Protein kinase C mediates insulin-inhibited Ca2+ transport and contraction of vascular smooth muscle

Insulin acutely inhibits contraction of primary cultured vascular smooth muscle (VSM) cells from canine femoral artery by inhibiting contractile agonist-induced Ca2+ influx. Insulin also inhibits contraction at step(s) distal to intracellular Ca2+ concentration (Ca2+i) by stimulating cyclic guanosine monophosphate (GMP) production. We wished to see whether these effects of insulin are mediated by protein kinase C (PKC). Ca2+ influx was assessed by measuring the rate of fluorescence quenching of intracellular fura 2 by extracellular Mn2+. We found that 10 μmol/L serotonin (5-HT) stimulated Mn2+ influx 3-fold, and 1 nmol/L insulin inhibited the 5-HT–stimulated component of Mn2+ influx by 63% (P< .05), but insulin had no effect in the presence of 1 μmol/L staurosporine, an inhibitor of PKC. In the absence of insulin, preincubating cells with 0.1 μmol/L phorbol 12-myristate 13-acetate (PMA) for 5 min inhibited the 5-HT–stimulated component of Mn2+ influx by 69% (P< .05). Insulin inhibited cell contraction induced by raising Ca2+i to supraphysiologic levels with ionomycin by 75% (P< .05). We also noted that 10−6 mol/L calphostin C, another PKC inhibitor, or 16-h preincubation with PMA completely blocked this effect of insulin. Finally, 10-min exposure to insulin or PMA increased cyclic GMP production in ionomycin-treated cells by 50% and 64%, respectively (both P< .05). We conclude that insulin inhibits VSM cell contraction by inhibiting 5-HT–stimulated Ca2+ influx and also at step(s) distal to Ca2+i by a PKC-dependent mechanism.