W-7 induces [Ca(2+)](i) increases in Madin-Darby canine kidney (MDCK) cells

The effect of W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride] on Ca(2+) signaling in Madin-Darby canine kidney cells was investigated. W-7 (0.1-1 mM) induced a [Ca(2+)](i) increase, which comprised an initial increase and a plateau. Ca(2+) removal inhibited the Ca(2+) signals by 80%, suggesting that W-7 activated external Ca(2+) influx and internal Ca(2+) release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (2 microM) and the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM) abolished the internal Ca(2+) release induced by 0.5 mM W-7; conversely, pretreatment with W-7 prevented thapsigargin and carbonylcyanide m-chlorophenylhydrazone from releasing internal Ca(2+). W-7 (0.2 mM) induced Mn(2+) quench of fura-2 fluorescence, which was inhibited by La(3+) (0.1 mM) by 80%. La(3+) (0.1 mM) partly inhibited 0.2 mM W-7-induced [Ca(2+)](i) increase. Addition of 5 mM Ca(2+) induced a significant [Ca(2+)](i) increase after pretreating with 0.2 to 1 mM W-7 in Ca(2+)-free medium for 5 min, suggesting that W-7 induced capacitative Ca(2+) entry. W-7 (0.5 mM) potentiated the capacitative Ca(2+) entry induced by 1 microM thapsigargin by 15%. Pretreatment with aristolochic acid (40 microM) to inhibit phospholipase A(2) reduced 0.5 mM W-7-induced internal Ca(2+) release and external Ca(2+) influx by 25 and 80%, respectively. Inhibition of phospholipase C with U73122 (2 microM) or inhibition of phospholipase D with propranolol (0.1 mM) had no effect on the internal Ca(2+) release induced by 0.5 mM W-7. It remains unclear whether W-7 induced [Ca(2+)](i) increases via inhibition of calmodulin. Three other calmodulin inhibitors (phenoxybenzamine, trifluoperazine, and fluphenazine-N-chloroethane) did not alter resting [Ca(2+)](i).