Comparative pharmacological analysis of Rho-kinase inhibitors and identification of molecular components of Ca²⁺ sensitization in the rat lower urinary tract

We aimed to compare the expression and function of molecular components of the RhoA/Rho-kinase signaling pathway in the contractile responses of detrusor, trigonal and urethral smooth muscle, using selective Rho-kinase inhibitors. Contractility studies and molecular approaches were employed to demonstrate the expression patterns and functional activity of the RhoA/Rho-kinase signaling pathway in the lower urinary tract. Frequency-response curves (1-32 Hz) and concentration-response curves (CRC) to carbachol (CCh, 0.01-30 μM), phenylephrine (PE, 0.01-300 μM) and endothelin-1 (ET-1, 0.01-100 nM) were significantly attenuated (p < 0.01) following incubation with the Rho-kinase inhibitors H-1152 (0.1-1 μM), Y-27632 (1-10 μM) or HA-1077 (10 μM). Addition of Rho-kinase inhibitors also markedly reduced (p < 0.01) the contractions evoked by either KCl (80 mM) or α,β-methylene ATP (α,β-mATP, 10 μM). Among the Rho-kinase inhibitors tested, H-1152 was approximately 9-16 times more potent than Y-27632 or HA-1077. In addition, basal tone of detrusor and trigonal strips was reduced following addition of Y-27632 (10 μM), H-1152 (1 μM) and HA-1077 (10 μM). The expression of RhoA, RhoGDI, leukemia-associated RhoGEF (LARG) and p115RhoGEF was similar among the detrusor, trigone and urethra, whereas Rho-kinase α, Rho-kinase β and PDZ-RhoGEF protein levels were significantly lower in the urethra. Components of the RhoA/Rho-kinase signaling are expressed in detrusor, trigonal and urethral smooth muscle and dynamically regulate contraction and tone. Manipulation of RhoGEF expression may provide further understanding of mechanisms involving Ca²⁺ sensitization in the lower urinary tract.