Structural identification of cytochrome P450-dependent arachidonate metabolites formed by rabbit medullary thick...

The medullary thick ascending limb of Henle's loop (mTALH) contributes importantly to the regulation of extracellular fluid volume and composition and metabolizes arachidonic acid (AA) chiefly by a cytochrome P450 monooxygenase pathway. Rabbit mTALH cells, when incubated with radiolabeled [14C]AA, form products that segregate into two peaks designated P1 and P2 based on their reverse-phase high pressure liquid chromatography retention times. We have now definitively identified their chemical structures. mTALH cells, isolated from the rabbit outer medulla, were homogenized and incubated with [14C]AA in the presence of NADPH. The AA metabolites in P1 and P2 were identified by gas chromatographic-mass spectrometric methods, including fast atom bombardment, negative ion electron capture, and electron ionization. All mass spectrometric data, the lack of UV chromophores, and comparisons with authentic standards were consistent with P1 containing two principal components: 19-hydroxy-5,8,11,14 eicosatetraenoic acid (19-HETE) and 20 - hydroxy - 5,8,11,14 - eicosatetraenoic acid (20-HETE), P2 contained primarily 1,20-eicosa-5,8,11,14-tetraenedioic acid (20-COOH-AA). The biological properties of P1 and P2 were compared with those of the authentic standards of 19- and 20-HETE and 20-COOH-AA. P1 dose dependently relaxed precontracted mesenteric arterial rings, as did authentic (19S)- and (19R)-HETE, whereas 20-HETE relaxed at lower and contracted at higher concentrations. As P1 contained a mixture of 19- and 20-HETE, each of these AA metabolites presumably contributed to the vascular relaxation produced by P1. Neither P2 nor 20-COOH-AA exhibited vasoactivity, but each demonstrated a similar potency in inhibiting rabbit medullary Na(+)-K(+)-ATPase activity. As previously reported, P2 was a more potent inhibitor of Na(+)-K(+)-ATPase than P1. The lesser inhibitory activity of P1 presumably reflects the presence of similar amounts of 19-HETE, the least active metabolite, and 20-HETE, which resembles 20-COOH-AA in its capacity to inhibit Na(+)-K(+)-ATPase. Thus, the biological activity of the less polar peak, P1, can be accounted for by 19- and 20-HETE, and that of P2, by 20-COOH-AA.