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Glutathione and glutathione-linked enzymes in normal human aortic smooth muscle cells: chemical inducibility and protection against reactive oxygen and nitrogen species-induced injury

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Abstract

Substantial evidence suggests a crucial role for glutathione (GSH) and GSH-linked enzymes in protecting against oxidative vascular disorders. However, studies on the chemical inducibility of these antioxidant defenses and their protective effects on oxidant injury in normal human vascular cells are currently lacking. Accordingly, this study was undertaken to investigate the inducibility of GSH, glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST) by the chemoprotective agent, 3H-1,2-dithiole-3-thione (D3T) in cultured normal human aortic smooth muscle cells (HASMCs). HASMCs expressed measurable levels/activities of GSH, GR, GPx, and GST. Incubation of HASMCs with low micromolar concentrations of D3T resulted in a marked elevation in total cellular GSH content and GR activity. The protein and mRNA expression of γ-glutamylcysteine ligase (GCL) and GR were also upregulated by D3T. In addition, D3T caused significant increases in mitochondrial GSH content and GR activity. In contrast, neither cellular GPx nor GST activity was altered after D3T treatment. Pretreatment of HASMCs with D3T afforded remarkable protection against reactive oxygen and nitrogen species (ROS/RNS)-mediated cell injury. Depletion of cellular GSH by pretreatment with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis led to marked potentiation of the ROS/RNS-induced cell injury. Moreover, co-treatment of HASMCs with BSO was found to completely abolish the D3T-mediated GSH elevation, and remarkably reverse D3T cytoprotection against the ROS/RNS-elicited injury. Taken together, this study demonstrates that both GSH/GCL and GR in normal HASMCs are inducible by D3T, and that upregulation of GSH biosynthesis appears to be the predominant mechanism underlying D3T-mediated cytoprotection against ROS/RNS-elicited injury to human vascular smooth muscle cells.

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Abbreviations

BSO:

Buthionine sulfoximine

CDNB:

1-Chloro-2,3-dinitrobenzene

D3T:

3H-1,2-dithiole-3-thione

DCIP:

2,6-Dichloroindophenol

DCF:

2,7-Dichlorodihydrofluorescein

FBS:

Fetal bovine serum; GPx, glutathione peroxidase

GR:

Glutathione reductase

GSH:

Reduced glutathione

GSSG:

Oxidized form of glutathione

GST:

Glutathione S-transferase

MTT:

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium

PBS:

Phosphate buffered saline

ROS:

Reactive oxygen species

RNS:

Reactive nitrogen species

GCL:

γ-Glutamylcysteine ligase

GCL-C:

γ-Glutamylcysteine ligase catalytic subunit

GCL-M:

γ-Glutamylcysteine ligase modulating subunit

SIN-1:

3-Morpholinosydnonimine

XO:

Xanthine oxidase

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Acknowledgments

This work was supported in part by NIH grants CA91895 and HL71190 (Y.L.). M.A.T. was supported by NIH grants ES03760, ES03819 and ES08078.

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Author notes

  1. Yunbo Li

    Present address: Edward Via Virginia College of Osteopathic Medicine, Virginia Tech Corporate Research Center, Research Building II, 1861 Pratt Drive, Blacksburg, VA, 24060, USA

Authors and Affiliations

  1. Davis Heart and Lung Research Institute, and Department of Internal Medicine and Division of Cardiovascular Medicine, The Ohio State University Medical Center, 473 West 12th Avenue, Columbus, OH, 43210, USA

    Hong Zhu, Li Zhang & Yunbo Li

  2. Brigham and Women’s Hospital, Cardiology Division, Harvard Medical School, 20 Shattuck Street, Thorn 1130, Boston, MA, 02115, USA

    Zhuoxiao Cao

  3. Division of Toxicology, Department of Environmental Health Sciences, The Johns Hopkins University Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD, 21205, USA

    Michael A. Trush

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  1. Hong Zhu
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Correspondence to Yunbo Li.

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Zhu, H., Cao, Z., Zhang, L. et al. Glutathione and glutathione-linked enzymes in normal human aortic smooth muscle cells: chemical inducibility and protection against reactive oxygen and nitrogen species-induced injury. Mol Cell Biochem 301, 47–59 (2007). https://doi.org/10.1007/s11010-006-9396-z

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