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

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. 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. Current methods for compound screening and pharmacological profiling rely on the use of labels of various nature: radioactive isotopes, fluorescent tags, dyes, and so on. In the past years, there has been an increasing interest in developing new technologies, the so-called label-free technologies, which do not require the use of specific markers. The main advantage of label-free technologies is to avoid possible interference of the marker with the physiological activity/status of the cell. Label-free devices are based on biosensors that, thanks to a transducer, are capable of transforming molecular interactions into electrical signals. The most widespread label-free technologies presently available are based on optical biosensing. However, more recently, instruments equipped with biosensors capable of exploiting acoustic resonance, differential calorimetry, and microcantilever or electrical impedance have been developed.1Cooper MA Non-optical screening platforms: the next wave in label-free screening?.Drug Discov Today. 2006; 1: 1068-1074Crossref Scopus (68) Google Scholar The impedance-based biosensors are of particular interest for their potential widespread application. At present, 4 different platforms based on cell impedance are on the market: ECIS (from Applied Biophysics, Troy, NY), Bionas 2500 (from Bionas, Rostack, Germany), Cell Key (from MDS Sciex, Concord, Ontario, Canada), and RT-CES from ACEA (now commercialized by Roche as xCELLigence, Roche Applied Science, Penzburg, Germany). These platforms were successfully applied to study cytotoxicity, cell adhesion, cell proliferation, cell migration, and receptor-mediated signaling.2Xi B Yu N Wang X Xu X Abassi YA The application of cell-based label-free technology in drug discovery.Biotechnol J. 2008; 3: 484-495Crossref PubMed Scopus (164) Google Scholar,3Peters MF Scott CW Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity.J Biomol Screen. 2009; 14: 246-255Abstract Full Text PDF PubMed Scopus (106) Google Scholar Considering the importance, from a drug discovery perspective, of G-protein-coupled receptors (GPCRs), the use of cell impedance–based technologies to monitor GPCR activity is of particular interest. The study of various GPCRs (H1, D1, D2, D5, 5-HT1A, 5-HT2B, M1, CB1, and MC4) demonstrated the possibility of evaluating different second-messenger coupling as well as identifying agonists and inverse agonists.3Peters MF Scott CW Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity.J Biomol Screen. 2009; 14: 246-255Abstract Full Text PDF PubMed Scopus (106) Google Scholar,4Yu N Atienza JM Bernard J Blanc S Zhu J Wang X et al.Real-time monitoring of morphological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors.Anal Chem. 2006; 78: 35-43Crossref PubMed Scopus (217) Google Scholar We decided to extend the evaluation of this technology by studying other GPCRs with the RT-CES platform. For this purpose, 2 receptors were chosen: cannabinoid receptor 2 (CB2) and metabotropic glutamate receptor 1 (mGluR1). The pharmacology of these 2 receptors is well known,5Howlett AC Barth F Bonner TI Cabral G Casellas P Devane WA et al.International Union of Pharmacology: XXVII. Classification of cannabinoid receptors.Pharmacol Rev. 2002; 54: 161-202Crossref PubMed Scopus (2302) Google Scholar,6Ferraguti F Crepaldi L Nicoletti F Metabotropic glutamate 1 receptor: current concepts and perspectives.Pharmacol Rev. 2008; 60: 536-581Crossref PubMed Scopus (167) Google Scholar as well as their intracellular downstream signaling pathways. The CB2 is Gi-coupled, and its activation triggers a decrease in intracellular cAMP level, whereas mGluR1 is Gq-coupled, and its stimulation is linked to phosphoinositide (PI) hydrolysis and intracellular Ca2+ mobilization. Using recombinant systems expressing either CB2 or mGluR1, we were able to demonstrate that cell impedance measurement is suitable to identify agonists, antagonists, and inverse agonists at these receptors. The results obtained with this technology are comparable with that obtained using classical assays detecting cAMP or calcium transient. 