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 mu M). Dye signal calibration within the astrocytes showed that the ratiometric dye signal changed monotonically with changes in [Na+](i) from 0 to 140 mM. 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 similar to 5 mM, suggesting activation of inward Na+-HCO3- cotransport by CO2/HCO3-. During furosemide (frusemide, 1 mM) or bumetanide (50 mu M) application, a slow decrease in [Na+](i) of similar to 2 mM was observed, indicating a steady inward transport of Na+ via Na+-K+-2Cl(-) cotransport under control conditions. Tetrodotoxin (100 mu M) 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 increase in [K+](o). These mechanisms are well suited to help buffer increases in [K+](o) associated with neural activity.
