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

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.

Inhibition of serotonin 5-hydroxytryptamine2c receptor function through heterodimerization: receptor dimers bind two molecules of ligand and one G-protein.

Although dimerization appears to be a common property of G-protein-coupled receptors (GPCRs), it remains unclear whether a GPCR dimer binds one or two molecules of ligand and whether ligand binding results in activation of one or two G-proteins when measured using functional assays in intact living cells. Previously, we demonstrated that serotonin 5-hydroxytryptamine2C (5-HT2C) receptors form homodimers (Herrick-Davis, K., Grinde, E., and Mazurkiewicz, J. (2004) Biochemistry 43, 13963-13971). In the present study, an inactive 5-HT(2C) receptor was created and coexpressed with wild-type 5-HT2C receptors to determine whether dimerization regulates receptor function and to determine the ligand/dimer/G-protein stoichiometry in living cells. Mutagenesis of Ser138 to Arg (S138R) produced a 5-HT2C receptor incapable of binding ligand or stimulating inositol phosphate (IP) signaling. Confocal fluorescence imaging revealed plasma membrane expression of yellow fluorescent protein-tagged S138R receptors. Expression of wild-type 5-HT2C receptors in an S138R-expressing stable cell line had no effect on ligand binding to wild-type 5-HT2C receptors, but inhibited basal and 5-HT-stimulated IP signaling as well as constitutive and 5-HT-stimulated endocytosis of wild-type 5-HT2C receptors. M1 muscarinic receptor activation of IP production was normal in the S138R-expressing cells. Heterodimerization of S138R with wild-type 5-HT2C receptors was visualized in living cells using confocal fluorescence resonance energy transfer (FRET). FRET was dependent on the donor/acceptor ratio and independent of the receptor expression level. Therefore, inactive 5-HT2C receptors inhibit wild-type 5-HT2C receptor function by forming nonfunctional heterodimers expressed on the plasma membrane. These results are consistent with a model in which one GPCR dimer binds two molecules of ligand and one G-protein and indicate that dimerization is essential for 5-HT receptor function.