1S-3R-ACPD ((±)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid); S-3,5-DHPG (3,5-dihydrophenylglycine); L-glutamate ((S)-1-Aminopropane-1,3-dicarboxylic acid); quisqualate ((L)-(+)-a-amino-3,5-dioxo-1,2,4-oxadiazolidine-2-propanoic acid); CP55940 ((-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol); JWH133 ((6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a- tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran); and AM630 (6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-[1H-indol-3-yl](4-methoxyphenyl)methanone). JNJ16259685 (3,4-dihydro-2H-pyrano[2,3-b]-quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone; YM298198 (6-Amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide hydrochloride); CPCCOEt (7- (Hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester); and BAY36-7620 [(3aS,6aS)-Hexahydro-5-methylene-6a-(2-naphthalenylmethy1)-1H-cyclopenta[c]furan-1-one]. All agonists and antagonists were purchased from Tocris Cookson (Bristol, UK), except for SCH1041222, which was synthesized by Schering Plough (Kenilworth, NJ). Parental Chinese hamster ovary (CHO) cell line (ATCC, Manassas, VA) was grown in F12 medium (Ham nutrient mixture) + 10% fetal bovine serum (FBS) + 100 µg mL–1 penicillin/streptomycin (P/S). The cell line expressing CB2 was maintained in the above medium containing G418 (600 µg mL–1) for clone selection, whereas the cell line expressing mGluR1 was maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented by 10% FBS, 100 µg mL–1 P/S, 100 µg mL–1 Glutamax, and 600 µg mL–1 G418. Cell lines were grown at 37°C with 5% CO2. For mGluR1 recombinant cell line, the complete medium was substituted 6 h after plating with a serum-free DMEM high glucose, 3 U mL–1 glutamic-pyruvic transaminase (GPT; Sigma, St. Louis, MO), and 5 mM sodium pyruvate (Invitrogen, Paisley, UK). Culture splitting was performed by detaching the cells with 0.5% trypsin/EDTA, and cells were plated at different concentration (see below) and maintained in the incubator at 37°C with 5% CO2. The method employed for the generation of hCB2 and hmGluR1 transfected cell lines and their characterization was reported previously (Mancini et al.7Mancini I Brusa R Quadrato G Foglia C Scandroglio P Silverman LS et al.Constitutive activity of cannabinoid receptor 2 plays an essential role in the protean agonism of (+)AM1241 and L768242.Br J Pharmacol. 2009; 158: 382-391Crossref PubMed Scopus (41) Google Scholar and submitted US patent, respectively). Experiments were performed using the E-Plates™ from ACEA (San Diego, CA). One day before the experiment, 50 µL of medium was added to each well and background recorded. Following background measurement, 50 µL of media containing the cell suspension was seeded on the E-Plate™, incubated at room temperature for 1 h and then placed on the device station, and hosted in an incubator at 37°C with 5% CO2. The cells were allowed to equilibrate for 24 h and impedance constantly monitored every 2 min for the first 2 h and every 30 min for the remaining time. To evaluate the effect of cell density on impedance responses and to identify the better signal-to-noise ratio, we tested cell densities ranging from 10,000 to 50,000 cells/well. In addition, experiments were performed to establish if DMSO, the solvent in which compounds were solubilized, could influence cell impedance. For this purpose, cell media were discharged and replaced with Hank’s balanced salt solution (HBSS) containing DMSO concentration ranging from 0.003% to 3%, and impedance was monitored every 70 s. To evaluate compounds activity, culture media was discarded, replaced with 180 or 160 µL of HBSS (for agonism and antagonism studies, respectively), and baseline was recorded for 20 min. Then, 20 µL of compounds, at the desired concentration, was gently added using a PlateMate™ 2 × 2 (Matrix, Thermo Fisher Scientific, Hudson, NH) and impedance recorded every 70 s until the end of the experiment. In experiments aimed at evaluating antagonism, antagonists (dissolved in 20 µL HBSS) were added first and cells incubated for 10 min, and then agonists (dissolved in 20 µL HBSS) were added. The final concentration of DMSO in the assay was kept at In the experiments performed with cells were with mL–1 for 24 h and then as The results were by the cell which is the of the cell before and after the of the cAMP were using the cAMP UK). cells were seeded in at a density of µL culture 24 h before the