BARIUM INFLUX MEDIATED BY THE CARDIAC SODIUM-CALCIUM EXCHANGER IN TRANSFECTED CHINESE-HAMSTER OVARY CELLS

We examined Ba2+ influx using isotopic and fura-2 techniques in transf ected Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca 2+ exchanger (CK1.4 cells). Ba2+ competitively inhibited exchange-me d iated Ca-45(2+) uptake with a K-i similar to 3 mM. Ba2+ uptake was sti mulated by pretreating the cells with ouabain and by removing extracel lular Na+, as expected for Na+/Ba2+ exchange activity. The maximal vel ocity of Ba2+ accumulation was estimated to be 50% of that for Ca2+. W hen the monovalent cation ionophore gramicidin was used to equilibrate internal and external concentrations of Na+, Ba2+ influx was negligib le in the absence of Na+ and increased to a maximum at 20-40 mM Na+. A t higher Na+ concentrations, Ba2+ influx declined, presumably due to t he competition between Na+ and Ba2+ for transport sites on the exchang er. Unlike Ca2+, Ba2+ did not appear to be taken up by intracellular o rganelles: Thus, Ba-133(2+) uptake in ouabain-treated cells was not re duced by mitochondrial inhibitors such as Cl-CCP or oligomycin-rotenon e. Moreover, intracellular Ca2+ stores that had been depleted of Ca2by pretreatment of the cells with ionomycin (a Ca2+ ionophore) remaine d empty during a subsequent period of Ba2+ influx. Ca2+ uptake or rele ase by intracellular organelles secondarily regulated exchange activit y through alterations in [Ca2+](i). Exchange-mediated Ba2+ influx was inhibited when cytosolic [Ca2+] was reduced to 20 nM or less and was a ccelerated at cytosolic Ca2+ concentrations of 25-50 nM. We conclude t hat (a) Ba2+ substitutes for Ca2+ as a transport substrate for the exc hanger, (b) cytosolic Ba2+ does not appear to be sequestered by intrac ellular organelles, and (c) exchange-mediated Ba2+ influx is accelerat ed by low concentrations of cytosolic Ca2+.