Tissue damage releases many endogenous mediators including ATP, serotonin (5-HT), histamine and prostaglandins such as PGE2. Many of these substances directly excite primary nerve endings to elicit either pain (ATP) or both pain and itch (histamine and 5-HT). Prostaglandins do not directly activate the nerve endings but are known to sensitise them to histamine and 5-HT. Although histamine is known to act on sensory neurones via the Hi histamine receptor, the receptor subtypes activated by ATP and 5-HT remain unclear. In addition, the mechanism by which prostaglandins sensitise the nerve endings is unknown. These issues have been investigated in cultured rat dorsal root ganglion neurones by using the ratiometric indicator Fura-2 to monitor changes in intracellular calcium. 5-HT evoked a rise in Ca2+ j in approximately a fifth of neurones. This proportion remained unchanged when cells were stimulated in a nominally calcium free solution or with the 5-HT2 receptor agonist, a-methyl 5-HT. Thus, sensory neurones responded to 5-HT via 5-HT2 receptors. PGE2 sensitised neurones to 5-HT as shown by a leftward shift in the dose response curves. A similar leftward shift was seen for the effect of PGE2 on the response to histamine. The effects of PGE2 could be mimicked by application of forskolin and were blocked by the protein kinase A (PKA) inhibitor, H89. This suggests that the sensitisation results from a phosphorylation reaction mediated by PKA. Three quarters of DRG neurones responded to ATP and a similar proportion responded to the P2Y agonist, 2-methyl thio ATP. However, although the response to the P2Y agonist was reduced by pretreatment with the PLC inhibitor U73122 it was not totally abolished, supporting the notion that P2X receptors were also involved. In conclusion, it appears that the calcium responses elicited by histamine and 5-HT in rat sensory neurons are primarily due to activation of G protein coupled receptors and that the sensitivity of these receptors was enhanced by PKA mediated phosphorylation.

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Small conductance calcium-activated K+ channels (SK channels) form a subfamily of K+ -selective, volt age-insensitive channels that have been recently cloned. This thesis is concerned primarily with an investigation of the pharmacology of these channels. Understanding their pharmacology is of potential clinical importance since SK channels participate in diverse physiological roles, for example, generating neuronal afterhyperpolarizations (that set tonic firing frequencies) and regulating smooth muscle contraction. This thesis consists of two parts. The first is concerned with expressing and establishing recording conditions for rSK3 in mammalian cell lines. This was made difficult because of channel run up and run down. However, some progress was made towards stabilizing channels through MgATP regulation and this allowed progress towards the second part (the major part) of this thesis; a functional characterization of site-directed point mutants that were created to better understand the pharmacology of these channels. First, two mutations were made to examine similarities between the pore structures of the KcsA and Shaker channels and the SK channels. The first of these mutants would be predicted to increase Tetraethylammonium (TEA) affinity to the sub-millimolar range, and the second to provide increased sensitivity to charybdotoxin (CTX) block. Both these predictions were fulfilled suggesting that the KcsA/Shaker pore structure can provide a reasonable model for the SK channel pore. However, an important difference was identified in the TEA sensitive mutant, indicating that although the channels are similar, they are not identical. Three UCL compounds were then studied; UCL 1848, UCL 1684, and UCL 1530 with six channel mutants. The effects of these mutations on blocker affinity provided the basis of a "map" of the channel residues interacting with UCL compounds and establishes that these blockers bind in the channel pore region. Further, experiments co-expressing wildtype (WT) and mutant subunits demonstrate that UCL compounds do not require all four "sensitive" subunits for block, suggesting an asymmetric interaction with the channel outer pore. Finally, some work has also been done to define the possible assembly patterns of SK subunits in forming heteromeric channels. Evidence is presented that SKI and SK3 can co-assemble. Overall, this thesis provides a starting point for understanding the pharmacology of small molecule SK channel blockers at the molecular level.

No description available.