experiment. the day of the experiment, the medium was discarded, and 10 of mM in and µL of were added to each well for To functional activity by receptor 10 of mM in was by µL of either or and µL of 10 final were incubated for 30 min at 37°C and 5% and then of the cAMP solution µL and µL was added. were incubated at room temperature in the The was with a 3 at 1 To of the compounds were from a in were seeded into at a density of in DMEM high supplemented with 10% FBS, 100 µg mL–1 P/S, and 100 µg The complete medium was substituted 6 h after plating with a new one containing DMEM high glucose, 3 U mL–1 St. Louis, MO), and 5 mM sodium pyruvate Following 24 h the cells were with a fluorescent calcium CA). The was in the assay which of HBSS with 20 mM solution To avoid mM was added to the calcium were with the for 2 h at 37°C in a calcium were using a stimulation baseline measurement or antagonist and were from of using the 4 Diego, CA). monitoring of cell impedance of cells expressing either the hCB2 or hmGluR1 a profile for each In both with in a at 20 h after whereas in the was after media at h after is that 5 h after complete medium was replaced with free medium containing and sodium pyruvate to hmGluR1 a and in by a decrease and of not is that DMSO cell The of and by Scopus Google Scholar and its to cell in culture an effect on impedance changes in the were after DMSO The DMSO concentration a effect on this effect was at a concentration and at the not Considering the effect of DMSO at and the need to compounds, experiments have been performed the DMSO concentration at To if stimulation of could in the RT-CES cells were with the CB2 a decrease of that a and then to with different profiles on concentration the in the impedance changes by receptor is possible to at different after stimulation and to the To establish was the to the were from RT-CES from 1 to after receptor over the that in the 5 and were and consistent with The for ligands for the CB2 PubMed Scopus Google Scholar before or after this an increasing from the In the experiments obtained from the recorded after compound were to data and To evaluate if the changes on profiles CB2 activation and were not to an the was applied to nontransfected parental JWH133 to cells in cellular impedance variation confirming a To establish the of in the of cell impedance triggered by cells were with the the JWH133 effect on cell confirming that the effect is ligands have been and results obtained cell impedance and cAMP assay The 3 ligands were a a and a of concentration activity obtained with cAMP assay are as of cAMP The is as a to CP55940 In cell impedance is to the cellular impedance after CP55940 at the concentration in with for cAMP assay, to CP55940 stimulation and to CP55940 was more in cellular impedance in intracellular cAMP and in RT-CES and cAMP assay, JWH133 potency was in both and 30 In the cAMP assay, JWH133 and CP55940 as agonists with the other in the cell impedance assay, JWH133 was an of of that of AM630 as an inverse in both cell impedance and increasing cAMP levels. In the cell impedance assay, AM630 a effect of whereas the cAMP assay a effect of To confirm that cellular impedance triggered by JWH133 were we AM630 to the JWH133 Application of AM630 to 1 a to the of the concentration activity of JWH133 with a consistent of the from confirming a effect of concentration activity that CB2 is in the cell Application of the mGluR1 quisqualate to in an and in cellular impedance cell was recorded 1 h after compound addition, and then the Following an to that for CB2 not the to for and to was Using this quisqualate potency was and comparable to data obtained using an hydrolysis MA and of a metabotropic glutamate receptor 1 coupling on with a glutamate Pharmacol. Google Scholar Experiments in the parental cell line using agonists and in The only exception was a of cell impedance stimulation with the glutamate concentration This to glutamate to mGluR1 a intracellular F Crepaldi L Nicoletti F Metabotropic glutamate 1 receptor: current concepts and perspectives.Pharmacol Rev. 2008; 60: 536-581Crossref PubMed Scopus (167) Google Scholar activation of mGluR1 is measuring intracellular calcium To evaluate if a functional assay performed using cell impedance have data consistent with a more classical assay, we mGluR1 ligands in in the 2 assays In both glutamate