Many endothelial cell (EC) functions depend on influx of extracellular Ca2+, which is triggered by a variety of mechanical and chemical signals. Here, we discuss possible pathways for this Ca2+ entry. The superfamily of cation channels derived from the "transient receptor potential" (TRP) channels is introduced. Several members of this family are expressed in ECs, and they provide pathways for Ca2+ entry. All TRP subfamilies may contribute to the Ca2+ entry channels or to the regulation of Ca2+ entry in EC. Members of Ca2+ entry channels in endothelium probably belong to the canonical TRP subfamily, TRPC. All TRPC1-6 have been discussed as Ca2+ entry channels that might be store-operated and/or receptor-operated. More importantly, knockout models of TRPC4 have proven that this channel is functionally involved in the regulation of endothelial-dependent vasorelaxation and in the control of EC barrier function. TRPC1 might be an important candidate for involvement of eodothelial growth factors. TRPC3 is unequivocally important for a sustained EC Ca2+ entry. ECs express different patterns of TRPCs, which may increase the variability of TRPC channel function by formation of different multiheteromers. Among the two other TRP subfamilies, TRPMV and TRPM, at least TRPV4 and TRPM4 are EC channels. TRPV4 is a Ca2+ entry channel that is activated by an increase in cell volume, which might be involved in mechano-sensing, by an increase in temperature, and perhaps by ligand-activation. TRPM4 is a nonselective cation channel, which is not Ca2+ permeable. It is probably modulated by NO and might be essential for regulating the inward driving force for Ca2+ entry. Possible modes of TRP channel regulation are described, involving (a) activation via the phospholipase (PL)Cβ and PLCγ pathways; (b) activation by lipids (diacylglycerol [DAG], arachidonic acid); (c) Ca2+ depletion of Ca2+ stores in the endoplasmic reticulum; (d) shear stress; and (e) radicals.

Increased production of amyloid beta (A-beta) peptide, via altered proteolytic cleavage of amyloid protein precursor (APP), and abnormalities in neuronal calcium homeostasis play central roles in the pathogenesis of Alzheimer's disease (AD). Notch1, a membrane receptor that controls cell fate decisions during development of the nervous system, has been linked to AD because it is a substrate for the gamma-secretase protein complex in which mutations cause early-onset inherited AD. Numb is an evolutionarily conserved endocytic adapter involved in the internalization of transmembrane receptors. Mammals produce four Numb isoforms that differ in two functional domains, a phosphotyrosine-binding domain (PTB) and a proline-rich region (PRR). Recent studies showed that the PTB domain of Numb interacts with the cytoplasmic tails of APP and Notch but the functional relevance of these interactions with respect to AD pathogenesis is not clear. In the current studies, we proposed to investigate the biological consequences of the interaction of the Numb proteins with APP and Notch in neural cells stably overexpressing each of the four human Numb proteins. In the first part of our studies, we found that expression of the Numb isoforms lacking the insert in the PTB (SPTB-Numb) caused the abnormal accumulation of cellular APP in the early endosomes, and increased the levels of C-terminal APP fragments and A-beta. By contrast, expression of the Numb isoforms with the insert in PTB (LPTB-Numb) leads to the depletion of cellular APP and coincides with significantly lower production of APP derivatives and A-beta. The contrasting effects of the Numb isoforms on APP metabolism were not attributed to differences in the expression of APP nor the activities of the various APP-processing secretases. In the second part of our studies, we found that expression of SPTB-Numb protein enhances neuronal vulnerability to serum deprivation-induced cell death by a mechanism involving the dysregulation of cellular calcium homeostasis. Neural cells expressing SPTB-Numb exhibited enhanced Notch activity, which markedly upregulated the expression of transient receptor potential canonical 6 (TRPC6) channels enhancing calcium entry in response to store depletion. We also found that serum deprivation increased TRPC6 expression, mediating the serum deprivation-induced death in neural cells. Interestingly, expression of LPTB-Numb protein suppressed serum deprivation-induced activation of Notch and the subsequent upregulation of TRPC6 and cell death. Finally, we showed that the Numb proteins differentially impact Notch activation by altering the endocytic trafficking and processing of Notch. Taken together, these studies demonstrate that aberrant expression of the Numb proteins may influence APP metabolism and Notch-mediated cellular responses to injury by altering their endocytic trafficking and processing.

