Characterisation of the functional role of OCTN1 in the pathogenesis of COPD
Citation:CLERKIN, CAOIMHE GERALDINE, Characterisation of the functional role of OCTN1 in the pathogenesis of COPD, Trinity College Dublin.School of Pharmacy & Pharma. Sciences.PHARMACY AND PHARMACEUTICAL SCIENCES, 2018
The organic cation transporter (OCTN1) has been reported to play a role in the pathogenesis and development of inflammatory diseases. However, this association remains conflicting. Without fully elucidating the physiological function of OCTN1, the link between the transporter and these diseases will never be understood. For the first time this research has identified the functional role of OCTN1, through the uptake and transport of Ergothioneine (ET) in the bronchial epithelial cell line, NCI-H441. Three novel single nucleotide polymorphisms (SNPs) of the gene responsible for the coding of OCTN1 i.e. SLC22A4 in the cell line NCI-H441 were identified. However, the functional impact of these mutations on the functional activity of OCTN1 is not yet fully understood. Furthermore, the transport of ET, the physiological substrate of OCNT1, was determined to be pH and temperature dependent. The antioxidant potential of ET was confirmed by counteracting reactive oxygen species (ROS) production intracellularly. It is suggested that ET carries out its cytoprotective properties by stabilising the intracellular pH. Once characterisation of OCTN1 in vitro was completed it was important to investigate its expression in patients suffering from Chronic Obstructive Pulmonary Disease (COPD). Bronchial epithelial samples were isolated and SLC22A4 was quantified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was identified that risk factors such as age, weight and cigarette smoking, upregulated the expression of the gene. Further, upregulation of the gene also occurred with reduced lung capacity, a key determinant of the pathogenesis and severity of COPD. This research for the first time has identified a significant association of OCTN1 function with COPD in the Irish population. As previously mentioned, OCTN1 is responsible for the uptake of ET. ET was confirmed to behave as an antioxidant, but its mechanism of action has never been fully elucidated. We identified structural changes that occur to ET once exposed to oxidative stressors, such as cigarette smoke extract (CSE). It was identified that ET is converted to hercynine (EH) and ergothioneine sulfonic acid (ESO3H). However, in vitro, only ESO3H was detected in a concentration dependent manner. The remaining presence of ET suggests an alternative mechanism of action. ET was identified to undergo phase II metabolism by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), to form dinitrophenyl-ergothioneine (DNP-ET). DNP-ET formation was identified to be catalysed by glutathione s-transferase (GST). However, there is some suggestion that alternative enzymes may play a role in this formation. The conjugate, DNP-ET was further identified to be effluxed from the cell by multi-drug resistant related protein (MRP) transporters. Hence, ET behaves in a similar way to glutathione (GSH) by carrying out its antioxidant properties. Finally, NCI-H441 was investigated to behave as a suitable model for in vitro analysis of transporters and channels in human alveolar epithelial primary cells (hAEpCs). For the majority of genes investigated, no change in the expression of ATP-Binding cassette (ABC) and Solute carriers (SLC) transporters occurred upon culturing under air-interface culture (AIC) or liquid covered culture (LCC) conditions. However, a markedly different expression in the majority of transporters did occur when comparing the expression of NCI-H441 with hAEpCs, but also during transdifferentiation of hAEpCs from alveolar type II (ATII) to an alveolar type I-like (ATI-like) phenotype. This may be as a result of NCI-H441 representing bronchial epithelium and not alveolar epithelium. Nevertheless, NCI-H441 does not represent the true phenotype of hAEpCs and so the need for a suitable in vitro model is still required. Collectively, these results have determined the functional role of OCTN1 in bronchial epithelium and have confirmed an association of an upregulation of OCTN1 and COPD in the Irish population.
Author: CLERKIN, CAOIMHE GERALDINE
Publisher:Trinity College Dublin. School of Pharmacy & Pharma. Sciences. Discipline of Pharmacy
Type of material:Thesis
Availability:Full text available