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

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 CO 2 /HCO 3 − and by butyric acid in the presence and absence of CO 2 /HCO 3 − . 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% CO 2 /26 mM HCO 3 − , 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 HCO 3 − 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 CO 2 /H + /HCO 3 − 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 HCO 3 − 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