<p>During the smoltification the Atlantic salmon (Salmo salar) develop different adaptations to survive in oceanic environment. One of the most important adaptations is the ability to excrete the surplus of salt through the gills. The excretion is controlled by an enzyme called Na+/K+-ATPase which is produced in an α-subunit by two gene isoforms called α1a and α1b. Enzyme activity is increasing during the smoltification process and is a strong indicator for that the process is taking place. The aim of this study was to investigate a landlocked strain of Atlantic salmon and see how the enzyme activity is developing in comparison with the gene expression of the mRNA that is coded for the enzyme. The study was carried out between March and May in the hatchery in Brattfors, Värmland. Fish were sampled at four occasions. The enzyme activity was compared between two groups of salmon where one group had full ration of food, 100% and the other group had a 15% food ration. The enzyme activity for the 100% group was then compared with the gene expression from the same group. The hypothesis was that food availability should effect smoltification and that the 15% group would have a faster increase in activity compared with the 100% group. There should also be some correlation between enzyme activity and gene expression. Na+/K+-ATPase enzyme activity showed no major differences between the groups except for a significant difference at the last sampling. Both groups had a large increase in activity from the second to the third sampling with a peak on 3.16 µmol ADP/mg/h at most. This was followed by a drop in activity at the last sampling date. The gene expression showed a fast increase of the α1b gene over the study with drop in the last sampling and the α1a gene had a constant increase from the first to the last sampling. The comparison with enzyme activity and gene expression showed a weak correlation. Compared with studies done on anadromous salmon and the land locked salmon in this study had a different development in gene expression. This could be explained that the different life strategies play an important role how the genes are expressed.</p> / <p>Under smoltifieringen utvecklar atlantlaxen (Salmo salar) olika anpassningar för att överleva i havsmiljö. En av de vikigaste anpassningarna är att utsöndra överskott av salt via gällarna. Exkretionen är kontrollerad av ett enzym som heter Na+/K+-ATPas som produceras i en α-subenhet av två isoformer av gener som heter α1a och α1b. Enzym aktiviteten ökar under smoltifieringen och är en stark indikator på att processen sker. Målet med denna studie var att undersöka en sjövandrande stam av atlantlax och se hur enzymaktiviteten utvecklas i jämförelse med gen expressionen av mRNA som kodar för produktionen av enzymet. Studien genomfördes vid fiskodlingen i Brattfors, Värmland där prover togs vid fyra tillfällen. Enzymaktiviteten jämfördes mellan två grupper av lax där en grupp fick full matranson 100 % och en grupp fick 15 % matranson. Senare jämfördes enzymaktiviteten för 100 % gruppen med gen expressionen inom samma grupp. Hypotesen var att tillgängligheten på mat skulle påverka smoltifieringen och att 15 % gruppen skulle ha en snabbare ökning i jämförelse med 100 % gruppen. Det skulle också vara en viss korrelation mellan enzymaktivitet och gen expression. Na+/K+-ATPas enzym aktiviteten visade inga större skillnader mellan grupperna förutom vid sista provtagningen. Båda grupperna hade en stor ökning från den andra till den tredje provtagningen med den högsta aktiviteten på 3.16 µmol ADP/mg/h. Detta följdes av ett fall i aktivitet vid sista provtagningen. Gen expressionen visade en snabb ökning av α1b genen över studien med en nedgång vid sista provtagningen och α1a hade en konstant men mindre ökning från första till sista provtagningen. Jämförelsen mellan enzymaktivitet och gen expression visade på en svag korrelation. Det fanns en skillnad i gen expression mellan studier gjorda på anadrom lax och sjövandrande lax i denna studie. Detta kan förklaras av att de olika livsstrategierna spelar en betydande roll i hur generna uttrycks.</p>