was the most compound and mGluR1 response. with glutamate, quisqualate efficacy in the Ca2+ assay, whereas a reduced efficacy in the cell impedance–based assay. in both 1S-3R-ACPD and S-3,5-DHPG as In both the of potency of the 4 agonists was quisqualate glutamate S-3,5-DHPG 1S-3R-ACPD of Agonists on and Ca2+ in a new To evaluate antagonism, we cells for 10 min with the antagonists J&J16259685, and and then with quisqualate at a concentration to its All antagonists, except for a of quisqualate both in the cell impedance and Ca2+ assay BAY36-7620 was to impedance changes by quisqualate, whereas Ca2+ with data reported in the The of potency for the other compounds was the in both and there was obtained using the impedance or Ca2+ assay of on and Ca2+ RT-CES was as in a new was as of in assays have been to study of downstream signaling as of and or activation of Wang X Xu X B Application of cell electronic technology to cell-based PubMed Scopus Google Scholar has been employed for this All these require the use of dyes, a pharmacological or a of the cells expressing the receptor of interest. a of these could to generation of and results more the pharmacology of the could the of or these label-free technologies have been developed. These technologies are based on the use of B Yu N Wang X Xu X Abassi YA The application of cell-based label-free technology in drug discovery.Biotechnol J. 2008; 3: 484-495Crossref PubMed Scopus (164) Google Scholar and do not require In the present we tested one of these technologies, the RT-CES that is based on the measurement of changes in cell YA Zhu J J Wang X Xu X monitoring of cell activation on cell sensor PubMed Scopus Google Scholar This technology was previously successfully applied to the study of 3 receptors (H1, D1, and linked to different intracellular signaling and N Atienza JM Bernard J Blanc S Zhu J Wang X et al.Real-time monitoring of morphological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors.Anal Chem. 2006; 78: 35-43Crossref PubMed Scopus (217) Google Scholar was to the available on this technology by an of the pharmacological profile of other 2 CB2 and a first we the variation of cell impedance after cell and to pharmacological This was possible this technology cell impedance variation over time. In both cell lines we an of cell impedance to cell adhesion, and from cell medium an to a reduced or cellular which in the media for 24 a more of and was only h The possibility to establish at which cells a is an advantage by this This could to the of the experiment, a better assay Considering that DMSO is the solvent most to compounds for in we variation of cell impedance at various DMSO in classical assays a in cell impedance. this technology is to of the cell DMSO is not an In assay a to avoid with compound assay is to use DMSO concentration This concentration a of impedance an compound this high to DMSO the of compounds that require concentration of solvent to into the of recombinant cells with CB2 or mGluR1 ligands in impedance changes. The of these impedance changes was by the of the compounds were tested in parental cell lines that do not CB2 or to agonists profiles that were different for the 2 receptors. at different receptors could in changes in cell impedance has previously been N Atienza JM Bernard J Blanc S Zhu J Wang X et al.Real-time monitoring of morphological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors.Anal Chem. 2006; 78: 35-43Crossref PubMed Scopus (217) Google Scholar has been that stimulation of cAMP with at the to an in N Atienza JM Bernard J Blanc S Zhu J Wang X et al.Real-time monitoring of morphological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors.Anal Chem. 2006; 78: 35-43Crossref PubMed Scopus (217) Google Scholar Following this could that a in cAMP in a the other was reported that stimulation of receptors and an of MF Scott CW Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity.J Biomol Screen. 2009; 14: 246-255Abstract Full Text PDF PubMed Scopus (106) Google Scholar,4Yu N Atienza JM Bernard J Blanc S Zhu J Wang X et al.Real-time monitoring of morphological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors.Anal Chem. 2006; 78: 35-43Crossref PubMed Scopus (217) Google Scholar In with this in stimulation of CB2 that to an intracellular cAMP decrease in an of cell impedance. a the effect of the CB2 confirming that was and linked to a cAMP second-messenger in by receptors is linked to and activation activation of different intracellular coupling with different Experiments using different recombinant cell lines and expressing that receptor coupling to could by cell intracellular in cells was whereas in was MF Scott CW Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity.J Biomol Screen. 