Sean A. Cullum
Kinetic analysis of endogenous β2-adrenoceptor-mediated cAMP GloSensorTM responses in HEK293 cells
Cullum, Sean A.; Veprintsev, Dmitry B.; Hill, Stephen J.
Authors
DMITRY VEPRINTSEV DMITRY.VEPRINTSEV@NOTTINGHAM.AC.UK
Professor of Molecular and Cellular Pharmacology
STEPHEN HILL STEVE.HILL@NOTTINGHAM.AC.UK
Professor of Molecular Pharmacology
Abstract
Background and Aim: Standard pharmacological analysis of agonist activity utilises measurements of receptor-mediated responses at a set time-point, or at the peak response level, to characterise ligands. However, the occurrence of non-equilibrium conditions may dramatically impact the properties of the response being measured. Here we have analysed the initial kinetic phases of cAMP responses to β2-adrenoceptor agonists in HEK293 cells expressing the endogenous β2-adrenoceptor at extremely low levels. Experimental Approach: The kinetics of β2-adrenoceptor agonist-stimulated cAMP responses were monitored in real-time, in the presence and absence of antagonists, in HEK293 cells expressing the cAMP GloSensor™ biosensor. Potency (EC50) and efficacy (Emax) values were determined at the peak of the agonist GloSensor™ response and compared to kinetic parameters L50 and IRmax values derived from initial response rates. Key Results: The partial agonists salbutamol and salmeterol displayed reduced relative IRmax values (with respect to isoprenaline) when compared with their Emax values. Except for the fast dissociating bisoprolol, preincubation with β2-adrenoceptor antagonists produced a large reduction in the isoprenaline peak response due to a state of hemi-equilibrium in this low receptor reserve system. This effect was exacerbated when IRmax parameters were measured. Furthermore, bisoprolol produced a large reduction in isoprenaline IRmax consistent with its short residence time. Conclusions and Implications: Kinetic analysis of real-time signalling data can provide valuable insights into the hemi-equilibria that can occur in low receptor reserve systems with agonist–antagonist interactions, due to incomplete dissociation of antagonist whilst the peak agonist response is developing.
Citation
Cullum, S. A., Veprintsev, D. B., & Hill, S. J. (2023). Kinetic analysis of endogenous β2-adrenoceptor-mediated cAMP GloSensorTM responses in HEK293 cells. British Journal of Pharmacology, 180(10), 1304-1315. https://doi.org/10.1111/bph.16008
Journal Article Type | Article |
---|---|
Acceptance Date | Dec 8, 2022 |
Online Publication Date | Dec 10, 2022 |
Publication Date | 2023-05 |
Deposit Date | Dec 18, 2022 |
Publicly Available Date | Dec 11, 2023 |
Journal | British Journal of Pharmacology |
Print ISSN | 0007-1188 |
Electronic ISSN | 1476-5381 |
Publisher | Wiley |
Peer Reviewed | Peer Reviewed |
Volume | 180 |
Issue | 10 |
Pages | 1304-1315 |
DOI | https://doi.org/10.1111/bph.16008 |
Keywords | Pharmacology |
Public URL | https://nottingham-repository.worktribe.com/output/14895187 |
Publisher URL | https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.16008 |
Files
CAMP Signalling Kinetics Paper BJP
(2.6 Mb)
PDF
Publisher Licence URL
https://creativecommons.org/licenses/by/4.0/
You might also like
Universal allosteric mechanism for G? activation by GPCRs
(2015)
Journal Article
Probing G?i1 protein activation at single-amino acid resolution
(2015)
Journal Article
Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor
(2016)
Journal Article
Diverse activation pathways in class A GPCRs converge near the G-protein-coupling region
(2016)
Journal Article
Downloadable Citations
About Repository@Nottingham
Administrator e-mail: discovery-access-systems@nottingham.ac.uk
This application uses the following open-source libraries:
SheetJS Community Edition
Apache License Version 2.0 (http://www.apache.org/licenses/)
PDF.js
Apache License Version 2.0 (http://www.apache.org/licenses/)
Font Awesome
SIL OFL 1.1 (http://scripts.sil.org/OFL)
MIT License (http://opensource.org/licenses/mit-license.html)
CC BY 3.0 ( http://creativecommons.org/licenses/by/3.0/)
Powered by Worktribe © 2024
Advanced Search