During the smoltification the Atlantic salmon (Salmo salar) develop different adaptations to survive in oceanic environment. One of the most important adaptations is the ability to excrete the surplus of salt through the gills. The excretion is controlled by an enzyme called Na+/K+-ATPase which is produced in an α-subunit by two gene isoforms called α1a and α1b. Enzyme activity is increasing during the smoltification process and is a strong indicator for that the process is taking place. The aim of this study was to investigate a landlocked strain of Atlantic salmon and see how the enzyme activity is developing in comparison with the gene expression of the mRNA that is coded for the enzyme. The study was carried out between March and May in the hatchery in Brattfors, Värmland. Fish were sampled at four occasions. The enzyme activity was compared between two groups of salmon where one group had full ration of food, 100% and the other group had a 15% food ration. The enzyme activity for the 100% group was then compared with the gene expression from the same group. The hypothesis was that food availability should effect smoltification and that the 15% group would have a faster increase in activity compared with the 100% group. There should also be some correlation between enzyme activity and gene expression. Na+/K+-ATPase enzyme activity showed no major differences between the groups except for a significant difference at the last sampling. Both groups had a large increase in activity from the second to the third sampling with a peak on 3.16 µmol ADP/mg/h at most. This was followed by a drop in activity at the last sampling date. The gene expression showed a fast increase of the α1b gene over the study with drop in the last sampling and the α1a gene had a constant increase from the first to the last sampling. The comparison with enzyme activity and gene expression showed a weak correlation. Compared with studies done on anadromous salmon and the land locked salmon in this study had a different development in gene expression. This could be explained that the different life strategies play an important role how the genes are expressed. / Under smoltifieringen utvecklar atlantlaxen (Salmo salar) olika anpassningar för att överleva i havsmiljö. En av de vikigaste anpassningarna är att utsöndra överskott av salt via gällarna. Exkretionen är kontrollerad av ett enzym som heter Na+/K+-ATPas som produceras i en α-subenhet av två isoformer av gener som heter α1a och α1b. Enzym aktiviteten ökar under smoltifieringen och är en stark indikator på att processen sker. Målet med denna studie var att undersöka en sjövandrande stam av atlantlax och se hur enzymaktiviteten utvecklas i jämförelse med gen expressionen av mRNA som kodar för produktionen av enzymet. Studien genomfördes vid fiskodlingen i Brattfors, Värmland där prover togs vid fyra tillfällen. Enzymaktiviteten jämfördes mellan två grupper av lax där en grupp fick full matranson 100 % och en grupp fick 15 % matranson. Senare jämfördes enzymaktiviteten för 100 % gruppen med gen expressionen inom samma grupp. Hypotesen var att tillgängligheten på mat skulle påverka smoltifieringen och att 15 % gruppen skulle ha en snabbare ökning i jämförelse med 100 % gruppen. Det skulle också vara en viss korrelation mellan enzymaktivitet och gen expression. Na+/K+-ATPas enzym aktiviteten visade inga större skillnader mellan grupperna förutom vid sista provtagningen. Båda grupperna hade en stor ökning från den andra till den tredje provtagningen med den högsta aktiviteten på 3.16 µmol ADP/mg/h. Detta följdes av ett fall i aktivitet vid sista provtagningen. Gen expressionen visade en snabb ökning av α1b genen över studien med en nedgång vid sista provtagningen och α1a hade en konstant men mindre ökning från första till sista provtagningen. Jämförelsen mellan enzymaktivitet och gen expression visade på en svag korrelation. Det fanns en skillnad i gen expression mellan studier gjorda på anadrom lax och sjövandrande lax i denna studie. Detta kan förklaras av att de olika livsstrategierna spelar en betydande roll i hur generna uttrycks.

