The in vivo role of endothelial nitric oxide synthase (eNOS) uncoupling mediating oxidative stress in ischemia/reperfusion (I/R) injury has not been well established. In vitro, eNOS coupling refers to the reduction of molecular oxygen to L-arginine oxidation and generation of L-citrulline and nitric oxide NO synthesis in the presence of an essential cofactor, tetrahydrobiopterin (BH(4)). Whereas uncoupled eNOS refers to that the electron transfer becomes uncoupled to L-arginine oxidation and superoxide is generated when the dihydrobiopterin (BH(2)) to BH(4) ratio is increased. Superoxide is subsequently converted to hydrogen peroxide (H(2)O(2)). We tested the hypothesis that promoting eNOS coupling or attenuating uncoupling after I/R would decrease H(2)O(2)/increase NO release in blood and restore postreperfused cardiac function. We combined BH(4) or BH(2) with eNOS activity enhancer, protein kinase C epsilon (PKC ε) activator, or eNOS activity reducer, PKC ε inhibitor, in isolated rat hearts (ex vivo) and femoral arteries/veins (in vivo) subjected to I(20 min)/R(45 min). When given during reperfusion, PKC ε activator combined with BH(4), not BH(2), significantly restored postreperfused cardiac function and decreased leukocyte infiltration (p < 0.01) while increasing NO (p < 0.05) and reducing H(2)O(2) (p < 0.01) release in femoral I/R veins. These results provide indirect evidence suggesting that PKC ε activator combined with BH(4) enhances coupled eNOS activity, whereas it enhanced uncoupled eNOS activity when combined with BH(2). By contrast, the cardioprotective and anti-oxidative effects of the PKC ε inhibitor were unaffected by BH(4) or BH(2) suggesting that inhibition of eNOS uncoupling during reperfusion following sustained ischemia may be an important mechanism.

Reduced nitric oxide (NO) bioavailability and increased oxidative stress are major factors mediating ischemia/reperfusion (I/R) injury. Tetrahydrobiopterin (BH4) is an essential cofactor of endothelial NO synthase (eNOS) to produce NO, whereas dihydrobiopterin (BH2) can shift the eNOS product profile from NO to superoxide, which is further converted to hydrogen peroxide (H2O2) and cause I/R injury. The effects of BH4 and BH2 on oxidative stress and postreperfused cardiac functions were examined in ex vivo myocardial and in vivo femoral I (20 min)/R (45 min) models. In femoral I/R, BH4 increased NO and decreased H2O2 releases relative to saline control, and these effects correlated with improved postreperfused cardiac function. By contrast, BH2 decreased NO release relative to the saline control, but increased H2O2 release similar to the saline control, and these effects correlated with compromised postreperfused cardiac function. In conclusion, these results suggest that promoting eNOS coupling to produce NO and decrease H2O2 may be a key mechanism to restore postreperfused organ function during early reperfusion.

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