REGULATION OF CYCLOOXYGENASE BY NITRIC OXIDE--IMPLICATIONS FOR ATHEROGENEIS

越来越多的证据表明， 血管壁产生的第二信使， 胆固醇运输和增殖状态改变， 动脉粥样硬化 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， 部分还原环氧合酶（前列腺素H合酶，PGHS） 表情 与PGI/2一样，NO也促进血管舒张并抑制平滑肌细胞增殖。 肌肉细胞（SMC）增殖;其影响在 动脉粥样硬化 这些观察结果支持了PGHS的假设， 调节可能与NO的产生有关。 我们有初步证据 这表明NO通过两种不同的方式促进类花生酸的产生， 机制：（i）PGHS-1的直接激活，和（ii）PGHS-1的上调。 有丝分裂原诱导型PGHS-2的表达。 本提案的总体目标是 确定NO与类花生酸代谢的关系 在正常和脂质负载条件下。 建议的实验 具体目标一将决定的机制，一氧化氮直接 激活PGHS-1。 这将使用各种 生物化学和生物物理技术，这将部分完成， 与Silverstein博士合作。 其次，由于PGHS-1是一种膜- 结合酶，我们假设PGHS-1活性受其 脂质环境 因此，我们将评估NO改变 PGHS-1在极性脂质存在下的活性和结构 来自正常和富含脂质的SMC的胆固醇。 具体目标2中提出的实验将通过以下方式确定机制： NO改变完整SMC中类花生酸的合成。 PGHS-1是 组成型表达，而PGHS-2是转录调节， NF-κ B、AP-1和SP-1的结合。 PGHS-2的一条重要路线 表达通过p21 ras信号级联发生，因为PDGF 通过p21 ras激活细胞，有效增加PGHS-2转录。 由于NO也以p21 ras依赖的方式激活NF-kB，因此NO可以诱导 PGHS-2的表达。 因此，实验 提出确定NO改变PGHS-1的生化机制 活性，以及PGHS-2 mRNA和蛋白表达和细胞 活动 这些研究将与项目合作进行 四、这些合作对成功完成 具体目标2中提出的实验。 这些研究的结果 应该有重要的影响，因为它们可能建议一种机制， NO可能通过促进PGI/2的表达而调节动脉粥样硬化性疾病， 生成，这反过来又可以抑制胆固醇酯沉积。

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.