@article { , title = {Kinetic analysis of endogenous β2-adrenoceptor-mediated cAMP GloSensorTM responses in HEK293 cells}, 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.}, doi = {10.1111/bph.16008}, eissn = {1476-5381}, issn = {0007-1188}, issue = {10}, journal = {British Journal of Pharmacology}, note = {Gold OA. Replace AAM with final version. Need to add volume, issue, pagination.}, pages = {1304-1315}, publicationstatus = {Published}, publisher = {Wiley}, url = {https://nottingham-repository.worktribe.com/output/14895187}, volume = {180}, keyword = {Pharmacology}, year = {2023}, author = {Cullum, Sean A. and Veprintsev, Dmitry B. and Hill, Stephen J.} }