Renal Arachidonic Acid Metabolism

Cells were isolated from the outer medulla of the rabbit kidney, primarily from the thick ascending limb of Henle's loop (mTALH). These mTALH cells are heavily invested with a cytochrome P450-Iinked monooxygenase that represents the third pathway by which arachidonic acid is metabolized. After cell separation, approximately 80% of the cells proved to be mTALH in origin, based on electron microscopic criteria and immunofluorescent localization of Tamm-Horsfall protein, a specific marker for mTALH cells. The specific activity of alkaline phosphatase, a marker for proximal tubular cells, decreased threefold after separation of mTALH cells from outer medullary cells, associated with a fourfold Increase in the capacity of the separated mTALH cells to metabolize arachidonic acid. Incubation of mTALH cells with C-arachidonic acid resulted in formation of oxygenated metabolites, identified as two peaks (Pt and P2), which accounted for 30 to 40% of the recovered radioactivity. Formation of prostaglandin E2 and F^, accounted for only 3 to 5%. The chromatographic retention times of Pi and P2 were different from products of lipoxygenases. An inhibitor of cytochrome P450-dependent enzymes, SKF-525A (50 fj.M), reduced product formation by mTALH cells by more than 70%, while induction of cytochrome P450 increased product formation. Formation of P, and P2 by cell-free homogenates of mTALH was totally dependent on the presence of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), which suggests a NADPHdependent cytochrome P450-linked monooxygenase pathway. Vasopressin and calcitonin (10" M to 10~ M) stimulated release of arachidonic acid metabolites from mTALH cells. P2 yielded a principal product that proved to be a potent inhibitor of Na,KATPase, whereas the major component of P, was a weak inhibitor. The latter material relaxed isolated blood vessels. In mTALH cells obtained from rabbits made hypertensive by aortic constriction, increased P, and P2 formation could be demonstrated within 7 days. (Hypertension 7 [Suppl I]: I-136-1-144, 1985)