Membrane-associated carbonic anhydrase in cultured rabbit nonpigmented ciliary epithelium.
OBJECTIVE
To measure the activity of membrane-associated carbonic anhydrase (CA) in cultured rabbit nonpigmented epithelial (NPE) cells, determine its identity and its sensitivity to extracellular 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.
Wu
Q
Q
Department of Pharmacology and Toxicology, University of Louisville, Kentucky 40292, USA.
Delamere
N A
NA
Pierce
W
W
D000086:Acetazolamide / Q000494:pharmacology; D000818:Animals; D015415:Biomarkers; D015153:Blotting, Western; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D004910:Erythrocyte Membrane / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D011817:Rabbits; D013552:Swine
D015415:Biomarkers; D002257:Carbonic Anhydrase Inhibitors; D008565:Membrane Proteins; D002256:Carbonic Anhydrases; D000086:Acetazolamide