Real time analysis of β2-adrenoceptor-mediated signaling kinetics in Human Primary Airway Smooth Muscle Cells reveals both ligand and dose dependent differencesReport as inadecuate




Real time analysis of β2-adrenoceptor-mediated signaling kinetics in Human Primary Airway Smooth Muscle Cells reveals both ligand and dose dependent differences - Download this document for free, or read online. Document in PDF available to download.

Respiratory Research

, 12:89

First Online: 01 December 2011Received: 22 March 2011Accepted: 02 July 2011

Abstract

Backgroundβ2-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.

MethodsCells 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.

ResultsTemporal 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.

ConclusionsWe 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.

AbbreviationshASMhuman airway smooth muscle

cAMPcyclic AMP

EpacExchange Protein directly Activated by Cyclic AMP

CFPCyan Fluorescent Protein

YFPYellow Fluorescent Protein

FRETFluorescence Resonance Energy Transfer.

Electronic supplementary materialThe online version of this article doi:10.1186-1465-9921-12-89 contains supplementary material, which is available to authorized users.

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Author: Charlotte K Billington - Ian P Hall

Source: https://link.springer.com/







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