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The Journal of Neuroscience, November 15, 2000, 20(22):8247-8253

Interstitial Carbonic Anhydrase (CA) Activity in Brain Is Attributable to Membrane-Bound CA Type IV

Chi-Kun Tong¹, Luc P. Brion³, Carlos Suarez³, and Mitchell Chesler^{1, 2}

Departments of ¹ Physiology and Neuroscience and ² Neurosurgery, New York University School of Medicine, New York, New York 10016, and ³ Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York 10461

We tested the hypothesis that extracellular membrane-bound carbonic anhydrase (CA) type IV is responsible for the regulation of interstitial pH (pH_o) transients in brain. Rat hippocampal slices were incubated in phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the link of CA IV to the external face of plasma membranes. Then evoked alkaline pH_o 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 adjacent, paired control slices. The effect of benzolamide (a poorly permeant CA inhibitor) on evoked pH_o shifts was diminished greatly in the PI-PLC-treated tissue, consistent with the washout of interstitial CA. Treatment of the incubation fluid with SDS abolished nearly all of the CA activity in fluid from controls, whereas an SDS-insensitive component remained in the fluid from PI-PLC-treated slices. These data suggested that CA type II (which is blocked by SDS) leaked from injured glial cells in both slice preparations, whereas CA type IV (which is insensitive to SDS) was liberated selectively into the fluid from PI-PLC-treated tissue. Western blot analysis was consistent with this interpretation, demonstrating a predominance of CA IV in the incubation fluid from PI-PLC-treated tissue and variable amounts of CA II in fluid from PI-PLC-treated and control slices. These results demonstrate that interstitial CA activity brain is attributable principally to membrane-bound CA IV.

Key words: hippocampal slice; carbonic anhydrase; benzolamide; phosphatidylinositol-glycan link; extracellular pH; alkaline shift

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Copyright © 2000 Society for Neuroscience  0270-6474/00/20228247-07$05.00/0

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