A depolarization‐stimulated, bafilomycin‐inhibitable H + pump in hippocampal astrocytes

Relatively little is known about the mechanisms of pH i regulation in mammalian glial cells. We analyzed pH i regulation in rat hippocampal astrocytes in vitro using the pH‐sensitive dye BCECF. All experiments were carried out in CO 2 /HCO 3 ‐free solutions. Recovery from NH + 4 ‐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 pH i 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 pH i 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 A 1 (BA 1 ). In normal Na + containing solutions, exposure to BA 1 caused a small acid shift in baseline pH i and slowed recovery rate from NH + 4 ‐induced acid loads by about 32%. The rate of Na + ‐independent pH i recovery was increased by depolarization with 50 mM [K + ] solution, and this effect was rapidly reversible and blocked by BA 1 . These results indicate that, in CO 2 /HCO − 3 ‐free solution, pH i regulation in hippocampal astrocytes was mediated by Na + − ‐H + exchange and by a BA 1 ‐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 pH i in brain cells. © 1993 Wiley‐Liss, Inc.