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 microM-1 mM. The rises induced by 10 microM-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 Ca(2+)-free medium. This capacitative Ca2+ entry was inhibited by econazole (25 microM) or SKF96365 (50 microM). 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.
Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, Taiwan, ROC. crian@isca.vghks.gov.tw
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Jan
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Wu
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Tseng
D000255:Adenosine Triphosphate / Q000494:pharmacology / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D004285:Dogs; D004464:Econazole / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D007093:Imidazoles / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D007093:Imidazoles; D004464:Econazole; D000255:Adenosine Triphosphate; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium; D016257:Fura-2