Intracellular pH regulation in primary rat astrocytes and C6 glioma cells

Abstract

We used the pH‐sensitive fluorescent dye BCECF to study intracellular pH (pH_i) regulation in primary cultures of rat astrocytes and C6 glioma cells. Both cell types contain three pH‐regulating transporters: (1) alkalinizing Na⁺/H⁺ exchange; (2) alkalinizing Na⁺ + HCO₃ −/Cl₋ exchange; and (3) acidifying Cl⁻/HCO₃⁻ exchange. Na⁺/H⁺ exchange was most evident in the absence of CO₂; recovery from acidification was Na⁺ dependent and amiloride sensitive. Exposure to CO₂ caused a cell alkalinization that was inhibited by DIDS, dependent on external Na⁺, and inhibited 75% in the absence of Cl⁻ (thus mediated by Na⁺ + HCO₃⁻/Cl⁻ exchange). When pH_i was increased above the normal steady‐state pH_i, a DIDS‐inhibitable and Na⁺ ‐independent acidifying recovery was evident, indicating the presence of Cl⁻ /HCO₃⁻ exchange. Astrocytes, but not C6 cells, contain a fourth pH‐regulating transporter, Na⁺ −HCO₃⁻ cotransport; in the presence of CO₂, depolarization caused an alkalinization of 0.12 ⁺₋ 0.01 (n = 8) and increased the rate of CO₂‐induced alkalinization from 0.23 ± 0.02 to 0.42 ± 0.03 pH unit/min. Since C6 cells lack the Na⁺ ‐HCO₃⁺ cotransporter, they are an inferior model of pH_i regulation in glia. Our results differ from previous observations in glia in that: (1) Na⁺ /H⁺ exchange was entirely inhibited by amiloride; (2) Na⁺ + HCO₃⁻/Cl⁻ exchange was present and largely responsible for CO₂−induced alkalinization; (3) Cl⁻ /HCO₃⁻ exchange was only active at pH_i values above steady state; and (4) depolarization‐induced alkalinization of astrocytes was seen only in the presence of CO₂.