title: Mechanisms of pHi regulation studied in individual neurons cultured from mouse cerebral cortex.
abstract: Maintenance and regulation of intracellular pH (pHi) was studied in single cultured mouse neocortical neurons using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Reversal of the Na+ gradient by reduction of the extracellular Na+ concentration ([Na+]o) resulted in rapid intracellular acidification, inhibited by 5'-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na+/H+ exchange. In the presence of EIPA and/or 4',4'-diisothiocyano-stilbene-2',2'-sulfonic acid (DIDS), an inhibitor of Na+-coupled anion exchangers and Na+-HCO3- cotransport, a slow decline of pHi was seen. Following intracellular acidification imposed by an NH4Cl prepulse, pHi recovered at a rapid rate, which was reduced by reduction of [Na+]o and was virtually abolished by EIPA and DIDS in combination. Creating an outward Cl- gradient by removal of extracellular Cl- significantly increased the rate of pHi recovery. In HCO3(-)-free media, the pHi recovery rate was reduced in control cells and was abolished at zero [Na+]o and by EIPA. After intracellular alkalinization imposed by an acetate prepulse, pHi recovery was unaffected by DIDS but was significantly reduced in the absence of extracellular Cl-, as well as in the presence of Zn2+, which is a blocker of proton channels. Together, this points toward a combined role of DIDS-insensitive Cl-/HCO3- and passive H+ influx in the recovery of pHi after alkalinization.
authors.affiliation: Department of Biochemistry, August Krough Institute, Copenhagen, Denmark.
authors.forename: S F
authors.initials: SF
authors.lastname: Pedersen
authors.forename: N K
authors.initials: NK
authors.lastname: Jørgensen
authors.forename: I
authors.initials: I
authors.lastname: Damgaard
authors.forename: A
authors.initials: A
authors.lastname: Schousboe
authors.forename: E K
authors.initials: EK
authors.lastname: Hoffmann
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000143:Acids / Q000494:pharmacology; D000468:Alkalies / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D005333:Fetus; D005452:Fluoresceins / Q000032:analysis / Q000378:metabolism; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D019346:Sodium Acetate / Q000494:pharmacology
chemical_list: D000143:Acids; D000468:Alkalies; D002712:Chlorides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D000643:Ammonium Chloride; D019346:Sodium Acetate; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C039614:ethylisopropylamiloride