<p>Chloride ion channels are macromolecular pores providing for passage of chloride ions (and certain other inorganic and organic anions) through the cell membrane, down their electrochemical gradients. Chloride channels are differentially expressed in various cells, to best suit specific cellular activities. They are present in practically all living cells, and regardless of cell specialization they play an important role in vital housekeeping functions of cell-volume and pH regulation and in membrane potential stabilization. Regulation of cell volume underlies the structural integrity and constancy of the intracellular milieu. A variety of metabolic pathways have been shown to be sensitive to cell volume, and alterations of cell volume and osmoregulation processes can influence various intracellular signaling and organizing factors.</p><p>Volume-regulated anion channels (VRACs) are believed to play a pivotal role in cell-volume regulating processes. In this report I present data from macroscopic patch-clamp studies of VRACs performed in a fibroblast cell line and from single channel studies of chloride channels (tentatively related to VRACs) in mouse brown adipocytes in primary culture.</p><p>One of the characteristic features of the VRACs is their dependence on the presence of cytoplasmic ATP. In whole-cell experiments, removal of ATP from the pipette solution almost completely prevented activation of VRACs, whereas substitution of ATP with the nonhydrolyzable analog ATPγS did not alter the activation of VRACs. The inhibitors of protein tyrosine kinases (PTK) tyrphostin A25 and B46 depressed VRAC currents in both cases (ATP and ATPγS), but a PTK ineffective analog (tyrphostin A1) did not affect VRAC currents. We infer that in the cell preparation we used, ATP has a dual role in VRAC regulation: it is required for channel-protein phosphorylation and it can influence channel activity through non-hydrolytic binding in a ligand-receptor manner. It can additionally be suggested that tyrosine-specific protein kinases can be involved in the regulation of VRACs, independently of the effects of ATP. We also studied cell cycle-related changes in activation of VRACs by osmotic swelling of cells chemically arrested at different phases of the cell cycle. We found no significant changes during most of the cell cycle, except short periods before and after mitosis and in the quiescent G0 state.</p><p>The single Cl^-channels of brown adipocytes resemble in their electrophysiological phenotype outwardly rectifying Cl^- channels (ORCCs). We investigated the sensitivity of these channels to intracellular Ca²⁺. It appeared that the commonly used Ca²⁺-chelators EGTA and BAPTA could influence the ORCCs currents by themselves, independently of their calcium chelating effects. In some channels, these chelators induced classical flickery-type block of activity, whereas in others there was quasi-blockage, i.e. a peculiar combination of flickery blockage and overall channel activation. The chloride channel blocking agents DIDS and SITS mimicked the true/quasi blockage of EGTA and BAPTA. These phenomena add to the structure-function characteristics of the ORCC molecule. Moderate inhibitory effect of Ca²⁺ within a physiological range of intracellular concentrations (sub-µM) was also detected; however, the biological relevance of this observation, as well as of these Cl^- channels in general, remains to be clarified.</p>

