Inhibition Of Protein Nitration In Human Corneal Epithelial And Endothelial Cells During Cold Storage.

Abstract

Abstract: : Purpose: We have previously demonstrated that nitric oxide (NO) production occurs in corneal tissue during storage prior to transplantation and that this production, which leads to protein nitration, can be inhibited with the addition of a nitric oxide synthase inhibitor to the corneal storage media. The aim of this study was to determine if the inhibition of NO production would lead to a decrease in protein nitration in corneal tissue during storage. Methods: Paired human corneas stored in Optisol–GS corneal storage media were obtained from the Cleveland Eye Bank. N_G–monomethylene–L–arginine (LMMA), a non–selective nitric oxide synthase inhibitor, was added to 20 ml of storage media of one cornea from each pair of corneas to a final concentration of 2 mM. Paired corneas were stored for 3 or 5 days, and bisected. The epithelial and endothelial cells of one half of each cornea were scraped off with a surgical blade and frozen in separate vials of phosphate buffered saline according to cell type. The remaining halves of each cornea were returned to their original storage media, stored for 4 more days, and then subjected to the same treatment as the other halves. Epithelial and endothelial cells of corneas from both the inhibitor and the control groups at Days 3, 5, 7, and 9 were pooled, and Western blot analysis was performed. Quantitation of these blots was performed by densitometry studies using Quantity One software. Results: Substantially less nitrated protein was found in epithelial cells which were exposed to LMMA than those in the control group at day 7 of storage. Endothelial cells exposed to LMMA demonstrated substantially less nitrated protein than the control group at day 9 of storage. No substantial differences occurred at any other time point between the inhibited and control groups for epithelial or endothelial cells. Conclusions: The addition of a nitric oxide synthase inhibitor to corneal storage media seems to decrease the amount of protein nitration during storage. This may correlate clinically to less damage to corneal cellular elements during storage due to the toxic effects of protein nitration.

Keywords: cornea: storage • nitric oxide • protein modifications–post translational