Pyridine nucleotides and phosphorylation potential of rabbit corneal epithelium and endothelium

In order to validate in situ corneal redox fluorometry, the redox state and phosphorylation potential of freeze trapped rabbit corneal epithelium and endothelium were studied using quantitative histochemical methods. The results were compared with noninvasive measurements using an optically sectioning fluorometer microscope. Enucleated rabbit eyes were either frozen in Freon-12, cooled by liquid nitrogen or exposed for 1 hr in 1 mM NaCN to block oxidation and then freeze trapped. Corneas were sectioned, freeze-dried, samples of individual layers dissected, weighed, and analyzed for: NADH, NAD+, NADPH, NADP+, ATP, ADP, and Pi. The aerobic epithelium showed a ratio for NAD+/NADH of 1.85 +/- 0.08 (9). In anoxia this ratio decreased to 1.06 +/- 0.07 (8). The NAD+/NADH ratio of aerobic endothelium was 3.25 +/- 0.28 (6); in anoxia this ratio was 0.68 +/- 0.14 (5). The values of phosphorylation potential ATP/(ADP X Pi)M-1 were: 447.9 +/- 40.2 (9) in aerobic epithelium, 378.2 +/- 24.7 (5) in anoxic epithelium; 308.4 +/- 25.2 (7) in aerobic endothelium and 225.4 +/- 19.1 (5) in anoxic endothelium. Aerobic-anoxic transitions alter the concentrations of NADH and NAD+ but did not affect the concentration of NADPH and NADP+. The microhistochemical data indicate that the redox state of rabbit epithelium is less sensitive to hypoxia than the endothelium. This difference between the two limiting layers is reflected in alterations of phosphorylation potential induced by hypoxia. The similarly high efficiencies of both layers in maintaining relatively high ATP levels during histotoxic hypoxia is most likely a result of compensatory ATP generation by enhanced glycolysis.