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postgraduate thesis: Roles of cystic fibrosis transmembrane conductance regulator, pannexins and connexins in ATP release from human and rat skeletal muscle

TitleRoles of cystic fibrosis transmembrane conductance regulator, pannexins and connexins in ATP release from human and rat skeletal muscle
Authors
Issue Date2017
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Wu, T. E. [胡子軒]. (2017). Roles of cystic fibrosis transmembrane conductance regulator, pannexins and connexins in ATP release from human and rat skeletal muscle. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractATP release has vital importance as an intracellular energy molecule and upon release from cells, as a purinergic signaling molecule. Working skeletal muscle releases ATP which is subsequently converted extracellularly to adenosine by ectoenzymes, producing vasodilation so as to increase oxygen supply for continuous muscle contraction. Previous studies have shown that acidosis leads to increased ATP release from muscle cells, which can mimic the exercise-induced ATP release; skeletal muscle CFTR was found to be activated during acidosis, and to play an essential role in the exercise or acidosis-induced ATP release. Patients with Cystic Fibrosis, where there is a reduced number of functional Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channels on the cell membranes, have reduced exercise tolerance. With a small pore, CFTR itself is not likely to conduct ATP directly, suggesting that other CFTR-regulated big channels are responsible for the release. In this study, large channel opening was measured by the uptake of Lucifer Yellow, a fluorescent dye; ATP release from cells was determined by measuring the accumulation of ATP in the bathing medium of cells, and protein expression of cells was studied by Western Blot. I investigated whether connexins and/or pannexins contributed to ATP release from cultured skeletal myoblasts, and whether they were involved in the acidosis-induced CFTR-regulated ATP release. Both rat L6 myoblasts and human skeletal muscle myoblasts (HSMMs) expressed CFTR, Connexin 43 (Cx43) and Pannexin 1 (Panx1), with a higher level of Panx1 expression in HSMMs. Acidosis could induce ATP release from, and LY uptake to, both HSMMs and L6 cells, which was inhibited by CFTR inhibitors, confirming the role of CFTR as a regulator of ATP release. In both cell types, the application of forskolin to activate CFTR, along with adenylyl-imidodiphosphate (AMP-PNP) to lock the CFTR pore open, also stimulated ATP release. In L6 cells, pannexin inhibitors significantly decreased the acidosis- or forskolin-AMP-PNP-induced LY uptake and ATP release; silencing of pannexin expression using siRNA suggested that Pannexin 3 (Panx3) channels mediated most of the ATP release, with a smaller contribution from Panx1; Cx43 contributed a little to basal ATP release, but not the CFTR-regulated acidosis-induced component. In HSMMs, either connexin or pannexin inhibitors could reduce, but not abolish, the ATP release induced by either acidosis or direct activation of CFTR, suggesting that both connexins and pannexins were involved in the activated-CFTR-induced ATP release mechanism. I attempted to develop a CFTR-and-Panx1-co-overexpression model from L6 cells through lentiviral transduction: this was only partially successful, since it overexpressed CFTR, but Panx1 overexpression was not stable. In this model, both pannexins and connexins were involved in the activated-CFTR-associated ATP release, which was similar to HSMMs. Further investigation is required to identify the mechanism by which CFTR is able to regulate the ATP release via pannexin or connexin channels in skeletal myoblasts.
DegreeMaster of Philosophy
SubjectPathophysiology - Cystic fibrosis
Adenosine triphosphate
Connexins
Dept/ProgramBiomedical Sciences
Persistent Identifierhttp://hdl.handle.net/10722/255423

 

DC FieldValueLanguage
dc.contributor.authorWu, Tsz-hin, Elton-
dc.contributor.author胡子軒-
dc.date.accessioned2018-07-05T07:43:30Z-
dc.date.available2018-07-05T07:43:30Z-
dc.date.issued2017-
dc.identifier.citationWu, T. E. [胡子軒]. (2017). Roles of cystic fibrosis transmembrane conductance regulator, pannexins and connexins in ATP release from human and rat skeletal muscle. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/255423-
dc.description.abstractATP release has vital importance as an intracellular energy molecule and upon release from cells, as a purinergic signaling molecule. Working skeletal muscle releases ATP which is subsequently converted extracellularly to adenosine by ectoenzymes, producing vasodilation so as to increase oxygen supply for continuous muscle contraction. Previous studies have shown that acidosis leads to increased ATP release from muscle cells, which can mimic the exercise-induced ATP release; skeletal muscle CFTR was found to be activated during acidosis, and to play an essential role in the exercise or acidosis-induced ATP release. Patients with Cystic Fibrosis, where there is a reduced number of functional Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channels on the cell membranes, have reduced exercise tolerance. With a small pore, CFTR itself is not likely to conduct ATP directly, suggesting that other CFTR-regulated big channels are responsible for the release. In this study, large channel opening was measured by the uptake of Lucifer Yellow, a fluorescent dye; ATP release from cells was determined by measuring the accumulation of ATP in the bathing medium of cells, and protein expression of cells was studied by Western Blot. I investigated whether connexins and/or pannexins contributed to ATP release from cultured skeletal myoblasts, and whether they were involved in the acidosis-induced CFTR-regulated ATP release. Both rat L6 myoblasts and human skeletal muscle myoblasts (HSMMs) expressed CFTR, Connexin 43 (Cx43) and Pannexin 1 (Panx1), with a higher level of Panx1 expression in HSMMs. Acidosis could induce ATP release from, and LY uptake to, both HSMMs and L6 cells, which was inhibited by CFTR inhibitors, confirming the role of CFTR as a regulator of ATP release. In both cell types, the application of forskolin to activate CFTR, along with adenylyl-imidodiphosphate (AMP-PNP) to lock the CFTR pore open, also stimulated ATP release. In L6 cells, pannexin inhibitors significantly decreased the acidosis- or forskolin-AMP-PNP-induced LY uptake and ATP release; silencing of pannexin expression using siRNA suggested that Pannexin 3 (Panx3) channels mediated most of the ATP release, with a smaller contribution from Panx1; Cx43 contributed a little to basal ATP release, but not the CFTR-regulated acidosis-induced component. In HSMMs, either connexin or pannexin inhibitors could reduce, but not abolish, the ATP release induced by either acidosis or direct activation of CFTR, suggesting that both connexins and pannexins were involved in the activated-CFTR-induced ATP release mechanism. I attempted to develop a CFTR-and-Panx1-co-overexpression model from L6 cells through lentiviral transduction: this was only partially successful, since it overexpressed CFTR, but Panx1 overexpression was not stable. In this model, both pannexins and connexins were involved in the activated-CFTR-associated ATP release, which was similar to HSMMs. Further investigation is required to identify the mechanism by which CFTR is able to regulate the ATP release via pannexin or connexin channels in skeletal myoblasts. -
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.subject.lcshPathophysiology - Cystic fibrosis-
dc.subject.lcshAdenosine triphosphate-
dc.subject.lcshConnexins-
dc.titleRoles of cystic fibrosis transmembrane conductance regulator, pannexins and connexins in ATP release from human and rat skeletal muscle-
dc.typePG_Thesis-
dc.description.thesisnameMaster of Philosophy-
dc.description.thesislevelMaster-
dc.description.thesisdisciplineBiomedical Sciences-
dc.description.naturepublished_or_final_version-
dc.date.hkucongregation2018-
dc.identifier.mmsid991044019382503414-

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