Ketoconazole blocks organic osmolyte efflux independently of its effect on arachidonic acid conversion

Ketoconazole, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, and gossypol are reported inhibitors of the lipoxygenase (LO) and cytochrome P-450 enzyme systems and are potent blockers of swelling-activated efflux of organic osmolytes and volume-sensitive anion channels in C6 glioma cells. To directly test the hypothesis that LO- or cytochrome P-450-derived products of arachidonic acid (AA) participate in the regulation of these volume-sensitive transport pathways, we incubated C6 cells with [1-14C]AA and observed the extent and profile of its conversion under basal conditions and after acute swelling. High-performance liquid chromatographic analysis revealed that most (70-80%) of the labeled AA remained unchanged with only 6-8% and 10-20% of label converted to LO- [12(S)- and 15(S)-hydroxyeicosatetraenoic acid (12- and 15-HETE)] and cyclooxygenase- [prostaglandin (PG) E2 and PGF2a] derived products, respectively. Leukotrienes and epoxyeicosatrienoic acid compounds were not produced. The conversion profile of [1-14C]AA was not altered substantially by cell swelling. Treatment of cells with the LO-derived products 5-, 12-, and 15-HETE or their immediate metabolic precursors, 5(S)-, 12(S)-, and 15(S)-hydroxyperoxyeicosatetraenoic acid, at 5 microM concentrations did not stimulate efflux of [3H]inositol. In addition, treatment with HETEs did not override the inhibition of efflux observed with the LO-cytochrome P-450 blocker ketoconazole. Whole cell patch-clamp experiments demonstrated that volume-sensitive anion channels, the postulated pathway for organic osmolyte efflux in C6 cells, are rapidly and reversibly blocked by ketoconazole in a fashion suggestive of direct inhibition rather than via interruption of a second messenger pathway.(ABSTRACT TRUNCATED AT 250 WORDS)