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

Abstract

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), GTPγS, and by the stable thromboxane analog, 9,11-dideoxy-9α,11α-methanoepoxy-PGF_2α (U-46619), were determined in α-toxin-permeabilized smooth muscles. Y-27632 relaxed (up to 99%) Ca²⁺-sensitization by GTPγS (10 μM) and U-46619 (1 μM), but not by PDBu (20 μM), and reduced GTPγS-induced myosin light chain (MLC₂₀) phosphorylation from 28% to 17% (P=0.002). GTPγS-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 α-toxin-permeabilized smooth muscle, Y-27632 inhibited AA (50 μM)-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.