Calcium is an important secondary messenger and plays a major role in cell function, including proliferation, cell growth, secretion and death .It also plays a critical role in uterine smooth muscle contraction and embryo implantation. This thesis is concerned with calcium homeostasis in epithelial tissue lining the oviduct and uterus which are key players in early reproductive events, being involved in gamete transport, sperm capacitation and providing the micro-environment for the gametes and early embryo. Calcium transport across epithelial cells is either via tight junctions or calcium channels, specifically, members of the transient receptor potential (TRP) channel superfamily and the Na+/Ca2+ exchanger. TRP channels are an important class of calcium channels with more than 28 identified members and their potential involvement in calcium transport in uterus and oviduct epithelia has yet to be determined. The aim of this study was to discover which TRPC isoforms are expressed in epithelial cells lining the female reproductive tract in the bovine and human. Gene expression of TRPC channels changes was measured throughout the oestrous cycle in bovine oviduct and uterine epithelial cells using Real-Time PCR, while immunohistochemistry, immunocytochemistry and western blotting were used to discover the localization of TRPC channels in oviduct/uterine epithelium and changes in protein expression of TRPC isoforms induced by sex hormones. . to The physiological role of TRPC isoforms in regulating intracellular calcium concentration in bovine oviduct epithelial cells was determined using a calcium assay approach and finally. the potential clinical relevance of a possible role of TRP channels in female reproduction was investigated.# OF 7 members of TRPC family, TRPC1, 2, 3, 4 and 6 were expressed in bovine oviduct and uterine epithelia. In human endometrium, TRPC1, 6 and 7 genes were detected. Expression levels of all TRPC isoforms present in both bovine oviduct and uterine epithelia changed throughout the oestrous cycle. 17β-estradiol, FSH and LH individually and in combination up-regulated gene expression of TRPC isoforms in bovine oviduct epithelial cells. However, progesterone inhibited the upregulatory effect of 17β-estradiol, FSH and LH on TRPCs gene expression. TRPC1 and TRPC6 which are the common TRPC isoforms in bovine oviduct/uterine epithelium and human endometrium were localized on the apical, basal and lateral membranes of the epithelial tissue in bovine oviduct/uterus and human endometrium. TRPC isoforms were physiologically active in bovine oviduct epithelial cells (BOEC). SKF96365 which is a general TRP channel blocker inhibited the calcium influx into BOEC. Furthermore, Hyperforin which is a TRPC6 channel activator increased the intracellular calcium concentration in BOEC. TRPC1, 6 and 7 expression in endometrium of patients being treated for infertility by IVF illustrated that gene expression of TRTPC1 and 6 were up regulated in the endometrium of the IVF patients compared to controls. However, gene expression of TRPC7 in IVF patients was downregulated compared to that of the endometrium of the control group. Gene expression of TRPC6 and 7 in endometrium of women with Poly Cystic Ovarian Syndrome (PCOS) who have higher level of LH and normal FSH level, alongside the absence of the post-ovulatory increase in progesterone secretion, were up -regulated compared to that of the control group. However, the expression level of TRPC1 in endometrium of PCOS patients was not significantly different compared to the control group. Gene expression of TRPC isoforms in the epithelia lining the female reproductive tract is possibly regulated by sex hormones via nuclear factor-kappa B (NF-КB) signalling pathway. However, further investigation is required to determine the mechanisms underlying the endocrine regulation of TRPC channels.

