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 microM), phenylephrine (PE, 0.01-300 microM) 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 microM), Y-27632 (1-10 microM) or HA-1077 (10 microM). Addition of Rho-kinase inhibitors also markedly reduced (p<0.01) the contractions evoked by either KCl (80 mM) or alpha,beta-methylene ATP (alpha,beta-mATP, 10 microM). 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 microM), H-1152 (1 microM) and HA-1077 (10 microM). The expression of RhoA, RhoGDI, leukemia-associated RhoGEF (LARG) and p115RhoGEF was similar among the detrusor, trigone and urethra, whereas Rho-kinase alpha, Rho-kinase beta 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 Ca(2+) sensitization in the lower urinary tract.