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.