Abstract
We have investigated the role of several protein kinases in carbachol-stimulated, M3 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 μM; calphostin C, 0.1–1 μM; and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (Gö 6850), 1–10 μM), by the tyrosine kinase inhibitor genistein or its inactive control daidzein (3–30 μM each), or by two inhibitors of activation of mitogen-activated protein kinase [10–100 μM2′-amino-3′-methoxyflavone (PD 98,059) and 3–30 μM 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 μM). Although high concentrations of wortmannin (3–30 μM) 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 μM) 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 μM), 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.
Footnotes
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This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (Mi 294/7-1). M.F. and T.S. were recipients of training fellowships from the Deutscher Akademischer Austauschdienst and the intramural grant program of the University of Essen Medical School (IFORES), respectively.
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DOI: 10.1124/jpet.103.058255.
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ABBREVIATIONS: PKC, protein kinase C; PI-3-kinase, phosphatidylinositol-3-kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinases; PD 98,059, 2′-amino-3′-methoxyflavone; LY 294,002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; LY 303,511, 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one; U 124, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; U 126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; Y 27,632, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide; DMSO, dimethylsulfoxide; Gö 6850, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide.
- Received August 6, 2003.
- Accepted September 24, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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