2009; 14: 246-255Abstract Full Text PDF PubMed Scopus (106) Google Scholar In addition, cell impedance assays could coupling to Using a with was possible to a in the of cannabinoid receptor 1 to MF Scott CW Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity.J Biomol Screen. 2009; 14: 246-255Abstract Full Text PDF PubMed Scopus (106) Google Scholar profiles obtained for the receptor in different cell and on second-messenger are before of using a cell impedance assay for evaluating a pharmacological profile is the possibility of measuring a changes over time. the of concentration activity was possible to that is only a In a classical end assay, this and assay is performed at a after the is of the compound potency could the possibility of monitoring compound potency in is an possibility by impedance-based To the of the technology results obtained using agonists, antagonists and inverse agonists at CB2 or mGluR1 were to that obtained with classical functional cAMP and Ca2+ The pharmacological profile obtained with the different assays was in and the of potency of agonists and antagonists was was possible to identify inverse activity of AM630 at These results confirm the of the to the activity of the different of ligands antagonists, and inverse The only exception was the mGluR1 antagonist In the assay, this compound an activity consistent with Ca2+ assay whereas in the impedance assay, was could to on a molecular pharmacology of this for the possibility that this compound is a protean in the of in of this a more is that the DMSO concentration in the cell impedance assay compound and its in pharmacology were for the CB2 CP55940 was more in the cell impedance assay with the Ca2+ assay, and the efficacy of JWH133 was reduced in the RT-CES assay with the cAMP assay. The for these are not a possible could from the of of receptor Full Text PDF PubMed Scopus Google Scholar that a acting on a receptor a signaling and or with efficacy and one triggered by other ligands at the receptor. ligands could have on the receptor different activity at different second-messenger pathways. this has been for of by acting at CB2 PubMed Scopus Google Scholar has been that CP55940 could not only the second-messenger linked to cAMP the of the cAMP second-messenger linked to and Ca2+ from the intracellular of by acting at CB2 PubMed Scopus Google Scholar activation of these different a effect on impedance in and To this experiments aimed at evaluating the activity of on the and Ca2+ and with the activity of CP55940 on these To the present study that is possible to use cell impedance as by the to study receptors linked to different pathways. This a advantage with systems not require the of an assay on different technologies to activity of receptors linked to different pathways. of second-messenger that could by impedance this a and is to the potential of impedance-based assays in this The use of impedance-based technology the advantage of monitoring of receptor response. This an to the the of pharmacological responses at the cellular and a more pharmacological characterization of In we have the of the 3 assays in is in the 3 assays with to of of and the other the impedance assay performed with the RT-CES is not available in the and the well is with the other 2 This is not that of impedance technology is at the and its use not is that decrease in with in technology and more widespread use of this assay. the impedance is comparable with that of a for the cAMP assay and an of with to the for the Ca2+ assay. In its of the RT-CES is an suitable for or compound profiling not for of Cell and Ca2+ of Cell of cAMP with of with of media to 6 2 to 24 is performed the day to 2 of media the and and and well well in media is performed the day the in a new technology could successfully applied to the pharmacological characterization of specific compounds could that could not with classical end

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.