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

Journal of Neuroinflammation

Alex Odermatt²  Adam Lister²  Denise V Kratschmar²  Boonrat Chantong¹ 

[1]Current address: Department of Preclinical Science and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Phutthamonthon, Nakhonpathom, Thailand

[2]Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, Basel, 4056, Switzerland

关键词: Intracellular calcium;    Endoplasmic reticulum stress;    Oxidative stress;    Inflammation;    mGluR5;    Glutamate receptor;    Microglia;   

Others  :  1150098
DOI  :  10.1186/s12974-014-0190-7

 received in 2014-06-13, accepted in 2014-10-30,  发布年份 2014

【 摘 要 】

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 AMPK? 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 ?M 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 AMPK? 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 G_i-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 G_i-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 G_i-proteins and subsequent activation of PLC, IP₃R and Ca²⁺ release from the ER.

【 授权许可】

   
2014 Chantong et al.; licensee BioMed Central Ltd.

【 图 表 】

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