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

Abstract

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)

Blockade of swelling‐induced chloride channels by phenol derivatives

“…The depolarization of PD is believed to be predominantly due to the activation of chloride channels after reducing extracellular osmolarity Paulmichl et al, 1989;Weiss & Lang, 1992 The experiments lead to the conclusion that gossypol and NDGA are able to block directly the swelling-induced chloride channels. For gossypol a similar mechanism was proposed by Strange and collaborators (Strange et al, 1993;McManus et al, 1994). As shown in Figure 3 NDGA added to the extracellular fluid is able to block the swelling-activated chloride current within seconds.…”

Reactive oxygen species are important mediators of taurine release from skeletal muscle cells

“…The data in the present paper indicate that 5-LO is present in the myotube cultures and that cell swelling, as well as direct stimulation of PLA 2 activity with melittin, leads to taurine release via a DIDS-sensitive transport pathway that requires 5-LO activity for activation (Table 1). It is noted that several inhibitors of arachidonic acid metabolism nonspecifically block volume-dependent anion channels, which are permeable to taurine (28). However, 5-LO inhibition in the case of HeLa cells was obtained by different types of inhibition, i.e., substrate inhibition, inhibition of the 5-LO binding to the 5-LO activating protein (FLAP), direct inhibition, and ROS scavenging by addition of antioxidants (21).…”

Mechanisms Sensing and Modulating Signals Arising From Cell Swelling

“…Volume regulatory K + channels are activated by PGE2 in ciliary epithelial cells [42]. Ketoconazole, a blocker of lipoxygenase-and cytochrome P-450 enzyme systems, inhibits the release of organic osmolytes from renal papillary cells, MDCK cells and C6 glioma cells [43][44][45]. Since in C6 cells this effect can not be reversed by addition of HETEs, this effect on osmolyte flux is most likely not due to an inhibition of epoxygenase, but rather due to a direct block of an efflux pathway.…”