Effect of L-buthionine-(S,R)-sulphoximine, an inhibitor of γ-glutamylcysteine synthetase on peroxynitrite- and endotoxic shock-induced vascular failure

Salvatore Cuzzocrea, Basilia Zingarelli, Michael O'Connor, Andrew L. Salzman, Csaba Szabo

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

1. Peroxynitrite, a cytotoxic oxidant formed from the reaction of nitric oxide (NO) and superoxide is a mediator of cellular injury in ischaemia/reperfusion injury, shock and inflammation. Here we investigated whether L-buthionine-(S,R)-sulphoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase, alters endothelial and vascular smooth muscle injury in response to peroxynitrite in vitro and during endotoxic shock in vivo. 2. In human umbilical vein endothelial cells and in rat aortic smooth muscle cells, BSO (1 mM, for 24 h) enhanced, whereas glutathione (3 mM) or glutathione ethyl ester (3mM) attenuated the peroxynitrite (100-1000 μM)-induced suppression of mitochondrial respiration (measured by the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan), formation of nitrotyrosine (detected by Western blotting), protein oxidation (measured by detection of 2,4 dinitrophenylhydrazine-reactive carbonyls), and DNA single strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) (measured by the incorporation of radiolabelled NAD+ into nuclear proteins and by the alkaline unwinding assay, respectively). Glutathione ethyl ester treatment reduced the BSO-induced enhancement of peroxynitrite induced cytotoxicity. 3. In rat isolated thoracic aortic rings, BSO treatment (in vivo, at 1 g kg-1 intraperitoneally (i.p.) for 24 h) enhanced, whereas pretreatment with glutathione (in vitro, 3 mM) attenuated the peroxynitrite-induced reduction of the contractions to noradrenaline, and the peroxynitrite-induced impairment of the endothelium-dependent relaxations to acetylcholine. 4. In BSO-pretreated rats, treatment with bacterial lipopolysaccharide (LPS, 15 mg kg-1, i.p., for 6 h) caused a more pronounced vascular hyporeactivity and endothelial dysfunction ex vivo. BSO pretreatment also increased the degree of nitrotyrosine staining (detected by imunohistochemistry) in the aorta after LPS treatment. 5. In conclusion, our results demonstrate that L-buthionine-(S,R)-sulphoximine, an inhibitor of γ-glutamylcysteine synthetase enhances peroxynitrite- and endotoxic shock-induced vascular failure. Based on these findings, we suggest that endogenous glutathione plays an important protective role against peroxynitrite- and LPS-induced vascular injury.

Original languageEnglish (US)
Pages (from-to)525-537
Number of pages13
JournalBritish Journal of Pharmacology
Volume123
Issue number3
DOIs
StatePublished - 1998
Externally publishedYes

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Glutamate-Cysteine Ligase
Peroxynitrous Acid
Septic Shock
Blood Vessels
Glutathione
Poly Adenosine Diphosphate Ribose
Enzyme Activation
buthionine
Vascular System Injuries
Human Umbilical Vein Endothelial Cells
Wounds and Injuries
Ligases
Nuclear Proteins
Reperfusion Injury
Vascular Smooth Muscle
Oxidants
Superoxides
NAD
Acetylcholine
Smooth Muscle Myocytes

Keywords

  • Antioxidants
  • Endothelium
  • Endothelium-derived factors
  • Free radicals
  • Shock

ASJC Scopus subject areas

  • Pharmacology

Cite this

Effect of L-buthionine-(S,R)-sulphoximine, an inhibitor of γ-glutamylcysteine synthetase on peroxynitrite- and endotoxic shock-induced vascular failure. / Cuzzocrea, Salvatore; Zingarelli, Basilia; O'Connor, Michael; Salzman, Andrew L.; Szabo, Csaba.

In: British Journal of Pharmacology, Vol. 123, No. 3, 1998, p. 525-537.

Research output: Contribution to journalArticle

Cuzzocrea, Salvatore ; Zingarelli, Basilia ; O'Connor, Michael ; Salzman, Andrew L. ; Szabo, Csaba. / Effect of L-buthionine-(S,R)-sulphoximine, an inhibitor of γ-glutamylcysteine synthetase on peroxynitrite- and endotoxic shock-induced vascular failure. In: British Journal of Pharmacology. 1998 ; Vol. 123, No. 3. pp. 525-537.
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AU - Zingarelli, Basilia

AU - O'Connor, Michael

AU - Salzman, Andrew L.

AU - Szabo, Csaba

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N2 - 1. Peroxynitrite, a cytotoxic oxidant formed from the reaction of nitric oxide (NO) and superoxide is a mediator of cellular injury in ischaemia/reperfusion injury, shock and inflammation. Here we investigated whether L-buthionine-(S,R)-sulphoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase, alters endothelial and vascular smooth muscle injury in response to peroxynitrite in vitro and during endotoxic shock in vivo. 2. In human umbilical vein endothelial cells and in rat aortic smooth muscle cells, BSO (1 mM, for 24 h) enhanced, whereas glutathione (3 mM) or glutathione ethyl ester (3mM) attenuated the peroxynitrite (100-1000 μM)-induced suppression of mitochondrial respiration (measured by the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan), formation of nitrotyrosine (detected by Western blotting), protein oxidation (measured by detection of 2,4 dinitrophenylhydrazine-reactive carbonyls), and DNA single strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) (measured by the incorporation of radiolabelled NAD+ into nuclear proteins and by the alkaline unwinding assay, respectively). Glutathione ethyl ester treatment reduced the BSO-induced enhancement of peroxynitrite induced cytotoxicity. 3. In rat isolated thoracic aortic rings, BSO treatment (in vivo, at 1 g kg-1 intraperitoneally (i.p.) for 24 h) enhanced, whereas pretreatment with glutathione (in vitro, 3 mM) attenuated the peroxynitrite-induced reduction of the contractions to noradrenaline, and the peroxynitrite-induced impairment of the endothelium-dependent relaxations to acetylcholine. 4. In BSO-pretreated rats, treatment with bacterial lipopolysaccharide (LPS, 15 mg kg-1, i.p., for 6 h) caused a more pronounced vascular hyporeactivity and endothelial dysfunction ex vivo. BSO pretreatment also increased the degree of nitrotyrosine staining (detected by imunohistochemistry) in the aorta after LPS treatment. 5. In conclusion, our results demonstrate that L-buthionine-(S,R)-sulphoximine, an inhibitor of γ-glutamylcysteine synthetase enhances peroxynitrite- and endotoxic shock-induced vascular failure. Based on these findings, we suggest that endogenous glutathione plays an important protective role against peroxynitrite- and LPS-induced vascular injury.

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KW - Endothelium

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