Expression of RhoA mRNA and activated RhoA in urothelium and smooth muscle, and effects of a Rho-kinase inhibitor on contraction of the porcine urinary bladder

Abstract

Aims: To investigate the concentration and activity of RhoA in detrusor and urothelium, as well as the effects of a Rho-kinase inhibitor, Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride], on contraction of the pig urinary bladder.

Methods: The concentration of RhoA mRNA was studied by the real-time RT-PCR and activated RhoA enzyme was studied by activation assay and Western blotting. In functional studies, the response to Y-27632 was examined in bladder strips with or without urothelium.

Results: The concentration of RhoA mRNA (n = 38) and activated RhoA enzyme (n = 19) were greater in urothelium than in detrusor. Tension decrease after administration of Y-27632 (1 nM to 100 microM) was significantly greater in tissues with urothelium than in tissues without urothelium, after pre-contraction with KCl (decrease by 52.0 +/- 4.6% vs. 28.0 +/- 6.8%, respectively; P = 0.0088) or with carbachol (decrease by 53.1 +/- 7.2% vs. 30.6 +/- 5.8%, respectively; P = 0.0035). Maximum contraction on CRC to carbachol was reduced significantly after administration of 3, 10, and 30 microM Y-27632 (to 72.2 +/- 6.8%, 43.9 +/- 7.1%, and 25.0 +/- 5.5%, respectively, of the control value) in strips with intact urothelium (n = 36), but was reduced only at 10 and 30 microM (to 66.7 +/- 8.3% and 85.6 +/- 2.6%, respectively) in tissues without urothelium (n = 20). Inhibitory effect of Y-27632 (3-30 microM) on the response to electrical field stimulation at 20 and 50 Hz was also greater in tissues with intact urothelium than in tissues without urothelium.

Conclusions: The concentration of RhoA mRNA and activated RhoA enzyme were greater in urothelium than in detrusor. Y-27632 showed a stronger inhibitory effect in detrusor with intact urothelium than in dose without urothelium.