L’hyperglycémie est détectée et intégrée au niveau de l’hypothalamus médio-basal (MBH) qui inhibe la prise alimentaire et déclenche la sécrétion d’insuline. Le MBH renferme des neurones spécialisés gluco-sensibles (GS) qui détectent directement ou indirectement des variations de la concentration extracellulaire en glucose. Dans une première étude, nous suggérons que la détection indirecte du glucose par les neurones GS hypothalamiques repose sur la libération d’endozépines par les astrocytes, un gliotransmetteur connu pour inhiber la prise alimentaire en réponse à l’hyperglycémie. Nous travaux montrent que les endozépines activent spécifiquement les neurones à pro-opiomélanocortine (POMC) du MBH pour générer leur effet anorexigène. Dans une seconde étude, nous montrons que la détection directe de l’hyperglycémie implique les neurones hypothalamiques dits « high gluco-excited » (HGE). Grâce à des approches pharmacologiques et génétiques, nous mettons en évidence que les canaux redox sensibles Transient Receptor Potential Canonical 3 et 4 (TRPC3/4) sont fondamentaux pour la détection du glucose par les neurones HGE in vitro, la stimulation de la sécrétion d’insuline et la diminution de la prise alimentaire en réponse à l’hyperglycémie cérébrale in vivo. De plus, nos travaux démontrent que les canaux TRPC3 du MBH jouent un rôle clef dans le contrôle de l’homéostasie énergétique. Les travaux de cette thèse permettent de mettre en évidence deux nouveaux mécanismes de détection hypothalamique de l’hyperglycémie : l’un reposant sur l’implication des canaux TRPC3/4 dans les neurones HGE et l’autre proposant les endozépines astrocytaires comme relai du signal « glucose » aux neurones POMC. / Hyperglycemia is detected and integrated by the mediobasal hypothalamus (MBH) which, in turn, inhibits food intake and triggers insulin secretion. The MBH houses specialized glucose-sensitive (GS) neurons, which directly or indirectly modulate their electrical activity in response to changes in glucose level. In a first study, we hypothesized that indirect detection of glucose by MBH GS neurons involves the secretion of endozepine by astrocytes, a gliotransmitter known to inhibit food intake in response to hyperglycemia. The present work shows that endozepines selectively activate anorexigenic MBH pro-opiomelanotortine (POMC) neurons. In the second study, we show that the direct detection of increased glucose level involves hypothalamic glucose-excited (HGE) neurons. Using pharmacological and genetic approaches, we demonstrate that the redox-sensitive Transient Receptor Potential Canonical 3 et 4 (TRPC3/4) channels are involved in MBH HGE response to glucose in vitro and increased insulin secretion and decreased food intake in response to cerebral hyperglycemia in vivo. We also obtained evidences that MBH TRPC3 channel is a critical new player for energy homeostasis. This thesis work identifies two new mechanisms involved in hypothalamic detection of hyperglycemia: the first based on the involvement of TRPC3/4 channels in HGE neurons and the second highlighting the astroglial endozepines as a relay of the “glucose” signal to POMC neurons.

Du, Juan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 110-134). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Kwan Hiu Yee. / "June 2006." / 2+ in the title is superscript. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 131-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Attempts were made to determine the pore properties, such as permeability, rectification and voltage-dependent block, of the putative TRPV4-TRPC1 channel. We demonstrated that this putative TRPV4-TRPC1 heterotetrameric channels displays distinct property different (although not drastically different) from TRPV4 homotetrameric channel with regard to I-V relation, kinetics of cation current, cations permeability and rectification properties. Together, the data from FRET and functional studies both suggest that heterologous expression of TRPV4 and TRPC1 can produce functional TRPV4-TRPC1 heterotetrameric channel. / Hemodynamic blood flow is one of most important physiological factors that control vascular tone. Flow shear stress acts on the endothelium to stimulate the release of vasodilators such as nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factors, causing endothelium-dependent vascular relaxation. In many cases, a key early signal in this flow-induced vascular dilation is Ca2+ influx in endothelial cells in response to flow. There is intense interest in searching for the molecular identity of the channels that mediate flow-induced Ca2+ influx. The present study aimed at identifying an interaction of TRPV4 with TRPC1, and investigating functional role of such a complex in flow-induced Ca2+ influx / In functional study, flow elicited a [Ca2+]i rise in TRPV4-expressing HEK cells. Co-expression of TRPC1 with TRPV4 markedly prolonged this [Ca2+]i transient, and it also enabled