Evaluation of Cannabinoid Receptor 2 and Metabotropic Glutamate Receptor 1 Functional Responses Using a Cell Impedance–Based Technology

Abstract

Recently, new technologies based on biosensors and called label free have been developed. These technologies eliminate the need for using markers and dyes. The authors applied one of these technologies, based on measurement of cell impedance variation, to study the pharmacological profiles of ligands for the cannabinoid receptor 2 (CB2), a Gi-coupled receptor, and for the metabopotropic glutamate receptor 1 (mGluR1), a Gq-coupled receptor. Reference agonists and antagonists/inverse agonists for the 2 receptors were applied to recombinant cell lines and impedance monitored over time. Agonists (JWH133 and CP55940 for CB2; quisqualate, glutamate, 1S-3R-ACPD, and S-3,5-DHPG for mGluR1) triggered a variation of impedance consistent in both potency and efficacy with data obtained using classical assays measuring cAMP or Ca2+ levels. This effect was not present in the parental nontransfected cell line, confirming specific receptor-mediated response. Application of antagonists (AM630 for CB2; YM298198, SCH1014222, J&J16259685, and CPCCOEt for mGluR1) reduced agonist-induced impedance changes. The only exception was the mGluR1 antagonist BAY367620 that, while active in the Ca2+ assay, was inactive in the impedance assay. Overall, these results confirm the possibility of using cell impedance–based technology to study the pharmacological profile of ligands acting at G-protein-coupled receptors coupled to different downstream signaling pathways.

Whole-cell biosensor for label-free detection of GPCR-mediated drug responses in personal cell lines

“…In our hands, LCL densities of 50,000 cells/well were sufficient for detection of a robust GPCR response (Fig. 4), a number that is merely 2.5-fold higher than common for adherent cells (Guo et al, 2012;Scandroglio et al, 2010;Stallaert et al, 2012) and very comparable to existing suspension cell protocols described above.…”

“…The initial experimental setup was based on previously published protocols for adherent cell lines (Guo et al, 2012;Scandroglio et al, 2010;Stallaert et al, 2012). While label-free assays are often deemed incompatible with suspension cells, some application examples exist for various label-free assays based on optical or impedance detection.…”

“…Such cellular parameters are also affected upon activation of GPCR signaling, thereby resulting in impedance changes and real-time monitoring of cellular signaling events . Typically, GPCR-mediated activation would result in an increase in cell adhesion and overall increase in CI, while a lower CI would indicate loss of adhesion (Scandroglio et al, 2010).…”

Constitutive activity of the metabotropic glutamate receptor 2 explored with a whole-cell label-free biosensor

“…In the field of class C GPCRs, this technique was never characterized extensively, yet it was used as a tool in two studies: Scandroglio et al [39] presented a small proof of concept study on the mGlu 1 receptor and recently we reported on the mGlu 2 receptor using the xCELLigence as a tool to assess the effect of residence time on functional efficacy of mGlu 2 PAMs [40]. Here, CHO-K1_hmGlu 2 cells stably expressing the hmGlu 2 receptor were used.…”

Molecular Insights into Metabotropic Glutamate Receptor Allosteric Modulation

“…Comprehensive investigation into stimulus-bias that factors in these contributing mechanisms and confounds is sorely needed. Annoura et al, 1996;Litschig et al, 1999;Scandroglio et al, 2010;Jiang et al, 2014 . mGlu 4 PAM: L-AP4 induced changes in VFT.…”