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Abstract
PURPOSE: To determine whether maintenance of corneal hydration is dependent on bicarbonate ions and whether these ions can be derived from metabolic or exogenous CO2, and to investigate the relationship of transendothelial fluid movement to control of hydration. METHODS: The thickness of intact or deepithelialized rabbit corneas was measured while superfused on the endothelial surface with either 33 mM HGO3-/5% CO2 buffered media or 10 mM HPO4- buffered media in the presence and absence of inhibitors of ion transport and respiration. The corneal surface was covered with either silicone oil ("normal" corneas) or with the same media used for superfusion ("swollen" corneas). ATP and Na+,K(+)-ATPase activity were measured in endothelia scraped from the tissues after superfusion. RESULTS: Intact and deepithelialized corneas covered with oil swelled at a negligible rate (4 to 8 microns/hour) in 33 mM HCO3- medium but at 45 to 60 microns/hour in HPO4- medium. Antimycin A altered neither of these swelling rates, but ethoxzolamide (0.1 mM) caused swelling in HCO3-/CO2 (approximately 12 microns/hour above controls) with no change of rate in HPO4-. Ouabain (0.1 mM) increased swelling to 45 to 50 microns/hour in HCO3-/CO2 but had no effect in HPO4-. Saturating the oil on deepithelialized corneas with 5% CO2, or putting HCO3-/CO2 medium on the epithelial surface of intact corneas, did not alter the swelling rates seen with HPO4- superfusion. The equilibrium thickness of deepithelialized corneas swollen with HCO3-/CO2 on both surfaces was 35 microns less than that of corneas swollen in HPO4-. The difference was abolished by ouabain, which caused corneas in HCO3-/CO2 to swell an additional 30 microns but did not alter the equilibrium thickness of corneas swollen in HPO4-. Ethoxzolamide and DIDS (0.2 mM) increased the thickness in HCO3-/CO2 but not in HPO4-. Na+,K(+)-ATPase activities of endothelia were similar after HCO3-/CO2 and HPO4- superfusions, but the concentration of ATP in the HPO4(-)-superfused tissues was increased 35%. CONCLUSIONS: Normal corneal thickness can be maintained in vitro only in media that contain HCO3- at concentrations of more than 20 mM. Neither metabolic CO2 nor CO2 present in air-equilibrated, nominally HCO3(-)-free media can supply this requirement for HCO3-, even though these sources support the presumably related processes of transendothelial fluid movement and intracellular pH regulation.