Asthma is characterised by airway inflammation, remodelling and bronchial hyperresponsiveness to normally innocuous stimuli. There is growing evidence that the airway smooth muscle (ASM) in asthmatic patients is altered and contributes to all three of these central pillars of the disease. Intracellular free calcium ions act as a secondary messenger within ASM in all of these processes and the expression of proteins regulating this tightly controlled process are altered in the disease. It has further been suggested that the long-term detrimental effect observed with some asthma therapies maybe associated with changes in calcium handling. The hypothesis of this study was that the calcium handling proteins SERCA2, TRPC3 and TRPC6 are altered by cytokines associated with asthma, or current therapies, leading to changes in calcium dynamics in the cell which contribute to the asthmatic phenotype. To investigate this hypothesis human primary healthy ASM cells were grown in culture and stimulated with either IL-13, TGF-β, TNF-α or a combination thereof or β2-adrenergic receptor agonists. The RNA and protein expression of the aforementioned calcium regulators were measured by qPCR and western blot and the calcium dynamics assessed by loading cells with fura-2. The impact of reduced SERCA2 expression, as observed in ASM derived from asthmatics, was investigated in a murine model of ovalbumin induced allergic airway disease using SERCA2+/- mice. End-points included lung function measurements and inflammatory cell and cytokine infiltration. The key results found were that TGF-β differentially upregulates the expression of TRPC6 splice variants in a Smad2/3 dependent manner resulting in a reduction in flufenamic acid induced calcium entry. TNF-α upregulates TRPC3 expression while concomitantly down-regulating TRPC6 possibly conferring a switch in signalling from receptor operated calcium entry to store operated calcium entry. A reduction in SERCA2 in an in vivo model of asthma results in enhanced neutrophilia and an increase in sensitivity to methacholine to increase total airway resistance and decrease dynamic compliance. Finally it appears that chronic administration of β2-adrenergic receptor agonists can lead to a variable decrease in SERCA2 expression however cAMP formation significantly increases it. Therefore β-arrestins may play a role in the detrimental effects observed with chronic dosing of this class of compounds. In conclusion the calcium handling proteins TRPC3 and TRPC6 are differentially altered by both TNF-α and TGF-β but unaffected by IL-13. The protein expression of SERCA2 on the other hand appears to be less regulated by the presence of asthmatic cytokines and its continued reduced expression in cultured asthmatic cells could be the result of genetic or epigenetic regulatory mechanisms.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:650777 |
| Date | January 2014 |
| Creators | Wright, David Blair |
| Contributors | Ward, Jeremy Patrick Thomas; Lee, Tak Hong |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://kclpure.kcl.ac.uk/portal/en/theses/altered-intracellular-calcium-ion-handling-in-airway-smooth-muscle-and-the-pathogenesis-of-asthma(aa468afa-a353-46e3-b1c9-8f7f86140dde).html |
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