Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury

Bruce R. Pitt, Margaret Schwarz, Elizabeth S. Woo, Emily Yee, Karla Wasserloos, Sothi Tran, Weili Weng, Robert J. Mannix, Simon A. Watkins, Yulia Y. Tyurina, Vladimir A. Tyurin, Valerian E. Kagan, John S. Lazo

Research output: Contribution to journalArticle

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Abstract

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein with extensive metal binding capacity and potential nonenzymatic antioxidant activity. Despite the sensitivity of vascular endothelium to either heavy metal toxicity or oxidative stress, little is known regarding the role of MT in endothelial cells. Accordingly, we determined the sensitivity of cultured sheep pulmonary artery endothelial cells (SPAEC) that overexpressed MT to tert-butyl hydroperoxide (t-BOOH), hyperoxia, or 2,2'-azobis(2,4- dimethylvaleronitrile) (AMVN; peroxyl radical generator). Nontoxic doses of 10 μM Cd increased MT levels from 0.21 ± 0.03 to 2.07 ± 0.24 μg/mg and resulted in resistance to t-BOOH and hyperoxia as determined by reduction of Alamar blue or [3H]serotonin transport, respectively. SPAEC stably transfected with plasmids containing either mouse or human cDNA for MT were resistant to both t-BOOH and hyperoxia. In addition, we examined transition metal-independent, noncytotoxic AMVN-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis- parinaric acid into phospholipids and high-performance liquid chromatography separation. SPAEC that overexpressed MT after gene transfer completely inhibited peroxyl oxidation of phosphatidylserine, phosphatidylcholine, and sphingomyelin (but not phosphatidylethanolamine) noted in wild-type SPAEC. These data show for the first time that MT can 1) protect pulmonary artery endothelium against a diverse array of prooxidant stimuli and 2) directly intercept peroxyl radicals in a metal-independent fashion, thereby preventing lipid peroxidation in intact cells.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume273
Issue number4 17-4
StatePublished - 1997

Fingerprint

Metallothionein
Oxidants
Endothelial Cells
Pulmonary Artery
Lung
Wounds and Injuries
Hyperoxia
Sheep
Metals
Lipid Peroxidation
tert-Butylhydroperoxide
Sphingomyelins
Phosphatidylserines
Vascular Endothelium
Phosphatidylcholines
Endothelium
Cysteine
Phospholipids
Serotonin
Oxidative Stress

Keywords

  • Cadmium
  • Hyperoxia
  • Lipid peroxidation
  • Organic hydroperoxides
  • Peroxyl radicals
  • Pulmonary artery endothelial cells

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Cell Biology
  • Physiology
  • Physiology (medical)

Cite this

Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury. / Pitt, Bruce R.; Schwarz, Margaret; Woo, Elizabeth S.; Yee, Emily; Wasserloos, Karla; Tran, Sothi; Weng, Weili; Mannix, Robert J.; Watkins, Simon A.; Tyurina, Yulia Y.; Tyurin, Vladimir A.; Kagan, Valerian E.; Lazo, John S.

In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 273, No. 4 17-4, 1997.

Research output: Contribution to journalArticle

Pitt, BR, Schwarz, M, Woo, ES, Yee, E, Wasserloos, K, Tran, S, Weng, W, Mannix, RJ, Watkins, SA, Tyurina, YY, Tyurin, VA, Kagan, VE & Lazo, JS 1997, 'Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury', American Journal of Physiology - Lung Cellular and Molecular Physiology, vol. 273, no. 4 17-4.
Pitt, Bruce R. ; Schwarz, Margaret ; Woo, Elizabeth S. ; Yee, Emily ; Wasserloos, Karla ; Tran, Sothi ; Weng, Weili ; Mannix, Robert J. ; Watkins, Simon A. ; Tyurina, Yulia Y. ; Tyurin, Vladimir A. ; Kagan, Valerian E. ; Lazo, John S. / Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury. In: American Journal of Physiology - Lung Cellular and Molecular Physiology. 1997 ; Vol. 273, No. 4 17-4.
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abstract = "Metallothionein (MT) is a low-molecular-weight cysteine-rich protein with extensive metal binding capacity and potential nonenzymatic antioxidant activity. Despite the sensitivity of vascular endothelium to either heavy metal toxicity or oxidative stress, little is known regarding the role of MT in endothelial cells. Accordingly, we determined the sensitivity of cultured sheep pulmonary artery endothelial cells (SPAEC) that overexpressed MT to tert-butyl hydroperoxide (t-BOOH), hyperoxia, or 2,2'-azobis(2,4- dimethylvaleronitrile) (AMVN; peroxyl radical generator). Nontoxic doses of 10 μM Cd increased MT levels from 0.21 ± 0.03 to 2.07 ± 0.24 μg/mg and resulted in resistance to t-BOOH and hyperoxia as determined by reduction of Alamar blue or [3H]serotonin transport, respectively. SPAEC stably transfected with plasmids containing either mouse or human cDNA for MT were resistant to both t-BOOH and hyperoxia. In addition, we examined transition metal-independent, noncytotoxic AMVN-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis- parinaric acid into phospholipids and high-performance liquid chromatography separation. SPAEC that overexpressed MT after gene transfer completely inhibited peroxyl oxidation of phosphatidylserine, phosphatidylcholine, and sphingomyelin (but not phosphatidylethanolamine) noted in wild-type SPAEC. These data show for the first time that MT can 1) protect pulmonary artery endothelium against a diverse array of prooxidant stimuli and 2) directly intercept peroxyl radicals in a metal-independent fashion, thereby preventing lipid peroxidation in intact cells.",
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AU - Tran, Sothi

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AU - Mannix, Robert J.

AU - Watkins, Simon A.

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AU - Tyurin, Vladimir A.

AU - Kagan, Valerian E.

AU - Lazo, John S.

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