Barium Influx Mediated by the Cardiac Sodium-Calcium Exchanger in Transfected Chinese Hamster Ovary Cells |
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Authors: | Madalina Condrescu Galina Chernaya Vijay Kalaria John P Reeves |
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Institution: | From the Department of Physiology, Pharmacology and Toxicology, University of Medicine and Dentistry of New Jersey, The New Jersey Medical School, Newark, New Jersey 07103 |
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Abstract: | We examined Ba2+ influx using isotopic and fura-2 techniques in transfected Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells). Ba2+ competitively inhibited exchange-me diated 45Ca2+ uptake with a K
i ∼ 3 mM. Ba2+ uptake was stimulated by pretreating the cells with ouabain and by removing extracellular Na+, as expected for Na+/Ba2+ exchange activity. The maximal velocity of Ba2+ accumulation was estimated to be 50% of that for Ca2+. When the monovalent cation ionophore gramicidin was used to equilibrate internal and external concentrations of Na+, Ba2+ influx was negligible in the absence of Na+ and increased to a maximum at 20–40 mM Na+. At higher Na+ concentrations, Ba2+ influx declined, presumably due to the competition between Na+ and Ba2+ for transport sites on the exchanger. Unlike Ca2+, Ba2+ did not appear to be taken up by intracellular organelles: Thus, 133Ba2+ uptake in ouabain-treated cells was not reduced by mitochondrial inhibitors such as Cl-CCP or oligomycin-rotenone. Moreover, intracellular Ca2+ stores that had been depleted of Ca2+ by pretreatment of the cells with ionomycin (a Ca2+ ionophore) remained empty during a subsequent period of Ba2+ influx. Ca2+ uptake or release by intracellular organelles secondarily regulated exchange activity through alterations in Ca2+]i. Exchange-mediated Ba2+ influx was inhibited when cytosolic Ca2+] was reduced to 20 nM or less and was accelerated at cytosolic Ca2+ concentrations of 25–50 nM. We conclude that (a) Ba2+ substitutes for Ca2+ as a transport substrate for the exchanger, (b) cytosolic Ba2+ does not appear to be sequestered by intracellular organelles, and (c) exchange-mediated Ba2+ influx is accelerated by low concentrations of cytosolic Ca2+. |
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Keywords: | fura-2 endoplasmic reticulum Ba uptake Na/Ca exchange CHO cells |
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