This thesis develops biophysically-based data-driven mathematical models of intracellular calciumdynamics in ventricularmyocytes for both normal and genetically modified mouse hearts, based on species- and temperature-consistent experimental data. The models were subsequently applied to quantitatively examine the changes in calcium dynamics in mice with cardiomyocyte-specific knockout (KO) of the cardiac sarco/endoplasmic reticulum ATPase (SERCA2) gene, to determine the contributing mechanisms which underlie the ultimate development of heart failure in these animals. In Chapter 1, with emphasis on calcium dynamics and calcium regulation in heart failure, an overview of cardiac electrophysiology, excitation-contraction coupling and mathematical models of cardiac electrophysiology is provided. In Chapter 2, models of calcium dynamics in the ventricular myocytes from the C57BL/6 mouse heart at a physiological temperature is developed and validated based on species- and temperature-consistent measurements. In Chapter 3, the C57BL/6 model framework is re-parameterised to experimental data from the control and SERCA2 KO mice at 4 weeks after gene deletion. The models are then used to quantitatively characterise changes in calcium dynamics in the KO animals and the role of the compensatory mechanisms. In Chapter 4, the model framework is extended to include differential distributions of ion channels in the sarcolemma and the calcium dynamics in the sub-sarcolemmal space, with parameters in these sub-components fitted to experimentally measured calcium dynamics from the control and KO cardiomyocytes at 7-week after gene deletion. Finally in Chapter 5, conclusions are drawn, the limitations of this study are discussed, and the future extensions to this study are described.

La mucoviscidose (Cystic Fibrosis, CF) est caractérisée par des mutations dans le gène CFTR codant pour un canal chlorure situé à la membrane apicale des cellules épithéliales. La mutation principalement retrouvée chez les patients (CF) est la mutation F508del qui entraine une rétention du canal dans le RE des cellules, et son absence à la membrane apicale. Cette mutation va avoir de nombreuses conséquences sur la signalisation cellulaire. Outre la perte d’activité de canal chlorure du CFTR, de nombreux canaux ioniques sont dérégulés entrainant notamment des défauts dans la sécrétion d’ions et d’eau par les épithéliums. Une forte dérégulation de l’homéostasie calcique des cellules exprimant la mutation F508del a également été montrée, et pourrait participer de manière importante aux différentes atteintes cellulaires observées dans ces cellules. L’objectif de cette thèse a été d’une part de poursuivre la description de la dérégulation de l’homéostasie calcique observée dans la mucoviscidose et d’autre part de caractériser l’implication d’une protéine du RE sensible au Ca2+, la caluménine, dans ces dérégulations et dans la biosynthèse du CFTR. Nous avons retrouvé dans notre modèle cellulaire de cellules épithéliales bronchiques CF un certain nombre des dérégulations de l’homéostasie calcique décrites précédemment et mis en évidence de nouvelles dérégulations. Ainsi une modulation de l’activité des pompes SERCA et PMCA due à la dérégulation de l’interaction entre le CFTR muté et ces protéines a été identifiée pour la première fois. Nous avons de plus démontré que la caluménine joue un rôle important de chaperonne du CFTR et que la modulation de son expression régule la biosynthèse du CFTR. Nos travaux ont de plus établi l’implication de la caluménine dans la régulation de l’activité des pompes SERCA dans les cellules épithéliales bronchiques. / Cystic Fibrosis (CF) is characterized by mutations in the CFTR gene that encodes for an apical plasma membrane chloride channel in epithelial cells. The most common mutation found in CF is the F508del mutation, which lead to the synthesis of a wrongly conformed CFTR protein sequestered in the endoplasmic reticulum (ER) and absent of the plasma membrane. CFTR ER retention has important consequences on cell signaling. Besides its Cl- channel activity, CFTR regulates many ionic channels, and its mutations lead to defects in epithelial ionic and water secretion. Moreover an important deregulation of Ca2+ homeostasis is observed in CF cells that impacts a large range of cellular functions. The aim of this work was firstly to pursue the description of Ca2+ homeostasis defects in CF cells, and secondly to characterize the implication of calumenin, an ER Ca2+-sensitive protein, in these deregulations and in CFTR biosynthesis. We observed in our CF epithelial cell models some of the previously described deregulations in Ca2+ homeostasis, and highlighted new deregulations. Indeed, we showed for the first time perturbations of SERCA and PMCA pumps activities in CF cells. These deregulations are mediated by a modified interaction of these pumps with CFTR. Moreover, we demonstrated that calumenin acts as a CFTR chaperone, and modulating calumenin expression modifies CFTR biosynthesis. Finally, we also characterized calumenin implication in epithelial cell Ca2+ homeostasis.