Insulin increases NADH/NAD+ redox state, which stimulates guanylate cyclase in vascular smooth muscle.
BACKGROUND
Insulin inhibits contraction and migration of primary confluent, cultured canine vascular smooth muscle cells (VSMCs) with inducible nitric oxide synthase (iNOS) by stimulating cyclic GMP (cGMP) production. The present study was performed to determine how insulin stimulates guanylate cyclase activity in these cells.


METHODS
Primary cultured VSMC were obtained from canine femoral arteries. Lactate and pyruvate levels were measured by enzymatic assays, cGMP production by radioimmunoassay, iNOS activity by conversion of arginine to citrulline, and cell contraction by photomicroscopy.


RESULTS
Insulin (1 nmol/L) increased cGMP production fivefold in VSMC with iNOS while raising the lactate-to-pyruvate ratio (LPR) from 3.1 +/- 0.5 to 10.0 +/- 1.6 (P < .05), indicating a rise in the ratio of reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD+) redox state of the cell. Insulin's stimulation of cGMP production was blocked by 0.1 mmol/L NG-monomethyl-L-arginine (L-NMMA) indicating dependence on iNOS activity, but insulin did not affect iNOS activity. Blocking insulin's increase in LPR by pyruvate (0.5 mmol/L) or oxaloacetate (0.5 mmol/L) completely inhibited the insulin-stimulated component of cGMP production. Pyruvate also blocked insulin's inhibition of serotonin-induced contraction in nonproliferated cells. In the absence of insulin, 5 mmol/L lactate or isocitrate increased the LPR by 420% +/- 47% and 167% +/- 20%, respectively (both P < .05), and stimulated cGMP production by 1,045% +/- 272% and 278% +/- 33%, respectively (both P < .05) by an L-NMMA-inhibitable mechanism. Although cGMP production in cells with iNOS was increased by insulin, the stimulation of cGMP production in cells without iNOS by 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) was not affected by insulin, suggesting that insulin does not stimulate guanylate cyclase activity directly.


CONCLUSIONS
We conclude that insulin increases cGMP production in VSMC with iNOS by raising the cell NADH/NAD+ redox state, which may increase the availability of iNOS-derived NO.
Kahn
Andrew M
AM
Department of Medicine. The University of Texas Health Science Center, Houston 77225, USA.
Allen
Julius C
JC
Zhang
Sui
S
D000818:Animals; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000096:biosynthesis; D004285:Dogs; D006162:Guanylate Cyclase / Q000378:metabolism*; D007191:Indazoles / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007523:Isocitrates / Q000494:pharmacology; D019344:Lactic Acid / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D062907:Oxaloacetic Acid / Q000494:pharmacology; D010084:Oxidation-Reduction; D019289:Pyruvic Acid / Q000494:pharmacology; D013268:Stimulation, Chemical
D007191:Indazoles; D007328:Insulin; D007523:Isocitrates; D009243:NAD; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D062907:Oxaloacetic Acid; D019344:Lactic Acid; D019289:Pyruvic Acid; C034219:isocitric acid; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester