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 hippocampa l astrocytes in vitro using the pH-sensitive dye BCECF. All experiment s were carried out in CO2/HCO3--free solutions. Recovery from NH4+-ind uced acid loads was strongly dependent on the presence of extracellula r 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+ inhi bitors, however, these astrocytes continued to show significant recove ry from acid loads. The mechanism of this amiloride-insensitive and Na +-independent pH(i) recovery process was sought and appeared to be a p roton pump. In the absence of Na+, recovery from an acid load was comp letely 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 slowe d recovery rate from NH4+-induced acid loads by about 32%. The rate of NA+-independent pH(i) recovery was increased by depolarization with 5 0 mM [K+] solution, and this effect was rapidly reversible and blocked by BA1. These results indicate that, in CO2/HCO3--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 activ ity was influenced by membrane potential, this acid exporting mechanis m could contribute to the depolarization-induced alkalinization that i s seen in astrocytes. Although v-type H+-ATPase had been previously is olated from the brain, this is the first report indicating that it has a role in regulating pH(i) in brain cells. (C) 1993 Wiley-Liss, Inc.