A ROLE FOR RHO-KINASE IN Ca2+-INDEPENDENT CONTRACTIONS INDUCED BY PHORBOL-12,13-DIBUTYRATE

1. Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca2+-depleted solutions. In the present study, we tested the hypothesis that Rho-kinase plays a role in Ca2+-independent contractions induced by PDBu in vascular smooth muscles. 2. In Ca2+-free solution, 0.1 and 1 mu mol/L PDBu induced contraction and myosin light chain (MLC20) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 mu mol/L), an inhibitor of Rho-kinase, but not ML7 (10 mmol/L), an inhibitor of myosin light chain kinase, inhibited Ca2+-independent contractions induced by PDBu. 3. In Ca2+-free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI-17 almost completely abolished by 1 mu mol/L Ro31-8220, an inhibitor of PKC. 4. In Ca2+-free solution, PDBu increased the amount of GTP-RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31-8220, but not by the Rho kinase inhibitor H1152. 5. In conclusion, RhoA/Rho-kinase plays an important role in Ca2+-independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca2+-independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP-RhoA and subsequent phosphorylation of MYPT1 and CPI-17.