Ca²⁺ sensitization and the regulation of contractility in rat anococcygeus and retractor penis muscle

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 Ca²⁺ 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. Ca²⁺ 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 Ca²⁺ sensitization pathway is expressed in anococcygeus and retractor penis muscles and enhances contractions produced by receptor-dependent and independent mechanisms.