ERECTILE DYSFUNCTION AND NITRIC OXIDE SYNTHASE IN AGING

勃起功能障碍，即阴茎不能[达到或维持] 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功能缺陷性 由于组织顺应性的丧失[和/或可能过度 交感神经音，]和/或，61减少一氧化氮(NO)的合成， 阴茎勃起的主要调节因子。我们实验室发现并克隆了 PnNOS，神经元型一氧化氮合酶(NNOS)的新变体，以及 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。一种可能的内皮型一氧化氮合酶(ENOS) 还检测到变异体。我们还证明了一种蛋白质抑制物 一氧化氮合酶活性(PIN)在大鼠和人阴茎中表达。]我们 还发现与衰老相关的大鼠勃起功能障碍可以 阴茎诱导型cDNA3基因治疗的改进 一氧化氮合酶(INOS)。这个项目的目标首先是确定 增龄对PnNOS及其突变体表达的影响 通过Western和Northern印迹、RT/PCR法、免疫细胞化学 和原位杂交，以及阴茎一氧化氮合酶的减少 非常老的动物的酶活性是由一氧化氮合酶结合到 抑制剂或通过一氧化氮合酶糖基化。海绵体的基因治疗 然后将使用几种方案对患有PnNOS的老年大鼠进行研究 并与L精氨酸共同构建cDNA，刺激一氧化氮合酶 活动。提高老龄大鼠勃起功能的实验研究 对海绵体神经的电场刺激(EFS)，以及 阴茎反射，将会被确定。[最后，老化对 PIN与PnNOS变体的结合水平将在 用免疫检测技术检测大鼠阴茎。一种抗PIN基因疗法 截短的PnNOS的cDNA不能结合PIN(PnNOSbeta) 将被测试以刺激不依赖于PIN的NOS活性。 或者，与PIN结合的nNOS蛋白结构域的cDNA会 被给予猝灭PIN效应，反义PIN寡核苷酸将 因可能下调PIN转换而接受调查。其他 将使用以下方法寻找PnNOS活性的可能蛋白质调节剂 随机展示的多肽文库]。长期目标是验证 这些药物在勃起医学治疗中的临床前应用 与衰老相关的功能障碍。

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.