Upregulation of endothelial and inducible nitric oxide synthase expression by reactive oxygen species.
BACKGROUND
The effect of reactive oxygen species (ROS) on nitric oxide synthase (NOS) expression remains uncertain. This study explored the effect of increased ROS activity on NOS expression in vitro in human coronary artery endothelial cells (HCAECs) grown in culture and in intact animals.


METHODS
Endothelial NOS (eNOS) expression and nuclear factor kappaB (NFkappaB) activation were determined in HCAECs grown in culture and exposed to oxidative stress with xanthine-xanthine oxidase (X-XO) generated superoxide, H(2)O(2), or glutathione depletion with buthionine sulfoximine (BSO) for 24 h. In parallel experiments, cells were treated with a nitric oxide (NO) scavenger (hemoglobin), and with an NO donor S-nitroso-N-acetyl penicillamine (SNAP)]. In addition, eNOS and inducible NOS (iNOS) expressions were determined in rats treated with either BSO or vehicle for 48 h.


RESULTS
Increases in ROS activity, achieved by exogenous superoxide and H(2)O(2) or by glutathione depletion, upregulated the expression of eNOS at both transcriptional and translational levels in HCAECs. Similar effects were seen with the non-radical NO scavenger, hemoglobin. The upregulatory action of hemoglobin on eNOS messenger RNA (mRNA) and protein expressions was overcome by the NO donor, SNAP, thereby suggesting that there is a negative feedback regulation of eNOS by NO. Nuclear translocation of NFkappaB (p65) was noted within 5 min of exposure to H(2)O(2) and at least 15 min after exposure to superoxide or BSO. Induction of oxidative stress by glutathione depletion led to upregulation of renal and aorta eNOS and iNOS in live animals.


CONCLUSIONS
An increase in ROS activity upregulates NOS expression in vitro in HCAECs grown in culture, and also in vivo in animals. This effect appears to be, in part, mediated by limiting the availability of NO, thereby exerting a negative feedback influence on NOS expression through activation of NFkappaB.
Zhen
Junhui
J
Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Lu
Hua
H
Wang
Xiu Q
XQ
Vaziri
Nosratola D
ND
Zhou
Xin J
XJ
D000818:Animals; D001011:Aorta / Q000201:enzymology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000201:enzymology; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D025461:Feedback, Physiological; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D008297:Male; D016328:NF-kappa B / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D015854:Up-Regulation
D006454:Hemoglobins; D016328:NF-kappa B; D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013481:Superoxides; D019328:Buthionine Sulfoximine; D026423:S-Nitroso-N-Acetylpenicillamine; D006861:Hydrogen Peroxide; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496318:Nos2 protein, rat; C496316:Nos3 protein, rat; D005978:Glutathione