Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus

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Abstract
We studied the regulation of intracellular pH (pHi) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pHi. Intrinsic buffering power (βI) was 10.5 mM (pH unit)−1 at pHi 7.0, and decreased linearly with pHi; the best-fit line to the data had a slope of −10.0 mM (pH unit)−2. In the absence of HCO3−, pHi 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 pHi 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pHi. Acid extrusion was inhibited 68% (pHi 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 pHi to increase from 6.82 to 6.90, due to a Na+-driven HCO3− transporter. The HCO3−-induced pHi increase was unaffected by amiloride, but was inhibited 75% (pHi 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pHi 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 CO2/HCO3−-induced pHi 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 pHi recovery from an acid load. For example, HCO3− shifted the plot of acid-extrusion rate vs. pHi 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 (pHi 6.20). When astrocytes were acid loaded in amiloride, with Li+ as the major cation, HCO3− failed to elicit a substantial increase in pHi. Thus, Li+ does not appear to substitute well for Na+ 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.