Abstract 5257: Inhibition of Myosin Light Chain Phosphatase by Rho Kinase (ROCK) Modulates Arteriolar Tone and Constriction of Coronary Arterioles
ROCK has been suggested to regulate smooth muscle contraction of conduit artery via multiple pathways including direct myosin light chain (MLC) phosphorylation or inhibition of MLC phosphatase (MLCP) activity. Although recent studies have implicated the involvement of ROCK in pathogenesis of cardiovascular disease, the role and expression of ROCK isoforms (ROCK1 and ROCK2) and their signaling pathway for vasomotor regulation in resistance arterioles remain unclear. Using an isolated and pressurized vessel approach, we found that the resting tone and the endothelin-1 (ET-1)-induced constriction of porcine coronary arterioles (60±4 μm resting diameter) were preferentially inhibited, in a dose-dependent manner, by a potent ROCK2 inhibitor H-1152 compared to a non-selective ROCK antagonist Y-27632. MLCP inhibitor calyculin A reversed the inhibitory effects of H-1152 and Y-27632, but ROCK inhibition failed to prevent or reverse calyculin A-induced arteriolar constriction. These results indicate that ROCK is involved in the development of basal tone and vasoconstriction to ET-1 and that MLCP is a major target of ROCK. Immunoblotting and immunohistochemical data showed that ROCK2 expression is 2-fold higher than ROCK1 in coronary arterioles with preferential media-layer distribution. Both ROCK1 and ROCK2 were also expressed in cultured porcine coronary arteriolar smooth muscle cells (PCASMC) with ROCK2 as the major isoform. In cultured PCASMC, MLC phosphorylation (ser19) elicited by ET-1, thromboxane analogue (U46619) and 80 mM KCl was sensitive to H-1152, supporting the involvement of ROCK2 in regulating coronary arteriolar tone via MLC phosphorylation. Consistent with our functional data, calyculin A increased MLC phosphorylation (ser19) in PCASMC, but ROCK2 inhibition (by H-1152) failed to reverse such effect. We conclude that ROCK2 is the major isoform mediating the development of basal tone and vasoconstrictor activity in coronary resistance arterioles primarily via regulation of MLCP activity.