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(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?