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.

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.