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CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures
BACKGROUND: G protein-coupled receptors (GPCRs) represent a physiologically and pharmacologically important family of receptors that upon coupling to Gα(S) stimulate cAMP production catalyzed by adenylyl cyclase. Thus, developing assays to monitor cAMP production is crucial to screen for ligands in...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4228090/ https://www.ncbi.nlm.nih.gov/pubmed/25366423 http://dx.doi.org/10.1186/s12964-014-0070-x |
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author | Trehan, Ashutosh Rotgers, Emmi Coffey, Eleanor T Huhtaniemi, Ilpo Rivero-Müller, Adolfo |
author_facet | Trehan, Ashutosh Rotgers, Emmi Coffey, Eleanor T Huhtaniemi, Ilpo Rivero-Müller, Adolfo |
author_sort | Trehan, Ashutosh |
collection | PubMed |
description | BACKGROUND: G protein-coupled receptors (GPCRs) represent a physiologically and pharmacologically important family of receptors that upon coupling to Gα(S) stimulate cAMP production catalyzed by adenylyl cyclase. Thus, developing assays to monitor cAMP production is crucial to screen for ligands in studies of GPCR signaling. Primary cell cultures represent a more robust model than cell lines to study GPCR signaling since they physiologically resemble the parent tissue. Current cAMP assays have two fundamental limitations: 1) absence of cAMP kinetics as competition-based assays require cell lysis and measure only a single time-point, and 2) high variation with separate samples needed to measure consecutive time points. The utility of real-time cAMP biosensors is also limited in primary cell cultures due to their poor transfection efficiency, variable expression levels and inability to select stable clones. We therefore, decided to develop an assay that can measure cAMP not only at a single time-point but the entire cAMP kinetics after GPCR activation in untransfected primary cells. RESULTS: CANDLES (Cyclic AMP iNdirect Detection by Light Emission from Sensor cells) assay for monitoring cAMP kinetics in cell cultures, particularly in primary cultures was developed. The assay requires co-culturing of primary cells with sensor cells that stably express a luminescent cAMP sensor. Upon GPCR activation in primary cells, cAMP is transferred to sensor cells via gap junction channels, thereby evoking a luminescent read-out. GPCR activation using primary cultures of rat cortical neurons and mouse granulosa cells was measured. Kinetic responses of different agonists to adrenergic receptors were also compared using rat cortical neurons. The assay optimization was done by varying sensor-test cell ratio, using phosphodiesterase inhibitors and testing cell-cell contact requirement. CONCLUSIONS: Here we present CANDLES assay based on co-culturing test cells with cAMP-detecting sensor cells. This co-culture setup allows kinetic measurements, eliminates primary cell transfections and reduces variability. A variety of cell types (rat cortical neurons, mouse granulosa cells and established cell lines) and receptors (adrenergic, follicle stimulating hormone and luteinizing hormone/chorionic gonadotropin receptors) were tested for use with CANDLES. The assay is best applied while comparing cAMP generation curves upon different drug treatments to untransfected primary cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12964-014-0070-x) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4228090 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-42280902014-11-12 CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures Trehan, Ashutosh Rotgers, Emmi Coffey, Eleanor T Huhtaniemi, Ilpo Rivero-Müller, Adolfo Cell Commun Signal Methodology BACKGROUND: G protein-coupled receptors (GPCRs) represent a physiologically and pharmacologically important family of receptors that upon coupling to Gα(S) stimulate cAMP production catalyzed by adenylyl cyclase. Thus, developing assays to monitor cAMP production is crucial to screen for ligands in studies of GPCR signaling. Primary cell cultures represent a more robust model than cell lines to study GPCR signaling since they physiologically resemble the parent tissue. Current cAMP assays have two fundamental limitations: 1) absence of cAMP kinetics as competition-based assays require cell lysis and measure only a single time-point, and 2) high variation with separate samples needed to measure consecutive time points. The utility of real-time cAMP biosensors is also limited in primary cell cultures due to their poor transfection efficiency, variable expression levels and inability to select stable clones. We therefore, decided to develop an assay that can measure cAMP not only at a single time-point but the entire cAMP kinetics after GPCR activation in untransfected primary cells. RESULTS: CANDLES (Cyclic AMP iNdirect Detection by Light Emission from Sensor cells) assay for monitoring cAMP kinetics in cell cultures, particularly in primary cultures was developed. The assay requires co-culturing of primary cells with sensor cells that stably express a luminescent cAMP sensor. Upon GPCR activation in primary cells, cAMP is transferred to sensor cells via gap junction channels, thereby evoking a luminescent read-out. GPCR activation using primary cultures of rat cortical neurons and mouse granulosa cells was measured. Kinetic responses of different agonists to adrenergic receptors were also compared using rat cortical neurons. The assay optimization was done by varying sensor-test cell ratio, using phosphodiesterase inhibitors and testing cell-cell contact requirement. CONCLUSIONS: Here we present CANDLES assay based on co-culturing test cells with cAMP-detecting sensor cells. This co-culture setup allows kinetic measurements, eliminates primary cell transfections and reduces variability. A variety of cell types (rat cortical neurons, mouse granulosa cells and established cell lines) and receptors (adrenergic, follicle stimulating hormone and luteinizing hormone/chorionic gonadotropin receptors) were tested for use with CANDLES. The assay is best applied while comparing cAMP generation curves upon different drug treatments to untransfected primary cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12964-014-0070-x) contains supplementary material, which is available to authorized users. BioMed Central 2014-11-04 /pmc/articles/PMC4228090/ /pubmed/25366423 http://dx.doi.org/10.1186/s12964-014-0070-x Text en © Trehan et al.; licensee BioMed Central Ltd. 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Trehan, Ashutosh Rotgers, Emmi Coffey, Eleanor T Huhtaniemi, Ilpo Rivero-Müller, Adolfo CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures |
title | CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures |
title_full | CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures |
title_fullStr | CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures |
title_full_unstemmed | CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures |
title_short | CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures |
title_sort | candles, an assay for monitoring gpcr induced camp generation in cell cultures |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4228090/ https://www.ncbi.nlm.nih.gov/pubmed/25366423 http://dx.doi.org/10.1186/s12964-014-0070-x |
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