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Title: An in vitro analysis of the expression and function of bronchial organic cation transporters
Author: Mukherjee, Manali
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2012
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The mounting popularity of pulmonary inhalation has instigated a growing interest in the membrane transporters expressed in the lung epithelium. Many of the commonly inhaled drugs have net positive charge at physiological pH, which makes them substrates for members of the Organic Cation Transporter (OCT) family. The five popular members, namely, OCT1, OCT2, OCT3, OCTNl and OCTN2, have important physiological and biopharmaceutical roles established in the liver, kidneys, brain and the intestine. In regard to the lungs, detailed investigations have been sparse. The primary aim of this study thus intended to examine the expression and functions of the OCTs constitutively expressed in the bronchial epithelium, and to unveil any latent physiological functions, possibly connected to the intrinsic protective mechanisms in an inflammatOlY event. To successfully execute an investigation of this type, it was impoltant that an in vitro model be selected that would resemble the bronchial epithelium in terms of barrier propelties, cellular morphology and the right expression profile of OCT subtypes. Based on the summative observations from the polymerase chain reaction, immunofluorescence and in-cell Western ™ studies; OCT1, OCT3, OCTNl and OCTN2 were found to be expressed in 21 day old air-liquid interfaced (All) Calu-3 layers and were collectively termed the 'bronchial OCTs'. Uptake studies with fluorescent OCT substrate demonstrated the presence of one or more functional OCT subtypes and OCT-inhaled drug interactions in the layers. To investigate the physiological roles of the bronchial OCTs, responses to different environment-derived extraneous insults were considered. OCT expression was up-regulated at both mRNA and protein levels by lipopolysaccharide-induced bronchial inflammation. An incident of wound/injury was replicated on the Transwell® grown monolayers via a simple pipette 'scrape' . Recovered layers showed increased expression levels of OCTs; while the consequent recovery itself was hindered in the presence of non-selective OCT inhibitors or after OCTl/OCTN2 siRNA targeted silencing protocols. Further on, clinically relevant risk factors like house dust mite (HDM) allergens showed significant changes in the OCT expression profile along with tight junction disruption in the Calu-3 layers. The study also generated preliminary data that suggested the OCTs to be involved in controlling the release of reactive oxygen species during an induced inflammatory event. For the first time an attempt was made to understand the physiological roles of the bronchial OCTs; where these constitutively expressed drug transporters were demonstrated to respond to common environmental inhaled insults. An unknown association with airway inflammation was established in vitro; along with a possible 'protective' function in counteracting environment-derived disease-associated extraneous agents. Further investigations into the underlying mechanisms responsible for the documented alterations might provide a new insight on common respiratory/ lung diseases and pave the way for better patient management through inhaled drug therapy. ii >0" :-'
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available