Apoptosis is a special form of cell death that if non-functional may lead to diseases such as cancer. A reduction of the intracellular potassium ion (K+) content is necessary for activating enzymes important for the execution of apoptosis. Pharmacological modulation of K+ fluxes to reduce intracellular K+ in cancer cells might therefore force the cells into apoptosis and decrease tumour cell mass. Human malignant pleural mesothelioma (MPM) is a form of cancer often caused by asbestos exposure. Although asbestos has been banned in the Western World, the incidence of MPM is expected to increase. Cisplatin is the first-line chemotherapy for MPM, but acquired resistance to the drug is a clinical problem. This thesis is mainly based on work with the human malignant pleural mesothelioma cell line (P31 wt) and a cisplatin-resistant sub-line (P31 res). The aim was to first characterize K+ fluxes in P31 wt and P31 res cells, and then manipulate them in order to reduce intracellular K+ and induce apoptosis with K+ manipulation alone or in combination with cisplatin. Characterization of K+ fluxes in P31 wt cells showed that: 1) ouabain, a digitalis-like drug, and specific blocker of the Na+, K+, ATPase pump, effectively inhibited K+ uptake, 2) bumetanide, a diuretic, and an inhibitor of the Na+, K+, 2Cl-¬-cotransporter, had a transient effect on K+ uptake, and 3) the antifungal drug amphotericin B stimulated K+ efflux. In order to determine intracellular K+ content, the potassium-binding fluorescent probe PBFI-AM was used in a 96-well plate assay. After a 3-h incubation with ouabain, with or without bumetanide, combined with amphotericin B, the intracellular K+ content was reduced in P31 wt cells but not in P31 res cells. Ouabain induced apoptosis in both P31 wt and P31 res cells. P31 res cells were sensitized to cisplatin by ouabain, since 10 mg/L cisplatin in combination with ouabain induced about the same percentage of apoptotic cells as 40 mg/L cisplatin. Apoptosis was executed via caspase-3 activation in both P31 wt and P31 res cells. Amphotericin B enhanced ouabain-induced apoptosis in P31 wt cells via caspase-9 activation, with increased caspase-3 activation and DNA fragmentation as consequences. Ouabain-induced apoptosis in P31 res cells was executed via increased expression of pro-apoptotic Bak. The combination of cisplatin with ouabain and amphotericin B was stressful to both P31 wt and P31 res cells, since SAPK/JNK a known factor in stress-induced apoptosis was activated. In conclusion, K+ flux manipulation with clinical used drugs can induce apoptosis per se and also enhance cisplatin-induced apoptosis in P31 wt and P31 res cells.

La Ciclosporina A (CsA) es un fármaco inmunosupresor que ha supuesto una revolución en el trasplante de órganos. A pesar de sus propiedades anti-inflamatorias, su uso se ha visto limitado por los efectos tóxicos que causa en algunos pacientes. La CsA inhibe la trascripción de genes involucrados en el sistema inmune. Se cree que esta inhibición es la causante de los efectos tóxicos producidos por el fármaco. El modo de acción de CsA está mediado por la unión de sus principales receptores intracelulares, las ciclofilinas (Cyps). Las ciclofilinas son proteínas que están conservadas en todas las especies y su principal función está involucrada en el plegamiento correcto de otras proteínas. Se ha descrito que los efectos tóxicos producidos por CsA podían estar mediados directamente por sus receptores las ciclofilinas. A pesar de que se descrito que las ciclofilinas están involucradas en diferentes funciones, no se les atribuye una función específica. En nuestro laboratorio hemos estado interesados en las posibles vías que involucran a las Cyps y si éstas tienen relación con lo efectos causados por CsA. En esta tesis se ha intentado identificar proteínas que interaccionan con CypA, CypB y FKBP12 y su implicación en los efectos tóxicos producidos por CsA. Hemos identificado una serie de interacciones y hemos estudiado más a fondo dos de ellas. Estas interacciones son las ocurridas entre la ciclofilina B y la subunidad beta de la bomba sodio/potasio, la ciclofilina A y la subunidad beta de la bomba sodio/potasio y por último la ciclofilina B y la subunidad b1 de la ATP sintetasa mitocondrial. Una vez identificadas estas interacciones hemos realizado ensayos funcionales con cada una de ellas. Por un lado hemos estudiado si la actividad de la bomba sodio/potasio estaba afectada en células renales humanas tratadas con CsA y células que habían sido silenciadas para la ciclofilina B y ciclofilina A. Por otro lado hemos realizado un estudio de la actividad y expresión de los complejos de la cadena respiratoria mitocondrial en células renales tratadas con CsA y células renales que habían sido silenciadas para los genes CypA y CypB. A parte de las