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

Abstract

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.

Protein Kinase C Epsilon PeptideInhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

“…+dP/dt max and -dP/dt min ). A putative mechanism for this effect in either I/R model is that treatment with Myr-PKCε-inhibits uncoupled eNOS activity, resulting in an increase in the bioavailability of NO and inhibition of degradation of NO produced from other sources such as hemoglobin and myoglobin nitrite reductase [13,34]. NO has been shown to promote a number of beneficial actions during normal cardiac activity and I/R injury, such as reduction in O 2 consumption, inhibition of Ca 2+ influx, and improvement in the coupling efficiency between ATP synthesis and O 2 consumption [46][47][48][49][50].…”

“…During reperfusion, eNOS uncoupling is promoted by BH 4 oxidation to dihydrobiopterin (BH 2 ). Since BH 2 and BH 4 have equal affinity for the eNOS oxygenase-binding domain, an increase in the BH 2 to BH 4 ratio during reperfusion shifts eNOS towards the uncoupled state [34]. Hence, inhibiting PKCε during reperfusion will attenuate ROS mediated insults arising from uncoupled eNOS activity, the mK ATP channel, and leukocyte endothelial adhesion and penetration.…”

“…Myr-PKCε-acts as a competitive translocation inhibitor by mimicking the receptor for activated C kinase (RACK) binding site on PKCε (see Figure 1) [30]. During reperfusion in this I/R model, three principle sources of ROS will be reduced directly and indirectly by inhibition of PKCε activity, opening of the mK ATP channels, uncoupled endothelial nitric oxide synthase (eNOS) activity, a switch from nitric oxide (NO) to ROS production, and intracellular adhesion molecule-1 (ICAM-1) mediated leukocyte ROS release (see Figure 2) [31][32][33][34][35]. We previously reported that PKCε activation upon reperfusion is not cardioprotective, unless eNOS uncoupling is reversed to its normal or coupled state by the addition of an essential cofactor, tetrahydrobiopterin (BH 4 ) [34].…”

“…Activated PKCε phosphorylation increases uncoupled eNOS activity and opens mitochondrial ATP-dependent potassium channels during reperfusion leading to increased O 2 -. induced exacerbation of I/R injury [34,38]. determined by carefully dissecting (under magnification) and weighing the corresponding tissue in both areas.…”

Vázizomkisvénák vazomotortónusának intrinszik szabályozómechanizmusai

“…Az I/R okozta oxidatív stressz emelkedett ROS (szuperoxid anion, H 2 O 2 , hidroxil-szabadgyök) -koncentrá-ció és/vagy csökkent antioxidáns hatás miatt alakul ki, amiért főleg a csökkent antioxidáns enzimszintek felelő-sek (glutation, E-vitamin, aszkorbinsav, glutation-peroxidáz, szuperoxid dizmutáz, kataláz) [57,58]. Feltéte-lezhető, hogy az oxidatív stressz szignifikáns szerepet játszhat a vénás mikroerek diszfunkciójának kialakulásá-ban is [55].…”