Intracellular pH regulation in cultured astrocytes from rat hippocampus. II. Electrogenic Na/HCO3 cotransport

Academic Article

Abstract

  • In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453-465.), we showed that a Na -driven influx of HCO / causes the increase in intracellular pH (pH(i)) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO /17 mM HCO / . In the present study, we used the pH-sensitive fluorescent indicator 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na -driven Cl-HCO exchanger, an electrogenic Na/HCO cotransporter, or an electroneutral Na/HCO cotransporter. To determine if the transporter is a Na -driven Cl-HCO exchanger, we depleted the cells of intracellular Cl by incubating them in a Cl -free solution for an average of ~11 min. We verified the depletion with the Cl -sensitive dye N-(6- methoxyquinolyl)acetoethyl ester (MQAE). In CL -depleted cells, the ph(i) still increases after one or more exposures to CO /HCO / . Furthermore, the pH(i) decrease elicited by external Na removal does not require external CL . Therefore, the transporter cannot be a Na -driven Cl-HCO exchanger. To determine if the transporter is an electrogenic Na/HCO cotransporter, we measured pHi and plasma membrane voltage (V(m)) while removing external Na , in the presence/absence of CO /HCO / and in the presence/absence of 400 μM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). The CO /HCO / solutions contained 20% CO and 68 mM HCO / , pH 7.3, to maximize the HCO / flux. In pH(i) experiments, removing external Na in the presence of CO /HCO / elicited an equivalent HCO / efflux of 281 μM s . The HCO / influx elicited by returning external Na was inhibited 63% by DIDS, so that the predicted DIDS-sensitive V(m) change was 3.3 mV. Indeed, we found that removing external Na elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO /HCO / . Thus, the Na/HCO cotransporter is electrogenic. Because a cotransporter with a Na :HCO / stoichiometry of 1:3 or higher would predict a net HCO / efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO cotransporter with a stoichiometry of 1:2. + - - + + - - - - - + - + + - - - - + - - -1 - + + - + - - 3 2 3 3 3 3 3 2 3 3 3 2 3 2 3 2 3 3 2 3 3 3 2 3 3 3 3 3
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    Author List

  • Bevensee MO; Apkon M; Boron WF
  • Start Page

  • 467
  • End Page

  • 483
  • Volume

  • 110
  • Issue

  • 4