A molecular technique for detecting the liberation of intracellular zinc in cultured neurons

J Neurosci Methods. 2004 Aug 30;137(2):175-80. doi: 10.1016/j.jneumeth.2004.02.018.

Abstract

We have previously reported that oxidative stimuli liberate Zn(2+) from metalloproteins, a phenomenon that can trigger neuronal cell death. Excessive intracellular Zn(2+) in many cell types triggers the expression of genes that encode metal binding proteins, such as metallothionein, via the activation and nuclear translocation of metal response element (MRE)-binding transcription factor-1 (MTF-1). Cd(2+) strongly induces nuclear translocation of MTF-1 in non-neuronal cells, but it does so by displacing Zn(2+) from its metal binding sites within the cell and increasing the intracellular concentration of this ion. Here, we describe the use of MRE-driven expression of a luciferase reporter gene as a sensitive molecular assay for detecting increases in intracellular zinc concentrations. MRE transactivation was induced in primary cortical neurons upon brief exposure to Zn(2+) or Cd(2+). Enhanced MRE transactivation was observed upon co-exposure of neurons to Cd(2+) together with NMDA, as this metal can permeate through the receptor channel. Luciferase expression was observed regardless of whether or not neurons had been co-transfected with an MTF-1-containing plasmid, suggesting the presence of an endogenous MTF-1-like protein. Indeed, RT-PCR revealed that MTF-1 mRNA is present in neurons. In contrast, MTF-1 deficient dko7 cells were only observed to have MRE transactivation when co-transfected with MTF-1. Our results indicate that Cd(2+) can effectively induce transactivation of MRE in neurons by liberating Zn(2+) from its intracellular binding sites.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cadmium / pharmacology
  • Cells, Cultured
  • Cerebral Cortex / cytology*
  • DNA-Binding Proteins
  • Dose-Response Relationship, Drug
  • Drug Interactions
  • Embryo, Mammalian
  • Enzyme Activation / drug effects
  • Excitatory Amino Acid Agonists / pharmacology
  • Gene Expression Regulation, Enzymologic / drug effects
  • Genes, Reporter / physiology
  • Intracellular Space / metabolism*
  • Luciferases / metabolism
  • Models, Neurological
  • Molecular Probe Techniques*
  • N-Methylaspartate / pharmacology
  • Neurons / metabolism*
  • RNA, Messenger / biosynthesis
  • Rats
  • Rats, Sprague-Dawley
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Transcription Factors / metabolism
  • Valine / analogs & derivatives*
  • Valine / pharmacology
  • Zinc / analysis*

Substances

  • DNA-Binding Proteins
  • Excitatory Amino Acid Agonists
  • RNA, Messenger
  • Transcription Factors
  • transcription factor MTF-1
  • Cadmium
  • N-Methylaspartate
  • 2-amino-5-phosphopentanoic acid
  • Luciferases
  • Valine
  • Zinc