interacciones encontradas, hemos visto que la actividad de la bomba Na/K está disminuida por el fármaco y que en esta disminución estaría involucrada la ciclofilina B, también hemos visto que los complejos de la cadena respiratoria mitocondrial estarían afectados en células tratadas con CsA y en células interferidas tanto para CypB como para CypA. Como conclusión podríamos sugerir que las ciclofilinas estarían involucradas en los efectos nefrotóxicos que produce la CsA. Estos efectos nefrotóxicos causados por el tratamiento con CsA podrían estar mediados por vias alternativas a la clásicamente descrita, como es la inhibición de la calcineurina. Dichas vías estarían integradas por nuevos efectores tales como Na/K-ATPasa, ATP sintetasa, así como otras putativas dianas que en el futuro serán estudiadas en nuestro laboratorio. / Cyclosporine A is an immunosuppressive drug that has revolutionized organ transplantation. Even though it has anti-inflammatory property, its used has been reduced due to the renal toxic effects caused in some patients. CsA inhibits transcription of genes involved in the immune system. The CsA mode of action involves the binding of its main receptors, the cyclophilins (Cyps). Cyps are proteins conserved in all species and their main function is the correct folding of immature proteins. It has been describes that the main toxic effects caused by CsA could be mediated directly by its receptors, the cyclophilins. Although Cyps are involved in many processes, there is not a specific function for them. In our lab, we have identified a number of proteins that interact with Cyclophilin A (CypA), Cyclophilin B (CypB) and FK-Binding Protein 12 (FKBP12) by yeast two hybrid assays. Some of those novel interactions were confirmed by pull-down and co-immunoprecipitation assays. Finally, we have also carried out functional assays in some of those interactions. As a conclusion, we could suggest that cyclophilins would be involved in the nephrotoxic effects caused by cyclosporine A. Those effects are mediated by alternative pathways. Those pathways would be integrated by novel effectors such as the sodium/potassium ATPase, mitochondrial ATP synthase and also other proteins that will be studied in our lab.

Most vertebrates suffer permanent damage after minutes of anoxia. Many insects however, have part of their life cycle in anoxia or constant hypoxia, such as during their egg-hatching phase, by living as deep burrowers, or at high altitudes. Insects are able to survive in anoxia from hours to days, or even months by developing various strategies through evolution. For example, the locusts (Locusta migratoria) enter a reversible coma during anoxia that is associated with an arrest of ventilation, and a reinitiation of ventilation when returned to normoxia. This coma is correlated with a surge in the concentration of extracellular potassium ions ([K+]o), and recovery from this reversible coma is dependent on re-establishing the functional [K+]o. Prior exposure to a sublethal heatshock (HS)-preconditioning grants locusts a temporary resilience to anoxia; however, the molecular mechanisms of this protection are still unclear. This project investigated the effects of HS-preconditioning on locusts’ ventilation, the total enzymatic activity of the Na+/K+-ATPase, as well as its distribution within the metathoracic ganglion and tested the hypothesis that HS-preconditioning alters locusts’ ventilation and increases the totally Na+/K+-ATPase activity and its concentration within neuronal membranes. I recorded electromyograms of locusts’ ventilatory motor patterns in the presence and absence of anoxic coma by placing a copper wire electrode on ventilatory muscles 161 or 173 in control and HS-preconditioned animals. In addition, I studied the enzymatic activity of the Na+/K+-ATPase using a pyruvate kinase/lactate dehydrogenase assay and the localization of the Na+/K+-ATPase using immunohistochemistry in control and HS-preconditioned locusts at different stages of coma. I found that the ventilatory cycle period was decreased and the ventilatory muscle burst duration was increased after recovery from anoxic coma in HS-preconditioned locusts. I also found that anoxia did not affect the activity or the localization of the Na+/K+-ATPase. However, HS-preconditioning increased the total activity of the Na+/K+-ATPase and the localization of the Na+/K+-ATPase within the neuronal membranes. From this project, I concluded that HSpreconditioning affected locusts’ ventilatory motor pattern after recover from anoxia and increased the total activity and the neuronal membrane localization of the Na+/K+-ATPase. / Thesis (Master, Biology) -- Queen's University, 2011-09-26 13:14:48.472