Chloride ion channels are macromolecular pores providing for passage of chloride ions (and certain other inorganic and organic anions) through the cell membrane, down their electrochemical gradients. Chloride channels are differentially expressed in various cells, to best suit specific cellular activities. They are present in practically all living cells, and regardless of cell specialization they play an important role in vital housekeeping functions of cell-volume and pH regulation and in membrane potential stabilization. Regulation of cell volume underlies the structural integrity and constancy of the intracellular milieu. A variety of metabolic pathways have been shown to be sensitive to cell volume, and alterations of cell volume and osmoregulation processes can influence various intracellular signaling and organizing factors. Volume-regulated anion channels (VRACs) are believed to play a pivotal role in cell-volume regulating processes. In this report I present data from macroscopic patch-clamp studies of VRACs performed in a fibroblast cell line and from single channel studies of chloride channels (tentatively related to VRACs) in mouse brown adipocytes in primary culture. One of the characteristic features of the VRACs is their dependence on the presence of cytoplasmic ATP. In whole-cell experiments, removal of ATP from the pipette solution almost completely prevented activation of VRACs, whereas substitution of ATP with the nonhydrolyzable analog ATPγS did not alter the activation of VRACs. The inhibitors of protein tyrosine kinases (PTK) tyrphostin A25 and B46 depressed VRAC currents in both cases (ATP and ATPγS), but a PTK ineffective analog (tyrphostin A1) did not affect VRAC currents. We infer that in the cell preparation we used, ATP has a dual role in VRAC regulation: it is required for channel-protein phosphorylation and it can influence channel activity through non-hydrolytic binding in a ligand-receptor manner. It can additionally be suggested that tyrosine-specific protein kinases can be involved in the regulation of VRACs, independently of the effects of ATP. We also studied cell cycle-related changes in activation of VRACs by osmotic swelling of cells chemically arrested at different phases of the cell cycle. We found no significant changes during most of the cell cycle, except short periods before and after mitosis and in the quiescent G0 state. The single Cl- channels of brown adipocytes resemble in their electrophysiological phenotype outwardly rectifying Cl- channels (ORCCs). We investigated the sensitivity of these channels to intracellular Ca2+. It appeared that the commonly used Ca2+-chelators EGTA and BAPTA could influence the ORCCs currents by themselves, independently of their calcium chelating effects. In some channels, these chelators induced classical flickery-type block of activity, whereas in others there was quasi-blockage, i.e. a peculiar combination of flickery blockage and overall channel activation. The chloride channel blocking agents DIDS and SITS mimicked the true/quasi blockage of EGTA and BAPTA. These phenomena add to the structure-function characteristics of the ORCC molecule. Moderate inhibitory effect of Ca2+ within a physiological range of intracellular concentrations (sub-µM) was also detected; however, the biological relevance of this observation, as well as of these Cl- channels in general, remains to be clarified.

NO / Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H2O2 in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 μM H2O2 increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH4Cl and elevated l-tyrosine, H2O2-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H2O2-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca2+-chelator BAPTA. Thus, BAPTA reduced the level of H2O2-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca2+ and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca2+ binding capacity and, in addition, correlated inversely with H2O2-induced increases in intracellular Ca2+. Our results show that melanin may have an important role in regulating intracellular Ca2+ homeostasis and it is suggested that melanin protects against H2O2-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca2+.

Des azobenzènes photochromiques ont été intégrés à des macrocycles synthétiques pour moduler photochimiquement la chélation de différents ions qui jouent un rôle essentiel dans les processus biologiques. La photocommutation de ces récepteurs a été étudiée (rendements quantiques, réversibilité, état photostationnaire) et les constantes de stabilité ont été déterminées. Le développement d’un récepteur biocompatible du Ca2+ (BAPTA) a permis de moduler la concentration de Ca2+ dans une solution aqueuse avec la lumière. Les molécules entrelacées sont également un sujet majeur dans le domaine de la chimie supramoléculaire. Une méthode de macrocyclisation photochimique, basée sur la dimérisation de l’anthracène, a été appliquée à des assemblages supramoléculaires dans le but de modifier leur topologie et de développer une nouvelle stratégie de photocaténation. En parallèle, un rotaxane a été réalisé en ajoutant des groupements encombrants sur les anthracènes par une réaction de Diels-Alder. / As the availability of ions plays a key role in biological processes, photochromic azobenzene macrocycles were synthetized to photochemically modulate chelation of different ions in various media. The photoswitching of these receptors was evaluated (quantum yields, reversibility, photostationary state) as well as binding constants. Integrating a biocompatible Ca2+ receptor (BAPTA) with azobenzene enabled the modulation of calcium concentration in aqueous solution and to reversibly switch the fluorescence emission of a molecular probe based on photoinduced electron transfer. Interlocked molecules are also a major topic in the field of supramolecular chemistry. A Photochemical macrocyclization method, based on the dimerization of anthracene, was applied to supramolecular assemblies in order to change their topology and develop a new strategy of photocatenation. In parallel, a rotaxane was achieved by adding bulky groups on anthracenes by a Diels-Alder reaction.

