Intracellular pH Regulation in Primary Rat Astrocytes and C6 Glioma Cells

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 ⁺ + HCO ₃ −/Cl ₋ exchange; and (3) acidifying Cl ⁻ /HCO ₃ ⁻ exchange. Na ⁺ /H ⁺ exchange was most evident in the absence of CO ₂ ; recovery from acidification was Na ⁺ dependent and amiloride sensitive. Exposure to CO ₂ 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 ⁺ + HCO ₃ ⁻ /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 ⁻ /HCO ₃ ⁻ exchange. Astrocytes, but not C6 cells, contain a fourth pH-regulating transporter, Na ⁺ −HCO ₃ ⁻ cotransport; in the presence of CO ₂ , depolarization caused an alkalinization of 0.12 ⁺ ₋ 0.01 (n = 8) and increased the rate of CO ₂ -induced alkalinization from 0.23 ± 0.02 to 0.42 ± 0.03 pH unit/min. Since C6 cells lack the Na ⁺ -HCO ₃ ⁺ 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 ⁺ + HCO ₃ ⁻ /Cl ⁻ exchange was present and largely responsible for CO ₂ −induced alkalinization; (3) Cl ⁻ /HCO ₃ ⁻ 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 CO ₂ .