Cytochrome P-450 arachidonate metabolites affect ion fluxes in rabbit medullary thick ascending limb

The medullary thick ascending limb of Henle's loop (mTALH) of the rabbit metabolizes arachidonic acid (AA) via a cytochrome P-450 (P-450) monooxygenase pathway to several products, of which the principal are 20- hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) and 1,20- eicosatetraenedioic acid (20-COOH-AA). To understand their mechanism of action on alkali cation metabolism in mTALH cells, we have compared their effects with those of ouabain and furosemide. Incubation of rabbit isolated mTALH cells with either 1 mM ouabain or furosemide decreased K⁺ content from a control of 1,015 ± 51 peq/μg protein to 717 ± 41 and to 548 ± 48 peq/μg protein, respectively, whereas they had opposite effects on Na⁺ content; from a control of 138 ± 22 peq/μg protein, ouabain increased Na⁺ content to 357 ± 37 peq/μg protein, and furosemide decreased it to 64 ± 23 peq/μg protein. Preincubation with either 20-HETE (1 μM) or 20-COOH-AA (1 μM) decreased Na⁺ and K⁺, resembling furosemide in their effects on Na⁺ and K⁺ content. In other experiments we used monensin-treated cells to determine ⁸⁶Rb uptake under conditions in which Na⁺ entry into the cell was not rate limiting. Under these conditions ouabain still inhibited ⁸⁶Rb uptake, and the effect of AA was blocked. A major action of AA metabolites on Na⁺-K⁺-adenosinetriphosphatase was thereby excluded. Furthermore, AA metabolites did not inhibit Ba²⁺-sensitive ⁸⁶Rb efflux, indicating that they do not act through K⁺ channels. We conclude that these P-450-AA metabolites have a furosemide-like effect; namely, they act principally to inhibit the Na⁺-K⁺-2Cl^- cotransporter of the mTALH cells.