Cultured rat astrocytes possess Na(+)-Ca2+ exchanger

Na(+)-Ca2+ exchange activity in its reverse mode was demonstrated in cultured rat astrocytes. Combination of ouabain (1 mM) and monensin (20 microM) caused a marked increase in 45Ca2+ uptake in astrocytes. 45Ca2+ uptake was also stimulated by lowering the external Na+ concentration. Ouabain plus monensin-stimulated 45Ca2+ uptake was blocked by 3,4-dichlorobenzamil (IC50, 16 microM), an inhibitor of Na(+)-Ca2+ exchanger, but not by nifedipine (0.1 microM). The stimulated-45Ca2+ uptake was observed even in K(+)-free medium, and external K+ at 5-10 mM caused a 2.2-fold increase in the uptake. Microspectrofluorimetry using the Ca(2+)-sensitive dye fura-2 showed that ouabain plus monensin increased intracellular Ca2+ concentration in single astrocytes. The Ca2+ signal was dependent on external Ca2+ (EC50, 1.4 mM), and blocked by 20 microM 3,4-dichlorobenzamil, but not by Ca2+ channel blockers (Cd2+, 20 microM; Ni2+, 100 microM). Antiserum of cardiac Na(+)-Ca2+ exchanger recognized 160 and 120-135 kDa proteins on SDS-polyacrylamide gel electrophoresis of astrocyte homogenate. Northern blot analysis revealed the presence of mRNA for the exchanger protein in astrocytes. These findings indicate that Na(+)-Ca2+ exchanger which is modulated by K+ is present in cultured rat astrocytes.