Barium Influx Mediated by the Cardiac Sodium-Calcium Exchanger in Transfected Chinese Hamster Ovary Cells

We examined Ba(2+) influx using isotopic and fura-2 techniques in transfected Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger (CK1.4 cells). Ba(2+) competitively inhibited exchange-me diated (45)Ca(2+) uptake with a K (i) ∼ 3 mM. Ba(2+) uptake was stimulated by pretreating the cells with ouabain and by removing extracellular Na(+), as expected for Na(+)/Ba(2+) exchange activity. The maximal velocity of Ba(2+) accumulation was estimated to be 50% of that for Ca(2+). When the monovalent cation ionophore gramicidin was used to equilibrate internal and external concentrations of Na(+), Ba(2+) influx was negligible in the absence of Na(+) and increased to a maximum at 20–40 mM Na(+). At higher Na(+) concentrations, Ba(2+) influx declined, presumably due to the competition between Na(+) and Ba(2+) for transport sites on the exchanger. Unlike Ca(2+), Ba(2+) did not appear to be taken up by intracellular organelles: Thus, (133)Ba(2+) uptake in ouabain-treated cells was not reduced by mitochondrial inhibitors such as Cl-CCP or oligomycin-rotenone. Moreover, intracellular Ca(2+) stores that had been depleted of Ca(2+) by pretreatment of the cells with ionomycin (a Ca(2+) ionophore) remained empty during a subsequent period of Ba(2+) influx. Ca(2+) uptake or release by intracellular organelles secondarily regulated exchange activity through alterations in [Ca(2+)](i). Exchange-mediated Ba(2+) influx was inhibited when cytosolic [Ca(2+)] was reduced to 20 nM or less and was accelerated at cytosolic Ca(2+) concentrations of 25–50 nM. We conclude that (a) Ba(2+) substitutes for Ca(2+) as a transport substrate for the exchanger, (b) cytosolic Ba(2+) does not appear to be sequestered by intracellular organelles, and (c) exchange-mediated Ba(2+) influx is accelerated by low concentrations of cytosolic Ca(2+).