{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T02:00:59Z","timestamp":1788487259861,"version":"build-2803163510"},"reference-count":57,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2023,1,6]],"date-time":"2023-01-06T00:00:00Z","timestamp":1672963200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000265","name":"Medical Research Council","doi-asserted-by":"publisher","award":["MR\/N013913\/1"],"award-info":[{"award-number":["MR\/N013913\/1"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000265","name":"Medical Research Council","doi-asserted-by":"publisher","award":["MR\/N020081\/1"],"award-info":[{"award-number":["MR\/N020081\/1"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["bpspubs.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["British J Pharmacology"],"published-print":{"date-parts":[[2023,5]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background and Aim<\/jats:title><jats:p>Standard pharmacological analysis of agonist activity utilises measurements of receptor\u2010mediated responses at a set time\u2010point, or at the peak response level, to characterise ligands. However, the occurrence of non\u2010equilibrium conditions may dramatically impact the properties of the response being measured. Here we have analysed the initial kinetic phases of cAMP responses to \u03b2<jats:sub>2<\/jats:sub>\u2010adrenoceptor agonists in HEK293 cells expressing the endogenous \u03b2<jats:sub>2<\/jats:sub>\u2010adrenoceptor at extremely low levels.<\/jats:p><\/jats:sec><jats:sec><jats:title>Experimental Approach<\/jats:title><jats:p>The kinetics of \u03b2<jats:sub>2<\/jats:sub>\u2010adrenoceptor agonist\u2010stimulated cAMP responses were monitored in real\u2010time, in the presence and absence of antagonists, in HEK293 cells expressing the cAMP GloSensor\u2122 biosensor. Potency (EC<jats:sub>50<\/jats:sub>) and efficacy (E<jats:sub>max<\/jats:sub>) values were determined at the peak of the agonist GloSensor\u2122 response and compared to kinetic parameters L<jats:sub>50<\/jats:sub> and IR<jats:sub>max<\/jats:sub> values derived from initial response rates.<\/jats:p><\/jats:sec><jats:sec><jats:title>Key Results<\/jats:title><jats:p>The partial agonists salbutamol and salmeterol displayed reduced relative IR<jats:sub>max<\/jats:sub> values (with respect to isoprenaline) when compared with their E<jats:sub>max<\/jats:sub> values. Except for the fast dissociating bisoprolol, preincubation with \u03b2<jats:sub>2<\/jats:sub>\u2010adrenoceptor antagonists produced a large reduction in the isoprenaline peak response due to a state of hemi\u2010equilibrium in this low receptor reserve system. This effect was exacerbated when IR<jats:sub>max<\/jats:sub> parameters were measured. Furthermore, bisoprolol produced a large reduction in isoprenaline IR<jats:sub>max<\/jats:sub> consistent with its short residence time.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions and Implications<\/jats:title><jats:p>Kinetic analysis of real\u2010time signalling data can provide valuable insights into the hemi\u2010equilibria that can occur in low receptor reserve systems with agonist\u2013antagonist interactions, due to incomplete dissociation of antagonist whilst the peak agonist response is developing.<\/jats:p><\/jats:sec>","DOI":"10.1111\/bph.16008","type":"journal-article","created":{"date-parts":[[2022,12,10]],"date-time":"2022-12-10T13:55:50Z","timestamp":1670680550000},"page":"1304-1315","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Kinetic analysis of endogenous \u03b2<sub>2<\/sub>\u2010adrenoceptor\u2010mediated cAMP GloSensor\u2122 responses in HEK293 cells"],"prefix":"10.1111","volume":"180","author":[{"given":"Sean A.","family":"Cullum","sequence":"first","affiliation":[{"name":"Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences University of Nottingham  Nottingham UK"},{"name":"Centre of Membrane Proteins and Receptors University of Birmingham and Nottingham  Nottingham UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dmitry B.","family":"Veprintsev","sequence":"additional","affiliation":[{"name":"Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences University of Nottingham  Nottingham UK"},{"name":"Centre of Membrane Proteins and Receptors University of Birmingham and Nottingham  Nottingham UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4424-239X","authenticated-orcid":false,"given":"Stephen J.","family":"Hill","sequence":"additional","affiliation":[{"name":"Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences University of Nottingham  Nottingham UK"},{"name":"Centre of Membrane Proteins and Receptors University of Birmingham and Nottingham  Nottingham UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,1,6]]},"reference":[{"key":"e_1_2_12_2_1","doi-asserted-by":"publisher","DOI":"10.1111\/bph.15538"},{"key":"e_1_2_12_3_1","doi-asserted-by":"publisher","DOI":"10.1111\/bph.15542"},{"key":"e_1_2_12_4_1","doi-asserted-by":"publisher","DOI":"10.1124\/mol.65.4.986"},{"key":"e_1_2_12_5_1","doi-asserted-by":"publisher","DOI":"10.1124\/mol.114.095364"},{"key":"e_1_2_12_6_1","doi-asserted-by":"publisher","DOI":"10.1124\/mol.118.112573"},{"key":"e_1_2_12_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.pupt.2012.05.007"},{"key":"e_1_2_12_8_1","doi-asserted-by":"publisher","DOI":"10.1021\/cb200248h"},{"key":"e_1_2_12_9_1","doi-asserted-by":"publisher","DOI":"10.1098\/rspb.1983.0093"},{"key":"e_1_2_12_10_1","doi-asserted-by":"publisher","DOI":"10.1096\/fj.11-201640"},{"issue":"1","key":"e_1_2_12_11_1","first-page":"182","article-title":"Stable activation and desensitization of beta 2\u2010adrenergic receptor stimulation of adenylyl cyclase