Involvement of Rho-kinase-mediated phosphorylation of myosin light chain in enhancement of cerebral vasospasm

Subarachnoid hemorrhage (SAH) often induces a long-term narrowing of the ce rebral artery called cerebral vasospasm, Myosin light chain (MLC) in the sp astic basilar artery was reported previously to be phosphorylated by Ca2+/c almodulin-dependent MLC kinase. Because Rho-kinase, which is activated by t he small GTPase Rho, phosphorylates not only MLC but also myosin phosphatas e at its myosin-binding subunit (MBS), thus inactivating myosin phosphatase , we examined whether Rho-kinase is involved in the development of vasospas m. Cerebral vasospasm was produced in the canine basilar artery by a 2-hemo rrhage method, and vasocontractions were induced by topical application of 80 mmol/L KCl or 0.5 mu mol/L serotonin to the canine basilar artery expose d transclivally. The phosphorylation of MLC in the basilar artery was incre ased concurrently with an enhancement in the intensity of vasospasm with th e passage of time after SAH. In addition, Rho-kinase in the basilar artery was activated concurrently with an increase in the phosphorylation of MBS a t Ser854 in vasospasm. The Rho-kinase activation levels in vasospasm on day s 0 and 2 were comparable to those in KCl- and serotonin-induced sustained vasocontraction, respectively, and those in vasospasm on day 7 were markedl y high. The topical application of Y-27632, a specific inhibitor of Rho-kin ase, to the exposed spastic basilar artery on day 7 induced a dose-dependen t dilation, and the intensities of vasospasm and the phosphorylation of MBS and MLC were simultaneously decreased by 10 mu mol/L Y-27632, although the decrease in MBS phosphorylation was more marked than the decrease in MLC p hosphorylation. These results indicate that the activation of Rho-kinase an d the phosphorylation of MLC and MBS occur concomitantly during vasospasm i nduced by SAH and suggest that Rho-kinase is involved in the enhancement of cerebral vasospasm in addition to Ca2+/calmodulin-dependent MLC kinase by increasing the phosphorylation of MLC directly or indirectly as a result of the inhibition of myosin phosphatase by its phosphorylation.