A physiological measure of carbonic anhydrase in müller cells

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Abstract

Carbonic anhydrase activity was characterized in freshly dissociated Müller cells of the salamander retina. Intracellular pH was monitored using ratio imaging of the indicator dye BCECF as extracellular Pco2 was varied. The extracellular solution was switched rapidly (141 ms rise time) from a HEPES buffered to a CO2‐HCO3 buffered solution (both pH 7.4). Introduction of CO2‐HCO3 produced a rapid cell acidification. Cell pH dropped from a steady‐state pH of 7.02 in HEPES solution to pH 6.81 in CO2‐HCO3. Methazolamide, a carbonic anhydrase inhibitor, dramatically reduced the initial rate of acidification, demonstrating that the acidification was produced by the carbonic anhydrase‐catalyzed hydration of CO2. The initial rate of acidification, 52.6 pH units per min (0.88 pH units per s), was reduced ∼ 150‐fold to 0.36 pH units per min by 10−3 M methazolamide. Half‐maximal inhibition occurred at a methazolamide concentration of 5.6. 10−7 M. The carbonic anhydrase inhibitor acetazolamide (10−3 M) also greatly reduced the rate of cell acidification. The latency to the onset of carbonic anhydrase inhibition was 660 ms for methazolamide and 7.5 s for acetazolamide. The carbonic anhydrase inhibitor benzolamide (10−4 M, 4 min exposure), which is poorly membrane permeant, had little effect on the rate of cell acidification, indicating that the site of carbonic anhydrase action was intracellular. The activity of Müller cell carbonic anhydrase may help to buffer extracellular CO2 variations in the retina. © 1994 Wiley‐Liss, Inc.

Original languageEnglish (US)
Pages (from-to)291-299
Number of pages9
JournalGlia
Volume11
Issue number4
DOIs
StatePublished - Aug 1994

Keywords

  • Carbon dioxide regulation
  • Glial cell
  • Intracellular pH
  • Retina

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