Skip to Content
Merck
  • Hypoxia and reoxygenation induce endothelial nitric oxide synthase uncoupling in endothelial cells through tetrahydrobiopterin depletion and S-glutathionylation.

Hypoxia and reoxygenation induce endothelial nitric oxide synthase uncoupling in endothelial cells through tetrahydrobiopterin depletion and S-glutathionylation.

Biochemistry (2014-04-25)
Francesco De Pascali, Craig Hemann, Kindra Samons, Chun-An Chen, Jay L Zweier
ABSTRACT

Ischemia-reperfusion injury is accompanied by endothelial hypoxia and reoxygenation that trigger oxidative stress with enhanced superoxide generation and diminished nitric oxide (NO) production leading to endothelial dysfunction. Oxidative depletion of the endothelial NO synthase (eNOS) cofactor tetrahydrobiopterin can trigger eNOS uncoupling, in which the enzyme generates superoxide rather than NO. Recently, it has also been shown that oxidative stress can induce eNOS S-glutathionylation at critical cysteine residues of the reductase site that serves as a redox switch to control eNOS coupling. While superoxide can deplete tetrahydrobiopterin and induce eNOS S-glutathionylation, the extent of and interaction between these processes in the pathogenesis of eNOS dysfunction in endothelial cells following hypoxia and reoxygenation remain unknown. Therefore, studies were performed on endothelial cells subjected to hypoxia and reoxygenation to determine the severity of eNOS uncoupling and the role of cofactor depletion and S-glutathionylation in this process. Hypoxia and reoxygenation of aortic endothelial cells triggered xanthine oxidase-mediated superoxide generation, causing both tetrahydrobiopterin depletion and S-glutathionylation with resultant eNOS uncoupling. Replenishing cells with tetrahydrobiopterin along with increasing intracellular levels of glutathione greatly preserved eNOS activity after hypoxia and reoxygenation, while targeting either mechanism alone only partially ameliorated the decrease in NO. Endothelial oxidative stress, secondary to hypoxia and reoxygenation, uncoupled eNOS with an altered ratio of oxidized to reduced glutathione inducing eNOS S-glutathionylation. These mechanisms triggered by oxidative stress combine to cause eNOS dysfunction with shift of the enzyme from NO to superoxide production. Thus, in endothelial reoxygenation injury, normalization of both tetrahydrobiopterin levels and the glutathione pool are needed for maximal restoration of eNOS function and NO generation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acetonitrile, anhydrous, 99.8%
Sigma-Aldrich
Methanol-12C, 99.95 atom % 12C
Supelco
Menadione (K3), analytical standard
Sigma-Aldrich
Potassium phosphate monobasic, 99.99% trace metals basis
Sigma-Aldrich
L-Cysteine, 97%
Sigma-Aldrich
Acetonitrile, ≥99.5%, ACS reagent
Sigma-Aldrich
Acetonitrile, HPLC Plus, ≥99.9%, poly-coated bottles
Supelco
Citric acid, Anhydrous, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Citric acid, ACS reagent, ≥99.5%
Sigma-Aldrich
L-Cysteine, ≥97%, FG
Sigma-Aldrich
2-Hydroxybutyric acid sodium salt, 97%
Supelco
L-Cysteine, certified reference material, TraceCERT®, Manufactured by: Sigma-Aldrich Production GmbH, Switzerland
Sigma-Aldrich
Acetonitrile, electronic grade, 99.999% trace metals basis
USP
Citric acid, United States Pharmacopeia (USP) Reference Standard
Supelco
Citric acid, certified reference material, TraceCERT®, Manufactured by: Sigma-Aldrich Production GmbH, Switzerland
Sigma-Aldrich
DAPI, for nucleic acid staining
Sigma-Aldrich
Citric acid, anhydrous, suitable for cell culture, suitable for plant cell culture
Sigma-Aldrich
Potassium phosphate monobasic, powder, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
Sigma-Aldrich
Menadione, meets USP testing specifications
Sigma-Aldrich
Menadione, crystalline
Sigma-Aldrich
Potassium phosphate monobasic, ReagentPlus®
Sigma-Aldrich
Potassium phosphate monobasic, for molecular biology, ≥98.0%
Sigma-Aldrich
Oxypurinol, ≥98% (HPLC)
Sigma-Aldrich
Citric acid, 99%
Sigma-Aldrich
L-Cysteine, from non-animal source, BioReagent, suitable for cell culture, ≥98%
Sigma-Aldrich
Citric acid, BioUltra, anhydrous, ≥99.5% (T)
SAFC
L-Cysteine
Sigma-Aldrich
L-Cysteine, BioUltra, ≥98.5% (RT)
Supelco
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9% (GC)
Supelco
Acetonitrile, analytical standard