Vol. 292, Issue 1, 358-365, January 2000

W-7 Induces [Ca²⁺]_i Increases 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, Taiwan, Republic of China (C.-R.J.)

The effect of W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride] on Ca²⁺ signaling in Madin-Darby canine kidney cells was investigated. W-7 (0.1-1 mM) induced a [Ca²⁺]_i increase, which comprised an initial increase and a plateau. Ca²⁺ removal inhibited the Ca²⁺ signals by 80%, suggesting that W-7 activated external Ca²⁺ influx and internal Ca²⁺ release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (2 µM) and the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (1 µM) abolished the internal Ca²⁺ release induced by 0.5 mM W-7; conversely, pretreatment with W-7 prevented thapsigargin and carbonylcyanide m-chlorophenylhydrazone from releasing internal Ca²⁺. W-7 (0.2 mM) induced Mn²⁺ quench of fura-2 fluorescence, which was inhibited by La³⁺ (0.1 mM) by 80%. La³⁺ (0.1 mM) partly inhibited 0.2 mM W-7-induced [Ca²⁺]_i increase. Addition of 5 mM Ca²⁺ induced a significant [Ca²⁺]_i increase after pretreating with 0.2 to 1 mM W-7 in Ca²⁺-free medium for 5 min, suggesting that W-7 induced capacitative Ca²⁺ entry. W-7 (0.5 mM) potentiated the capacitative Ca²⁺ entry induced by 1 µM thapsigargin by 15%. Pretreatment with aristolochic acid (40 µM) to inhibit phospholipase A₂ reduced 0.5 mM W-7-induced internal Ca²⁺ release and external Ca²⁺ influx by 25 and 80%, respectively. Inhibition of phospholipase C with U73122 (2 µM) or inhibition of phospholipase D with propranolol (0.1 mM) had no effect on the internal Ca²⁺ release induced by 0.5 mM W-7. It remains unclear whether W-7 induced [Ca²⁺]_i increases via inhibition of calmodulin. Three other calmodulin inhibitors (phenoxybenzamine, trifluoperazine, and fluphenazine-N-chloroethane) did not alter resting [Ca²⁺]_i.