Nous cherchons à développer une sonde permettant le relargage sélectif de cations métalliques. Un tel système permettrait de capter et relarguer des cations sans contamination ni perturbation importante de l'environnement proche de l'électrode. Par la suite le dispositif pourrait être miniaturisé et utilisé in vivo. La stratégie adoptée consiste à concevoir des composés possédant un centre redox à proximité d'un ligand ayant une grande affinité vis-à-vis des cations métalliques, en particulier pour le calcium. Des groupes fonctionnels, séparés par un espaceur modulable, permettent le greffage à la surface de l'électrode. Lors de l'oxydation du centre redox initialement neutre, on peut s'attendre à ce que la constante de complexation soit diminuée de manière importante, principalement à cause de la répulsion électrostatique qui tend à éjecter le cation de la cavité. Lors de mon travail de thèse, quatre nouvelles familles de ligands possédant une phénylènediamine comme centre redox ont été synthétisées et étudiées. Parmi ces composés, les dérivés de type " demi-BAPTA " ont permis de démontrer le concept de relargage de calcium pour la première fois en milieu aqueux, à pH physiologique. Cependant, la constante d'affinité étant trop faible pour des applications in vivo, des composés basés sur le ligand BAPTA ont été préparés et s'avèrent prometteurs

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)- benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells and OC2 human oral cancer cells was unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended MDCK and OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. m-3M3FBS at concentrations between 0.1-20 £gM increased [Ca2+]i in a concentration-dependent manner in MDCK cells, however in OC2 cells, m-3M3FBS at concentrations between 10-60 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signals were reduced partly by removing extracellular Ca2+ in the two cell types. m-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, m-3M3FBS pretreatment abolished the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin, cyclopiazonic acid or 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, cyclopiazonic acid or BHQ partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. Collectively, in MDCK and OC2 cells, m-3M3FBS induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels and other unidentified Ca2+ channels. Additionally, 5-100 £gM of m-3M3FBS killed cells in a concentration-dependent manner in OC2 cells. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 £gM) induced apoptosis in a Ca2+-independent manner. We were also interested in knowing whether BAPTA suppressed cell death during oxidative stress in MDCK cells. BAPTA loading altered tBHP (tert-butyl hydroperoxide) and H2O2-induced cell death in a concentration-dependent manner. This suggests that the cell death induced by tBHP and H2O2 appears to be Ca2+-dependent in MDCK cells. The tBHP and H2O2-induced cell death was not suppressed by 2 £gM U73122 (PLC inhibitor), 50 £gM zVAD-fmk (caspase inhibitor), 2 £gM cyclosporin A (a potent inhibitor of the MPTP), 20 £gM PD98059 (ERK inhibitor) or 2 £gM SP600125 (JNK inhibitor). This suggests that the tBHP and H2O2-induced MDCK cells death was not via the PLC, MPTP, caspase, ERK or JNK pathways. Propidium iodide staining, caspase-3 activity assay and cell morphology data suggest that tBHP and H2O2-induced cell death was necrosis, not via apoptosis, and the cell death appears to be caspase-independent and Ca2+-dependent. The effect of the antidepressant paroxetine on [Ca2+]i in OC2 human oral cancer cells is unclear. This study also explored whether paroxetine changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Paroxetine at concentrations between 100-1000 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 50% by removing extracellular Ca2+. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished paroxetine¡Vinduced [Ca2+]i rise. Inhibition of PLC with U73122 did not alter paroxetine-induced [Ca2+]i rise. Paroxetine at 10-50 £gM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca2+ was chelated with BAPTA. Propidium iodide staining suggests that apoptosis played a role in the death. Collectively, in OC2 cells, paroxetine induced [Ca2+]i rise by causing PLC-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Paroxetine also induced cell death in a Ca2+-independent manner.

