title: A depolarization-stimulated, bafilomycin-inhibitable H+ pump in hippocampal astrocytes.
abstract: Relatively little is known about the mechanisms of pHi regulation in mammalian glial cells. We analyzed pHi regulation in rat hippocampal astrocytes in vitro using the pH-sensitive dye BCECF. All experiments were carried out in CO2/HCO3(-)-free solutions. Recovery from NH4(+)-induced acid loads was strongly dependent on the presence of extracellular Na+ and was inhibited by amiloride and its more specific analog EIPA, indicating the presence of Na(+)-H+ exchange in these cells. Removing bath Na+ or adding amiloride caused resting pHi to shift in the acid direction. Even in the absence of bath Na+ or presence of Na+/H+ inhibitors, however, these astrocytes continued to show significant recovery from acid loads. The mechanism of this amiloride-insensitive and Na(+)-independent pHi recovery process was sought and appeared to be a proton pump. In the absence of Na+, recovery from an acid load was completely blocked by the highly specific blocker of vacuolar-type (v-type) H+ ATPase, bafilomycin A1 (BA1). In normal Na+ containing solutions, exposure to BA1 caused a small acid shift in baseline pHi and slowed recovery rate from NH4(+)-induced acid loads by about 32%. The rate of Na(+)-independent pHi recovery was increased by depolarization with 50 mM [K+] solution, and this effect was rapidly reversible and blocked by BA1. These results indicate that, in CO2/HCO3(-)-free solution, pHi regulation in hippocampal astrocytes was mediated by Na(+)-H+ exchange and by a BA1-inhibitable proton pump. Because the proton pump's activity was influenced by membrane potential, this acid exporting mechanism could contribute to the depolarization-induced alkalinization that is seen in astrocytes. Although v-type H(+)-ATPase had been previously isolated from the brain, this is the first report indicating that it has a role in regulating pHi in brain cells.
authors.affiliation: Department of Neurology, Yale University School of Medicine, New Haven, CT 06510.
authors.forename: C A
authors.initials: CA
authors.lastname: Pappas
authors.forename: B R
authors.initials: BR
authors.lastname: Ransom
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D004558:Electric Stimulation; D005452:Fluoresceins / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects*; D006851:Hydrochloric Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D018942:Macrolides; D008564:Membrane Potentials / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects*
chemical_list: D000900:Anti-Bacterial Agents; D005452:Fluoresceins; D018942:Macrolides; D009419:Nerve Tissue Proteins; D000644:Quaternary Ammonium Compounds; D017923:Sodium-Hydrogen Exchangers; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; D000584:Amiloride; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases; D006851:Hydrochloric Acid