Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : I. Role of HCO(3)(−)

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 (β(I)) 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 HCO(3) (−), 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 CO(2)/HCO(3) (−)-free to a CO(2)/HCO(3) (−)-containing bath solution caused mean steady state pH(i) to increase from 6.82 to 6.90, due to a Na(+)-driven HCO(3) (−) transporter. The HCO(3) (−)-induced pH(i) increase was unaffected by amiloride, but was inhibited 75% (pH(i) 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pH(i) 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO(2)/HCO(3) (−)-induced pH(i) increase was blocked when external Na(+) was replaced with N-methyl-d-glucammonium (NMDG(+)). In the presence of HCO(3) (−), the Na(+)-driven HCO(3) (−) transporter contributed to the pH(i) recovery from an acid load. For example, HCO(3) (−) 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, HCO(3) (−) increased acid extrusion 3.8-fold (pH(i) 6.20). When astrocytes were acid loaded in amiloride, with Li(+) as the major cation, HCO(3) (−) failed to elicit a substantial increase in pH(i). Thus, Li(+) does not appear to substitute well for Na(+) on the HCO(3) (−) transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na(+)-driven HCO(3) (−) transporter are the predominant acid extruders in astrocytes.