Membrane-associated carbonic anhydrase in cultured rabbit nonpigmented ciliary epithelium

Purpose. To measure the activity of membrane-associated carbonic anhydrase (CA) in cultured rabbit nonpigmented epithelial (NPE) cells, determine its identity and its sensitivity to extra-cellular trypsin, and compare the ability of acetazolamide and a cell-impermeant dextran-bound CA inhibitor to change cytoplasmic pH. Methods. Studies were conducted using a cell line derived from rabbit NPE. The cells were lysed and separated into soluble and insoluble fractions by differential centrifugation. CA activity in these fractions was determined using a CO₂ hydration assay. In studies with intact cells, a membrane-impermeable high-molecular-weight dextran- bound inhibitor (DBI) was synthesized and used to selectively bind and inhibit the extracellular-facing membrane-bound CA. Measurements of CA activity in intact red blood cells were conducted to confirm DBI remains extracellular. Acetazolamide, a membrane-permeable CA inhibitor, was used to inhibit total CA activity. Intracellular pH was determined using the pH- dependent absorbance of the fluorescent dye BCECF-AM. Results. A low-speed pellet enriched with plasma membrane material accounted for 22.3 ± 6.1% (n = 18) of the total CA activity in the cultured NPE. When intact cells were exposed to trypsin-EDTA, a 28% reduction of membrane-associated CA activity was observed; DBI inhibited this CA activity loss. Cytosolic CA activity was inhibited by 0.2% sodium dodecyl sulfate (SDS). In contrast, membrane- associated CA was SDS resistant, a characteristic of the CA-IV isozyme. By Western blot, CA-IV immunoreactive polypeptide was detected in the cultured cells and also in native rabbit and porcine ciliary epithelium. Inhibition of total CA activity with acetazolamide and inhibition of extracellular-facing membrane-associated CA with DBI caused an identical intracellular pH decrease in intact NPE cells. Conclusions. Expression of the CA-IV isozyme could account for the significant fraction of CA activity in the cultured NPE, which is membrane associated and SDS resistant. Sensitivity to tryptic hydrolysis suggests the membrane-associated CA partially faces extracellularly. As judged by responses to an extracellular CA inhibitor, the membrane-associated CA has a functional role in maintaining cytoplasmic pH.