MEMBRANE-ASSOCIATED CARBONIC-ANHYDRASE IN CULTURED RABBIT NONPIGMENTED CILIARY EPITHELIUM

Purpose. To measure the activity of membrane-associated carbonic anhyd rase (CA) in cultured rabbit nonpigmented epithelial (NPE) cells, dete rmine its identity and its sensitivity to extracellular trypsin, and c ompare the ability of acetazolamide and a cell-impermeant dextran-boun d CA inhibitor to change cytoplasmic pH. Methods. Studies were conduct ed using a cell line derived from rabbit NPE. The cells were lysed and separated into soluble and insoluble fractions by differential centri fugation. CA activity in these fractions was determined using a CO2 hy dration assay. In studies with intact cells, a membrane-impermeable hi gh-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 membr ane-permeable CA inhibitor, was used to inhibit total CA activity. Int racellular pH was determined using the pH-dependent absorbance of the fluorescent dye BCECF-AM. Results. A low-speed pellet enriched with pl asma membrane material accounted for 22.3 +/- 6.1% (n = 18) of the tot al CA activity in the cultured NPE. When intact cells were exposed to trypsin-EDTA, a 28% reduction of membrane-associated CA activity was o bserved; DBI inhibited this CA activity loss. Cytosolic CA activity wa s inhibited by 0.2% sodium dodecyl sulfate (SDS). In contrast, membran e-associated CA was SDS resistant, a characteristic of the CA-IV isozy me. By Western blot, CA-IV immunoreactive polypeptide was detected in the cultured cells and also in native rabbit and porcine ciliary epith elium. Inhibition of total CA activity with acetazolamide and inhibiti on of extracellular-facing membrane-associated CA with DBI caused an i dentical intracellular pH decrease in intact NPE cells. Conclusions, E xpression of the CA-IV isozyme could account for the significant fract ion of CA activity in the cultured NPE, which is membrane associated a nd SDS resistant. Sensitivity to tryptic hydrolysis suggests the membr ane-associated CA partially faces extracellularly. As judged by respon ses to an extracellular CA inhibitor, the membrane-associated CA. has a functional role in maintaining cytoplasmic pH.