Pyridine nucleotides of rabbit cornea with histotoxic anoxia: chemical analysis, non-invasive fluorometry and physiological correlates.
The pyridine nucleotides from both the epithelium and the endothelium of rabbit cornea were measured by the cycling assay. Sodium azide (10 mM) applied for 1 hr to induce histotoxic anoxia decreased the endothelial NAD+/NADH ratio from 4.62 to 1.49 and decreased the epithelial NAD+/NADH ratio from 2.56 to 1.08. The larger NAD+/NADH ratio for the endothelium as compared to the epithelium corresponds to a more oxidized state. The corresponding ratios for NADP+/NADPH were 1.2 for the endothelium and 0.70 for the epithelium. Sodium azide had no effect on the NADP+/NADPH ratio for the endothelium, but decreased the epithelial ratio to 0.62. Pyridine nucleotide fluorescence was measured with a difference corneal fluorometer on the perfused whole cornea preparation and the perfused everted corneal preparation. Sodium azide (10 mM) for 30 min resulted in a 19.4 +/- 0.7% increase in the pyridine nucleotide fluorescence from the whole corneal preparation and a 4.5 +/- 0.6% increase from the everted endothelial preparation. Corneal anoxia induced by stopping the perfusion on the endothelial side resulted in a 18.7 +/- 0.6% increase in pyridine nucleotide fluorescence for the whole corneal preparation. Sodium azide (10 mM) resulted in a 35% decrease in the transendothelial potential difference and a 76% decrease in the rate of transendothelial fluid transport. A comparison is made between invasive chemical analysis and real time, non-invasive fluorometry to measure histotoxic corneal anoxia.
D000818:Animals; D001386:Azides / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D003315:Cornea / Q000187:drug effects / Q000378:metabolism*; D004727:Endothelium / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010100:Oxygen; D010477:Perfusion; D011817:Rabbits; D019810:Sodium Azide; D013050:Spectrometry, Fluorescence
D001386:Azides; D009243:NAD; D009249:NADP; D019810:Sodium Azide; D010100:Oxygen