Understanding trans plasma membrane electron transport in epithelial cells
Publication Date
July 22, 2019
Creator
Abstract
Trans plasma membrane electron transport systems (tPMETS) are present throughout all kingdoms of life, and function to transport electrons across the plasma membranes of living organisms. Since the 1970s, research into tPMETS has grown rapidly, and it is now known that they underpin many important physiological and pathological functions within cells. Within lung epithelial type cells there are still tPMETS to be investigated that have been identified in other cell types, and confirmation of certain tPMETS’ presence and function within different epithelial cell types is also not fully established. The comparison of particular tPMET mechanisms between cell types is also not well understood. This work applies a well-known linear sweep voltammetry technique to study and compare tPMETS within the human lung epithelial derived cell lines, Calu-3, H1299 and A549, and in doing so identify potential differences in tPMET activity between cells originating from the same tissue and organ.
The results gathered demonstrate that linear sweep voltammetry at a microelectrode is a suitable experimental technique for the highly sensitive measurements of tPMET activity using the redox mediator ferricyanide. The novel application of this technique identified differences within the tPMET activity of the lung derived epithelial cell lines tested, with no strong link to metabolic or proliferative rates. Inhibition assays concluded that dehydroascorbate was transported across the cytoplasmic membrane into the cell by transmembrane glucose transporters (GLUTs), and upon intracellular reduction ascorbate was effluxed across the cytoplasmic membrane via anion channels in Calu-3 and H1299 cells, but this was not observed in A549 cells. Interaction with the redox mediator ferricyanide and removal of extracellular ascorbate using ascorbate oxidase yielded the identification of this transport mechanism as an ascorbate shuttle-based tPMET, which was most proficient in A549 and Calu-3 cells, whilst least proficient in H1299 cells. Quantitative reverse transcriptase polymerase chain reaction showed the presence of the gene encoding for a known tPMET enzyme, Duodenal cytochrome b (Dcytb), in the tested cells. Immunocytochemistry confirmed the presence of this enzyme both at the cell plasma membrane and intracellularly. Efficient transfection and partial knockdown of the Dcytb encoding gene (CYBRD1) and protein was only achieved in A549 cells. This resulted in a reduction in tPMET activity and highlighted Dcytb as a component of the tPMET system in A549 cells.
The work suggests that the mechanisms of tPMET activity in lung derived epithelial cells are diverse and potentially interlinked. It is proposed that the differences in the use of ascorbate shuttling and ascorbate-mediated enzyme tPMET may be due to the characteristics of the cells. A549 and Calu-3 cells are both cell types which produce secretions for the lung lining, whilst H1299 cells are farthest from a typical lung phenotype. This may lead to the higher levels of ascorbate efflux-attributable tPMET activity in A549 and Calu-3 cells in line with the observed results. The type II alveolar cells have been shown to concentrate high levels of ascorbate intracellularly when incubated with external ascorbate or dehydroascorbate. This may explain the increased ability for ascorbate-mediated electron transport (both shuttle and enzymatic) of A549 cells compared to Calu-3 and H1299 cells. The type II cell is also present at the surface of the alveolar epithelium, which again may provide an explanation for the higher tPMET capacity observed in A549 cells, potentially to deal with incoming foreign matter.
Importantly, the results demonstrate that both tPMET activity and preference for specific tPMET systems differ between cell lines originating from human lung epithelial cell populations. This may have implications for the targeting of tPMET in particular pathologies, and raises the possibility of screening cells for their preferred tPMETS usage.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Pharmacy (UK)
eprints ID
55991
UoN Repository URI
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Sherman_Harry_4232247_Understanding Trans Plasma Membrane Electron Transport in Epithelial Cells.pdf
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