Ca2+ dynamics affect many critical cellular processes. In the bag cell neurons of Aplysia californica, intracellular Ca2+ is elevated during a prolonged period of firing known as the afterdischarge. This consists of a fast and slow phase of firing, which triggers peptide secretion and culminates in egg-laying. The present study examines how Ca2+ influx and release shape neurosecretion and membrane activity. Using capacitance tracking as an index of secretion, a 5 Hz, 1 min train, to mimic the fast phase, induced a clear elevation in the membrane surface area of cultured bag cell neurons. The capacitance change was abolished by replacing external Ca2+ with Ba2+ or addition of the Ca2+ channel blocker, Ni2+. Additionally, the response was reduced by either strong buffering of intracellular Ca2+ or pretreatment with N-ethylmaleimide, an alkylating agent that disrupts vesicular transport. Depleting mitochondrial Ca2+ with the protonophore, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP), also elevated capacitance, while depleting endoplasmic reticulum Ca2+ with the Ca2+-ATPase inhibitor, cyclopiazonic acid, did not. Similarly, FCCP alone depolarized bag cell neurons. In a concentration-dependent manner, FCCP elicited an inward current that was insensitive to Ni2+, associated with an increase in conductance, and a linear current/voltage relationship that reversed around -40 mV. Removal of extracellular Ca2+ reduced the current and left-shifted the reversal, consistent with opening a Ca2+-permeable, voltage-independent, non-selective cation channel. The current was decreased when intracellular Ca2+ was strongly buffered, while fura-imaging demonstrated that FCCP elevated intracellular Ca2+ with a similar time course, suggesting a dependence on intracellular Ca2+. Although both oligomycin A and bafilomycin A, inhibitors of mitochondrial ATP sythetase and V-type H+-ATPase, respectively, gradually increased Ca2+, neither produced a current. The FCCP-induced Ca2+ elevation and the current were also diminished by disabling the mitochondrial permeability transition pore with N-ethylmaleimide. The data suggests that a cation current is preferentially gated by Ca2+ released from the mitochondria, rather than disruption of ATP production. This current could provide depolarizing drive for the afterdischarge. While Ca2+ entry appears to be responsible for initiating neurosecretion, mitochondrial Ca2+ may support prolonged peptide release during and subsequent to the afterdischarge. / Thesis (Master, Physiology) -- Queen's University, 2009-09-18 19:30:32.957

BACKGROUND: Isolated GnRH Deficiency (IGD) is a rare Mendelian disorder, characterized by absent puberty and infertility. Over 30 causal IGD genes have been discovered, yet only 30-35% of IGD cases have a proven genetic etiology, highlighting the importance of new discovery methods. Homozygosity mapping, traditionally used to detect autozygous segments in inbred populations, has recently proven useful for gene discovery in outbred populations. METHODS: Strict quality control (QC) metrics for the SNP genotyped cohort and controls were used. Overall, 653 European IGD probands and 1,911 European controls remained. After pruning, 206,012 SNPs remained. Runs of Homozygosity (ROH) were found and a burden analysis was performed. Areas of known IGD genes, Expression Quantitative Trait Loci (eQTLs) of known genes and novel regions predicted to be associated with IGD were explored. RESULTS: While there was no statistical difference between the ROH burden in cases vs. controls, there was an unexpected trend towards a greater burden in the controls. Of the known gene locations and their eQTL regions, there was a trend in cases having a higher representation of ROHs within these regions. Four ROH regions unique to cases only were also discovered. None of the individuals had a variant in the coding genes found in these regions. CONCLUSIONS: SNP markers are found in coding regions and non-coding regions. This allows for a preview of potential disease causing variation in the non-coding region. Even though no coding gene of interest was found, the non-coding parts of the identified regions are of significant interest.

