Schneider Tim, Fetscher Charlotte, Krege Susanne, Michel Martin C
Department of Medicine, University of Essen, Germany.
J Pharmacol Exp Ther. 2004 Jun;309(3):1148-53. doi: 10.1124/jpet.103.063735. Epub 2004 Feb 9.
The present study was designed to reexamine the muscarinic acetylcholine receptor subtype mediating carbachol-induced contraction of human urinary bladder and to investigate the underlying signal transduction. Based upon the nonselective tolterodine, the highly M(2)-selective (R)-4-[2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl]piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M(3)-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M(3) receptors. The phospholipase C inhibitor 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122) (1-10 microM) did not significantly affect carbachol-stimulated bladder contraction. The phospholipase D inhibitor butan-1-ol relative to its negative control butan-2-ol (0.3% each) caused small but detectable inhibition of carbachol-induced bladder contraction. The Ca(2+) entry blocker nifedipine (10-100 nM) strongly inhibited carbachol-induced bladder contraction. In contrast, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SK&F 96,365) (1-10 microM), an inhibitor of store-operated Ca(2+) channels, caused little inhibition. The protein kinase C inhibitor bisindolylmaleimide I (1-10 microM) did not significantly affected carbachol-induced bladder contraction. 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 human urinary bladder via M(3) receptors largely depends on Ca(2+) entry through nifedipine-sensitive channels and activation of a rho kinase, whereas phospholipase D and store-operated Ca(2+) channels contribute only in a minor way. Surprisingly, phospholipase C or protein kinase C do not seem to be involved to a relevant extent.
本研究旨在重新审视介导卡巴胆碱诱导人膀胱收缩的毒蕈碱型乙酰胆碱受体亚型，并研究其潜在的信号转导机制。基于非选择性的托特罗定、高度选择性的M(2)型(R)-4-[2-[3-(4-甲氧基苯甲酰氨基)-苄基]-哌啶-1-基甲基]哌啶-1-羧酸酰胺（Ro-320-6206），以及高度选择性的M(3)型达非那新和3-(1-氨基甲酰基-1,1-二苯基甲基)-1-(4-甲氧基苯乙基)吡咯烷（APP），收缩是通过M(3)受体发生的。磷脂酶C抑制剂1-(6-[([17β]-3-甲氧基雌甾-1,3,5[10]-三烯-17-基)氨基]己基)-1H-吡咯-2,5-二酮（U 73,122）（1-10微摩尔）对卡巴胆碱刺激的膀胱收缩没有显著影响。相对于其阴性对照丁醇-2（各0.3%），磷脂酶D抑制剂丁醇-1对卡巴胆碱诱导的膀胱收缩有轻微但可检测到的抑制作用。钙离子通道阻滞剂硝苯地平（10-100纳摩尔）强烈抑制卡巴胆碱诱导的膀胱收缩。相比之下，1-[β-[3-(4-甲氧基苯基)丙氧基]-4-甲氧基苯乙基]-1H-咪唑盐酸盐（SK&F 96,365）（1-10微摩尔），一种储存-操作性钙离子通道抑制剂，几乎没有抑制作用。蛋白激酶C抑制剂双吲哚马来酰亚胺I（1-10微摩尔）对卡巴胆碱诱导的膀胱收缩没有显著影响。相比之下，rho相关激酶抑制剂反式-4-[(1R)-1-氨基乙基]-N-4-吡啶基环己烷甲酰胺（Y 27,632）（1-10微摩尔）浓度依赖性地有效减弱了卡巴胆碱反应。我们得出结论，卡巴胆碱通过M(3)受体诱导人膀胱收缩主要依赖于通过硝苯地平敏感通道的钙离子内流和rho激酶的激活，而磷脂酶D和储存-操作性钙离子通道仅起次要作用。令人惊讶的是，磷脂酶C或蛋白激酶C似乎在很大程度上不参与其中。
