CORNEAL ARACHIDONATE METABOLITES VIA CYTOCHROME P450

这是一项继续描述眼部生物合成和 新型细胞色素P450(P450)花生四烯酸(AA)的代谢 代谢物，测定其生物活性并评价其 对角膜功能的贡献以及在正常和病理生理条件下 (炎症)状态和房水动力学。在前述期间 在授权期内，我们证明了人、兔和人的角膜上皮细胞 牛眼通过P450途径将AA代谢成几个含氧的 代谢物。其中两个经GC/MS分离纯化和鉴定 与生物测定相结合的分析：12(R)HETE和12(R)DH-HETE。我们发现 12(R)HETE是一种有效的Na，K-ATPase抑制剂，并证明了它 降低兔眼内压(IOP)，表明它是一种 房水动力学的内源性角膜调节剂。角膜 透明和房水分泌物是过程的例子 依赖于Na，K-ATPase活性。我们假设12(R)HETE是一个 内源性Na，K-ATPase抑制物在眼内调节这些过程 因此有助于维持角膜的透明度和 眼压。另一方面，12(R)DH-HETE是一种强大的促炎药物 化合物；它扩张血管，增加膜的通透性， 刺激中性粒细胞迁移并产生新生血管。其效果 12(R)二氢呋喃脱氢表雄酮在兔眼上的作用 炎症性刺激。血管扩张，血房水破裂 屏障和新生血管是众所周知的眼部后果 发炎。我们假设通常会发生的炎症 角膜损伤后，部分是通过释放 12(R)由角膜上皮产生的脱氢表雄酮。这些炎症 后果是眼睛中许多病理过程的共同事件 超过了对角膜上皮的损伤。它将会引起人们对 未来将调查12(R)DH-HETE在以下情况下的参与情况 葡萄膜炎、老年性黄斑变性和糖尿病视网膜病变。至 评估这些新的代谢物在眼功能中的重要性， 有关生物化学和作用机制的若干问题 这些化合物必须得到解决。特别是关于 导致它们形成的酶步骤，它们在体内的代谢降解 眼组织以及其他眼组织是否有能力 在对他们的治疗进行评估之前，必须回答他们的问题 势是可以被画出来的。

This is a proposal to continue characterizing the ocular biosynthesis and metabolic fate of the novel cytochrome P450 (P450) arachidonic acid (AA) metabolites, determining their biological activities and evaluating their contribution to corneal function and under normal and pathophysiological (inflammation) states and to aqueous humor dynamics. During the preceding grant period, we demonstrated that corneal epithelium of human, rabbit and bovine eyes metabolizes AA via the P450 pathway to several oxygenated metabolites. Two of them have been purified and identified by GC/MS analysis coupled to bioassays: 12(R)HETE and 12(R)DH-HETE. We found that 12(R)HETE is a potent Na, K-ATPase inhibitor and demonstrated that it reduces intraocular pressure (IOP) in rabbits, suggesting it is an endogenous corneal modulator of aqueous humor dynamics. Corneal transparency and aqueous humor secretion are examples of processes that depend on Na,K-ATPase activity. We hypothesized that 12(R)HETE as an endogenous inhibitor of Na, K-ATPase modulates these processes in the eye and therefore contribute to the maintenance of corneal transparency and IOP. 12(R)DH-HETE, on the other hand, is a powerful pro-inflammatory compound; it dilates blood vessels, increases membrane permeability, stimulates neutrophil migration and produces neovascularization. The effect of 12(R)DH-HETE on the rabbit eye mimic the response of the eye to an inflammatory stimulus. Vasodilatation, breakdown of the blood aqueous barrier and neovascularization are well known consequences of ocular inflammation. We hypothesize that inflammation that typically occurs following injury of the cornea is mediated, in part, by the release of 12(R)DH-HETE produced by the corneal epithelium. These inflammation consequences are events common to many pathological processes in the eye beyond injury to the corneal epithelium. It will be of interest in the future to investigate the involvement of 12(R)DH-HETE in conditions such as uveitis, age-related macular degeneration and diabetic retinopathy. To assess the importance of these novel metabolites in ocular functions, certain questions regarding the biochemistry and the mechanism of action of these compounds have to be addressed. In particular, the questions of the enzymic steps leading to their formation, their metabolic degradation in ocular tissues and whether other ocular tissues have the capacity to produce them have to be answered before an evaluation on their therapeutic potential can be drawn.