ERECTILE DYSFUNCTION AND NITRIC OXIDE SYNTHASE IN AGING

勃起功能障碍，阴茎无法[达到或维持] 一种僵硬的勃起，与衰老有关。 在衰老大鼠模型中， 阴茎海绵体平滑肌松弛缺陷 由于组织顺应性的丧失，[和/或，可能过度 交感神经紧张，]和/或，61减少一氧化氮（NO）的合成， 阴茎勃起的主要介质。 我们的实验室发现并克隆了 PnNOS是神经元型一氧化氮合酶（nNOS）的一种新变体， 显示它是大鼠阴茎中唯一表达的nNOS [两者都是完整的， 长度和较短的蛋白质变体。 一种推定的内皮NOS（eNOS） 也发现了变异。 我们还表明，蛋白质抑制剂 NOS活性（PIN），在大鼠和人类阴茎中表达。 我们 还发现，大鼠中与衰老相关的勃起功能障碍可以 通过用阴茎可诱导的cDNA构建体进行基因治疗来改善 NOS（iNOS）。 该项目的目的是首先确定是否 衰老影响PnNOS和NOS（eNOS）变体的表达模式 Western和北方印迹、RT/PCR、免疫细胞化学 原位杂交，以及阴茎NOS的减少是否 在很老的动物中，酶活性是由NOS结合到 抑制剂或通过NOS糖化。 阴茎海绵体的基因治疗 然后将使用几种方案研究老年大鼠PnNOS 和cDNA构建体与L-精氨酸结合，以刺激NOS 活动 增加老年大鼠勃起反应的能力 海绵体神经的电场刺激（EFS），以及 阴茎反射将被确定 [最后，衰老对 PIN与PnNOS变体结合的水平将在 免疫检测技术检测大鼠阴茎。 抗PIN基因疗法 与不能结合PIN的截短型PnNOS的cDNA（PnNOS β） 将被测试以刺激PIN非依赖性NOS活性。 或者，结合PIN的nNOS蛋白结构域的cDNA将 以淬灭PIN效应，并且反义PIN寡核苷酸将 接受调查，以确定PIN翻译是否可能下调。其他 PnNOS活性的推定蛋白质调节剂将使用 随机展示肽文库]。 长期目标是验证 在临床前使用这些药物治疗勃起 与衰老有关的功能障碍。

Erectile dysfunction, the inability of the penis to [attain or maintain] a rigid erection, is associated with aging. In the aging rat model, a defective relaxation of the penile corpora cavernosal smooth muscle results from a loss of tissue compliance, [and/or, possibly excessive sympathetic tone,] and/or, 61 reduced synthesis of nitric oxide (NO), the main mediator of penile erection. Our laboratory found and cloned PnNOS, a novel variant of neuronal nitric oxide synthase (nNOS), and showed it is the only nNOS expressed in the rat penis [both as full- length and shorter protein variants. A putative endothelial NOS (eNOS) variant was also detected. We have also shown that a protein Inhibitor of NOS activity (PIN), is expressed in the rat and human penis.] We also found that the aging-associated erectile dysfunction in the rat can be ameliorated by gene therapy with cDNA constructs of penile inducible NOS (iNOS). The aims of this project are first to determine whether aging affects the expression pattern of PnNOS and NOS (eNOS) variants in the penis by western and northern blots, RT/PCR, immunocytochemistry and in situ hybridization, and whether the reduction in penile NOS enzyme activity in very old animals is caused by NOS binding to inhibitors or by NOS glycation. Gene therapy of the corpora cavernosa in aged rats with PnNOS Will then be investigated, using several regimes and cDNA constructs in conjunction with L-arginine, to stimulate NOS activity. The ability to increase the erectile response of aged rats to electrical field stimulation (EFS) of the cavernosal nerve, and penile reflexes, will be determined. [Finally, the effects of aging on the levels of PIN binding to PnNOS variants will be determined in the rat penis with immunodetection techniques. An anti-PIN gene therapy with the cDNA for the truncated PnNOS unable to bind PIN (PnNOSbeta) will be tested to stimulate PIN-independent NOS activity. Alternatively, the cDNA for the nNOS protein domain that binds PIN will be given to quench PIN effects, and antisense PIN oligonucleotides will be investigated for a possible down-regulation of PIN translation. Other putative protein modulators of PnNOS activity will be searched for using the random display peptide library]. The long-term goal is to validate pre-clinically the use of these agents for medical treatment of erectile dysfunction associated with aging.