High effective cytosolic H+ buffering in mouse cortical astrocytes attributable to fast bicarbonate transport

Glia. 2015 Sep;63(9):1581-94. doi: 10.1002/glia.22829. Epub 2015 Mar 27.

Abstract

Cytosolic H(+) buffering plays a major role for shaping intracellular H(+) shifts and hence for the availability of H(+) for biochemical reactions and acid/base-coupled transport processes. H(+) buffering is one of the prime means to protect the cell from large acid/base shifts. We have used the H(+) indicator dye BCECF and confocal microscopy to monitor the cytosolic H(+) concentration, [H(+)]i, in cultured cortical astrocytes of wild-type mice and of mice deficient in sodium/bicarbonate cotransporter NBCe1 (NBCe1-KO) or in carbonic anhydrase isoform II (CAII-KO). The steady-state buffer strength was calculated from the amplitude of [H(+)]i transients as evoked by CO2/HCO3(-) and by butyric acid in the presence and absence of CO2/HCO3(-). We tested the hypotheses if, in addition to instantaneous physicochemical H(+) buffering, rapid acid/base transport across the cell membrane contributes to the total, "effective" cytosolic H(+) buffering. In the presence of 5% CO2/26 mM HCO3(-), H(+) buffer strength in astrocytes was increased 4-6 fold, as compared with that in non-bicarbonate, HEPES-buffered solution, which was largely attributable to fast HCO3 (-) transport into the cells via NBCe1, supported by CAII activity. Our results show that within the time frame of determining physiological H(+) buffering in cells, fast transport and equilibration of CO2/H(+)/HCO3(-) can make a major contribution to the total "effective" H(+) buffer strength. Thus, "effective" cellular H(+) buffering is, to a large extent, attributable to membrane transport of base equivalents rather than a purely passive physicochemical process, and can be much larger than reported so far. Not only physicochemical H(+) buffering, but also rapid import of HCO3(-) via the electrogenic sodium-bicarbonate cotransporter NBCe1, supported by carbonic anhydrase II (CA II), was identified to enhance cytosolic H(+) buffer strength substantially.

Keywords: carbonic anhydrase; pH; sodium-bicarbonate cotransport; sodium/hydrogen exchange.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Astrocytes / metabolism*
  • Bicarbonates / metabolism
  • Biological Transport / physiology*
  • Cells, Cultured
  • Cerebral Cortex / metabolism*
  • Cytosol / metabolism*
  • Mice, 129 Strain
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Oocytes
  • Protons*
  • Sodium-Bicarbonate Symporters / genetics
  • Sodium-Bicarbonate Symporters / metabolism*
  • Xenopus laevis

Substances

  • Bicarbonates
  • Protons
  • Slc4a4 protein, mouse
  • Sodium-Bicarbonate Symporters