β2-adrenoceptor inverse agonists signalling in human airway cells

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Bawa-Sani, HG, 2019. β2-adrenoceptor inverse agonists signalling in human airway cells. PhD, Nottingham Trent University.

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Abstract

Bronchial asthma is characterised by airway inflammation, obstruction and reversible airway-hyper responsiveness (Lötvall et al., 2011). One common treatment is the use of bronchodilator (particularly β agonists).Several studies have reported that the bronchodilators were less effective in chronic use, with worsening symptoms of poor asthma control (Bond et al., 2007). In the quest for new modalities of treatment, antagonists (or inverse agonists) were found to be beneficial to patients on chronic administration. Previously, the agonists were shown to activate the receptor stimulating cyclic AMP production while inverse agonists do not, but act as "biased" agonists in promoting β-arrestin-dependent signalling through the β–adrenoceptor (Nguyen 2008, van der Westhuizen 2014). Because these cell signalling pathways are associated with modulation of intracellular β2-adrenoceptor activity, it is plausible to hypothesize that the β2-adrenoceptor agonists and inverse agonists directly affect β2-adrenoceptor activity. In view of the this, the thesis main aims were to investigate whether the β2-adrenoceptor population are sensitive to inhibition by β2-adrenoceptor inverse agonists and whether they are biased agonist which stimulate G-protein independent signalling pathways and stimulate the functional effects of the β2-adrenoceptors on airway cell biology.

In the first part of the investigation, the production of cyclic AMP in Calu-3 and BEAS2B-R1 cells and real-time analysis of cellular impedance was investigated in BEAS2B-R1, Calu-3 and CHO-β2 cells. Phosphorylation of ERK1/2 in Calu-3 and CHO-β2 cells was assessed using western blotting techniques. The second part of investigation assessed the functional effect β2-adrenoceptor stimulation using MTT reduction, MTT real time glo, Neutral red, incuCyte real time assays and cell counting. The third part assessed the effect of β2-adrenoceptor stimulation in wound healing using mechanical, chemical and automated methods of wounding. The fourth part assessed the effect of TEER in Calu-3 cells and was investigated under air liquid interface experiments (ALI).

The β2-adrenergic stimulation with isoprenaline and formoterol increased the cyclic AMP in Calu-3 cells and cellular impedance in BEAS2BR-1cells. Cellular impedance assay using the xCELLigence system decreased in BEAS2BR-1 and increased in Calu-3 cells and CHO-β2 cells with formoterol and propranolol. The MEK 1 inhibitor, PD98059 did not decrease cellular impedance with both formoterol and propranolol. Formoterol and carvedilol increased ERK 1/2 phosphorylation in CHO-β2 cells. The assessment of the functional effects of β2-adrenoceptor stimulation showed increased cell viability in response to propranolol and nadolol in Calu-3 cells using MTT reduction assay. Responses to propranolol in neutral red assay increased cell viability in BEAS2BR-1 cells. Wound healing and repair assessed with incuCyte S3 system was inhibited by carvedilol and salmeterol and forskolin. The presence of tight junctions in Calu-3 cells was confirmed by sequential changes TEER measurements. The development of tight junctions was found to increase TEER in response to formoterol and propranolol. Overall, the findings highlight the β2-adrenoceptor activity in the cellular function of β2-adrenoceptor inverse agonist and shows the molecular mechanism involved via cyclic AMP and ERK1/2 dependent pathways.

Item Type:	Thesis
Description:	Abridged version
Creators:	Bawa-Sani, H.G.
Date:	December 2019
Rights:	This research is the intellectual property of the author. The use of my work written is this document is permitted for private study or personal use which must not be sold for any purpose. However only 5% may be copied. For other use, of any part of this research must be properly referenced to include, the author, title, degree and page number. Any query should be directed to owners in the intellectual property rights.
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	07 Apr 2021 13:26
Last Modified:	16 Mar 2022 03:00
URI:	https://irep.ntu.ac.uk/id/eprint/42655