Hi 152

a p21-Activated kinase.

with various Ki values in ATP-competitive fashion with a range of Ki values. The Ki values for Rho kinase and PKC were comparatively low, 0.35 and 0.90 mM, respectively. In an isolated arterial strip stimulated with PGF2a, we determined that HA1077 inhibits the phosphorylation of the regulatory myosin light chain (MLC20), in particular the diphosphorylation step was more sensitive than the monophosphorylation reaction (Seto et al. 1991).

HA1077 was an effective inhibitor of arterial contraction and MLC20 diphosphorylation at sub-micromolar concentrations, but the Ki for MLCK was 55 mM. This fact indicates that the inhibition of MLCK by HA1077 was not likely to be related to its pharmacological action. Subsequently, we and others have demonstrated that MLC diphosphorylation was associated with an inhibition of myosin-phosphatase through the phosphorylation of the myosin binding subunit (MBS 130 kDa) of protein phosphatase 1 by Rho kinase (Kimura et al. 1996; Nagumo et al. 2000). In fact, we demonstrated that HA1077 inhibits PGF2a-induced arterial contraction co-incident with inhibition of MBS-phosphatase (Ito et al. 2003). We believed it was likely that Rho kinase inhibition was likely to be responsible for the mechanism of action of HA1077 and that it could explain its pharmacological activities. Moreover, we discovered that HA1077 also inhibits the phosphorylation of calponin in a porcine coronary artery stimulated with PGF2a (Nagumo et al. 1998). At present, calponin phosphorylation is recognized to be alternatively catalyzed by both PKC and Rho kinase (Kaneko et al. 2000). The regulation of myosin

Pgf2a Mechanism Action
Fig. 7 Site of action of the Rho-kinase inhibitor HA1077 in arterial smooth muscle. Arrowheads show the target proteins, Rho-kinase and PKC. Myo-Pase, myosin phosphatase 1 with 130 kDa myosin binding subunit; CN, calponin; CPI-17, 17-kDa inhibitor protein of type 1 phosphatase; DA, diacylglycerol

phosphatase activity and calponin phosphorylation, which is dependent on Rho kinase or/and PKC, seems to control the calcium sensitivity of smooth muscle contraction. Taken together, HA1077 may have two sites of action in the mechanism of inhibition of smooth muscle contraction (Fig. 7).

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