this [Ca2+]i transient to be negatively modulated by protein kinase G (PKG). Furthermore, this [Ca2+]i rise was inhibited by an anti-TRPC1 blocking antibody T1E3 and a dominant negative construct TRPC1Delta567-793. Physical interaction of TRPV4 with TRPC1 and functional role of such a complex were also found in the primary cultured rat mesenteric artery endothelial cells (MAECs) and human umbilical vein endothelial cells (HUVECs). A TRPC 1-specific siRNA was used to knock-down TRPC1 protein levels in HUVECs. Interestingly, this siRNA not only reduced the magnitude of flow-induced [Ca2+]i rise, but also accelerated the decay of flow-induced [Ca2+]i transient. Pressure myograph was used to investigate the functional role of such a complex in flow-induced vascular dilation. T1E3 also decreased flow-induced vascular dilation. Thogether, the data from endothelial cells are consistent with those in overexpressed HEK cells, supporting the notion that TRPC 1 interacts with TRPV4 to prolong the flow-induced[Ca2+]i transient, and that TRPV4-TRPC1 complex plays an important role in flow-induced vascular dilation. / In summary, my study demonstrated that TRPV4 is capable of assembling with TRPC1 to form a functional TRPV4-TRPC1 heteromeric channel. TRPV4-TRPC1 heteromeric channel can rapidly translocate to the plasma membrane after Ca 2+ depletion in intracellular stores. This TRPV4-TRPC1 heteromeric channel plays an important role in flow-induced endothelial Ca2+ influx and its associated vascular relaxation. / Ion channels are delivered to the plasma membrane via vesicle trafficking. Thus the vesicle trafficking is a key mechanism to control the amount of TRP channel proteins in the plasma membrane, where they perform their function. TRP channels in vivo are often composed of heteromeric subunits. However, up to the present, there is lack of knowledge on trafficking of heteromeric TRP channels via vesicular translocation. In the present study, we examined the effect of Ca2+ store depletion on the translocation of TRPV4-TRPC1 heteromeric channels to the plasma membrane. Experiments using total internal fluorescence reflection microscopy (TIRFM) and biotin surface labeling showed that depletion of intracellular Ca2+ stores triggered a rapid translocation of TRPV4-TRPC1 channel proteins into the plasma membrane. Fluorescent Ca2+ measurement and patch clamp studies demonstrated that store Ca2+ depletion augmented several TRPV4-TRPC1 complex-related functions, which include store-operated Ca2+ influx and cation current as well as 4alpha-PDD-stimulated Ca2+ influx and cation current. The translocation required stromal interacting molecule 1 (STIM1). Furthermore, TRPV4-TRPC1 complex is more favorably translocated to the plasma membrane than TRPC1 or TRPV4 homomers. Similar mechanisms were identified in native endothelial cells, where the TRPV4-TRPC I complex is a key component mediating flow-induced Ca2+ influx and subsequent vascular relaxation. / With the use of fluorescence resonance energy transfer (FRET), co-immunoprecipitation and subcellular colocalization methods, it was found that TRPC1 interacts physically with TRPV4 to form a heteromeric channel complex. In addition, our experimental results indicate that C-terminal and N-terminal domains of both channels are required for their interaction. / Ma, Xin. / Adviser: Yao Xiaodiang. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 109-121). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Ma, Yan. / "Ca" in the title is subscript. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 143-166). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Feeding is one of the most important behaviors for an animal’s survival. At a gross level, it is known that the nervous system plays a major role in the expression of this complex behavior, yet a detailed understanding of the neural circuits directing feeding behavior remains unknown. Here we identify a command neuron in Drosophila melanogaster whose artificial activation, using dTrpA1, a heat-activated cation channel, induces the appearance of complete feeding behavior. We use behavioral, genetic, cellular and optical imaging techniques to show that the induced behavior is composed of multiple motor programs and can function to uptake exogenous, even noxious, material. Furthermore, we resolve the neuron’s location to the subesophageal ganglion, characterize its pre and post-synaptic sites, and determine its responsiveness to sucrose stimulation. Interestingly, the neuron’s dendritic field is proximal to sweet sensing axon terminals and its baseline activity corresponds to the fly’s satiation state, suggesting a potential point of integration between sensory, motor and motivational systems. The identification of a command neuron for feeding in a genetically tractable organism provides a useful model to develop a deeper understanding of the neural control of this ubiquitous and evolutionarily ancient behavior.