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Title: TRPV channels in the retinal microvascular endothelium
Author: McNaughten, Jennifer Elizabeth
ISNI:       0000 0004 5371 2351
Awarding Body: Queen's University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2014
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The aim of this study was to fully characterise the molecular and functional expression of TRPV channels in the retinal microvascular endothelium. Retinal microvascular endothelial cells (RMECs) were cultured from bovine retina. mRNA transcripts and protein expression was detected for TRPV1, 2, 3 and 4 using RT-PCR and western blots respectively, while fura-2-based Ca2+ microfluorimetry indicated TRPV channels were functional. Application of a TRPV1 agonist (resiniferatoxin; 10nM) to RMECs caused a transient rise in Ca2+. This was successfully abolished by pre-incubation with either of the TRPV1 antagonists capsazepine (5μM) or AMG9810 (1OμM). Ca2+ transients were also observed in response to 119-THC (1 OIJM) and were reduced after incubation with the TRPV2 inhibitor tranilast (75IJM). The TRPV3 agonist carvacrol (100pM) induced consistent calcium responses in RMECs while GSK1016790A (GSK; 100nM), a TRPV4 agonist, elicited transient rises in Ca2+ which were inhibited by HC067047 (1μM). Sub-cellular localisation of TRPV channels was examined in isolated RMECs and bovine retinal wholemounts using immunohistochemistry. Generally in RMECs, cytoplasmic staining for each TRPV channel was predominantly localised within a broad peri-nuclear zone. Interestingly, all TRPV channels, particularly TRPV3 and 4, were also detected in RMEC nuclei. Given the observed sub-cellular expression of TRPV4 in RMECs, the functionality of this channel was investigated further with confocal Ca2+ imaging. Application of GSK or 8'9 EET (an arachidonic acid metabolite) elicited transient Ca2+ signals in nuclear, mitochondrial and cytosolic regions of the cell. Inhibition of responses by HC067047 indicated TRPV4 activation was responsible and a possible involvement in EET-signalling. Low concentrations of GSK (1OμM) and 8'9 EET (1 nM) evoked isolated nuclear Ca2+ responses suggesting another role for TRPV4 in nuclear specific Ca2+ signalling. Detection of TRPV4 protein expression in nuclear protein extracts provided further evidence for such a role. Studies in this thesis have therefore reported functional TRPV expression, as well as highlighting potential roles for these channels, in RMECs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available