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)
McManus
M
M
Department of Medicine, Children's Hospital, Boston, Massachusetts.
Serhan
C
C
Jackson
P
P
Strange
K
K
D000818:Animals; D000838:Anions / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism*; D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology; D002851:Chromatography, High Pressure Liquid; D015777:Eicosanoids / Q000378:metabolism / Q000494:pharmacology; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D007294:Inositol / Q000378:metabolism; D007473:Ion Channels / Q000037:antagonists & inhibitors; D007654:Ketoconazole / Q000494:pharmacology*; D020024:Leukotriene Antagonists; D009995:Osmosis / Q000187:drug effects; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
D000838:Anions; D015777:Eicosanoids; D007473:Ion Channels; D020024:Leukotriene Antagonists; D016718:Arachidonic Acid; D007294:Inositol; D011451:Prostaglandin-Endoperoxide Synthases; D007654:Ketoconazole