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.
MI/R results in endothelial and cardiac contractile dysfunction in the presence of polymorphonuclear leukocytes (PMNs). The role of eNOS in reperfusion injury was studied in a rat PMN‐induced MI/R injury model. Isolated hearts subjected to ischemia (20 min) and reperfused (45 min) with activated PMNs were studied. Cell permeable protein kinase C (PKC) epsilon activator (ε+) or inhibitor (ε‐) peptides were used to increase or decrease eNOS activity respectively. Tetrahydrobiopterin (BH4) or dihydrobiopterin (BH2) are cofactors of eNOS that regulate nitric oxide or superoxide (SO) release respectively. The combination of PKC ε+ (10µM)/BH4 (5µM) (n=7) given during reperfusion significantly restored post‐reperfusion cardiac function compared to I/R PMN hearts (n=11, P <0.01) or PKC ε+ (n=6) or BH4 (n=6) by itself. By contrast, the combination of PKC ε+/BH2 (100µM) (n=6) compromised post‐reperfusion cardiac function suggesting that increased eNOS activity compromises cardiac function when eNOS becomes uncoupled by BH2 or can be cardioprotective when eNOS is coupled to BH4. Moreover, the combination of PKC ε‐ (5µM)/BH2 (n=6) is cardioprotective compared to I/R PMN hearts (P <0.01) or BH2 (n=7) by itself suggesting that decreased eNOS activity during reperfusion attenuates SO release from eNOS. This study was supported by NHLBI Grant 2R15HL‐076235‐02A1 and the Center for the Chronic Disorders of Aging at PCOM.
The roles of coupled or uncoupled eNOS mediating nitric oxide (NO) or hydrogen peroxide (H2O2) release respectively in I/R have not been evaluated in vivo. The effects of PKC epsilon activator or inhibitor peptides, which can increase or decrease eNOS activity respectively, combined with BH4 or BH2 were tested in isolated perfused rat hearts (ex vivo) and femoral arteries/veins (in vivo) that were subjected to I(20 min)/R(45 min). When given during reperfusion, the PKC epsilon activator combined with BH4 restored post‐reperfused cardiac function (p<0.05), increased NO release (p<0.05) and reduced H2O2 release in femoral veins (p<0.01) compared to controls, but when combined with BH2 resulted in compromised post‐reperfused cardiac function, decreased NO and increased H2O2 release in femoral veins compared to controls (p<0.05). The results suggest that the combination of PKC epsilon activator and BH4 promoted eNOS to function in its coupled state producing NO. However, the combination of PKC epsilon inhibitor with BH2 or BH4 significantly improved post‐reperfused cardiac function (p<0.05), reduced H2O2 release (p<0.05) and increased NO release (p<0.05) in femoral veins compared to controls. These results suggest that inhibition of eNOS uncoupling following I/R attenuates H2O2 release. This study was supported by NHLBI Grant 2R15HL‐76235‐02 and the Center for the Chronic Disorders of Aging at PCOM.
In the presence of polymorphonuclear leukocytes (PMNs) during reperfusion, following myocardial ischemia, cardiac contractile dysfunction is observed. Isolated ischemic (20 min) and reperfused (45 min) rat hearts in the presence of activated PMNs were studied. A cell permeable peptide (PKC ε+) that enhances interaction of PKC ε with endothelial nitric oxide synthase (eNOS) to increase nitric oxide (NO) release, and BH4, an essential cofactor of eNOS, were given during the first five min of reperfusion. In hearts given PKC ε+ (10 μM) and BH4 (5 μM) (n=7) with PMNs, left ventricular developed pressure (LVDP) significantly recovered to 92% ± 11% of baseline values compared to I/R + PMN hearts (n=5) (48% ± 7%) (p<0.05) and the maximal rate of LVDP (+dP/dtmax) significantly recovered to 83% ± 7% compared to I/R + PMN hearts (34% ± 4%) (p<0.01) at 45 min reperfusion. The cardioprotective effects of PKC ε+ and BH4 were associated with an approximate 50% decrease in PMN infiltration in post‐reperfused hearts as confirmed by myeloperoxidase assay. These results suggest that the combination of PKC ε+ and BH4 attenuates PMN‐induced cardiac contractile dysfunction after I/R, in part by promotion of eNOS to increase NO release and inhibition of PMN infiltration in post‐reperfused hearts. This study was supported by NHLBI Grant 1R15HL‐76235‐01 and the Center for the Chronic Disorders of Aging at PCOM.
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