Regulation of airway smooth muscle contraction by PGE2

Morgan, Leanna M. (2022) Regulation of airway smooth muscle contraction by PGE2. Doctoral thesis, Dundalk Institute of Technology.

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Abstract

Protease-activated receptor 2 (PAR2)is the most abundant PAR in the human lung and is expressed in bronchial epithelium and smooth muscle. There are conflicting reports on the precise role of PAR2 in the airways and studies have shown it can induce both bronchoconstriction and bronchodilation effects. While PAR2 is present on airway smooth muscle cells, it has been suggested that the inhibitory effects of PAR2 in the airways could be mediated via endogenous prostanoids.Prostaglandin E2(PGE2)is the most abundantly produced prostanoid in the body andexerts its biological effects through activation of four prostanoid E receptors(EPRs). Activation of EP1and EP3Rspromotes airway inflammation and cough, whereas EP2and EP4Rsinducepotent bronchodilation (Aso et al. 2013; Sastre and del Pozo 2012; Tilley et al.2003), however, the precise cellular mechanisms underlying the effects of PGE2in airway smooth musclehave not been fully elucidated. The aims of this study were to: 1) investigate the mechanisms underlying PAR2 mediated relaxations in murine airway smooth muscle; 2) identifythe EP receptor subtype responsible for the inhibitoryeffects of PGE2in murine airway smooth muscle;3) examine the underlying cellular processesand possible roleof K+channels inPGE2-induced relaxationsof airway smooth muscleand4) investigate if PGE2affected calcium signalling in isolated airway smooth muscle cells. Isometric tension recordings were obtainedfrom murine bronchial rings and intracellular calcium measurements were recordedfrom isolated airway smooth muscle cells using confocal microscopy. Transcriptional expression ofEPRsubtypes in whole bronchial tissueand isolatedairway smooth muscle cellswas investigatedusing RT-PCR and RT-quantification was determinedusing qPCR. The key findings of this study were: 1)PAR2 activation elicited potent relaxation of bronchial rings pre-contracted with EFS and the cholinergic agonist CCh (1PM) via releaseof endogenous PGE2; 2) Exogenous application of PGE2relaxedbronchial rings in a concentration-dependent manner;3) The inhibitoryeffects ofPGE2were reduced in the presence of an EP2R antagonist but not an EP4Rantagonist;4) PGE2-induced inhibitionof EFS-evoked contractions were also reduced by an EP2R antagonist, but not an EP4R antagonist, however 7inhibitory effects of the EP4R antagonist were unmasked when it was applied in the presence of the EP2R antagonist;5) PGE2-induced relaxations of CCh and EFS-evoked contractions were reduced in the presence of the BK channel blocker,iberiotoxinbut not by the Kv7 channel blocker,XE-991;6) PGE2reduced the frequency and amplitude of CCh-induced calcium oscillations in isolated murine airway smooth muscle cells;7) These effects were mimicked by the EP2R agonist, (R)-Butaprost, the AC activator, forskolin,the exchange protein activated by cAMP (EPAC) activator, 007-AM,the PKA activator, 6-MB-cAMP, and were partially reduced by the PKA inhibitor, Rp-8-CPT-cAMP; 8) CCh-induced calcium oscillations were inhibited by the IP3R inhibitor, 2-APB, the RyRinhibitor, tetracaine and, to a lesser extent, removal of calcium from the extracellular solution; 9) Caffeine-induced calcium transients in isolated airway smooth muscle cells were not affected by addition of PGE2; 10) All EPR subtypes were expressed in whole bronchial tissue, but EP4R was not detected in isolated airway smooth muscle cells.Taken together, these data suggest a role for EP2Rs in PGE2-induced inhibition of cholinergic responses in murine airway smooth muscle. These effects may involve activation of BK channels and inhibition of calcium oscillations through upregulation of PKA, and possibly EPAC, to inhibit IP3R evoked calcium release.

Item Type:	Thesis (Doctoral)
Uncontrolled Keywords:	Smooth muscle
Subjects:	Science > Biology Science
Research Centres:	UNSPECIFIED
Depositing User:	Sean McGreal
Date Deposited:	23 May 2022 11:18
Last Modified:	23 May 2022 11:18
License:	Creative Commons: Attribution-Noncommercial-Share Alike 4.0
URI:	https://eprints.dkit.ie/id/eprint/790

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