The thiol-modifying agent N-ethylmaleimide elevates the cytosolic concentration of free Zn2+ but not of Ca2+ in murine cortical neurons

The membrane permeant alkylating agent N-ethylmaleimide (NEM) regulates numerous biological processes by reacting with thiol groups. Among other actions, NEM influences the cytosolic concentration of free Ca2+ ([Ca2+](i)). Depending on the cell type and the concentration used, NEM can promote the release of Ca2+, affect its extrusion, stimulate or block its entry. However, most of these findings were obtained in experiments that employed fluorescent Ca2+ probes and one major disadvantage of such experimental setting derives from the lack of specificity of the probes as all the so-called Ca2+-sensitive indicators also bind metals like Zn2+ or Mn2+ with higher affinities than Ca2+. In this study, we examined the effects of NEM on the [Ca2+](i), homeostasis of murine cortical neurons. We performed live-cell Ca2+ and Zn2+ imaging experiments using the fluorescent probes Fluo-4, FluoZin-3 and RhodZin-3 and found that NEM does not affect the neuronal [Ca2+](i), homeostasis but specifically increases the cytosolic and mitochondrial concentration of free Zn2+([Zn2+](i)). In addition, NEM triggers some neuronal loss that is prevented by anti-oxidants such as N-acetylcysteine or glutathione. NEM-induced toxicity is dependent on changes in [Zn2+](i) levels as chelation of the cation with the cell-permeable heavy metal chelator, N,N,N'N'-tetrakis(-)[2-pyridylmethyl]-ethylenediamine (TPEN), promotes neuroprotection of cortical neurons exposed to NEM. Our data indicate that NEM affects [Zn2+](i), but not [Ca2+](i), homeostasis and shed new light on the physiological actions of this alkylating agent on central nervous system neurons. (C) 2010 Elsevier Ltd. All rights reserved.