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: 7017111
• 负责人: R Clinton Webb
• 金额: $ 34.91万
• 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/7017111
• 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，超过3000万男性患有勃起功能障碍。阴茎海绵体血管的收缩和扩张决定阴茎勃起。在没有唤醒刺激的情况下，Ca 2+敏化RhoA/Rho激酶信号传导维持血管收缩，保持阴茎不勃起。在唤醒时，从神经和内皮细胞释放的一氧化氮（NO）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以刺激环GMP（cGMP）的增加和随后cGMP依赖性蛋白激酶（cGK）的活化。在阴茎中，已提出cGK通过激活膜K+通道以引起超极化、抑制膜Ca 2+通道以减少激活剂Ca+和刺激肌浆网Ca 2+摄取以螯合阳离子来诱导扩张。然而，最近的工作表明，高水平的激活剂Ca 2+在收缩过程中不被维持，并且必须克服RhoA/Rho激酶的Ca 2+增敏作用才能引起扩张。我们假设cGK抑制RhoA易位到膜，导致Rho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激酶信号通路的组分的长期表达与NO生物利用度呈负相关。这些假设将通过3个具体目标进行检验：1）确定NO/cGMP/cGK信号传导是否拮抗RhoA激活以引起阴茎扩张和勃起; 2）确定内皮型一氧化氮合酶（eNOS）基因转移到阴茎是否将下调RhoA/Rho激酶信号传导以增强勃起;和3）确定降低的NO生物利用度（药理学阻断和去神经支配）是否导致RhoA/Rho-激酶途径的上调和勃起功能障碍。该方法将利用大鼠和小鼠勃起模型。实验将确定NO/cGMP/CGK对完整阴茎和分离海绵体条中RhoA/Rho激酶途径的生化、药理学和生理学测量的影响。显性负性RhoA和内皮NOS基因转移到阴茎将提供一个强大的工具来操纵RhoA/Rho激酶途径的活性。在离体海绵体条（完整和渗透）中的收缩力测量将提供Ca 2+致敏及其通过cGMP/CGK调节的证据。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Ca 2+增敏机制的长期变化如何导致勃起功能障碍。