Neurons perform complex computations, communications and precise transmissions of information in the form of action potentials (APs). The high level of heterogeneity and complexity at all levels of organization within a neuron and the functional requirement of highly permeable cell membranes leave neurons exposed to damage when energy levels are insufficient for the active maintenance of ionic gradients. When energy is limiting the ionic gradient across a neuron’s cell membrane risks being dissipated which can have dire consequences. Other researchers have advocated “generalized channel arrest” and/or “spike arrest” as a means of reducing the neuronal permeability allowing neurons to adjust the demands placed on their electrogenic pumps to lower levels of energy supply. I investigated the consequences of hypoxia on the propagation of a train of APs down the length of a fast conducting axon capable of transmitting APs at very high frequencies. Under normoxic conditions I found that APs show conduction velocities and instantaneous frequencies nearly double that of neurons experiencing energy limiting hypoxic conditions. I show that hypoxia affects AP conduction differently for different lengths of axon and for APs of different instantaneous frequencies. Action potentials of high instantaneous frequency in branching lengths of axon within ganglia were delayed more significantly than those in non-branching lengths contained within the connective and fail preferentially in branching axon. I found that octopamine attenuates the effects of hypoxia on AP propagation for the branching length of axon but has no effect on the non-branching length of axon. Additionally, for energetically stable cells, application of the anti-diabetic medication metformin or the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 resulted in a reduced performance similar to that seen in neurons experiencing energetic stress. Furthermore both metformin and ZD7288 affect the shape of individual APs within an AP train as well as the original temporal sequence of the AP train, which encodes behaviourally relevant information. I propose that the reduced performance observed in an energetically compromised cell represents an adaptive mechanism employed by neurons in order to maintain the integrity of their highly heterogeneous and complex organization during periods of reduced energy supply. / Thesis (Master, Biology) -- Queen's University, 2011-10-07 14:41:46.972

Previous studies have shown that ischemia-reperfusion (I/R) injury is associated with cardiac dysfunction and depression in sarcolemmal Na+-K+-ATPase activity. This study was undertaken to evaluate the role of proteases in these alterations by subjecting rat hearts to different times of global ischemia, and reperfusion after 45 min of ischemia. Decreases in Na+-K+-ATPase activity at 60 min of global ischemia were associated with augmented activities of both calpain and MMPs and depressed protein content of β1- and β2-subunits, without changes in α1- and α2-subunits of the enzyme. However, reperfusion of ischemic heart produced depression in Na+-K+-ATPase activity, no change in the augmented calpain activity, but decreases in augmented MMP-2 activity and Na+-K+-ATPase content. MDL28170, a calpain inhibitor, was more effective in attenuating I/R-induced alterations than doxycycline, an MMP inhibitor. Incubation of control SL preparation with calpain, unlike MMP-2, depressed Na+-K+-ATPase activity and decreased α1, α2 and β2 without changes in β1. These results support the view that activation of calpain is involved in depressing Na+-K+-ATPase activity and degradation of its subunits in hearts subjected to I/R injury.

Previous studies have shown that ischemia-reperfusion (I/R) injury is associated with cardiac dysfunction and depression in sarcolemmal Na+-K+-ATPase activity. This study was undertaken to evaluate the role of proteases in these alterations by subjecting rat hearts to different times of global ischemia, and reperfusion after 45 min of ischemia. Decreases in Na+-K+-ATPase activity at 60 min of global ischemia were associated with augmented activities of both calpain and MMPs and depressed protein content of β1- and β2-subunits, without changes in α1- and α2-subunits of the enzyme. However, reperfusion of ischemic heart produced depression in Na+-K+-ATPase activity, no change in the augmented calpain activity, but decreases in augmented MMP-2 activity and Na+-K+-ATPase content. MDL28170, a calpain inhibitor, was more effective in attenuating I/R-induced alterations than doxycycline, an MMP inhibitor. Incubation of control SL preparation with calpain, unlike MMP-2, depressed Na+-K+-ATPase activity and decreased α1, α2 and β2 without changes in β1. These results support the view that activation of calpain is involved in depressing Na+-K+-ATPase activity and degradation of its subunits in hearts subjected to I/R injury.

No description available.

