Homocysteine induces oxidative stress by uncoupling of NO synthase activity through reduction of tetrahydrobiopterin.
Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.
Topal
Gökce
G
Département de Signalisation cellulaire et athérosclérose précoce, UMR CNRS 7131- Université Paris 6, Hôpital Broussais, 102 rue Didot, 75014 Paris, France.
Brunet
Annie
A
Millanvoye
Elisabeth
E
Boucher
Jean-Luc
JL
Rendu
Francine
F
Devynck
Marie-Aude
MA
David-Dufilho
Monique
M
D000975:Antioxidants / Q000494:pharmacology; D001120:Arginine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D001205:Ascorbic Acid / Q000737:chemistry; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology; D004996:Ethidium / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006710:Homocysteine / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D018384:Oxidative Stress; D010766:Phosphorylation; D011622:Pterins / Q000737:chemistry; D026361:Reactive Nitrogen Species; D013481:Superoxides / Q000737:chemistry; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors
D000975:Antioxidants; D005456:Fluorescent Dyes; D011622:Pterins; D026361:Reactive Nitrogen Species; D006710:Homocysteine; D013481:Superoxides; D001708:Biopterins; D009569:Nitric Oxide; C018524:N,N-dimethylarginine; C017226:7,8-dihydrobiopterin; D001120:Arginine; C016727:sepiapterin; D007770:L-Lactate Dehydrogenase; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D013917:Thrombin; C003402:sapropterin; D004996:Ethidium; D001205:Ascorbic Acid; D019331:NG-Nitroarginine Methyl Ester