A cell-based assay to assess the persistence of action of agonists acting at recombinant human β2 adrenoceptors

IntroductionThe aim was to establish a robust, 96-well, cell-based assay to assess the potency and persistence of action of agonists acting at human recombinant β2 adrenoceptors expressed in CHO (Chinese Hamster Ovary) cells and to compare this with published duration of action data in guinea pig isolated trachea and human bronchus.MethodsCells were treated with either: (i) β-adrenoceptor agonist for 30 min, washed and cyclicAMP (cAMP) measured 30 min later—termed ‘washed’ cells or, (ii) treated with solvent for 30 min, washed, and then treated with β-adrenoceptor agonist for 30 min and cAMP measured—termed ‘unwashed’ cells. The ‘washed’ EC50 was divided by the ‘unwashed’ EC50 to determine a rightward shift concentration ratio, which was indicative of the persistence of action at the receptor.ResultsAt the β2 adrenoceptor salmeterol, carmoterol and indacaterol were resistant to washing with a concentration ratio of < 5, indicating a long persistence of action, whereas formoterol, isoprenaline and salbutamol were washed out with a ratio of 32, > 294 and > 800 respectively, suggesting a shorter persistence of action. At β1 and β3 adrenoceptors all compounds washed out. The persistent effects of salmeterol at β2 following washing could be reversed by the selective β2 antagonist ICI 118551, suggesting continued receptor activation.DiscussionThe data presented agree well with published data assessing duration of action of β2 agonists in human isolated bronchus and guinea pig isolated trachea. Key features are: (a) it is a 96-well format which can be used to assess many compounds in a single experiment, (b) both potency and persistence of agonist action are assessed in the same assay, (c) any effects of concentration on the persistence of action can be highlighted, and (d) it allows triage of compounds prior to tissue bath studies thus reducing the use of animal tissue.