Intracellular pH regulation in cultured astrocytes from rat hippocampus. II. Electrogenic Na/HCO3 cotransport

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