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

Abstract Insulin acutely inhibits contraction of primary cultured vascular smooth muscle (VSM) cells from canine femoral artery by inhibiting contractile agonist-induced Ca 2 + influx. Insulin also inhibits contraction at step(s) distal to intracellular Ca 2 + concentration (Ca 2 + i ) by stimulating cyclic guanosine monophosphate (GMP) production. We wished to see whether these effects of insulin are mediated by protein kinase C (PKC). Ca 2 + influx was assessed by measuring the rate of fluorescence quenching of intracellular fura 2 by extracellular Mn 2 + . We found that 10 μmol/L serotonin (5-HT) stimulated Mn 2 + influx 3-fold, and 1 nmol/L insulin inhibited the 5-HT–stimulated component of Mn 2 + 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 Mn 2 + influx by 69% ( P .05). Insulin inhibited cell contraction induced by raising Ca 2 + 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 Ca 2 + influx and also at step(s) distal to Ca 2 + i by a PKC-dependent mechanism.