by salmeterol: Evidence for quasi\u2010irreversible binding to an exosite","volume":"49","author":"Clark R. B.","year":"1996","journal-title":"Molecular Pharmacology"},{"key":"e_1_2_12_12_1","doi-asserted-by":"publisher","DOI":"10.1111\/bph.15868"},{"key":"e_1_2_12_13_1","doi-asserted-by":"publisher","DOI":"10.1021\/cb8000414"},{"key":"e_1_2_12_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1050-1738(98)00022-X"},{"key":"e_1_2_12_15_1","doi-asserted-by":"publisher","DOI":"10.1111\/bph.14988"},{"key":"e_1_2_12_16_1","doi-asserted-by":"publisher","DOI":"10.1124\/mol.63.6.1256"},{"key":"e_1_2_12_17_1","doi-asserted-by":"publisher","DOI":"10.1124\/mol.62.5.1094"},{"key":"e_1_2_12_18_1","doi-asserted-by":"publisher","DOI":"10.1124\/mol.114.096800"},{"key":"e_1_2_12_19_1","doi-asserted-by":"publisher","DOI":"10.1096\/fj.202002268R"},{"key":"e_1_2_12_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bcp.2017.10.013"},{"key":"e_1_2_12_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/prp2.779"},{"key":"e_1_2_12_22_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.8b00333"},{"key":"e_1_2_12_23_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jtbi.2018.02.014"},{"key":"e_1_2_12_24_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-67844-3"},{"key":"e_1_2_12_25_1","doi-asserted-by":"publisher","DOI":"10.3389\/fncel.2021.814547"},{"key":"e_1_2_12_26_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.bjp.0703535"},{"key":"e_1_2_12_27_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.bjp.0701658"},{"key":"e_1_2_12_28_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.272.38.23871"},{"key":"e_1_2_12_29_1","doi-asserted-by":"publisher","DOI":"10.1016\/0024-3205(93)90728-L"},{"key":"e_1_2_12_30_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsptsci.8b00057"},{"key":"e_1_2_12_31_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms10842"},{"key":"e_1_2_12_32_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrd2518"},{"key":"e_1_2_12_33_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tips.2007.12.002"},{"key":"e_1_2_12_34_1","doi-asserted-by":"publisher","DOI":"10.1124\/pr.110.004309"},{"key":"e_1_2_12_35_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-018-0145-x"},{"key":"e_1_2_12_36_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.ne.04.030181.002223"},{"key":"e_1_2_12_37_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.physiol.69.022405.154712"},{"key":"e_1_2_12_38_1","doi-asserted-by":"publisher","DOI":"10.1165\/rcmb.2006-0158OC"},{"key":"e_1_2_12_39_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1071550"},{"key":"e_1_2_12_40_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1476-5381.1993.tb12833.x"},{"key":"e_1_2_12_41_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ejphar.2012.11.001"},{"key":"e_1_2_12_42_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bmcl.2015.12.024"},{"key":"e_1_2_12_43_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature10361"},{"issue":"4","key":"e_1_2_12_44_1","first-page":"596","article-title":"Equilibrium and kinetic studies of the interactions of salmeterol with membrane bilayers","volume":"42","author":"Rhodes D. G.","year":"1992","journal-title":"Molecular Pharmacology"},{"key":"e_1_2_12_45_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00210-010-0533-6"},{"key":"e_1_2_12_46_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrd.2016.230"},{"key":"e_1_2_12_47_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(19)68694-1"},{"key":"e_1_2_12_48_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tips.2011.05.002"},{"key":"e_1_2_12_49_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature13430"},{"key":"e_1_2_12_50_1","doi-asserted-by":"publisher","DOI":"10.1124\/mol.117.111062"},{"key":"e_1_2_12_51_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1476-5381.1956.tb00006.x"},{"key":"e_1_2_12_52_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1476-5381.2011.01639.x"},{"key":"e_1_2_12_53_1","doi-asserted-by":"publisher","DOI":"10.1124\/mol.113.090209"},{"key":"e_1_2_12_54_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1476-5381.2009.00296.x"},{"key":"e_1_2_12_55_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1440-1681.2005.04222.x"},{"key":"e_1_2_12_56_1","doi-asserted-by":"publisher","DOI":"10.1124\/mol.65.1.196"},{"key":"e_1_2_12_57_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2021.04.006"},{"key":"e_1_2_12_58_1","doi-asserted-by":"publisher","DOI":"10.1111\/bph.14684"}],"container-title":["British Journal of Pharmacology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/bph.16008","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1111\/bph.16008","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/bpspubs.onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/bph.16008","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,8,22]],"date-time":"2023-08-22T03:59:06Z","timestamp":1692676746000},"score":1,"resource":{"primary":{"URL":"https:\/\/bpspubs.onlinelibrary.wiley.com\/doi\/10.1111\/bph.16008"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,1,6]]},"references-count":57,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2023,5]]}},"alternative-id":["10.1111\/bph.16008"],"URL":"https:\/\/doi.org\/10.1111\/bph.16008","archive":["Portico"],"relation":{},"ISSN":["0007-1188","1476-5381"],"issn-type":[{"value":"0007-1188","type":"print"},{"value":"1476-5381","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,1,6]]},"assertion":[{"value":"2022-08-01","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2022-12-02","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2023-01-06","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}