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Des études récentes montrent que l'homéostasie calcique intracellulaire, ainsi que l'expression et l'activité de canaux ioniques jouent un rôle essentiel dans le contrôle de la prolifération cellulaire aussi bien dans un contexte physiologique que dans certains cancers. Cependant, aucune approche proposant les canaux ioniques comme cible thérapeutique n'est actuellement envisagée dans le cadre des traitements des cancers de la prostate. Dans ce travail nous avons mis en évidence l'expression, la fonctionnalité et l'implication des canaux potassiques calcium-activés (IKca1) dans la prolifération des lignées cellulaires cancéreuses de la prostate humaine. Ces études ont également montré que l'activation du canal lKca1 favorise l'entrée de calcium via un canal calcique de la famille des TRP, le canal TRPV6, impliqué dans l'entrée passive de calcium dans les cellules cancéreuses prostatiques. De plus, par des études de co-immunoprécipitations, nous avons montré que le canal IKca1 et le canal calcique TRPV6 sont associés formant ainsi un complexe moléculaire fonctionnel permettant l'entrée de calcium et la prolifération des cellules cancéreuses prostatiques humaines. Par ailleurs, une suppression du canal IKcat induit la différenciation neuroendocrine des cellules cancéreuses prostatiques humaines. Ceci suggère un rôle essentiel joué par le canal IKca1 pour favoriser la croissance ou induire la différenciation cellulaire. Nos études ont également mis en évidence une surexpression de l'ARNm et de la protéine d'IKca1 dans les tissus prostatiques atteints d'un cancer de la prostate humaine. Ces données permettraient de proposer ces canaux ioniques comme marqueurs de cancer et/ou comme cibles thérapeutiques potentielles dans le traitement des cancers de la prostate humaine. / Recent studies show that the intracellular calcium homeostasis, as well as expression and the activity of ionic channels play an essential role in the control of cell proliferation as weIl in a physiological context as in certain cancers. However, no approaches offering ionic channels as therapeutic target is nowadays envisaged as part of the treatments of the cancers of the prostate. ln the present work, we showed the expression, functionality and involvement of calcium-activated potassium channels (IKCa1) in the proliferation of the human prostate cancer cells. These studies also showed that the activation of the IKca1 channel favours the calcium entry via a member of the TRP family of calcium channels, TRPV6, in the prostate cancer cells. Besides, by studies of co-immunoprécipitations, we showed that the IKca1 potassium channel and the TRPV6 calcium channel form a functional molecular complex allowing the calcium entry and the proliferation of the prostate cancer cells. Moreover, a down-regulation of the 1Kca1 channel leads to the neuroendocrine differentiation of the human prostate cancer cells. This suggests an essential role played by the 1Kca1 channel to favour growth or lead to cell differentiation. Our immunohistotluorescence studies also showed an overexpression of IKca1 mRNA and protein in human prostate cancer compared to non-tumor tissues. These data would allow to propose these ion channels as markers and/or as potential therapeutic targets in the treatment of the human prostate cancers.

The neurohormonal mechanisms underlying the regulation of extracellular osmolality are of critical physiological importance. These mechanisms act to maintain the osmolality of human plasma close to a “set-point” of about 290 milliosmoles per litre. The magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON), synthesize and secrete the neurohypophysial hormones vasopressin (VP) and oxytocin (OT). The primary hormonal regulator of osmolality is VP, which is released by the MNCs as a function of plasma osmolality and acts by controlling water reabsorption at the kidneys. MNCs decrease their volume and thus plasma membrane tension in response to acute increases in external osmolality and lack the compensatory mechanisms that limit volume changes in most cell types. This enables them to transduce changes in osmolality into changes in excitability via a mechanosensitive cation channel. It has been shown in vivo that sustained increases in plasma osmolality, however, cause marked hypertrophy of the MNCs that is part of a structural and functional adaptation that is thought to enable the MNCs to secrete large quantities of VP for prolonged periods. The mechanism of this important structural and functional adaptation of MNCs is difficult to address in an in vivo preparation and so an in vitro model of acutely isolated MNCs was used to pharmacologically assess the hypertrophy. It was observed that MNCs exposed to sustained hypertonic solutions, underwent an immediate shrinkage followed by a hypertrophy over 90 minutes and quickly recovered when reintroduced to isotonic conditions. This effect was found to depend on the size of the increase in osmolality, as smaller increases in osmolality resulted in smaller shrinkage and hypertrophy of the MNCs. Hypertrophy was shown to be independent of cell volume regulatory processes as inhibitors of the Na+-K+-Cl- cotransporter did not affect hypertrophy. Hypertrophy was also shown to be dependent on activation of phospholipase C (PLC) and protein kinase C (PKC), as adding inhibitors of these enzymes to the hypertonic solution prevented hypertrophy. Hypertrophy could occur in isotonic conditions by inducing cell depolarization, increasing intracellular calcium ([Ca2+]i) and by activating PKC, thus showing each of these processes are involved in hypertrophy. Recovery from hypertrophy depends upon dynamin-mediated endocytosis as blocking dynamin function prevented recovery. In addition, exposing the MNCs to hypotonic solution resulted in an immediate enlargement followed by a sustained decrease in cell size. Finally, exposing acutely isolated MNCs to hypertonic solution for two hours resulted in a 37% increase in the immunolabeling of the L-type Ca2+ channel CaV1.2 subunit. This increase in CaV1.2 immunolabeling does not depend on action potential firing as adding tetrodotoxin (TTX) to the hypertonic solution failed to prevent the increase. This project will help to elucidate the mechanisms underlying this interesting example of neuroendocrine adaptation and will help us to understand the regulation of body fluid balance during chronic challenges as seen in the elderly and chronically ill.

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