Intracellular pH regulation in cultured astrocytes from rat hippocampus .1. Role of HCO3-

We studied the regulation of intracellular pH (pH(i)) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pH(i). Intrinsic buffering power (beta(1)) was 10.5 mM (pH unit)(-1) at pH(i) 7.0, and decreased linearly with pH(i); the best-fit line to the data had a slope of -10.0 mM (pH unit)(-2). In the absence of HCO3-, pH(i) recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pH(i) 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH(i). Acid extrusion was inhibited 68% (pH(i) 6.23) by substituting Li+ for Na+ in the bath solution. Switching from a CO2/HCO3--free to a CO2/HCO3--containing bath solution caused mean steady state pH(i) to increase from 6.82 to 6.90, due to a Na+-driven HCO3-transporter. The HCO3--induced pH(i) increase was unaffected by amiloride, but was inhibited 75% (pH(i) 6.85) by 400 mu M 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and 65% (pH(i) 6.55-6.75) by pretreating astrocytes for up to similar to 6.3 h with 400 mu M 4-acetamide-4'-isothiocy anatostilbene-2,2'-disulfonic acid (SITS). The CO2/HCO3--induced pH(i) increase was blocked when external Na+ was replaced with N-methyl-D-glucammonium (NMDG(+)). In the presence of HCO3-, the Na+-driven HCO3- transporter contributed to the pH(i) recovery from an acid load. For example, HCO3- shifted the plot of acid-extrusion rate vs. pH(i) by 0.15-0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO3- increased acid extrusion 3.8-fold (pH(i) 6.20). When astrocytes were acid loaded in amiloride, with Li+ as the major cation, HCO3- failed to elicit a substantial increase in pH(i). Thus, Li+ does not appear to substitute well for Nai on the HCO3- transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na+-driven HCO3- transporter are the predominant acid extruders in astrocytes.