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

采用放射配体结合实验、实时RT-PCR、Western blotting和免疫组化等方法研究猪和人尿路上皮和逼尿肌中毒碱受体亚型、肾上腺素受体亚型mRNA和蛋白的表达以及RhoA/Rho激酶的表达及其活性。我们发现M2毒蕈碱受体亚型主要存在于猪膀胱尿路上皮和逼尿肌中。而M3毒蕈碱受体亚型多见于尿路上皮和膀胱平滑肌。β受体亚型中，β3-肾上腺素受体主要见于猪和人的逼尿肌。RhoA在猪和人尿路上皮中的表达较在膀胱光滑体中的表达少。Western blotting检测的RhoA在尿路上皮中的活性也高于在猪膀胱平滑肌中的活性。在功能研究中，对Y-27632和法舒地尔（rho激酶抑制剂）的反应在膀胱中进行了检测。Y-27632 （InM-100μM）在尿路上皮完整组织、KCl预收缩组（分别降低52.0±4.6% vs 28±6.8%,p=0.0088）和carbachol预收缩组（分别降低53.1±7.2% vs 30.6±5.81%,p=0.0035）中的大厦下降显著高于未加尿路上皮组织。在尿路上皮完整带（n=36）中，给药3、10和30μM Y-27632显著降低CRC对氯巴酚的最大收缩（分别为对照值的72.2±6.8%、43.9±7.1%和25.015.5%），但仅在10和30μM时降低（分别为66.7±825%和85.6±2.6%）；Y-27632 （3 ~ 30μM）对完整尿路上皮组织条20Hz和50hz电场刺激反应的抑制作用也大于不含尿路上皮组织条。由此可见，尿路上皮中RhoA mRNA的表达和活化RhoA的表达均高于膀胱平滑肌。Y-27632对含完整尿路上皮的组织制剂的抑制作用强于不含尿路上皮的组织制剂。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