A further investigation of ATP-induced calcium mobilization in MDCK cells

We have previously reported that La3+ inhibited the ATP-induced rise in intracellular Ca2+ levels ([Ca2+](i)) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. Here we further investigated the ATP-induced Ca2+ signal, ATP caused a rise in [Ca2+](i) dose-dependently between 1 mu M-1 mM, The rises induced by 10 mu M-1 mM ATP were inhibited by Ca2+ removal. The pleateau phase of the ATP response was primarily maintained by Ca2+ influx because it was reduced or eliminated by Ca2+ removal, ATP failed to elevate [Ca2+](i) after the endoplasmic reticulum Ca2+ store had been depleted by 2,5-di-tert-butylhydroquinone or cyclopiazonic acid, suggesting that the ATP-induced Ca2+ influx was capacitative Ca2+ entry. Capacitative Ca2+ entry was directly measured by addition of 5 mM CaCl2 to cells pretreated with ATP (0.1 mM) in Ca2+-free medium. This capacitative Ca2+ entry was inhibited by econazole (25 mu M) or SKF96365 (50 mu M). The ATP response was significantly enhanced by extracellular alkalization to pH 8 or pretreatment with gly-phe-beta-naphthylamide. Pretreatment with carbonylcyanide m-chlorophenylhydrazone (CCCP) or extracellular Na+ removal had no enhancement, implicating that efflux via plasmalemmal Ca2+ pumps (but not Na+/Ca2+ exchange) and buffering by lysosomes (but not mitochondria) might be involved in the decay of the ATP response.