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Title: Functional roles of different TRPV1 populations in calcium signalling of rat sensory neurons and HEK 293 cells expressing rat TRPV1
Author: Wong, Benjamin
Awarding Body: University of Hertfordshire
Current Institution: University of Hertfordshire
Date of Award: 2012
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The TRPV1 vanilloid receptor is thought to be expressed on both plasmalemmal (TRPV1PM) and intracellular (TRPV1ER) membranes. Membrane-impermeable TRPV1PM restricted antagonists (SB-497794-D and ruthenium red) were used to investigate the functional roles of TRPV1PM and TRPV1ER in capsaicin-activated calcium signalling of TRPV1-expressing cells (rat DRG neurons and rat TRPV1-expressing HEK 293 cells). The ability of TRPV1PM restricted antagonists to completely block capsaicin activation of TRPV1PM when TRPV1-expressing cells were challenged with capsaicin enabled the remaining capsaicin-activated TRPV1ER response to be assessed. The intracellular calcium imaging study that investigated the effect of 10 μM SB-497794-D against 1 μM capsaicin on rat TRPV1-expressing HEK 293 cells, in the presence of extracellular calcium, showed no contribution from TRPV1ER. In contrast, SB-497794-D (10 and 50 μM) and ruthenium red (10 and 100 μM) produced similar effects against 1 μM capsaicin on rat DRG neurons; these intracellular calcium imaging studies revealed that in the presence of extracellular calcium, the 1 μM capsaicin-induced intracellular calcium rise of rat DRG neurons was 70 % attributable to TRPV1PM, whereas the remaining 30 % calcium response was not blocked and hence mediated by TRPV1ER. However, in the absence of extracellular calcium, capsaicin (1 and 20 μM) failed to induce a TRPV1ER-mediated intracellular calcium response in the rat DRG neurons, suggesting that the TRPV1ER response is regulated by extracellular calcium. Intriguingly, the organ bath study that investigated the effect of 50 μM SB-497794-D against capsaicin-induced contraction of rat bladder strips demonstrated the ability of SB-497794-D to completely inhibit capsaicin-induced contraction of rat bladder strips. This suggests that TRPV1ER has no contribution to the capsaicin responses in this intact sensory nerve preparation. The clinical demonstration of azelastine to inhibit capsaicin-induced cough in cough patients suggests that this histamine-1 (H1) receptor antagonist has off-target antagonist effects at the putative TRPV1 cough receptor. Interestingly, two other H1 receptor antagonists (dexbrompheniramine maleate and chlorpheniramine) have recently been reported to have TRPV1 antagonist effects. FLIPR (Fluorescence Imaging Plate Reader) studies investigating the effect of azelastine against capsaicin on human and rat TRPV1, each expressed separately in HEK 293 cells, were conducted to address whether azelastine exerts off-target TRPV1 antagonism. Furthermore, the individual effects of other H1 receptor antagonists (dexbrompheniramine maleate, chlorpheniramine, mepyramine and olopatadine) against capsaicin on human and rat TRPV1 were investigated with the FLIPR assay to determine whether these H1 receptor antagonists have off-target TRPV1 antagonist effects. The FLIPR assay demonstrated the off-target TRPV1 antagonism of four H1 receptor antagonists (azelastine, dexbrompheniramine maleate, chlorpheniramine and mepyramine, but not olopatadine) against capsaicin activation of human and rat TRPV1. The off-target TRPV1 antagonism of dexbrompheniramine maleate was further demonstrated by the ability of dexbrompheniramine maleate (at 100 μM) to completely block the full capsaicin concentration-response curve for capsaicin-induced TRPV1-mediated contraction of rat bladder strips. Thus, it is likely that dexbrompheniramine maleate inhibited capsaicin activation of TRPV1PM in the bladder innervating primary sensory neurons to block the capsaicin-induced contraction of rat bladder strips.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available