The effects of modulating eNOS activity and coupling in ischemia/reperfusion (I/R)

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 ₂O ₂). We tested the hypothesis that promoting eNOS coupling or attenuating uncoupling after I/R would decrease H ₂O 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 ₂O 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.