REGULATION OF ACTIVITY AND EXPRESSION OF NO SYNTHASE

拟议研究的主要目标是阐明 一氧化氮合酶调节一氧化氮（NO）产生的机制 哺乳动物细胞近年来，NO研究领域几乎呈爆炸式增长。 多年来，自然界中最简单的分子之一NO已经发展成为 一个最普遍和多方面的生物介质， 生理学和病理生理学。鉴于广泛的行动， 内源性产生的NO作为血管扩张剂、血小板抑制剂和 白细胞功能，非肾上腺素能-非胆碱能神经递质 神经元和靶细胞损伤的病理生理介质， NO的生物合成和代谢是关键过程， 认识NO在生物组织中是化学不稳定的， 因此，影响NO生物学作用的因素将 在很大程度上取决于影响NO生成的因素。 中心假设是，NO的生物合成是由血管平滑肌 肌肉和内皮细胞，肺泡巨噬细胞和肝细胞， 受到内源性因素的严格调控， NOS活性和转录表达。最近的研究表明， 实验表明，NO、亚硝基 化合物、四氢生物蝶呤的可用性和氧化还原剂， 干扰核因子κ B活化的化合物 （NF-κ B）抑制诱导型NOS的表达 巨噬细胞 进一步的实验表明，NO可以下调 诱导型NOS mRNA的转录。因此，建议 研究集中在NOS活性和表达的调节， 哺乳动物细胞为实现这一目标，提出了五项具体目标： (1)为了阐明NO抑制神经元和 （2）探讨巨噬细胞诱导型NOS表达的原因 对NO的抑制作用有抵抗力;（3）阐明 钙调素促进以下物质的催化反应的机制 NOS的各种亚型;（4）确定所需的条件， 通过NOS同种型产生活性氧;和（5） 阐明NF-κ B和活性氧作为介导剂的作用， 诱导型一氧化氮合酶表达中早期反应基因的激活 巨噬细胞和肝细胞。拟议的研究代表了一个持续的 长期的努力，以了解生物因素的影响， NO在哺乳动物细胞中在健康和疾病中的作用。

The principal objective of the proposed research is to elucidate the mechanisms of regulation of nitric oxide (NO) production by NO synthase in mammalian cells. The field of NO research has virtually exploded in recent years and NO, one of the simplest molecules in nature, has developed into one of the most ubiquitous and multifaceted biological mediators in physiology and pathophysiology. In view of the widespread actions of endogenously generated NO as a vasodilator, inhibitor of platelet and leukocyte function, neurotransmitter in nonadrenergic-noncholinergic neurons, and pathophysiological mediator of target cell injury, the biosynthesis and metabolism of NO are critical processes that require understanding. NO is chemically labile in biological tissues and, therefore, the factors that influence the biological actions of NO will depend largely on the factors that affect the production of NO. The central hypothesis is that the biosynthesis of NO by vascular smooth muscle and endothelial cells, alveolar macrophages, and hepatocytes is tightly regulated by endogenous factors influencing both the enzymatic activity and transcriptional expression of NOS. Recent studies from this laboratory indicate that NOS activity can be altered by NO, nitroso compounds, tetrahydrobiopterin availability, and redox agents, and that compounds which interfere with the activation of nuclear factor kappa B (NF-kB) inhibit expression of inducible NOS in cytokine-activated macrophages. Additional experiments indicate that NO can down-regulate the transcription of mRNA for inducible NOS. Therefore, the proposed studies focus on the regulation of NOS activity and expression in mammalian cells. Five specific aims are proposed to achieve the objective: (1) to elucidate the mechanism by which NO inhibits neuronal and endothelial NOS; (2) to determine the reasons why macrophage inducible NOS is resistant to the inhibitory action of NO; (3) to elucidate the mechanism(s) by which calmodulin promotes the catalytic reaction of various isoforms of NOS; (4) to determine the conditions required for the production of reactive oxygen species by NOS isoforms; and (5) to elucidate the roles for NF-kB and reactive oxygen species as mediators of early response gene activation in the expression of inducible NOS in macrophages and hepatocytes. The proposed research represents a continuing long-term effort to understand the biological factors that influence the actions of NO in mammalian cells in both health and disease.