Nitric Oxide Inhibits RhoA/Rho-kinase in Penile Erection

描述(申请人提供)：美国有3000多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。在没有唤醒刺激的情况下，钙敏感的RhoA/Rho-Kinase信号维持血管收缩，使阴茎保持不勃起。在觉醒时，神经和内皮细胞释放的一氧化氮(NO)会导致扩张和勃起。虽然NO刺激勃起，但其细胞机制尚不清楚。NO与可溶性鸟苷环化酶结合，刺激cGMP(CGMP)增加，进而激活cGMP依赖的蛋白激酶(CGK)。在阴茎中，CGK被认为通过激活膜K通道引起超极化，通过抑制膜钙通道减少激活剂钙，以及刺激肌浆网钙摄取来隔离阳离子来诱导扩张。然而，最近的研究表明，在收缩过程中不会保持高水平的激活剂钙，而必须克服RhoAJRho-Kinase的钙敏化效应才能引起扩张。我们假设CGK抑制RhoA转位到膜上，导致Rho-Kinase活性降低，并消除其对肌球蛋白轻链(MLC)磷酸酶的抑制作用。这种去抑制作用会导致MLC磷酸化、平滑肌松弛和勃起减少。我们进一步假设，RhoA/Rho-Kinase信号通路各组成部分的长期表达与NO的生物利用度成反比。这些假说将通过3个特定的目标进行验证：1)确定NO/cGMP/CGK信号是否拮抗RhoA激活引起的勃起和勃起；2)确定内皮型一氧化氮合酶(ENOS)基因转移到阴茎是否下调RhoA/Rho-Kinase信号以增强勃起；以及3)确定NO生物利用度降低(药物阻断和去神经)是否导致RhoA/Rho-Kinase途径上调和勃起功能障碍。该方法将利用大鼠和小鼠的勃起模型。这些实验将确定NO/cGMP/CGK对完整阴茎和离体海绵体条中RhoA/Rho-Kinase途径的生化、药理学和生理学测量的影响。将显性负性RhoA和内皮型一氧化氮合酶基因转移到阴茎，将为调控RhoA/Rho-Kinase通路的活性提供强有力的工具。海绵体离体条(完整的和通透性的)的收缩力测量将为钙增敏及其被cGMP/CGK调节提供证据。这些研究将确定正常状态下NO介导的海绵体血管扩张的分子基础，以及钙增敏机制的长期变化如何导致勃起功能障碍。

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.