REGULATION OF CYCLOOXYGENASE BY NITRIC OXIDE--IMPLICATIONS FOR ATHEROGENEIS

有越来越多的证据表明， 由调节血管壁产生的第二信使 胆固醇的运输和增殖状态在 动脉硬化。这些第二信使包括前列环素(PGI/2)和 一氧化氮(NO)。动脉粥样硬化中PGI/2合成减少，原因是 部分为还原的环氧合酶(前列腺素H合成酶，PGHS) 表情。与PGI/2一样，NO也促进血管扩张，抑制血管平滑 肌肉细胞(SMC)增殖；其影响在 动脉硬化。这些观察结果支持了PGHS的前提 监管可能与不生产联系在一起。我们有初步证据 证明NO通过两种不同的方式促进二十烷类化合物的生成 机制：(I)直接激活PGHS-1，以及(Ii)上调 丝裂原诱导的PGHS-2的表达。这项提案的总体目标是 是确定一氧化氮和二十烷类化合物代谢之间的关系 在正常和载脂条件下。建议进行的实验 具体目的之一将直接确定NO的作用机制 激活PGHS-1。这将使用各种不同的 生化和生物物理技术，这将在#年部分完成 与西尔弗斯坦博士的合作。接下来，由于PGHS-1是一种膜- 结合酶，我们假设PGHS-1的活性是由它的 脂质环境。因此，我们将评估NO改变的能力 极性脂和磷脂存在时PGHS-1的活性和结构 胆固醇来源于正常和富含脂肪的SMC。 在特定目的二中提出的实验将通过以下方式确定机制 这不会改变完整SMC中二十烷类化合物的合成。PGHS-1 IS 组成性表达，而PGHS-2受转录调控 核因子-kB、AP-1和SP-1的结合。PGHS-2的一条重要途径 表达通过p21ras信号级联发生，因为PDGF 通过p21ras激活细胞，有效地促进PGHS-2转录。 由于NO还以p21ras依赖的方式激活NF-kB，所以NO可能诱导 PGHS-2的表达是通过这种机制实现的。因此，实验是 建议确定NO改变PGHS-1的生化机制 活性，以及PGHS-2mRNA和蛋白的表达和细胞 活动。这些研究将与Project合作进行 四、这些合作对成功完成 在特定目标下提出的实验2.这些研究的结果 应该有重要的影响，因为他们可能会提出一种机制，通过 其中NO可能通过促进PGI/2来调节动脉粥样硬化性疾病 生成，这反过来可能会抑制胆固醇酯沉积。

There is accumulating evidence indicating that the synthesis and effect of second messengers generated by the blood vessel wall which regulates cholesterol trafficking and proliferative state are altered in atherosclerosis. These second messengers include prostacyclin (PGI/2) and nitric oxide (NO). PGI/2 synthesis is reduced in atherosclerosis, due in part to reduced cyclooxygenase (prostaglandin H synthase, PGHS) expression. Like PGI/2, NO also promotes vasodilation and inhibits smooth muscle cell (SMC) proliferation; its effects are reduced in atherosclerosis. These observations support the premise that PGHS regulation may be linked to NO production. We have preliminary evidence demonstrating that NO promotes eicosanoid generation by two distinct mechanisms: (i) direct activation of PGHS-1, and (ii) upregulation of the expression of mitogen-inducible PGHS-2. The overall goal of this proposal is to determine the relationships between NO and eicosanoid metabolism under normal and lipid-loaded conditions. Experiments proposed in SPECIFIC AIM ONE will determine the mechanism by which NO directly activates PGHS-1. This will be investigated using a variety of biochemical and biophysical techniques, which will be done in part in collaboration with Dr. Silverstein. Next, since PGHS-1 is a membrane- bound enzyme, we hypothesize that PGHS-1 activity is regulated by its lipid environment. Thus, we will evaluate the ability of NO to alter PGHS-1 activity and structure in the presence of polar lipids and cholesterol derived from normal and lipid-enriched SMC. Experiments proposed in SPECIFIC AIM TWO will determine the mechanisms by which NO alters eicosanoid synthesis in intact SMC. PGHS-1 is constitutively expressed, while PGHS-2 is transcriptionally regulated by the binding of NF-kB, AP-1, and SP-1. An important route of PGHS-2 expression occurs through the p21ras signaling cascade, since PDGF which activates cells via p21ras, potently increases PGHS-2 transcription. Since NO also activates NF-kB in a p21ras-dependent manner, NO may induce PGHS-2 expression by this mechanism. Accordingly, experiments are proposed to determine the biochemical mechanisms by which NO alters PGHS-1 activity, as well as PGHS-2 mRNA and protein expression and cellular activity. These studies will be performed in collaboration with Project IV; these collaborations are crucial to successful completion of experiments proposed in Specific Aim 2. The results of these studies should have important implications, since they may suggest a mechanism by which NO may modulate atherosclerotic disease, that is by promoting PGI/2 generation, which in turn may inhibit cholesteryl ester deposition.