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

摘要

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. GLIA 2015;63:1581-1594