Interstitial carbonic anhydrase (CA) activity in brain is attributable to membrane-bound CA type IV

We tested the hypothesis that extracellular membrane-bound carbonic anhydra se (CA) type IV is responsible for the regulation of interstitial pH (pH(o) ) transients in brain. Rat hippocampal slices were incubated in phosphatidy linositol-specific phospholipase C (PI-PLC), which cleaves the link of CA I V to the external face of plasma membranes. Then evoked alkaline pHo shifts were studied in a recording chamber, using pH microelectrodes. Incubation fluid was saved for later analysis. The ability to buffer a rapid alkaline load was reduced markedly in PI-PLC-treated tissue as compared with adjacen t, paired control slices. The effect of benzolamide (a poorly permeant CA i nhibitor) on evoked pHo shifts was diminished greatly in the PI-PLC-treated tissue, consistent with the washout of interstitial CA. Treatment of the i ncubation fluid with SDS abolished nearly all of the CA activity in fluid f rom controls, whereas an SDS-insensitive component remained in the fluid fr om PI-PLC-treated slices. These data suggested that CA type II (which is bl ocked by SDS) leaked from injured glial cells in both slice preparations, w hereas CA type IV (which is insensitive to SDS) was liberated selectively i nto the fluid from PI-PLC-treated tissue. Western blot analysis was consist ent with this interpretation, demonstrating a predominance of CA IV in the incubation fluid from PI-PLC-treated tissue and variable amounts of CA II i n fluid from PI-PLC-treated and control slices. These results demonstrate t hat interstitial CA activity brain is attributable principally to membrane- bound CA IV.