Reversed electrogenic sodium bicarbonate cotransporter 1 is the major acid loader during recovery from cytosolic alkalosis in mouse cortical astrocytes

Abstract

Key points: The regulation of H+i from cytosolic alkalosis has generally been attributed to the activity of Cl--coupled acid loaders/base extruders in most cell types, including brain cells. The present study demonstrates that outwardly-directed sodium bicarbonate cotransport via electrogenic sodium bicarbonate cotransporter 1 (NBCe1) mediates the major fraction of H+i regulation from cytosolic alkalosis in mouse cortical astrocytes. Cl--coupled acid-loading transporters play only a minor role in the regulation of H+i from alkalosis in mouse cortical astrocytes. NBCe1-mediated H+i regulation from alkalosis was dominant, with the support of intracellular carbonic anhydrase II, even when the intra- and extracellular [HCO3-] was very low (<1mM), as in nominally CO2/HCO3- free condition. A reversed NBCe1 in astrocytes may also be significant for stabilizing extracellular pH in brain tissue. Recovery of intracellular pH from cytosolic alkalosis has been attributed primarily to Cl- coupled acid loaders/base extruders such as Cl-/HCO3- or Cl-/OH- exchangers. We have studied this process in cortical astrocytes from wild-type and transgenic mouse models with gene deletion for the electrogenic sodium bicarbonate cotransporter 1 (NBCe1) and for carbonic anhydrase (CA) isoform II. An acute cytosolic alkalosis was induced by the removal of either CO2/HCO3- or butyric acid, and the subsequent acid loading was analysed by monitoring changes in cytosolic H+ or Na+ using ion-sensitive fluorescent dyes. We have identified that NBCe1 reverses during alkalosis and contributes more than 70% to the rate of recovery from alkalosis by extruding Na+ and HCO3-. After CA inhibition or in CAII-knockout (KO) cells, the rate of recovery was reduced by 40%, and even by 70% in the nominal absence of CO2/HCO3-. Increasing the extracellular K+ concentration modulated the rate of acid loading in wild-type cells, but not in NBCe1-KO cells. Removing chloride had only a minor effect on the recovery from alkalosis. Reversal of NBCe1 by reducing pH/[HCO3-] was demonstrated in astrocytes and in Xenopus oocytes, in which human NBCe1 was heterologously expressed. The results obtained suggest that reversed NBCe1, supported by CAII activity, plays a major role in acid-loading cortical astrocytes to support recovery from cytosolic alkalosis.