HCO3--independent pH Regulation in Astrocytes in Situ Is Dominated by V-ATPase

The mechanisms of HCO3--independent intracellular pH (pH(i)) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pHi in cultured astrocytes was 6.82 +/- 0.06 and inhibition of the V-ATPase H+ pump by Cl- removal or via the selective inhibitor bafilomycin had only a small effect upon resting pHi and recovery following an acid load. In contrast, resting pH(i) in RON astrocytes was 7.10 +/- 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3-6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pHi recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO3--independent pHi regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.