Abstract 4881: Depletion of the Endothelial Nitric Oxide Synthase (eNOS) Substrate NADPH Triggers Endothelial Dysfunction in the Post-Ischemic Heart: Role of Glucose 6 Phosphate Dehydrogenase (G6PD)

Reperfusion injury causes dysfunction in the coronary vascular endothelium; however, current understanding of this process is incomplete. Delineation of the mechanisms involved is critical for advancing treatment of myocardial infarction. Previously, we showed that endothelial dysfunction is triggered by loss of the eNOS cofactor, tetrahydrobiopterin (BH 4 ), and BH 4 repletion partially restored endothelial-dependent vasodilation. We hypothesize that the oxidative stress induced by ischemia/reperfusion (I/R) injury may also result in loss of the essential eNOS substrate NADPH, which is also utilized for BH 4 resynthesis, thus impairing eNOS function. We observe in the isolated rat heart that NADPH declines by ~70% after 30 min I and 30 min R (from 0.41±0.02 to 0.13±0.04 μ M/gram tissue, by HPLC, n=4, p<0.01). The importance of this NADPH depletion was demonstrated by the marked rescue of coronary flow, with 80.0±4.0% increase, following NADPH repletion (175 μ M liposomal formulation infused for 10 min) in hearts subjected to 30 min of I and R. Further studies were performed to understand the mechanism of NADPH depletion. In the presence of oxidative stress, NADPH is rapidly oxidized and glucose 6 phosphate dehydrogenase (G6PD), is required for its regeneration. We observe that myocardial G6PD activity was decreased ~43% during ischemia (Table ), but this activity partially recovered after 30 min R. Interestingly, with addition of excess exogenous glucose-6-phosphate (G6P) the activity did not significantly change from control levels after 30 min of I and after 30 min R only a modest 20% fall was seen (Table ). Thus, the ability of G6PD to resynthesize NADPH remained largely intact but was limited by lack of its substrate G6P. In conclusion, we found that repletion of NADPH post ischemia results in a near total restoration of endothelial dependent coronary flow, and that ischemic depletion of G6P may be partially responsible for the limited bioavailability of NADPH. Glucose-6-Phosphate Dehydrogenase Enzyme Activity