Activation of microglia with zymosan promotes excitatory amino acid release via volume-regulated anion channels: the role of NADPH oxidases

J Neurochem. 2008 Sep;106(6):2449-62. doi: 10.1111/j.1471-4159.2008.05553.x. Epub 2008 Jul 9.


Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study, we used an electrophysiologic approach and a D-[(3)H]aspartate (glutamate) release assay to explore the ROS-dependent regulation of glutamate-permeable volume-regulated anion channels (VRACs). Exposure of rat microglia to hypo-osmotic media stimulated Cl(-) currents and D-[(3)H]aspartate release, both of which were inhibited by the selective VRAC blocker, DCPIB. Exogenously applied H(2)O(2) potently increased swelling-activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger, MnTMPyP, and by two inhibitors of NADPH oxidase (NOX), diphenyliodonium and thioridazine. However, zymosan-stimulated glutamate release was insensitive to other NOX blockers, apocynin and HEBSF. This pharmacologic profile pointed to the potential involvement of apocynin-insensitive NOX4. Using RT-PCR we confirmed that NOX4 is expressed in rat microglial cells along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2, we stimulated this isoform using protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate or inhibited it with the broad spectrum PKC blocker, Gö6983. Both agents potently modulated endogenous ROS production by NOX2 but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release and that its activity is regulated by endogenous ROS originating from NOX4.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Aspartic Acid / metabolism
  • Cells, Cultured
  • Encephalitis / metabolism
  • Enzyme Inhibitors / pharmacology
  • Excitatory Amino Acids / metabolism*
  • Glutamic Acid / metabolism
  • Isoenzymes / drug effects
  • Isoenzymes / metabolism
  • Microglia / drug effects
  • Microglia / metabolism*
  • NADPH Oxidase 4
  • NADPH Oxidases / drug effects
  • NADPH Oxidases / metabolism*
  • Oxidants / pharmacology
  • Oxidative Stress / drug effects
  • Oxidative Stress / physiology
  • Protein Kinase C / drug effects
  • Protein Kinase C / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Reactive Oxygen Species / metabolism
  • Voltage-Dependent Anion Channels / drug effects
  • Voltage-Dependent Anion Channels / metabolism*
  • Zymosan / pharmacology*


  • Enzyme Inhibitors
  • Excitatory Amino Acids
  • Isoenzymes
  • Oxidants
  • Reactive Oxygen Species
  • Voltage-Dependent Anion Channels
  • Aspartic Acid
  • Glutamic Acid
  • Zymosan
  • NADPH Oxidase 4
  • NADPH Oxidases
  • Nox4 protein, rat
  • Protein Kinase C