title: Real time analysis of β(2)-adrenoceptor-mediated signaling kinetics in human primary airway smooth muscle cells reveals both ligand and dose dependent differences.
abstract: BACKGROUND
  β2-adrenoceptor agonists elicit bronchodilator responses by binding to β2-adrenoceptors on airway smooth muscle (ASM). In vivo, the time between drug administration and clinically relevant bronchodilation varies significantly depending on the agonist used. Our aim was to utilise a fluorescent cyclic AMP reporter probe to study the temporal profile of β2-adrenoceptor-mediated signaling induced by isoproterenol and a range of clinically relevant agonists in human primary ASM (hASM) cells by using a modified Epac protein fused to CFP and a variant of YFP.
  
  
  METHODS
  Cells were imaged in real time using a spinning disk confocal system which allowed rapid and direct quantification of emission ratio imaging following direct addition of β2-adrenoceptor agonists (isoproterenol, salbutamol, salmeterol, indacaterol and formoterol) into the extracellular buffer. For pharmacological comparison a radiolabeling assay for whole cell cyclic AMP formation was used.
  
  
  RESULTS
  Temporal analysis revealed that in hASM cells the β2-adrenoceptor agonists studied did not vary significantly in the onset of initiation. However, once a response was initiated, significant differences were observed in the rate of this response with indacaterol and isoproterenol inducing a significantly faster response than salmeterol. Contrary to expectation, reducing the concentration of isoproterenol resulted in a significantly faster initiation of response.
  
  
  CONCLUSIONS
  We conclude that confocal imaging of the Epac-based probe is a powerful tool to explore β2-adrenoceptor signaling in primary cells. The ability to analyse the kinetics of clinically used β2-adrenoceptor agonists in real time and at a single cell level gives an insight into their possible kinetics once they have reached ASM cells in vivo.
authors: Division of Therapeutics and Molecular Medicine, Nottingham Respiratory Biomedical Research Unit, Floor D, South Block, University Hospital of Nottingham, The University of Nottingham, Nottingham, NG7 2UH, UK. charlotte.billington@nottingham.ac.uk
authors: Charlotte K
authors: CK
authors: Billington
authors: Ian P
authors: IP
authors: Hall
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology*; D015374:Biosensing Techniques; D001980:Bronchi / Q000187:drug effects* / Q000378:metabolism; D001993:Bronchodilator Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D006801:Humans; D007700:Kinetics; D008024:Ligands; D018613:Microscopy, Confocal; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000187:drug effects* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection
chemical_list: C553443:ADRB2 protein, human; D058666:Adrenergic beta-2 Receptor Agonists; D001993:Bronchodilator Agents; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP