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

Insulin acutely inhibits contraction of primary cultured vascular smooth mu scle (VSM) cells from canine femoral artery by inhibiting contractile agoni st-induced Ca2+ influx. Insulin also inhibits contraction at step(s) distal to intracellular Ca2+ concentration (Ca-i(2+)) by stimulating cyclic guano sine monophosphate (GMP) production. We wished to see whether these effects of insulin are mediated by protein kinase C (PKC), Ca2+ influx was assesse d by measuring the rate of fluorescence quenching of intracellular fura 2 b y extracellular Mn2+. We found that 10 mu mol/L serotonin (5-HT) stimulated Mn2+ influx 3-fold, and 1 nmol/L insulin inhibited the 5-HT-stimulated com ponent of Mn2+ influx by 63% (P < .05), but insulin had no effect in the pr esence of 1. mu mol/L staurosporine, an inhibitor of PKC. In the absence of insulin, preincubating: cells with 0.1 mu mol/L phorbol 12-myristate 13-ac etate (PMA) for 5 min inhibited the 5-HT-stimulated component of Mn2+ influ x by 69% (P < .05). Insulin inhibited cell contraction induced by raising C a-i(2+) 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 prein cubation with PMA completely blocked this effect of insulin. Finally, 10-mi n exposure to insulin or PMA increased cyclic GMP production in ionomycin-t reated cells by 50% and 64%, respectively (both P < .05). We conclude that insulin inhibits VSM cell contraction by inhibiting 5-HT-stimulated Ca2+ in flux and also at step(s) distal to Ca-i(2+) by a PKC-dependent mechanism. ( C) 2000 American Journal of Hypertension, Ltd.