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

M受体亚型在介导逼尿肌收缩中的作用：使用尿道条的纵向和横向条，计算正常和cAMP升高的组织(用氯化钾收缩，用异丙肾上腺素松弛)中M受体亚型对卡巴胆碱反应的亲和力。猪的尿路似乎主要有M_2受体。正常尿路收缩在环状肌中主要由M_2和M_3受体介导，而在纵肌中主要由M_3受体介导。在cAMP升高后，两种组织的M_2受体均参与收缩，但环行肌中M_2受体的参与程度更大。β_3肾上腺素受体在调节膀胱和尿路平滑肌松弛中的作用：猪和人。：从猪逼尿肌和尿道肌制备细胞膜，并与[~3H]-二氢丙烯醇…进行竞争实验。用非选择性β受体拮抗剂L作为特异性放射配基，确定β受体亚型的存在。在功能实验中，使用离体逼尿肌条来确定激动剂的效力和拮抗剂的亲和力。β_3-肾上腺素能受体是猪膀胱和尿路中主要的β-肾上腺素能受体亚型。从功能上讲，β肾上腺素受体介导的反应是通过β_2和β_3受体亚型介导的。在猪和人中，β_3肾上腺素能受体更多地参与膀胱体的松弛，而β_2肾上腺素能受体更多地参与猪的三角肌和尿道肌的松弛。在体外，Rho/Rho激酶主要存在于控制人膀胱收缩的尿路上皮细胞中：人膀胱穹隆区域的逼尿肌标本来自于膀胱癌全切除患者。用相对多重RT-PCR和Western blotting分析Rho-Kinase的mRNA和蛋白水平。使用与Western blotting相同的特异性抗体进行免疫组织化学，以支持Western blotting获得的数据。获得卡巴胆碱的累积浓度-反应曲线(CRC)，然后用含有10μM的Y 27632或赋形剂(时间对照)的克雷布斯溶液平衡30 mm。在相对多重RT-PCR中，RhoA和Rho激酶在尿路上皮中的含量高于在平滑肌中的含量。Western blotting还显示Rho激酶蛋白在尿路上皮中的表达显著高于在平滑肌中的表达(分别为p&lt；0.0001和p&lt；0.0001)。免疫组织化学显示，尿上皮细胞对Rho激酶抗体呈中至强反应，而平滑肌细胞呈弱至中度反应。Rho和Rho激酶似乎主要存在于尿路上皮中。尿路上皮存在时，卡巴胆碱的最大反应降低。Rho/Rho激酶通路似乎参与了人膀胱平滑肌在尿路上皮存在时的收缩。较少

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