No description available.

No description available.

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.

No description available.

No description available.

No description available.

No description available.

Nerve growth factor (NGF), one of the major inflammatory mediators released following tissue injury, causes thermal hyperalgesia by sensitisation of the heat- and capsaicin-gated ion channel, TRPV1. The molecular mechanisms by which NGF causes sensitisation of TRPV1 remain controversial. In this thesis, the functional effect of NGF on TRPV1 was investigated in HEK 293 cells and cultured mouse dorsal root ganglion (DRG) neurons using calcium imaging. The results support the hypothesis that sensitisation of TRPV1 by NGF is mediated by two pathways. The major pathway is activated by the Y760 site TrkA, which stimulates P13 kinase with Src kinase being involved at a subsequent stage. Src kinase phosphorylates TRPV1 at a single tyrosine residue, Y200, leading to trafficking and insertion of the channel into the surface membrane and thus enhancing the membrane ionic currents. The second and more minor pathway is the PLCγ / PKCε signalling pathway which causes phosphorylation TRPV1 at the S502 and S801 sites. The actions of different PKC isoforms in modulating TRPV1 are largely unknown. PKCβ was found to be located in the membrane of small DRG neurones from wild-type mice but in the cytoplasm of neurones from TRPV1 knockout mice, suggesting a physical interaction between PKCβ and TRPV1. Immunoprecipitation and <i>in vitro</i> GST pull down experiments confirmed that PKCβ binds predominantly to the N-terminal of TRPV1. The protein-protein interaction between PKCβ and TRPV1 suggests that PKCβ might play a specific role in modulation of TRPV1. These findings provide new insights into the molecular mechanisms underlying TRPV1 modulation by inflammatory mediators and PKC isoforms.

Oligodendrocytes and their precursors express a defined repertoire of integrin receptors, consisting of the heterodimers αvβ1, αvβ3, αvβ5, αvβ8 and a6β1. A role for the αvβ1 receptor has recently been defined in the migratory behaviour of oligodendrocyte precursors, and this project describes and extension of such work to examine the involvement of integrins in cellular differentiation and survival in this lineage. Using enriched populations of rodent oligodendrocyte precursors in simplified in vitro cell culture system several observations have been apparent: Firstly, there is evidence that β1 integrins are involved not only in migration of the precursors, but also in survival of the cells at all stages. In addition, morphological differentiation of the cells, which involves the elaboration of a complex meshwork of processes and subsequent myelin sheet formation, also turns out to be enhanced by a substrate of laminin-2, which is a potential ligand for the α6β1 integrin; correspondingly, it is shown that β1 integrins are also required for optimal morphological differentiation of the cells. Cellular differentiation is marked by a switch between an immature pattern of integrin expression (αvβ1, αvβ8 and α6β1) and a mature pattern of expression (αvβ3, αvβ5, αvβ8 and α6β1), and this switch is shown to be inhibited by the fibroblast growth factor, FGF-2. Upon withdrawal of FGF-2 therefore, the cells differentiate, exhibiting a burst of tyrosine phosphorylation; a group of tyrosine phosphorylated proteins are demonstrated to be associated with the β3 integrin subunit at this state, a subunit whose expression was inhibited by FGF-2. Finally, the different subunits β1, α6 and β5 show distinct spatial patterns of expression in differentiating cells, and evidence is presented to suggest the presence of an additional β1-associated α subunit, that is thus far unidentified.

The fusion of neurotransmitter-filled synaptic vesicles with the pre-synaptic membrane in response to calcium influx is exquisitely regulated. Synaptic vesicle exocytosis is energetically demanding and the neuronal SNARE proteins syntaxin, SNAP25 and synaptobrevin have come to prominence as the driving engines behind this process. Resident on both vesicular and pre-synaptic membranes they form a stable four-helical bundle, the assembly of which contributes to membrane fusion. However, SNAREs do not act in isolation during synaptic vesicle exocytosis but are instead regulated by a complex web of interactions with other proteins including synaptotagmin, a calcium-sensing component of the vesicle itself, and Munc18, a highly-conserved cytosolic protein. In addition, changes in the lipid environment surrounding the SNAREs play a critical role. In this thesis I report the results of two lines of investigation, into both synaptotagmin’s and Munc18’s action. Firstly, I consider the significance of the cytoplasmic double C2 domain structure of synaptotagmin. Using several strategies including a novel real-time absorbance assay, I show that these tandem C2 domains, but neither domain alone, rapidly cross-link lipid membranes in the presence of calcium. This property is conserved. Cross-linking ability can be masked in full-length synaptotagmin, via an electrostatic interaction with the membrane in which it is embedded. Finally, I address the mechanism of arachidonic acid action on syntaxin/Munc18, showing that this lipid activates Munc18-bound syntaxin and that a Munc18/syntaxin/SNAP25 assembly exists in brain. Arachidonic acid also activates free syntaxin, defining a molecular target for the reported role of this lipid in the promotion of vesicle fusion. My data are incorporated into a revised model of the protein-lipid interactions underlying synaptic vesicle exocytosis.