Ionomycin enhances Ca2+ influx by stimulating store-regulated cation entry and not by a direct action at the plasma membrane.
In fura-2-loaded ECV304 cells ionomycin elicited a saturable biphasic change in intracellular Ca2+ concentration ([Ca2+]i), where the initial phase represented mobilization of intracellular stores and the sustained component represented Ca2+ influx. To examine whether ionomycin could stimulate influx via a store-dependent mechanism. Mn2+ entry was monitored by the quenching of fura-2 fluorescence: influx was enhanced even after ionomycin wash-out, provided that internal stores were not refilled with Ca2+. Moreover, the maximal rate of histamine-stimulated Mn2+ entry was unaffected by ionomycin, suggesting a common route of entry. The Ca(2+)-entry blocker SK&F 96365 inhibited both the ionomycin-induced Mn2+ entry and the sustained [Ca2+]i response to the ionophore (leaving the initial peak [Ca2+]i response unaffected). In other experiments, although addition of ionomycin further increased the plateau phase induced by 100 microM histamine, the increase was completely abolished by pretreatment with the store Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). Furthermore, in store-depleted cells, re-addition of 1 mM extracellular Ca2+ (in the presence of CPA plus histamine) led to a rapid rise in [Ca2+]i, dependent on Ca2+ influx, with kinetics that were not enhanced by ionomycin. These data suggest that ionomycin acts primarily at the level of the internal Ca2+ stores, so that, at the concentrations used here (< or = 1 microM), it increases Ca2+ (and Mn2+) influx via activation of endogenous entry pathways and not by plasmalemmal translocation.
Vascular Biology Research Centre, King's College London, University of London, Kensington, U.K.
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Morgan
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Jacob
D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002451:Cell Compartmentation / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004730:Endothelium, Vascular / Q000166:cytology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology*; D008345:Manganese / Q000378:metabolism; D011738:Pyrilamine / Q000494:pharmacology
D007093:Imidazoles; D007211:Indoles; D008345:Manganese; D015759:Ionomycin; D006632:Histamine; D000252:Calcium-Transporting ATPases; D011738:Pyrilamine; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium; C000543:cyclopiazonic acid