Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus: I. Role of HCO3

MO Bevensee, RA Weed, WF Boron - The Journal of general physiology, 1997 - rupress.org
MO Bevensee, RA Weed, WF Boron
The Journal of general physiology, 1997rupress.org
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 …
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.
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