Novel effects of 5,8,11-eicosatriynoic acid, a lipoxygenase inhibitor, on Ca2+ mobilization in Madin Darby canine kidney cells.

Abstract

The effect of 5,8,11-eicosatriynoic acid, a widely used lipoxygenase inhibitor, on Ca2+ fate in Madin Darby canine kidney cells was examined by using fura-2 as a Ca2+ probe. At concentrations between 2-100 microM 5,8,11-eicosatriynoic acid increased [Ca2+]i concentration-dependently with an EC50 of 20 microM . Extracellular Ca2+ removal decreased the Ca2+ signals, indicating that 5,8,11-eicosatriynoic acid triggered Ca2+ release and Ca2+ influx. 5,8,11 -Eicosatriynoic acid (30 microM) induced a [Ca2+]i increase in Ca2+-free medium after pretreatment with carbonylcyanide m-chlorophenylhydrazone (2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor for 20 min. Conversely, 5,8,11-eicosatriynoic acid pretreatment almost abolished the Ca2+ release induced by carbonylcyanide m-chlorophenylhydrazone and thapsigargin. These results suggest that 30 microM 5,8,11-eicosatriynoic acid released Ca2+ from the endoplasmic reticulum, mitochondria and other stores. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 2-50 microM 5,8,11-eicosatriynoic acid for 10 min. in Ca2+-free medium concentration-dependently. Pretreatment with 10 microM La3+ abolished 30 microM 5,8,11-eicosatriynoic acid -induced [Ca2+]i increases, but adding La3+ during the decay phase had no effect. 5,8,11-Eicosatriynoic acid-induced Ca2+ release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), but was decreased by 60% by 40 microM aristolochic acid. Several other lipoxygenase inhibitors such as baicalein (50 microM), 5.8.11.14-eicosatetraynoic acid (ETYA; 0.1-0.2 mM), caffeic acid (5-50 microM), esculetin (5-50 microM), alpha-pentyl-3-(2-quinolinylmethoxy)-benzenemethanol (REV-5901; 0.1-0.2 mM) and alpha-pentyl-4-(2-quinolinylmethoxy)-benzenemethanol (L-655238; 80-100 microM) had no effect on [Ca2+]i. Collectively, the data suggest that the lipoxygenase inhibitor 5,8,11-eicosatriynoic acid induced a [Ca2+]i increase in renal tubular cells concentration-dependently, by releasing intracellular Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing extracellular Ca2+ influx in a La3+-sensitive manner.