title: Inhibition of metabotropic glutamate receptor 5 induces cellular stress through pertussis toxin-sensitive Gi-proteins in murine BV-2 microglia cells.
abstract: 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 Ca(2+) ([Ca(2+)]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 [Ca(2+)]i, following Ca(2+) 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 Ca(2+) 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 [Ca(2+)]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 Ca(2+)-dependent pathway in microglia. The induction of cellular stress and inflammatory mediators involves PTX-sensitive Gi-proteins and subsequent activation of PLC, IP3R and Ca(2+) release from the ER.
authors.affiliation: Current address: Department of Preclinical Science and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Phutthamonthon, Nakhonpathom, Thailand. boonrat.cha@mahidol.ac.th.
authors.forename: Boonrat
authors.initials: B
authors.lastname: Chantong
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland. denise.kratschmar@unibas.ch.
authors.forename: Denise V
authors.initials: DV
authors.lastname: Kratschmar
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland. adam.lister@unibas.ch.
authors.forename: Adam
authors.initials: A
authors.lastname: Lister
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland. alex.odermatt@unibas.ch.
authors.forename: Alex
authors.initials: A
authors.lastname: Odermatt
mesh_terms: D000818:Animals; D002460:Cell Line; D000091342:Endoplasmic Reticulum Chaperone BiP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D037342:Pertussis Toxin / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism; D064529:Receptor, Metabotropic Glutamate 5 / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C578936:Grm5 protein, mouse; C000717248:Hspa5 protein, mouse; D017382:Reactive Oxygen Species; D064529:Receptor, Metabotropic Glutamate 5; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go