Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : II. Electrogenic Na/HCO(3) Cotransport

In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453–465.), we showed that a Na(+)-driven influx of HCO(3) (−) causes the increase in intracellular pH (pH(i)) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO(2)/17 mM HCO(3) (−). In the present study, we used the pH-sensitive fluorescent indicator 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na(+)-driven Cl-HCO(3) exchanger, an electrogenic Na/HCO(3) cotransporter, or an electroneutral Na/HCO(3) cotransporter. To determine if the transporter is a Na(+)-driven Cl-HCO(3) exchanger, we depleted the cells of intracellular Cl(−) by incubating them in a Cl(−)-free solution for an average of ∼11 min. We verified the depletion with the Cl(−)-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl(−)-depleted cells, the pH(i) still increases after one or more exposures to CO(2)/HCO(3) (−). Furthermore, the pH(i) decrease elicited by external Na(+) removal does not require external Cl(−). Therefore, the transporter cannot be a Na(+)-driven Cl-HCO(3) exchanger. To determine if the transporter is an electrogenic Na/ HCO(3) cotransporter, we measured pH(i) and plasma membrane voltage (V(m)) while removing external Na(+), in the presence/absence of CO(2)/HCO(3) (−) and in the presence/absence of 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). The CO(2)/HCO(3) (−) solutions contained 20% CO(2) and 68 mM HCO(3) (−), pH 7.3, to maximize the HCO(3) (−) flux. In pH(i) experiments, removing external Na(+) in the presence of CO(2)/HCO(3) (−) elicited an equivalent HCO(3) (−) efflux of 281 μM s(−1). The HCO(3) (−) influx elicited by returning external Na(+) was inhibited 63% by DIDS, so that the predicted DIDS-sensitive V(m) change was 3.3 mV. Indeed, we found that removing external Na(+) elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO(2)/ HCO(3) (−). Thus, the Na/HCO(3) cotransporter is electrogenic. Because a cotransporter with a Na(+):HCO(3) (−) stoichiometry of 1:3 or higher would predict a net HCO(3) (−) efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO(3) cotransporter with a stoichiometry of 1:2.