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CELLULAR AND MITOCHONDRIAL METABOLISM

Nitric oxide-mediated protection of endothelial cells from hydrogen peroxide is mediated by intracellular zinc and glutathione

¹Institute of Molecular Medicine, Research Group Immunobiology, Medical Faculty of Heinrich-Heine-University of Düsseldorf, Düsseldorf; ²Department of Internal Medicine I, Cardio-Bio-Tec Research Group, and ³Department of Plastic Surgery, Hand and Reconstructive Surgery, Burn Center, Medical Faculty, RWTH Aachen University, Aachen; and ⁴Institute of Biochemistry and Molecular Biology II and ⁵Institute of Biochemistry and Molecular Biology I, Medical Faculty of Heinrich-Heine-University of Düsseldorf, Düsseldorf, Germany

Submitted 17 December 2008 ; accepted in final form 28 January 2009

Oxidative stress may cause endothelial dysfunction and vascular disease. It has been shown that NO protects endothelial cells (EC) against H₂O₂-induced toxicity. In addition, it is known that NO within cells induces a zinc release from proteins containing zinc-sulfur complexes. The aim of this study was to investigate whether zinc released intracellularly by NO plays a signaling role in the NO-mediated protection against H₂O₂ in rat aortic EC. Our results show that the NO-mediated protection toward H₂O₂ depends on the activities of glutathione peroxidase and glutamate cysteine ligase (GCL), the rate-limiting enzyme of glutathione (GSH) de novo biosynthesis. Moreover, NO increases the synthesis of the antioxidant GSH by inducing the expression of the catalytic subunit of GCL (GCLC). Chelating intracellular "free" zinc abrogates the NO-mediated increase of GCLC and of cellular GSH levels. As a consequence, the NO-mediated protection against H₂O₂-induced toxicity is impaired. We also show that under proinflammatory conditions, both cellular NO synthesis and intracellular "free" zinc are required to maintain the cellular GSH levels. Using RNA interference and laser scanning microscopy, we found that the NO-induced expression of GCLC depends on the activation of the transcription factor Nrf2 but not on the activity of the "zinc-sensing" transcription factor MTF-1. These findings show that intracellular "free" zinc plays a signaling role in the protective activity of NO and could explain why maintenance of an adequate zinc status in the endothelium is important to protect from oxidative stress and the development of vascular disease.

inducible nitric oxide synthase; zinc signaling; oxidative stress; glutamate cysteine ligase; Nrf2