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.