Signal Transduction Underlying Carbachol-Induced Contraction of Rat Urinary Bladder. II. Protein Kinases

Abstract

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 M; calphostin C, 0.1-1 M; and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (Gö 6850), 1-10 M), by the tyrosine kinase inhibitor genistein or its inactive control daidzein (3-30 M each), or by two inhibitors of activation of mitogenactivated protein kinase [10 -100 M 2Ј-amino-3Ј-methoxyflavone (PD 98,059) and 3-30 M 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 M). Although high concentrations of wortmannin (3-30 M) 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 M) 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 M), 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, rhoassociated kinases appear to play an important role in the regulation of bladder contraction.Muscarinic acetylcholine receptors are the physiologically most important mechanism to mediate contraction of the urinary bladder (Andersson, 1993). Although rat bladder expresses more M 2 than M 3 receptors, contractile responses to the exogenous muscarinic agonist carbachol and to endogenous agonists released by field stimulation occur predominantly, if not exclusively, via M 3 receptors in rats (Longhurst et al

M2 and M3 Muscarinic Receptor Activation of Urinary Bladder Contractile Signal Transduction. I. Normal Rat Bladder

“…Our data differ from other reports of PI-PLC mediating rat and human bladder contraction (Fleichman et al, 2004;Schneider et al, 2004a,b). These reports concluded that bladder contraction via M 3 receptors largely depends on calcium entry through nifedipine-sensitive channels and activation of ROCK, whereas phospholipase D and store-operated calcium channels contribute in a minor way, and phospholipase C or PKC do not seem to be involved.…”

Does Cyclic AMP Mediate Rat Urinary Bladder Relaxation by Isoproterenol?

“…Recently, we have intensively characterized the signaling pathways underlying muscarinic receptor-mediated contraction of rat and human urinary bladder (Fleichman et al, 2004;Schneider et al, 2004a,b). In the present study, we have investigated the signal transduction underlying rat urinary bladder relaxation by the ␤-adrenergic agonist isoproterenol.…”

Pharmacology of the Lower Urinary Tract: Basis for Current and Future Treatments of Urinary Incontinence

“…They also demonstrated high levels of Rho-kinase isoforms (I and II) in the bladder. Supporting a role for Rho-kinase in detrusor contraction, Fleichman et al (2004) demonstrated in the rat bladder that carbachol-induced contraction did not involve protein kinase C, phosphatidylinositol-3-kinase, tyrosine kinases, or extracellular signal-regulated kinases, but that Rhoassociated kinases were involved. In the human detrusor, Schneider et al (2004) confirmed that the muscarinic receptor subtype mediating carbachol-induced contraction was the M 3 receptor: They also demonstrated that the phospholipase C inhibitor U-73122 [1-[6-[[17␤-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione] did not significantly affect carbachol-stimulated bladder contraction, despite blocking IP 3 generation.…”