Nitric Oxide Inhibits RhoA/Rho-kinase in Penile Erection

一氧化氮抑制阴茎勃起中的 RhoA/Rho 激酶

• 批准号: 6731122
• 负责人: R Clinton Webb
• 金额: $ 35.75万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-04 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6731122
• 关键词: G protein coupled receptor kinase; biological signal transduction; cGMP dependent protein kinase; calcium flux; cyclic GMP; enzyme activity; enzyme inhibitors; gene delivery system; gene targeting; guanine nucleotide binding protein; immunocytochemistry; laboratory mouse; laboratory rat; myosin light chain kinase; nitric oxide; nitric oxide synthase; penis disorder; penis erection; phosphorylation; plasmids; protein transport; reproductive system pharmacology; sarcoplasmic reticulum; transfection /expression vector; vasodilation; western blottings

DESCRIPTION (provided by applicant): Over 30 million men suffer from erectile dysfunction in the United States. Constriction and dilation of the cavernosal vasculature determines penile erection. In the absence of arousal stimuli, Ca2+-sensitizing RhoA/Rho-kinase signaling maintains vasoconstriction, keeping the penis non-erect. Upon arousal, nitric oxide (NO), released from nerves and endothelial cells, induces dilation and erection. Although NO stimulates erection, the cellular mechanism of NO is unknown. NO binds soluble guanylate cyclase to stimulate an increase in cyclic GMP (cGMP) and the subsequent activation of cGMP-dependent protein kinase (cGK). In the penis, cGK has been proposed to induce dilation through activation of membrane K+ channels to cause hyperpolarization, inhibition of membrane Ca2+ channels to decrease activator Ca+, and stimulation of sarcoplasmic reticular Ca2+ uptake to sequester the cation. However, recent work suggests that high levels of activator Ca2+ are not maintained during constriction and it is the Ca2+-sensitizing effect of RhoAJRho-kinase that must be overcome to cause dilation. We hypothesize that cGK inhibits RhoA translocation to the membrane leading to a reduction in Rho-kinase activity and removal of its inhibitory action on myosin light chain (MLC) phosphatase. This dis-inhibition leads to reduced MLC phosphorylation, smooth muscle relaxation and erection. We further hypothesize that the long-term expression of components of the RhoA/Rho-kinase signaling pathway are inversely related to NO bioavailability. These hypotheses will be tested by 3 specific aims: 1) to determine if NO/cGMP/cGK signaling antagonizes RhoA activation to evoke dilation and penile erection; 2) to determine if gene transfer of endothelial nitric oxide synthase (eNOS) to the penis will down-regulate RhoA/Rho-kinase signaling to augment erection; and 3) to determine if reduced NO bioavailability (pharmacological blockade and denervation) leads to up-regulation of the RhoA/Rho-kinase pathway and erectile dysfunction. The approach will utilize rat and mouse models of erection. The experiments will determine the effect of NO/cGMP/cGK on biochemical, pharmacological and physiological measures of the RhoA/Rho-kinase pathway in the intact penis and in isolated cavernosal strips. Gene transfer of dominant-negative RhoA and endothelial NOS to the penis will provide a powerful tool to manipulate the activity of the RhoA/Rho-kinase pathway. Contractile force measurements in isolated cavernosal strips (intact and permeablizied) will provide evidence for Ca2+ sensitization and its regulation by cGMP/cGK. These studies will define the molecular basis for NO-mediated cavernosal vasodilation in the normal state and how long-term changes in the Ca 2+ sensitizing mechanism contribute to erectile dysfunction.

描述（由申请人提供）：在美国有超过3000万男性患有勃起功能障碍。海绵体血管的收缩和扩张决定了阴茎的勃起。在缺乏觉醒刺激的情况下，Ca2+敏感的RhoA/ rho激酶信号维持血管收缩，保持阴茎不勃起。在觉醒时，一氧化氮（NO），从神经和内皮细胞释放，诱导扩张和勃起。虽然NO刺激勃起，但NO的细胞机制尚不清楚。NO结合可溶性鸟苷酸环化酶，刺激环GMP （cGMP）的增加，并随后激活cGMP依赖性蛋白激酶（cGK）。在阴茎中，已经提出cGK通过激活膜K+通道引起超极化，抑制膜Ca2+通道以减少激活剂Ca+，以及刺激肌浆网状Ca2+摄取以隔离阳离子来诱导扩张。然而，最近的研究表明，在收缩过程中，高水平的激活剂Ca2+不能维持，而必须克服rhoajrho激酶的Ca2+敏化效应才能引起扩张。我们假设cGK抑制RhoA向膜的易位，导致RhoA激酶活性降低，并消除其对肌球蛋白轻链（MLC）磷酸酶的抑制作用。这种去抑制导致MLC磷酸化减少，平滑肌松弛和勃起。我们进一步假设RhoA/ rho激酶信号通路组分的长期表达与NO生物利用度呈负相关。这些假设将通过3个特定目的进行验证：1)确定NO/cGMP/cGK信号是否拮抗RhoA激活，从而引起阴茎扩张和勃起；2)研究内皮型一氧化氮合酶（eNOS）基因向阴茎的转移是否会下调RhoA/ rho激酶信号从而增强勃起；3)确定NO生物利用度降低（药物阻断和去神经支配）是否导致RhoA/ rho激酶途径上调和勃起功能障碍。该方法将利用大鼠和小鼠勃起模型。本实验将确定NO/cGMP/cGK对完整阴茎和离体海绵体条RhoA/ rho激酶途径生化、药理学和生理指标的影响。显性阴性RhoA和内皮NOS基因转移到阴茎将为RhoA/ rho激酶途径的活性调控提供强有力的工具。在孤立的海绵体条（完整的和渗透的）收缩力测量将为Ca2+敏化及其由cGMP/cGK调节提供证据。这些研究将确定正常状态下no介导的海绵体血管舒张的分子基础，以及ca2 +敏化机制的长期变化如何导致勃起功能障碍。