title: A cell-based assay to assess the persistence of action of agonists acting at recombinant human beta(2) adrenoceptors.
abstract: BACKGROUND
  The 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 beta(2) 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.
  
  
  METHODS
  Cells were treated with either: (i) beta-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 beta-adrenoceptor agonist for 30 min and cAMP measured-termed 'unwashed' cells. The 'washed' EC(50) was divided by the 'unwashed' EC(50) to determine a rightward shift concentration ratio, which was indicative of the persistence of action at the receptor.
  
  
  RESULTS
  At the beta(2) 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 beta(1) and beta(3) adrenoceptors all compounds washed out. The persistent effects of salmeterol at beta(2) following washing could be reversed by the selective beta(2) antagonist ICI 118551, suggesting continued receptor activation.
  
  
  CONCLUSIONS
  The data presented agree well with published data assessing duration of action of beta(2) 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.
authors.affiliation: Allergy and Respiratory Biology, Pfizer Global Research and Development, Sandwich, KentCT13 9NJ, UK. susan.summerhill@pfizer.com
authors.forename: Susan
authors.initials: S
authors.lastname: Summerhill
authors.forename: Timothy
authors.initials: T
authors.lastname: Stroud
authors.forename: Roshini
authors.initials: R
authors.lastname: Nagendra
authors.forename: Christelle
authors.initials: C
authors.lastname: Perros-Huguet
authors.forename: Michael
authors.initials: M
authors.lastname: Trevethick
mesh_terms: D058665:Adrenergic beta-1 Receptor Agonists; D058666:Adrenergic beta-2 Receptor Agonists; D000420:Albuterol / Q000031:analogs & derivatives / Q000494:pharmacology; D000662:Amphetamines / Q000494:pharmacology; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001980:Bronchi / Q000187:drug effects / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000032:analysis / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004983:Ethanolamines / Q000494:pharmacology; D000068759:Formoterol Fumarate; D006168:Guinea Pigs; D006801:Humans; D006912:Hydroxyquinolines / Q000494:pharmacology; D066298:In Vitro Techniques; D007189:Indans / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D011412:Propanolamines / Q000494:pharmacology; D015363:Quinolones / Q000494:pharmacology; D018342:Receptors, Adrenergic, beta-1 / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000819:agonists* / Q000378:metabolism; D000068299:Salmeterol Xinafoate; D013997:Time Factors; D014132:Trachea / Q000187:drug effects / Q000378:metabolism
chemical_list: D058665:Adrenergic beta-1 Receptor Agonists; D058666:Adrenergic beta-2 Receptor Agonists; D000662:Amphetamines; D004983:Ethanolamines; D006912:Hydroxyquinolines; D007189:Indans; D011412:Propanolamines; D015363:Quinolones; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; C026777:ICI 118551; D000068299:Salmeterol Xinafoate; C510790:indacaterol; C074413:carmoterol; D000242:Cyclic AMP; D007545:Isoproterenol; D000420:Albuterol; D000068759:Formoterol Fumarate