A novel mechanism for the Ca(2+)-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase.

Mechanisms of Ca 2+ sensitization of both myosin light chain (MLC) phosphorylation and force development by protein kinase C (PKC) were studied in permeabilized tonic smooth muscle obtained from the rabbit femoral artery. For comparison, the Ca 2+ sensitizing effect of guanosine 5'-O-(v-thiotriphosphate) (GTP~/S) was examined, which had been previously shown to inhibit MLC phospha- tase in phasic vascular smooth muscle. We now report that PKC activators (phorbol esters, short chain synthetic diacylglycerols and a diacylglycerol kinase inhibitor) and GTP~/S significantly increase both MLC phosphorylation and force develop- ment at constant (Ca2+). Major phosphorylation site occurring in the presence of phorbol-12,13-dibutyrate (PDBu) or GTP~S at constant (Ca ~+) is the same serine residue (Ser-19) as that phosphorylated by MLC kinase in response to increased Ca ~+ concentrations. In an ATP- and Ca2+-free solution containing 1-(5-chloro- naphthalene-l-sulfonyl)-lH-hexahydro-l,4-diazepine (ML-9), to avoid the kinase activity, both PDBu and GTP~/S significantly decreased the rate of MLC dephos- phorylation to half its control value. However, PDBu inhibited the relaxation rate more than did GTP~/S. In the presence of microcystin-LR to inhibit the phosphatase activity, neither PDBu nor GTP~/S affected MLC phosphorylation and force devel- opment. These results indicate that PKC, like activation of GTP binding protein, increases Ca 2+ sensitivity of both MLC phosphorylation and force production through inhibition of MLC phosphatase.