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 .