Inhibition of carbonic anhydrase activity in cultured bovine corneal endothelial cells by dorzolamide.
OBJECTIVE
Fluid transport by the corneal endothelium is dependent on the presence of HCO(3)(-) and the activity of carbonic anhydrase (CA)-II and -IV, the cytoplasmic and membrane-bound CAs, respectively. This study was conducted to examine the inhibition of CA activity in cultured bovine corneal endothelial cells (BCECs) by dorzolamide, a topical CA inhibitor used in glaucoma therapy.


METHODS
BCECs were grown on glass coverslips and then perfused with HCO(3)(-)-free Ringer's. The inward flux of CO(2) was induced by exposure to CO(2)-HCO(3)(-) Ringer's and the opposing outward flux by returning to HCO(3)(-)-free Ringer's. Consequent transients in intracellular pH (pH(i)) were measured using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). During the inward flux of CO(2,) the maximum rate of change of pH(i) was taken as a quantitative measure of the overall CA activity in BCECs.


RESULTS
Exposure to CO(2)-HCO(3)(-) Ringer's led to a transient decrease in pH(i) (component A), followed by a rapid increase to a new steady state (component B). However, when the CO(2)-HCO(3)(-) Ringer's was removed, the pH(i) increased transiently (component C) and then rapidly returned to the original pH(i) (component D). Component A, caused by an inward flux of CO(2) and its subsequent hydration by CA-II, was blocked by dorzolamide in a dose-dependent manner with an 50% inhibitory concentration (IC)(50) of 2.4 micro M (95% confidence interval: 0.5 -10.85 microM). However, the inhibition of the outward flux of CO(2), inward flux of HCO(3)(-), and outward flux of HCO(3)(-) (associated with components C, B, and D, respectively) was not dose dependent. Cells that were exposed to 500 nM of the drug for longer than 30 minutes did not show a significantly greater inhibition of any of the components. Dorzolamide and acetazolamide (500 microM) did not show additive inhibition of any of the components (P = 0.13; n = 6).


CONCLUSIONS
Dorzolamide significantly inhibits CA activity in BCECs at micromolar levels. Because these levels are encountered in the cornea and aqueous humor after topical administration, dorzolamide may compromise corneal hydration control, especially when the functional reserve of corneal endothelium is low. Dorzolamide does not appear to accumulate in the cells, because the inhibition of CA-II did not increase after prolonged exposure to the drug.
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D002254:Carbonates / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007552:Isotonic Solutions / Q000737:chemistry / Q000494:pharmacology; D009994:Osmolar Concentration; D000077331:Ringer's Solution; D013449:Sulfonamides / Q000008:administration & dosage / Q000494:pharmacology*; D013876:Thiophenes / Q000008:administration & dosage / Q000494:pharmacology*
D001639:Bicarbonates; D002254:Carbonates; D002257:Carbonic Anhydrase Inhibitors; D007552:Isotonic Solutions; D013449:Sulfonamides; D013876:Thiophenes; D000077331:Ringer's Solution; C062765:dorzolamide