Intracellular sodium homeostasis in rat hippocampal astrocytes

1. We determined the intracellular Na⁺ concentration (Na⁺]₁ 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 μM). Dye signal calibration within the astrocytes showed that the ratiometric dye signal changed monotonically with changes in [Na⁺]₁ 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⁺]₁ 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⁺]₁, 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 ~5 mM, suggesting activation of inward Na⁺-HCO₃^-, cotransport by CO₂/HCO₃^-. During furosemide (frusemide, 1 mM) or bumetanide (50 μM) application, a slow decrease in [Na⁺]₁, of ~2 mM was observed, indicating a steady inward transport of Na⁺ via Na⁺-K⁺-2Cl^- cotransport under control conditions. Tetrodotoxin (100 μM) did not influence [Na⁺]₁ in the majority of cells (85%), suggesting that influx of Na⁺ through voltage-gated Na⁺ channels contributed to baseline [Na⁺]₁ 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⁺]₁ of about 2 and 4 mM min-1, respectively. This indicated that Na⁺,K+^--ATPase activity was critical in maintaining low [Na⁺]₁ in the face of a steep electrochemical gradient, which would favour a much higher [Na⁺]₁.

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⁺]₁, were sensitive to bumetanide. A reduction of [K⁺](o) by 1mM increased [Na⁺]₁ by 3.0 ± 2.3 mM . In contrast, changing extracellular Na⁺ concentration by 20 mM resulted in changes in [Na⁺]₁ of less than 3 mM.

6. These results implied that in hippocampal astrocytes low baseline [Na⁺]₁ 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.