The effects of the Rho-kinase inhibitor Y-27632 on arachidonic acid-, GTPγS-, and phorbol ester-induced Ca2+-sensitization of smooth muscle

The effects of the Rho-kinase inhibitor, Y-27632 [1] on Ca2+-sensitization of force induced by arachidonic acid (AA), phorbol 12,13-dibutyrate (PDBu), GTP gamma S, and by the stable thromboxane analog, 9,11-dideoxy-9 alpha,11 alpha-methanoepoxy-PGF(2 alpha) (U-46619), were determined in alpha-toxin-permeabilized smooth muscles. Y-27632 relaxed (up to 99%) Ca2+-sensitization by GTP gamma S (10 mu M) and U-46619 (1 mu M), but not by PDBu (20 mu M), and reduced GTP gamma S-induced myosin light chain (MLC20) phosphorylation from 28% to 17% (P = 0.002), GTP gamma S-induced force sensitization was inhibited by Y-27632 more potently when the inhibitor was added during the plateau of force than prior to stimulation. In alpha-toxin-permeabilized smooth muscle, Y-27632 inhibited AA (50 mu M)-induced Ca2+-sensitization of force (by 66 +/- 1.3%) and reduced MLC20 phosphorylation, In contrast, Y-27632 did not relax force Ca2+-sensitized by AA in smooth muscle permeabilized with Triton X-100, We conclude that (i) AA induces Ca2+-sensitization through dual mechanisms, one mediated by Rho-kinase (or a related kinase), and (ii) Rho-kinase is not required for phorbol ester-induced Ca2+-sensitization. (C) 1998 Federation of European Biochemical Societies.