The thiol-modifying agent N-ethylmaleimide elevates the cytosolic concentration of free Zn(2+) but not of Ca(2+) 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 Ca(2+) ([Ca(2+)]i). Depending on the cell type and the concentration used, NEM can promote the release of Ca(2+), affect its extrusion, stimulate or block its entry. However, most of these findings were obtained in experiments that employed fluorescent Ca(2+) probes and one major disadvantage of such experimental setting derives from the lack of specificity of the probes as all the so-called "Ca(2+)-sensitive" indicators also bind metals like Zn(2+) or Mn(2+) with higher affinities than Ca(2+). In this study, we examined the effects of NEM on the [Ca(2+)]i homeostasis of murine cortical neurons. We performed live-cell Ca(2+) and Zn(2+) imaging experiments using the fluorescent probes Fluo-4, FluoZin-3 and RhodZin-3 and found that NEM does not affect the neuronal [Ca(2+)]i homeostasis but specifically increases the cytosolic and mitochondrial concentration of free Zn(2+)([Zn(2+)]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 [Zn(2+)]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 [Zn(2+)]i but not [Ca(2+)]i homeostasis and shed new light on the physiological actions of this alkylating agent on central nervous system neurons.
Gibon
Julien
J
UMR CNRS 5249, Grenoble, France.
Tu
Peng
P
Frazzini
Valerio
V
Sensi
Stefano L
SL
Bouron
Alexandre
A
D000814:Aniline Compounds; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011083:Polycyclic Compounds; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014966:Xanthenes; D015032:Zinc / Q000378:metabolism*
D000814:Aniline Compounds; C409648:Fluo 4; C451182:FluoZin-3; D011083:Polycyclic Compounds; C484817:RhodZin-3; D013439:Sulfhydryl Reagents; D014966:Xanthenes; D015032:Zinc; D005033:Ethylmaleimide; D002118:Calcium