SUMMARY 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 (P t and P 2 ), which accounted for 30 to 40% of the recovered radioactivity. Formation of prostaglandin E 2 and F^, accounted for only 3 to 5%. The chromatographic retention times of Pi and P 2 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 P 2 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" l0 M to 10~7 M) stimulated release of arachidonic acid metabolites from mTALH cells. P 2 yielded a principal product that proved to be a potent inhibitor of Na,K-ATPase, 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 P 2 formation could be demonstrated within 7 days.

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