title: High effective cytosolic H+ buffering in mouse cortical astrocytes attributable to fast bicarbonate transport.
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.
authors.affiliation: Abteilung Für Allgemeine Zoologie, Fachbereich Biologie, University of Kaiserslautern, Kaiserslautern, D-67653, Germany.
authors.forename: Shefeeq M
authors.initials: SM
authors.lastname: Theparambil
authors.affiliation: Abteilung Für Allgemeine Zoologie, Fachbereich Biologie, University of Kaiserslautern, Kaiserslautern, D-67653, Germany.
authors.forename: Joachim W
authors.initials: JW
authors.lastname: Deitmer
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009865:Oocytes; D011522:Protons; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D001639:Bicarbonates; D011522:Protons; C498899:Slc4a4 protein, mouse; D027982:Sodium-Bicarbonate Symporters
keywords: carbonic anhydrase; pH; sodium-bicarbonate cotransport; sodium/hydrogen exchange