A role for rho-kinase in Ca²⁺-independent contractions induced by phorbol-12,13-dibutyrate

Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca ²⁺-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. In Ca²⁺-free solution, 0.1 and 1 μmol/L PDBu induced contraction and myosin light chain (MLC ₂₀) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 μmol/L), an inhibitor of Rho-kinase, but not ML7 (10 μmol/L), an inhibitor of myosin light chain kinase, inhibited Ca²⁺-independent contractions induced by PDBu. 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 μmol/L Ro31-8220, an inhibitor of PKC. 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. 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.