Intracellular pH regulation in cultured astrocytes from rat hippocampus. I. Role Of HCO3-.
We studied the regulation of intracellular pH (pH) 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. Intrinsic buffering power (beta) was 10.5 mM (pH unit) at pH 7.0, and decreased linearly with pH; the best-fit line to the data had a slope of -10.0 mM (pH unit). In the absence of HCO, pH 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 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH. Acid extrusion was inhibited 68% (pH 6.23) by substituting Li for Na in the bath solution. Switching from a CO/HCO-free to a CO/HCO-containing bath solution caused mean steady state pH to increase from 6.82 to 6.90, due to a Na-driven HCO transporter. The HCO-induced pH increase was unaffected by amiloride, but was inhibited 75% (pH 6.85) by 400 microM 4, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and 65% (pH 6.55-6.75) by pretreating astrocytes for up to approximately 6.3 h with 400 microM 4-acetamide-4'-isothiocyanatostilbene-2, 2'-disulfonic acid (SITS). The CO/HCO-induced pH increase was blocked when external Na was replaced with -methyl--glucammonium (NMDG). In the presence of HCO, the Na-driven HCO transporter contributed to the pH recovery from an acid load. For example, HCO shifted the plot of acid-extrusion rate vs. pH by 0.15-0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO increased acid extrusion 3.8-fold (pH 6.20). When astrocytes were acid loaded in amiloride, with Li as the major cation, HCO failed to elicit a substantial increase in pH. Thus, Li does not appear to substitute well for Na on the HCO transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na-driven HCO transporter are the predominant acid extruders in astrocytes.
Bevensee
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Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Weed
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Boron
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D000818:Animals; D001253:Astrocytes / Q000502:physiology*; D001639:Bicarbonates / Q000378:metabolism*; D002021:Buffers; D002245:Carbon Dioxide / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D006624:Hippocampus / Q000166:cytology / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D051381:Rats; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D013267:Stilbenes / Q000494:pharmacology
D001639:Bicarbonates; D002021:Buffers; D017923:Sodium-Hydrogen Exchangers; D013267:Stilbenes; D002245:Carbon Dioxide