Signal transduction underlying carbachol-induced contraction of rat urinary bladder. II. Protein kinases

We have investigated the role of several protein kinases in carbachol-stimulated, M(3) muscarinic receptor-mediated contraction of rat urinary bladder. Concentration-response curves for the muscarinic receptor agonist carbachol were generated in the presence of multiple concentrations of inhibitors of various protein kinases, their inactive controls, or their vehicles. Bladder contraction was not significantly inhibited by three protein kinase C inhibitors (chelerythrine, 1-10 microM; calphostin C, 0.1-1 microM; and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (Gö 6850), 1-10 microM), by the tyrosine kinase inhibitor genistein or its inactive control daidzein (3-30 microM each), or by two inhibitors of activation of mitogen-activated protein kinase [10-100 microM 2'-amino-3'-methoxyflavone (PD 98,059) and 3-30 microM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U 124)] or their negative control 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U 126) (3-30 microM). Although high concentrations of wortmannin (3-30 microM) inhibited bladder contraction, this was not mimicked by another inhibitor of phosphatidylinositol-3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294,002) (3-30 microM) and, hence, was more likely due to direct inhibition of myosin light chain kinase by wortmannin than to an involvement of phosphatidylinositol-3-kinase. In contrast, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y 27,632) (1-10 microM), an inhibitor of rho-associated kinases, concentration-dependently and effectively attenuated the carbachol responses. We conclude that carbachol-induced contraction of rat urinary bladder does not involve protein kinase C, phosphatidylinositol-3-kinase, tyrosine kinases, or extracellular signal-regulated kinases; in contrast, rho-associated kinases appear to play an important role in the regulation of bladder contraction.