title: 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.
authors.affiliation: Department of Pathology, Microbiology, Immunology and Forensic Medicine, Philadelphia College of Osteopathic Medicine, 4170 City Avenue, Philadelphia, PA 19131-1694, USA.
authors.forename: Kerry-Anne A
authors.initials: KA
authors.lastname: Perkins
authors.forename: Sailesh
authors.initials: S
authors.lastname: Pershad
authors.forename: Qian
authors.initials: Q
authors.lastname: Chen
authors.forename: Sloane
authors.initials: S
authors.lastname: McGraw
authors.forename: Jovan S
authors.initials: JS
authors.lastname: Adams
authors.forename: Christopher
authors.initials: C
authors.lastname: Zambrano
authors.forename: Samuel
authors.initials: S
authors.lastname: Krass
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Emrich
authors.forename: Brandon
authors.initials: B
authors.lastname: Bell
authors.forename: Michael
authors.initials: M
authors.lastname: Iyamu
authors.forename: Catherine
authors.initials: C
authors.lastname: Prince
authors.forename: Helen
authors.initials: H
authors.lastname: Kay
authors.forename: Jane Chun-wen
authors.initials: JC
authors.lastname: Teng
authors.forename: Lindon H
authors.initials: LH
authors.lastname: Young
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D005268:Femoral Vein / Q000187:drug effects / Q000378:metabolism; D006321:Heart / Q000187:drug effects / Q000503:physiopathology; D006861:Hydrogen Peroxide / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D009200:Myocardial Contraction / Q000187:drug effects; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D009206:Myocardium / Q000473:pathology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000502:physiology*; D018384:Oxidative Stress / Q000187:drug effects; D051744:Protein Kinase C-epsilon / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D001708:Biopterins; D009569:Nitric Oxide; C017226:7,8-dihydrobiopterin; D006861:Hydrogen Peroxide; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D051744:Protein Kinase C-epsilon; C003402:sapropterin; D019331:NG-Nitroarginine Methyl Ester