Doctor of Philosophy (Medicine) / Glutamate transporters play a role in removing extracellular excitatory neurotransmitter, L-glutamate into the cells. The rate of the uptake depends on the density of the transporters at the membrane. Some studies claimed that glutamate transporters could transit between the cytoplasm and the membrane on a time-scale of minutes. The present study examined the distribution of glutamate transporter GLAST predominantly expressed in rat cortical cultured astrocytes between the membrane and the cytoplasm by using deconvolution microscopy and then analyzing the images. The regulation of the distribution of GLAST was studied in the presence of glutamate transporter substrate (D-aspartate), purinergic receptor activators (α,β-methylene ATP, adenosine), neuroleptic drugs (clozapine, haloperidol), ammonia (hyperammonia) and Na+/K+-ATPase inhibitors (ouabain, digoxin and FCCP). It was demonstrated that the translocation of GLAST towards the plasma membrane was induced by D-aspartate, α,β-methylene ATP, adenosine, clozapine and ammonia (at 100 μM and very high concentrations of 10 mM). However, the inhibition of Na+/K+-ATPase activity had an opposite effect, resulting in redistribution of GLAST away from the membrane. It has previously been claimed that the membrane-cytoplasm trafficking of GLAST was regulated by phosphorylation catalysed by protein kinase C delta (PKC-delta). Involvement of this mechanism has, however, been put to doubt when rottlerin, a PKC-delta inhibitor, used to test the hypothesis showed to inhibit Na+/K+-ATPase-mediated uptake of Rb+, suggesting that rottlerin influenced the activity of Na+/K+-ATPase. As Na+/K+-ATPase converts ATP to energy and pumps Na+, K+ ions, thus helping to maintain normal electrochemical and ionic gradients across the cell membrane. Its inhibition also reduced D-aspartate transport and could impact on the cytoplasm-to-membrane traffic of GLAST molecules. Furthermore, rottlerin decreased the activity of Na+/K+-ATPase by acting as a mitochondrial inhibitor. The present study has focused on the inhibition of Na+/K+-ATPase activity by rottlerin, ouabain and digoxin in homogenates prepared from rat kidney and cultured astrocytes. The activity of Na+/K+-ATPase was measured by the absorption of inorganic phosphate product generated from the hydrolysis of ATP and the fluorescent transition of the dye RH421 induced by the movement of Na+/K+-ATPase. This approach has a potential to test whether the rottlerin effect on Na+/K+-ATPase is a direct inhibition of the enzyme activity. Rottlerin has been found to block the activity of Na+/K+-ATPase in a dose-dependent manner in both rat kidney and astrocyte homogenates. Therefore, rottlerin inhibited the activity of Na+/K+-ATPase directly in a cell-free preparation, thus strongly indicating that the effect was direct on the enzyme. In parallel experiments, ouabain and digoxin produced similar inhibitions of Na+/K+-ATPase activity in rat kidney while digoxin blocked the activity of Na+/K+-ATPase to a greater extent than ouabain in rat cortical cultured astrocytes. In a separate set of experiments, Na+/K+-ATPase in the astrocytic membrane was found to be unsaturated in E1(Na+)3 conformation in the presence of Na+ ions and this could explain the differences between the effects of digoxin and ouabain on the activity of Na+/K+-ATPase in rat astrocytes. In addition, it was found that at low concentrations of rottlerin, the activity of Na+/K+-ATPase was increased rather than inhibited. This effect was further investigated by studying rottlerin interactions with membrane lipids. The activity of Na+/K+-ATPase has been reported to be regulated by membrane lipids. The enzyme activity can be enhanced by increasing fluidity of the lipid membrane. I have, therefore, proposed that rottlerin binds to the membrane lipids and the effects of rottlerin on Na+/K+-ATPase are mediated by changes in the properties (fluidity) of the membrane. The hypothesis was tested by comparing rottlerin and a detergent, DOC (sodium deoxycholate), for their binding to the lipids by using a DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) monolayer technique. DOC has been shown to both increase and inhibit activity of Na+/K+-ATPase in a manner similar to that displayed by rottlerin. The effects of rottlerin and DOC on the DMPC monolayers were studied by measuring the surface pressure of DMPC monolayers and surface area per DMPC molecule. I established that both rottlerin and DOC decreased the surface pressure of DMPC monolayers and increased the surface area per DMPC molecule. This indicates that both rottlerin and DOC penetrated into the DMPC monolayers. If rottlerin can interact with the lipids, changes in fluidity of the lipid membrane cannot be ruled out and should be considered as a possible factor contributing to the effects of rottlerin on the activity of Na+/K+-ATPase. Overall, the study demonstrates that rottlerin is not only a PKC-delta inhibitor but can have additional effects, both on the enzyme activities (Na+/K+-ATPase) and/or on lipid-containing biological structures such as membranes. The findings have implication not only for studies where rottlerin was used as a supposedly specific PKC-delta inhibitor but also for mechanisms of its toxicity.