Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells

In smooth muscle, a Rho-regulated system of myosin phosphatase exists; howe ver, it has yet to be established whether Rho kinase, one of the downstream effecters of Rho, mediates the regulation of myosin phosphatase activity i n vivo. In the present study, we demonstrate in permeabilized vascular smoo th muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homo piperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca2+-induced 20-kDa myosin light chain (MLC20) phosphorylation due to abrogating Rho-mediated i nhibition of MLC20 dephosphorylation. By an immune complex phosphatase assa y, we found that guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) stimulat ion of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-bin ding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent mann er. HA-1077 abolished both of the Rho-mediated events. Moreover, we observe d that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTP gamma S-induced p hosphorylation of MBS. These results provide direct in vivo evidence that R ho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC20 phosphorylation in smooth muscle and reveal the usefu lness of HA-1077 as a Rho kinase inhibitor.