Real time analysis of ß(2)-adrenoceptor-mediated signaling kinetics in human primary airway smooth muscle cells reveals both ligand and dose dependent differences.
Respir Res
; 12: 89, 2011 Jul 02.
Article
em En
| MEDLINE
| ID: mdl-21722392
ABSTRACT
BACKGROUND:
ß2-adrenoceptor agonists elicit bronchodilator responses by binding to ß2-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 ß2-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 ß2-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 ß2-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 ß2-adrenoceptor signaling in primary cells. The ability to analyse the kinetics of clinically used ß2-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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Brônquios
/
Broncodilatadores
/
Transdução de Sinais
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Receptores Adrenérgicos beta 2
/
Miócitos de Músculo Liso
/
Agonistas de Receptores Adrenérgicos beta 2
Limite:
Humans
Idioma:
En
Revista:
Respir Res
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Reino Unido