Membrane-bound carbonic anhydrase in human retinal pigment epithelium.
OBJECTIVE
Inhibition of carbonic anhydrase (CA) by acetazolamide causes a decrease in the standing potential of the retinal pigment epithelium (RPE) and an increase in the rate of subretinal fluid absorption, and it may improve cystoid macular edema. These effects are thought to be mediated by the RPE. Given the solubility coefficient of acetazolamide, the drug is most likely to act by direct inhibition of membrane-bound CA (CA IV). To identify a substrate for acetazolamide in the RPE, the distribution of CA activity and the isoform of CA in the RPE membrane were investigated.


METHODS
Carbonic anhydrase activity was determined by Hansson's technique in fresh human eyes from donors of both sexes and different ages. The presence of the membrane-bound isoform CA IV was investigated immunohistochemically at the light and electron microscopic level, as well as by Western blotting in fresh RPE, and in adult and fetal RPE cultures.


RESULTS
Hansson's histochemical method demonstrated CA activity on the apical and basolateral cell membrane of the RPE. Using the gamma-globulin fraction of a polyclonal antibody against pure CA IV, immunocytochemistry showed labeling for CA IV on the apical RPE membrane or morphologically polarized human adult and fetal RPE cultures. Gel electrophoresis and Western blotting demonstrated a major immunoreactive band at 55 kDa in homogenates, which was consistently reduced to approximately 35 kDa by incorporation of 0.1% Triton X-100 detergent.


CONCLUSIONS
These results suggest that the clinical effects of carbonic anhydrase inhibitors on RPE function may be mediated via membrane-bound carbonic anhydrase activity in RPE and that CA IV is responsible for activity on the apical surface.
Wolfensberger
T J
TJ
Department of Clinical Ophthalmology, Moorfields Eye Hospital, London, United Kingdom.
Mahieu
I
I
Jarvis-Evans
J
J
Boulton
M
M
Carter
N D
ND
Nógrádi
A
A
Hollande
E
E
Bird
A C
AC
D000293:Adolescent; D000328:Adult; D000368:Aged; D001485:Basement Membrane / Q000196:embryology / Q000201:enzymology / Q000648:ultrastructure; D002256:Carbonic Anhydrases / Q000032:analysis*; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D005260:Female; D005333:Fetus; D006801:Humans; D007527:Isoenzymes / Q000032:analysis*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000196:embryology / Q000201:enzymology* / Q000648:ultrastructure; D012160:Retina / Q000196:embryology / Q000201:enzymology* / Q000648:ultrastructure
D007527:Isoenzymes; D008565:Membrane Proteins; D002256:Carbonic Anhydrases