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

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 μM 5,8,11-eicosatriynoic acid increased [Ca²⁺]_i concentration-dependently with an EC₅₀ of 20 μM. 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 μM) induced a [Ca²⁺]_i increase in Ca²⁺-free medium after pretreatment with carbonylcyanide m-chlorophenylhydrazone (2 μM), a mitochondrial uncoupler, and thapsigargin (1 μM), 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 μM 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 μM 5,8,11-eicosatriynoic acid for 10 min. in Ca²⁺-free medium concentration-dependently. Pretreatment with 10 μM La³⁺ abolished 30 μM 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 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole- 2,5-dione (U73122), but was decreased by 60% by 40 μM aristolochic acid. Several other lipoxygenase inhibitors such as baicalein (50 μM), 5.8.11.14-eicosatetraynoic acid (ETYA; 0.1-0.2 mM), caffeic acid (5-50 μM), esculetin (5-50 μM), α-pentyl-3-(2-quinolinylmethoxy)-benzenemethanol (REV-5901; 0.1-0.2 mM) and α-pentyl-4-(2-quinolinylmethoxy)-benzenemethanol (L-655238; 80-100 μM) 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.