Determination of controlling factor of detrusor contraction in urothelium and the role of Rho Kinase

采用放射配基结合实验、实时荧光定量RT-PCR、Western blotting和免疫组织化学等方法，研究了猪和人膀胱逼尿肌M受体亚型、肾上腺素受体亚型和RhoA/Rho激酶的mRNA和蛋白表达及其活性。结果表明，M2受体亚型主要存在于猪膀胱的尿道和逼尿肌中。在β受体亚型中，β3肾上腺素受体主要分布在猪和人的逼尿肌中，RhoA在猪和人的膀胱逼尿肌中的表达比在膀胱平滑肌中的表达要晚。在功能研究中，我们检测了Rho激酶抑制剂Y-27632和法舒地尔对猪膀胱平滑肌RhoA活性的影响。 ...更多信息 有或没有尿道炎的组织条。Y-27632给药后体重下降在用KCl（分别降低52.0±4.6% vs 28± 6.8%; p=0.0088）或卡巴胆碱（分别降低53.1±7.2% vs 30.6± 5.81%; p=0.0035）预收缩后，具有完整尿路上皮的组织中的（InM-100μM）显著大于无尿路上皮的组织。给予3、10和30μM Y-27632后，CRC对卡巴胆碱的最大收缩显著降低（分别为对照值的72.2± 6.8%、43.9± 7.1%和25.0 ± 5.5%），但仅在10和30 μM时降低（分别为66.7±825%和85.6± 2.6%）Y-27632（3- 30μM）对20 Hz和50 Hz电场刺激反应的抑制作用在具有完整尿道炎的组织条中也大于无尿道炎的组织。总之，RhoA mRNA和活化RhoA在膀胱平滑肌中的表达均高于在尿道平滑肌中的表达。Y-27632对有完整尿路上皮的组织标本的抑制作用强于无尿路上皮的组织标本。少

The mRNA and protein expression of muscarinic receptor subtypes, adorenoceptor subtypes as well as RhoA/Rho kinase and its activity were investigated by radioligand binding experiments, realtime RT-PCR, Western blotting and immunohistochemistry in urothelium and detrusor muscle of the pig and human. We have hind that M2 muscarinic, receptor subtypes were predominantly present in both urothelium and detrusor of the pig bladder. However M3 muscarinic receptor subtypes were more hind in urothelium and smooth muscle of the human bladder Among β-receptor subtypes, β3-adrenoceptor were predominantly found in detrusor muscle of the pig and human.The expression of RhoA was more hind in the urothelium than in the smooth mule of the bladder in pig and human. The activity of RhoA determined by Western blotting were also greater in the urothelium than in the smooth muscle of the pig bladder.In functional studies, the responses to Y-27632 and fasudil, Rho-kinase inhibitors, were examined in bladder … More tissue strips with or without urothelium. Mansion decrease after administration of Y-27632 (InM-100μM) was significantly greater in tissues with intact urothelium than in tissues without urothelium, after pre-contraction with KCl (decrease by 52.0±4.6% vs 28±6.8%, respectively; p=0.0088) or with carbachol (decrease by 53.1±7.2% vs 30.6±5.81%, respectively; p=0.0035). Maximum contraction on CRC to carbachol was reduced significantly alter administration of 3, 10 and 30μM Y-27632 (to 72.2±6.8%, 43.9±7.1%, and 25.015.5%, respectively of the control value) in strips with intact urothelium (n=36), but was reduced only at 10 and 30 μM (to 66.7±825% and 85.6±2.6%, respectively) in tissues without urothelium Inhibitory effect of Y-27632 (3- 30μM) on the response to electrical field stimulation at 20Hz and 50 Hz was also greater in tissue strips with intact urothelium than in tissues without urothelium.In conclusion, the expression of RhoA mRNA and activated RhoA were greater in urothelium than in bladder smooth muscle. Y-27632 showed a stronger inhibitory effect on tissue preparations with intact urothelium than co those without urothelium. Less