<p> Calcium (Ca2+) is an important signaling molecule and hence its movement across cell membranes must be tightly regulated. The intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle and endothelium controls the coronary tone. After stimulation, decreasing the [Ca2+]i back to resting levels is achieved mainly by the sodium calcium exchanger (NCX), the plasma membrane calcium pump (PMCA) or the sarcoendoplasmic reticulum calcium pump (SERCA). The present study will focus on NCX and its interactions with SERCA in the smooth muscle and endothelium of pig coronary artery.</p> <p> Aim 1 of my thesis is determination of activity levels of NCX in smooth muscle cells (SMC) and endothelial cells (EC). The NCX activity in cultured cells was approximately 5 times greater in EC than in SMC. The NCX inhibitors KB-R7943 and SEA 0400 blocked the NCX mediated Ca2+ entry, as did collapsing the Na+ gradient with monensin. NCX1 is the isoform largely responsible for NCX activity in SMC and EC. NCX activity was also assayed as the Ca2+ efflux in cultured cells and as Ca2+ uptake in plasma membrane vesicles isolated from freshly isolated smooth muscle.</p> <p> Aim 2 is to assess the existence of a functional NCX mediated Ca2+ entry linked to SERCA in SMC. In the absence of thapsigargin, BAPTA loading SMC increased the NCX mediated uptake. Thapsigargin did not affect the Ca2+ uptake in BAPTA loaded cells but it inhibited the Ca2+ uptake in cells that were not loaded with BAPTA. These data are consistent with a model in which SER acts as a sink for the NCX mediated Ca2+ entry. However, with BAPTA chelation and the resulting lower intracellular Ca2+, the need for SER to act as a sink is eliminated, and NCX is driven in full force. EC did not demonstrate a NCX-SERCA linkage.</p> <p> Arterial SMC and EC differ in their structure and function. The function of SMC is the generation of tone which is achieved by the Ca2+ dependent contractile filaments. Since these filaments are distributed throughout the cell, Ca2+ must be transported to and removed from deep within the cell. As a result, the SER may play a large role in Ca2+ regulation in the SMC. Furthermore, SMC also contain higher levels of high affinity Ca2+ pumps (SERCA and PMCA) and thus Ca2+ is more tightly regulated. Endothelial cells release nitric oxide in response to an increase in [Ca2+]i, which relaxes the smooth muscle. The endothelial nitric oxide sythase produces nitric oxide and is located adjacent to the PM in EC. The SER that removes Ca2+ from deep within the cell cytosol may play a small role in Ca2+ dependent modulation of the endothelial nitric oxide synthase activity. Based on the Western blot data, EC contain a greater amount of the high capacity NCX, thus the larger quantities of Ca2+ can be removed from the cell and the vicinity of endothelial nitric oxide synthase.</p> / Thesis / Master of Science (MSc)

The primary focus of this dissertation is the development of novel fluorescent near-infrared molecules for various applications. In Chapter 1, a compound dU-BZ synthesized via Sonogashira coupling reaction methodology is described. A deoxyuridine building block was introduced to enhance hydrophilic properties and reduce toxicity, while an alkynylated benzothiazolium dye was incorporated for near-IR emission and reduce photodamage and phototoxicity that is characteristic of common fluorphores that are excited by UV or visible light. A 30-fold enhancement of fluorescence intensity of dU-BZ was achieved in a viscous environment. Values of fluorescence quantum yields in 99% glycerol/1% methanol (v/v) of varying temperature from 293 K to 343 K, together with fluorescence quantum yields, radiative and nonradiative rate constants and fluorescence lifetimes in glycerol/methanol solutions of varying viscosities from 4.8 to 950 cP were determined. It was found that both fluorescence quantum yields and fluorescence lifetimes increased with increasing viscosity, which is consistent with results predicted by theory. This suggests that the newly designed compound dU-BZ is capable of functioning as a probe of local microviscosity, and was later confirmed by in vitro bioimaging experiments. In Chapter 2, a new BAPTA (O,O*-bis(2-aminophenyl)ethyleneglycol-N,N,N*,N*-tetra acetic acid) and BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-based calcium indicator, BAPBO-3, is reported. A new synthetic route was employed to simplify both synthesis and purification, which tend to be low yielding and cumbersome for BAPTA derivatives. Upon excitation, a 1.5-fold increase in fluorescence intensity in buffer containing 39 ?? Ca2+ and a 3-fold increase in fluorescence intensity in buffer containing 1 M Ca2+ was observed; modest but promising fluorescence turn-on enhancements. In Chapter 3, a newly-designed unsymmetrical squaraine dye, SQ3, was synthesized. A one-pot synthesis was employed resulting in a 10% yield, a result that is generally quite favorable for the creation of unsymmetrical squaraines Photophysical and photochemical characterization was conducted in various solvents, and a 678 nm absorption maximum and a 692 nm emission maximum were recorded in DMSO solution with a fluorescence quantum yield of 0.32. In vitro cell studies demonstrated that SQ3 can be used as a near-IR probe for bioimaging.