Proceedings of The Physiological Society

University of Cambridge (2008) Proc Physiol Soc 11, PC133

Poster Communications

Peroxynitrite prevents Zn2+-induced cell death by reactivating glutathione reductase

J. An1, S. Hong1, D. Shin1, J. Seo1

1. Departmenr of Oral Biology, BK 21 Project for Yonsei Dental Sciences, Yonsei University College of Dentistry, Seoul, South Korea.

Peroxynitrite is a nitric oxide-derived highly reactive cytotoxic molecule. Recently, however, we found that 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, prevented Zn2+-induced cell death in differentiated PC12 cells. In the present study, therefore, we have investigated the mechanism responsible for the protective effect of SIN-1 on the Zn2+-induced cell death. Treatment of PC12 cells with zinc chloride (10 μM) and pyrithione (5 μM), a Zn2+ ionophore, caused cell death in differentiated PC12 cells. The Zn2+-induced cell death was inhibited by the pretreatment with SIN-1 over the concentration range 100 - 500 μM. Because SIN-1 produces both nitric oxide (NO) and peroxynitrite, we examined whether the protective effect of SIN-1 against PC12 cell death was attributed to NO or peroxynitrite. Pretreatment of cells with various NO donors, such as SNP and SNAP, was not able to attenuate the Zn2+-induced cell death. In addition to this, peroxynitrite scavengers, such as uric acid, trolox and L-methionine, abolished the protective effect of SIN-1, suggesting that peroxynitrite was responsible for the neuroprotective effect of SIN-1 in differentiated PC12 cells. To investigate the mechanism by which peroxynitrite exerted its protective effect, the intracellular glutathione (GSH) and oxidized GSH (GSSG) levels were measured. An exposure of cells to Zn2+-pyrithione increased the GSSG/GSH ratio. Zn2+ was also shown to inhibit the GSH reductase (GR) activity, which catalyzes the reduction of GSSG to the thiol form of GSH. However, the addition of 300 μM SIN-1 almost completely restored the GR activity and the GSSG/GSH ratio. Furthermore, exogenously applied GSH rescued the PC12 cells from Zn2+-induced cell death, indicating that disruption of GSH homeostasis plays a critical role in the Zn2+-induced cell death. Therefore, we suggest that Zn2+ induces cell death, at least in part, by increasing the GSSG/GSH ratio through the inhibition of GR activity, and peroxynitrite attenuates the Zn2+-induced cell death by re-activating the GR activity.

Where applicable, experiments conform with Society ethical requirements