Substrate availability for both endothelial nitric oxide synthase (eNOS) and glucose‐6‐phosphate dehydrogenase (G6PD) result in enzyme dysfunction as a result of ischemia/reperfusion injury: the role of NADPH in endothelial dysfunction.

Abstract

Ischemia/Reperfusion (IR) injury causes dysfunction in the coronary vascular endothelium. Endothelial nitric oxide synthase is a critical enzyme involved in endothelial function. Previously, we showed endothelial dysfunction occurs without direct damage to eNOS, but rather via depletion of the essential eNOS cofactor, tetrahydrobiopterin (BH4). Repletion of BH4 lost during IR partially restored endothelial function. Recently, we have found that 30 min of IR injury also results in the depletion of the eNOS substrate NADPH, to ~47% of pre‐injury levels. In isolated hearts repletion of NADPH restored endothelial function (as measured by coronary flow) to near pre‐ischemic levels, highlighting the significance the IR‐induced decrease in NADPH. Our current work shows levels of NADPH are altered throughout IR injury, rising ~20% above control values at 10 min ischemia (increasing from 49.2±2.2 to 61.2±3.5 nmoles/gram tissue), and then decreasing to below baseline (31.8±7.8 nmoles/gram tissue) 20 min post‐ischemia. Glucose‐6‐phosphate dehydrogenase (G6PD) is the rate limiting enzyme in the production of NADPH. We observed that while total G6PD activity is not changed during IR injury, it is limited by substrate availability. In conclusion, the modulation of NADPH throughout IR injury, partially due to a change in substrate availability of G6PD, gives new insights to the intracellular response to IR injury.