CORNEAL ARACHIDONATE METABOLITES VIA CYTOCHROME P450

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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.