REGULATION OF EXPRESSION OF NO SYNTHASE

一氧化氮(NO)是调节哺乳动物生理和病理生理过程的关键分子，其中许多涉及血管系统。NO的许多生理作用都是由环状GMP介导的，并且NO和环状GMP的作用时间都相对较短。NO是不稳定的，不会被传统机制储存、释放或失活。NO与活性氧物种以及与含铁和硫分子的反应会导致NO的快速失活。由于特定的磷酸二酯酶的水解，环状GMP也是不稳定的。因此，NO被自然发生的化学过程迅速失活，环状GMP被细胞中的磷酸二酯酶迅速失活，产生不到1秒的半衰期。因此，NO的作用高度依赖于其在一氧化氮合酶(NOS)作用下由L精氨酸生物合成。因此，NO生物合成的调控是决定NO药效学的最重要的过程。拟议研究的主要目标是阐明几种潜在的新机制，通过这些机制可以控制生物合成。这些靶向机制包括NOS基因表达的改变、NOS蛋白的表达和稳定性、精氨酸酶的表达和催化活性、精氨酸底物的可获得性以及NOS产生的活性氧物种。需要检验的中心假设是，细胞NO的产生受到各种内源性因素的严格调控，这些因素影响着NOS亚型的转录、翻译和翻译后的表达。为了实现这一目标，人们提出了三个具体的目标：(1)阐明NO下调诱导型一氧化氮合酶(INOS)和内皮型一氧化氮合酶(ENOS)表达的分子机制；(2)阐明精氨酸酶上调iNOS和eNOS表达的机制；(3)阐明精氨酸酶在调控iNOS和eNOS产生超氧阴离子和过氧化氢中的作用。这项拟议的研究代表了一项持续的长期努力，目的是阐明影响血管和NO在健康和疾病中的相关作用的生物因素。

Nitric oxide (NO) is a key molecule mediating physiological and pathophysiological processes in mammals, many of which involve the vascular system. Many physiological actions of NO are mediated by cyclic GMP, and both NO and cyclic GMP have relatively short durations of action. NO is labile and is not stored, released, or inactivated by conventional mechanisms. Reactivity of NO with reactive oxygen species and with iron and sulfur containing molecules causes rapid inactivation of NO. Cyclic GMP is also unstable due to hydrolysis by specific phosphodiesterases. Thus, NO is rapidly inactivated by naturally-occurring chemical processes and cyclic GMP is rapidly inactivated by phosphodiesterases in cells, yielding half lives for each of less than 1-second. Therefore, the actions of NO are highly dependent on its biosynthesis from L-arginine by NO synthase (NOS). Accordingly, the regulation or modulation of NO biosynthesis is the most important process dictating the pharmacodynamics of NO. The principal objective of the proposed studies is to elucidate several potentially novel mechanisms by which NO biosynthesis may be controlled. These targeted mechanisms involve changes in NOS mRNA expression, NOS protein expression and stability, arginase expression and catalytic activity, arginine substrate availability, and the production of reactive oxygen species by NOS. The central hypothesis to be tested is that cellular NO production is tightly regulated by various endogenous factors affecting transcriptional, translational and posttranslational expression of NOS isoforms. Three Specific Aims are proposed to achieve this objective: (1) to elucidate the molecular mechanisms by which NO downregulates the expression of inducible NOS (iNOS) and endothelial NOS (eNOS); (2) to elucidate the mechanisms by which arginase upregulates the expression of iNOS and eNOS; and (3) to elucidate the role of arginase in modulating the production of superoxide anion and hydrogen peroxide by iNOS and eNOS. The proposed research represents a continuing long-term effort to elucidate the biological factors influencing the vascular and related actions of NO in health and disease.