Ca 2+ sensitization and the regulation of contractility in rat anococcygeus and retractor penis muscle

Abstract

Stimulation of the RhoA/Rho-kinase (ROK) signaling represents a key step in the maintenance of agonist-induced contraction of smooth muscle. We aimed to demonstrate Ca 2+ sensitization in rat anococcygeus and retractor penis muscles and to identify the molecular expression of major components of this pathway. Both anococcygeus and retractor penis showed a similar expression of RhoA, ROKα, and ROKβ at the protein level as well as the mRNA for RhoGEFs. Cumulative addition of the ROK inhibitors H-1152 (0.001–3 μM), Y-27632 (0.01–30 μM) or HA-1077 (0.01–30 μM) caused sustained relaxations of precontracted smooth muscle strips. Ca 2+ sensitization induced by phenylephrine, norepinephrine and carbachol was markedly antagonized by all three ROK inhibitors. In addition, the contractile response to KCl-induced depolarization was highly sensitive to these ROK inhibitors. H-1152 was approximately 8–20 more potent than Y-27632 and HA-1077 to inhibit contraction. Electrical field stimulation (EFS, 1–32 Hz) caused transient contractions in both anococcygeus and retractor penis muscle, which were blocked by tetrodotoxin (1 μM), phentolamine (1 μM) or bretylium tosylate (30 μM). Similarly, H-1152 (0.1–1 μM), Y-27632 (1–10 μM) or HA-1077 (1–10 μM) significantly reduced EFS-evoked contractions in a concentration-dependent manner. The results indicate that the RhoA/ROK-mediated Ca 2+ sensitization pathway is expressed in anococcygeus and retractor penis muscles and enhances contractions produced by receptor-dependent and independent mechanisms.