Role of Rho-kinase and protein kinase C during contraction of hypertrophic detrusor in mice with partial urinary bladder outlet obstruction.

Abstract

To study muscarinic/purinergic receptor activation and Rho-kinase/protein kinase C (PKC) signalling during smooth muscle contraction in normal and hypertrophic mouse urinary bladders. Partial urinary outflow obstruction was induced in adult female (10-12 weeks) C57Bl/6 mice and comparisons were made with sham-operated controls. Bladder preparations were examined in vitro. Expression of signalling proteins was examined using Western blot analysis. Obstructed bladders increased more than threefold in weight and were found to have enhanced muscarinic and attenuated purinergic components during nerve-induced contractions. The contractile response to carbachol was shifted towards lower concentrations of carbachol for the peak response and had a markedly enhanced sustained component. The amplitude of the α,β-methylene ATP-induced responses was lowered. Rho-kinase inhibitor Y27632 (10 µM) inhibited peak and sustained contractile responses to carbachol in control bladders (peak by 38%; plateau 57%) and obstructed bladders (peak 37% plateau 47%). PKC inhibitor GF109203X (1 µM) inhibited carbachol contractions in controls (peak by 29%; plateau 29%) and obstructed bladders (peak 17%; plateau 12%). Inhibition by a similar extent was observed after nerve stimulation. Sensitivity to Ca(2+) in high-K(+) depolarized intact tissues increased in obstructed bladders. This increased receptor-independent Ca(2+)-sensitivity was abolished by Y27632. Tissue contents of the myosin-binding phosphatase subunit MYPT-1 and catalytic phosphatase subunit PP1β, were decreased and the contents of RhoGDI, RhoA and CPI-17 increased. A decrease in the Rho-kinase isoform ROCK-1 was observed. Based on these results, one can speculate that Rho-kinase inhibition would preferentially target the pathological phasic activity in the urinary bladder rather than inhibit the physiological receptor-mediated bladder emptying.