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The role of autonomic nerve receptor subtypes and G-proteins for the cause of overactive bladder

自主神经受体亚型和 G 蛋白在膀胱过度活动症病因中的作用

基本信息

批准号：
14571516
负责人：
YAMANISHI Tomonori
金额：
$ 2.11万
依托单位：
Dokkyo University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

The role of muscarinic receptor subtypes in mediating contraction of detrusor muscles : The affinities of muscarinic antagonists against carbachol responses were calculated in normal and cAMP-elevated tissues (contraction with KCl and relaxation with isoprenaline) using longitudinal and transverse strips of urethra. The pig urethra appears to possess predominantly M_2 muscarinic receptors. Contraction of the normal urethra appears to be predominantly mediated by M_2 and M_3-receptors in circular muscle, but by M_3-receptors in longitudinal muscle. Following cAMP-elevation, a contribution to contraction of M_2-receptors appeared to be demonstrated in both tissues, but the involvement of the M_2-receptors appeared greater in the circular muscle.The role of β_3-Adrenoceptors in mediating relaxation of bladder and urethral smooth muscle : of the pig and human. : Cell membranes were prepared from detrusor and urethral muscle of the pig and competition experiments with [^3H]-dihydroalprenolo … More l(DHA), a non-selective β-adrenoceptor antagonist was used as a specific radioligand to determine the presence of β-adrenoceptor subtypes. In functional experiments, isolated detrusor muscle strips were used to determine the potency of agonists and the affinity of antagonists. β_3-adrenoceptars are the predominant β-adrenoceptor subtype present in the pig bladder and urethra. Functionally, β-adrenoceptor mediated responses of this tissue are mediated via both the β_2- and β_3-adrenoceptor subtypes. β_3-adrenoceptors appear to be more involved in relaxation of bladder body in the pig and human, whereas β_2-adrenoceptors appear to be more involved in trigonal and urethral smooth muscle of the pig.Rho/Rho kinase is predominantly present in the urothelium controlling the contraction of the human urinary bladder in vitro : Samples of detrusor muscle from the dome region of the human urinary bladder were obtained from patients undergoing total cystectomy for bladder cancer. The mRNA and protein levels of Rho-kinase were analyzed using the relative multiplex RT-PCR and Western blotting. Immunohistochemistry, using the same specific antibodies as antibodies for Western blotting, was performed to support the data obtained by Western blotting. Cumulative concentration-response curves(CRCs) to carbachol were obtained, and were then equilibrated for 30 mm with Krebs solution containing the 10μM of Y 27632 or vehicle (time control). On the relative multiplex RT-PCR,RhoA and Rho kinase were more abundant in the urothelium than in the smooth muscle. Western blotting also demonstrated significantly greater expression of Rho kinase protein expression in the urothelium than in the smooth muscle (p<0.0001 and p<0.0001, respectively). Urothelial cells showed moderate to strong reaction for Rho kinase antibodies, and smooth muscle cells showed weak to moderate reaction on immunohistochemistrical study. Rho and Rho kinase appear to be predominantly present in the urothelium. The maximum response to carbachol was decreased in the presence of urothelium. The Rho/Rho kinase pathway appears to be involved in the contraction of human bladder smooth muscle in the presence of urothelium. Less

毒蕈碱受体亚型在介导逼尿肌收缩中的作用：在正常和camp升高的组织（KCl收缩和异丙肾上腺素松弛）中，通过尿道纵向和横向条计算毒蕈碱拮抗剂对氯巴托反应的亲和力。猪尿道似乎主要具有M_2毒蕈碱受体。正常尿道的收缩主要由圆形肌的M_2和m_3受体介导，而由纵向肌的m_3受体介导。camp升高后，两种组织都表现出对m2受体收缩的贡献，但m2受体的参与在圆形肌肉中表现得更大。β_3-肾上腺素能受体在猪和人膀胱尿道平滑肌松弛中的作用。用非选择性β-肾上腺素能受体拮抗剂[^3H]-二氢阿普萘诺多1 （DHA）作为特异性放射配体，测定β-肾上腺素能受体亚型的存在，制备猪逼尿肌和尿道肌细胞膜。在功能实验中，分离的逼尿肌条被用来测定激动剂的效力和拮抗剂的亲和力。β_3-肾上腺素受体是猪膀胱和尿道中主要的β-肾上腺素受体亚型。在功能上，β-肾上腺素能受体介导的反应是通过β_2-和β_3-肾上腺素能受体亚型介导的。β_2-肾上腺素受体主要参与猪和人膀胱体的松弛，而β_2-肾上腺素受体主要参与猪的三角肌和尿道平滑肌。Rho/Rho激酶主要存在于尿路上皮中，在体外控制人类膀胱的收缩：从接受膀胱癌全膀胱切除术的患者身上获得了人类膀胱穹窿区域的逼尿肌样本。采用相对多重RT-PCR和Western blotting分析rho激酶mRNA和蛋白水平。使用与Western blotting抗体相同的特异性抗体进行免疫组化，以支持Western blotting获得的数据。获得了对苯酚的累积浓度-响应曲线（CRCs），然后用含有10μM Y 27632或载药（时间对照）的Krebs溶液平衡30 mm。相对多重RT-PCR结果显示，Rho激酶和RhoA激酶在尿路上皮中的含量高于平滑肌。Western blotting也显示Rho激酶蛋白在尿路上皮中的表达显著高于平滑肌（p<0.0001和p<0.0001）。免疫组化结果显示，尿路上皮细胞对Rho激酶抗体表现为中~强反应，平滑肌细胞对Rho激酶抗体表现为弱~中反应。Rho和Rho激酶似乎主要存在于尿路上皮中。尿路上皮的存在降低了对氯乙醇的最大反应。Rho/Rho激酶通路似乎参与尿路上皮存在时膀胱平滑肌的收缩。少