A role for glutathione and glutathione reductase in control of corneal hydration

We have assessed the importance of the glutathione redox system of the corneal endothelial cells in the control of stromal hydration. The ability of freshly isolated corneas to maintain normal hydration during perfusion, while the activity of glutathione reductase was inhibited with 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU), was tested under a number of conditions. BCNU at 125 microM led to mild swelling and at 500 microM to more severe swelling. Swelling was also severe when 50 microM H2O2 was added together with 125 microM BCNU. The concentration of total glutathione (GSH + GSSG) in the endothelia of these corneas was decreased and the fraction found in the oxidized form was higher than in controls without BCNU. Glutathione reductase activity was inhibited in the perfused endothelia by 90%. With the addition of 0.5 mM GSSG during perfusion, swelling due to 125 microM BCNU was at a slightly lower rate and reductase activity was inhibited only 79%. Complete protection was afforded against 125 microM BCNU induced swelling by 1.0 mM GSH, corneas maintaining normal thickness for over 4 hr. The endothelial surface of the perfused corneas was shown by scanning electron microscopy to be little disturbed by the low concentration of BCNU, but at high concentration, or with H2O2 added, the cells were collapsed and had a heavily pitted appearance. We conclude that when the cornea is under oxidative stress (e.g. in the presence of H2O2) a rapid turnover of endothelial GSH via glutathione reductase and the hexose monophosphate shunt is required. Under conditions of less stress, the reduced need for GSH to counteract oxidative threats can be supplied either exogenously or by the partially inhibited reductase.