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

Abstract

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 CO₂/HCO₃-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 A₁ (BA₁). In normal Na⁺containing solutions, exposure to BA₁ 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 BA₁. These results indicate that, in CO₂/HCO⁻₃-free solution, pH_i 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 pH_i in brain cells.