism; D000242:Cyclic AMP / Q000032:analysis*; D006152:Cyclic GMP / Q000032:analysis*; D015870:Gene Expression; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D015759:Ionomycin / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000201:enzymology; D009569:Nitric Oxide / Q000096:biosynthesis / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D009599:Nitroprusside / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D014158:Transcription, Genetic
D007328:Insulin; D012333:RNA, Messenger; D009599:Nitroprusside; D002956:Citrulline; D009569:Nitric Oxide; D015759:Ionomycin; D000242:Cyclic AMP; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D006152:Cyclic GMP; D002118:Calcium