Intracellular sodium homeostasis in rat hippocampal astrocytes.

1. We determined the intracellular Na+ concentration ([Na+]i) and mechanisms of its regulation in cultured rat hippocampal astrocytes using fluorescence ratio imaging of the Na+ indicator SBFI‐AM (acetoxymethylester of sodium‐binding benzofuran isophthalate, 10 microM). Dye signal calibration within the astrocytes showed that the ratiometric dye signal changed monotonically with changes in [Na+]i from 0 to 140 nM. The K+ sensitivity of the dye was negligible; intracellular pH changes, however, slightly affected the ‘Na+’ signal. 2. Baseline [Na+]i was 14.6 +/‐ 4.9 mM (mean +/‐ S.D.) in CO2/HCO3(‐)‐containing saline with 3 mM K+. Removal of extracellular Na+ decreased [Na+]i in two phases: a rapid phase of [Na+]i reduction (0.58 +/‐ 0.32 mM min‐1) followed by a slower phase (0.15 +/‐ 0.09 mM min‐1). 3. Changing from CO2/HCO3(‐)‐free to CO2/HCO3(‐)‐buffered saline resulted in a transient increase in [Na+]i of approximately 5 mM, suggesting activation of inward Na(+)‐HCO3‐ cotransport by CO2/HCO3‐. During furosemide (frusemide, 1 mM) or bumetanide (50 microM) application, a slow decrease in [Na+]i of approximately 2 mM was observed, indicating a steady inward transport of Na+ via Na(+)‐K(+)‐2Cl‐ cotransport under control conditions. Tetrodotoxin (100 microM) did not influence [Na+]i in the majority of cells (85%), suggesting that influx of Na+ through voltage‐gated Na+ channels contributed to baseline [Na+]i in only a small subpopulation of hippocampal astrocytes. 4. Blocking Na+, K(+)‐ATPase activity with cardiac glycosides (ouabain or strophanthidin, 1 mM) or removal of extracellular K+ led to an increase in [Na+]i of about 2 and 4 mM min‐1, respectively. This indicated that Na+, K(+)‐ATPase activity was critical in maintaining low [Na+]i in the face of a steep electrochemical gradient, which would favour a much higher [Na+]i. 5. Elevation of extracellular K+ concentration ([K+]o) by as little as 1 mM (from 3 to 4 mM) resulted in a rapid and reversible decrease in [Na+]i. Both the slope and the amplitude of the [K+]o‐induced reductions in [Na+]i were sensitive to bumetanide. A reduction of [K+]o by 1 mM increased [Na+]i by 3.0 +/‐ 2.3 mM. In contrast, changing extracellular Na+ concentration by 20 mM resulted in changes in [Na+]i of less than 3 mM. 6. These results implied that in hippocampal astrocytes low baseline [Na+]i is determined by the action of Na(+)‐HCO3‐ cotransport, Na(+)‐K(+)‐2Cl‐ cotransport and Na+, K(+)‐ATPase, and that both Na+, K(+)‐ATPase and inward Na(+)‐K(+)‐2Cl cotransport are activated by small, physiologically relevant increases in [K+]o. These mechanisms are well suited to help buffer increases in [K+]o associated with neural activity.