20-HETE is a major cytochrome p450 (CYP) arachidonic acid (AA) metabolite in renal structures including proximal tubules, TALH and microvessels. 20- HETE has potent effects on the vasculature (vasoconstriction of renal arterioles) and tubules (inhibition of ion transport and K+ channel activity). It may, therefore, affect pro- and/or anti-hypertensive mechanisms. For example, an increase in 20-HETE synthesis in renal microvessels would bring about an increased vasoconstriction mechanisms influencing prohypertensive mechanism characterized by elevated vascular tone, increased GFR and RBF and sodium retention. On the other hand, increased tubular 20-HETE synthesis (proximal, TALH, macula densa) would be conducive to an increase in antihypertensive mechanisms operating at the level of regulation of salt and water balance and characterized by natriuresis and diuresis. The SHR is particularly interesting since it displays high (over expression) renal 20-HETE production. Furthermore, the development of hypertension in this model occurs between 5 and 9 weeks of age having been preceded by a many-fold increase in renal 20-HETE synthesis between 3 and 5 weeks. Inhibition of 2O-HETE synthesis in this stage is associated with blood pressure reduction suggesting a role for 20-HETE in the development of hypertension. This unique increase of 20- HETE production suggests the involvement of a distinct CYP isozyme(s) that is under regulatory control and that specifically metabolizes AA to 20- HETE. The CYP4A isoforms (4A1, 4A2 and 4A3), all of which are present in the rat kidney, are believed to be capable of metabolizing AA at the omega-carbon to yield 20-HETE. These isoforms, although sharing 66-98% homology, are localized to different renal structures and are exposed to different regulatory mechanisms. 20-HETE synthesis may be derived from different CYP4A isozymes in tubules and vessels. Alternatively, it may be formed by the same isozyme or a mixture of all three that are influenced by different regulatory elements. To understand the regulatory mechanisms underlying the renal synthesis of this important eicosanoid, we will characterize and correlate CYP4A protein and mRNA expression to AA omega- hydroxylation in tubular and vascular preparations from hypertensive and normotensive rats and study the influence of dietary salt. The contribution of CYP4A protein to 20-HETE synthesis in the kidney will be assessed by molecular cloning and expression of each isozyme following with analysis of its catalytic activity. The results of the proposed study will provide the biochemical basis for evaluating the functional significance of renal 20-HETE.
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