Inhibition of metabotropic glutamate receptor 5 induces cellular stress through pertussis toxinsensitive Gi-proteins in murine BV-2 microglia cells

Background: activation of metabotropic glutamate receptor 5 (mglur5) by (rs)-2-chloro-5-hydroxyphenylglycine (chpg) was shown to suppress microglia activation and decrease the release of associated pro-inflammatory mediators. in contrast,the consequences of mglur5 inhibition are less well understood. here,we used bv-2 cells,retaining key characteristics of primary mouse microglia,to examine whether mglur5 inhibition by 2-methyl-6- (phenylethynyl)-pyridine (mpep) enhances cellular stress and production of inflammatory mediators. methods: bv-2 cells were treated with mpep,followed by determination of cellular stress using fluorescent dyes and high-content imaging. the expression of inflammatory mediators,endoplasmic reticulum (er)-stress markers and phosphorylated ampkα was analyzed by quantitative pcr,elisa and western blotting. additionally,phospholipase c (plc) activity,cellular atp content and changes in intracellular free ca2+ ([ca2+]i) were measured using luminescence and fluorescence assays. results: treatment of bv-2 microglia with 100 μm mpep increased intracellular reactive oxygen species (ros),mitochondrial superoxide,mitochondrial mass as well as inducible nitric oxide synthase (inos) and il-6 expression. furthermore,mpep reduced cellular atp and induced ampkα phosphorylation and the expression of the er-stress markers chop,grp78 and grp96. the mpep-dependent effects were preceded by a rapid concentration-dependent elevation of [ca2+]i,following ca2+ release from the er,mainly via inositol triphosphate-induced receptors (ip3r). the mpep-induced er-stress could be blocked by pretreatment with the chemical chaperone 4-phenylbutyrate and the ca2+ chelator bapta-am. pretreatment with the ampk agonist aicar partially abolished,whilst the inhibitor compound c potentiated,the mpep-dependent er-stress. importantly,the plc inhibitor u-73122 and the gi-protein inhibitor pertussis toxin (ptx) blocked the mpep-induced increase in [ca2+]i. moreover,pretreatment of microglia with aicar,bapta-am,u-73122 and ptx prevented the mpep-induced generation of oxidative stress and inflammatory mediators,further supporting a role for gi-protein-mediated activation of plc. conclusions: the results emphasize the potential pathophysiological role of mglur5 antagonism in mediating oxidative stress,er-stress and inflammation through a ca2+-dependent pathway in microglia. the induction of cellular stress and inflammatory mediators involves ptx-sensitive gi-proteins and subsequent activation of plc,ip3r and ca2+ release from the er. © 2014 chantong et al.