Vol. 294, Issue 1, 96-102, July 2000

MK-886, a Leukotriene Biosynthesis Inhibitor, as an Activator of Ca²⁺ Mobilization in Madin-Darby Canine Kidney (MDCK) Cells¹

Chung-Ren Jan and Ching-Jiunn Tseng

Department of Medical Education and Research, Veterans General Hospital-Kaohsiung (C.-R.J., C.-J.T.); and Department of Biology and Institute of Life Sciences, National Sun Yat-sen University (C.-R.J.), Kaohsiung, Taiwan

The effect of 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (MK-886), a leukotriene biosynthesis inhibitor, on Ca²⁺ mobilization in Madin- Darby canine kidney cells has been examined by fluorimetry using fura-2 as a Ca²⁺ indicator. MK-886 at 0.5 to 25 µM concentration dependently increased [Ca²⁺]_i. The [Ca²⁺]_i increase comprised an immediate initial rise and a slowly decaying phase. Ca²⁺ removal inhibited the Ca²⁺ signals by reducing both the initial rise and the decay phase, suggesting that MK-886 activated Ca²⁺ influx and Ca²⁺ release. In Ca²⁺-free medium, 10 µM MK-886 still increased [Ca²⁺]_i after pretreatment with carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 µM), a mitochondrial uncoupler, and thapsigargin (1 µM), an endoplasmic reticulum Ca²⁺ pump inhibitor. Conversely, pretreatment with MK-886 abolished CCCP- and thapsigargin-induced Ca²⁺ release. This suggests that 10 µM MK-886 released Ca²⁺ from the endoplasmic reticulum, mitochondria, and other stores. The addition of 3 mM Ca²⁺ increased [Ca²⁺]_i after pretreatment with 10 µM MK-886 for 700 s in Ca²⁺-free medium, indicating that MK-886 induced capacitative Ca²⁺ entry. This capacitative Ca²⁺ entry was partly inhibited by SKF96365 (50 µM), by econazole (25 µM), and by inhibiting phospholipase A₂ with aristolochic acid (40 µM) but not by inhibiting phospholipase D with 0.1 mM propranolol. MK-886 (10 µM)-induced Ca²⁺ release was not altered by inhibiting phospholipase C with U73122 (2 µM) but was inhibited by 50% by suppressing phospholipase D and phospholipase A₂ with propranolol (0.1 mM) and aristolochic acid (40 µM), respectively.