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

Academic Article

Abstract

  • 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 ) was 10.5 mM (pH unit) 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) . In the absence of HCO / , 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 /HCO / -free to a CO /HCO / -containing bath solution caused mean steady state pH(i) to increase from 6.82 to 6.90, due to a Na -driven HCO / transporter. The HCO / -induced pHi 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 /HCO / - induced phi increase was blocked when external Na was replaced with N- methyl-D-glucammonium (NMDG ). In the presence of HCO / , the Na - driven HCO / transporter contributed to the pH(i) recovery from an acid load. For example, HCO / 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 / increased acid extrusion 3.8-fold (pH(i) 6.20). When astrocytes were acid loaded in amiloride, with Li as the major cation, HCO / failed to elicit a substantial increase in pH(i). 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. 1 1 3 2 3 2 3 3 3 2 3 3 3 3 3 3 3 3 - -2 - + + - - + - - - + + - + - - - + - + + - + -
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    Author List

  • Bevensee MO; Weed RA; Boron WF
  • Start Page

  • 453
  • End Page

  • 465
  • Volume

  • 110
  • Issue

  • 4