Molecular pharmacology of the capsaicin receptor (TRPV1) in the airways

Sadofsky, Laura R.

Postgraduate Medical School
December 2005

Thesis or dissertation


Rights
© 2005 Laura Rachel Sadofsky. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.
Abstract

The capsaicin receptor (vanilloid receptor I, transient receptor potential vanilloid 1 or TRPV I) is a member of the transient receptor potential (TRP) family of proteins. This cation channel is sensitive to a range of inflammatory mediators such as some lipoxygenase products, as well as the tussive agents capsaicin, resiniferatoxin and protons. It has been proposed that TRPV I is a cough receptor and may be important in airways inflammation.

Rat TRPVI (rTRPVI) and human TRPVI (hTRPVl) permanently expressing cell lines were generated and successfully characterised by agonist triggered changes in intracellular calcium levels. Thapsigargin and/or removal of extracellular calcium revealed that, both rTRPVI and hTRPVI are not only expressed on the cell surface but on thapsigargin sensitive and insensitive intracellular stores respectively.

Citric acid, an agent routinely used in the clinic for inhalation cough challenges, was investigated for its ability to activate TRPVl permanently expressed in a cell line. rTRPV I was activated by citric acid in a concentration and pH dependent manner. Citric acid activation of TRPVI was inhibited by iodoresiniferatoxin but not capsazepine. Mutation of the TRPVI putative proton binding site (E648 to A648) abolished citric acid activation of the channel without reducing the capsaicin evoked response. Thus, citric acid activates rTRPV I by a proton dependent mechanism.

The role of N-linked glycosylation and sialylation on rTRPVI and hTRPVI was investigated. Treatment of rTRPVl with neuraminidase or tunicamycin dramatically reduced the channels' maximal responses to capsaicin. In addition mutation of the rTRPVI N-linked glycosylation site (N604 to Q604) or expression ofrTRPVI in the glycosylation mutant cell line, Lec2, also resulted in a striking reduction in the receptors' maximal calcium response to capsaicin. Flow cytometry data indicated that these differences in TRPVI function were unlikely to be linked to differences in receptor cell surface expression. Human TRPV I also displayed significant reductions in responsiveness to capsaicin following either neuraminidase or tunicamycin treatment. Thus, receptor sialylation regulates TRPVI activation by capsaicin.

Finally, TRPVI expression on human primary bronchial fibroblasts (HPBF) was investigated. Negligible endogenous TRPVI expression was detected in HPBF. Interestingly, the inflammatory mediators tumour necrosis factor (TNF-a), lipopolysaccharide (LPS) and interleukin Ia (IL-Ia) all induced TRPVI expression in HPBF, as assessed by RT-PCR, flow cytometry and calcium signalling. TRPVI functional expression was observed as early as 6 hrs (for TNF-a) post challenge and remained elevated upto the final time point tested (96 hrs for IL-Ia). Thus, TRPVI may play an important role in the inflammatory process.

In conclusion, TRPV I may play an important role in conditions where cough and inflammation have been implicated.

Publisher
Postgraduate Medical School, The University of Hull
Ethos identifier
uk.bl.ethos.440131
Qualification level
Doctoral
Qualification name
PhD
Language
English
Extent
11 MB
Identifier
hull:8630
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