Purpose. To examine differences between carbonic anhydrase (CA) isozymes in the plasma membrane and cytosol of non-pigmented epithelial (NPE) cells as regards susceptibility to sulfonamide inhibitors and functional roles for the isozymes. Methods. Studies were conducted using SV40 virus transformed rabbit NPE provided by Dr. Coca-Prados (Yale). The isozymes cytosolic CA-II, plasma membrane bound CA-IV and mitochondrial CA-V were separated by differential centrifugation. A CO2 hydration assay was used to determine CA activity. Different CA inhibitors (CAI) were tested as inhibitors of these isozymes. Intracellular pH (pHi) was determined using the pH-dependent absorbance of the fluorescent dye BCECF. Results. CA-II was 45±6%, CA-IV 32±4% and CA-V 23±3% (n=7) of the total CA activity. The sensitivities of the isozymes to CAI were different. Ethyloxaloylazolamide, was 140 times more potent as an inhibitor of CA-IV and CA-V than for CA-II. A high MW dextran-bound inhibitor (DBI) was used as a pharmacological probe to selectively bind and inhibit CA-IV. DBI inhibited the CA-IV activity loss caused by tryptic hydrolysis. Acetazolamide (Actz.), which inhibits both CA-II & IV, was found to inhibit the pHi change caused by rapid change in bathing medium [HCO3-/CO2) or [NH4Cl]. Inhibition of only CA-IV with DBI had no effect on the response to HCO3-/CO2 withdrawal. Both Actz & DBI inhibited the change in pHi caused by the Na-H inhibitor, amiloride. Conclusions. The three cellular fractions were found to contain CA isozymes which have different sensitivities to CAIs. CA-II may participate in pHi regulation. The tryptic hydrolysis results suggest CA-IV is partially faced extracellularly. The specific role of CA-IV remains unclear although it seems that it may be linked to the function of Na-H exchanger.
|Original language||English (US)|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Feb 15 1996|
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience