Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea-pig ileum

1. Ca2+ sensitization of smooth muscle contraction involves the small GTPas e RhoA, inhibition of myosin light chain phosphatase (MLCP) and enhanced my osin regulatory light chain (LC20) phosphorylation. A potential effector of RhoA is Rho-associated kinase (ROK). 2. The role of ROK in Ca2+ sensitization was investigated in guinea-pig ile um. 3. Contraction of permeabilized muscle strips induced by GTP gamma S at pCa 6.5 was inhibited by the kinase inhibitors Y-27632, HA1077 and H-7 with IC 50 values that correlated with the known K-i values for inhibition of ROK. GTP gamma S also increased LC20 phosphorylation and this was prevented by H A1077. Contraction and LC50 phosphorylation elicited at pCa 5.75 were, howe ver, unaffected by HA1077. 4. Pre-treatment of intact tissue strips with HA1077 abolished the tonic co mponent of carbachol-induced contraction and the sustained elevation of LC2 0 phosphorylation, but had no effect on the transient or sustained increase in [Ca2+](1) induced by carbachol. 5. LC20 phosphorylation and contraction dynamics suggest that the ROK-media ted increase in LC20 phosphorylation is due to MLCP inhibition, not myosin light chain kinase activation. 6. In the absence of Ca2+, GTP gamma S stimulated S-35 incorporation from [ S-35]ATP gamma S into the myosin targeting subunit of MLCP (MYPT). The enha nced thiophosphorylation was inhibited by HA1077. No thiophosphorylation of LC20 was detected. 7. These results indicate that ROK mediates agonist-induced increases in my osin phosphorylation and force by inhibiting MLCP activity through phosphor ylation of MYPT. Under Ca2+-free conditions, ROK does not appear to phospho rylate LC20 in situ, in contrast to its ability to phosphorylate myosin in vitro. In particular, ROK activation is essential for the tonic phase of ag onist-induced contraction.