Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

O rim é tanto um órgão-alvo como a principal fonte de produção de ET-1, peptídio que regula a excreção de Na+ e água por este órgão que expressa os seus respectivos receptores, ETA e ETB, além dos trocadores NHE1 e NHE3 que são essenciais para o equilíbrio ácido base e hidroeletrolítico das células. Assim, o objetivo deste estudo foi investigar, em células IRPTC, o papel de ET-1 na atividade dos trocadores NHE1 e NHE3. Nossos resultados indicam que o tratamento agudo com ET-1 (10-9 M) aumenta a velocidade de recuperação do pHi (dpHi/dt) nos dois primeiros minutos após o pulso ácido, sugerindo aumento na atividade dos trocadores NHE1 e NHE3, que ocorre via ativação dos receptores ETA e ETB e parece ser secundária à atividade da p90RSk e p38MAPK. O tratamento crônico com ET-1 (10-9 M) reduz a dpHi/dt nos dois primeiros minutos após o pulso ácido, o que sugere redução na atividade dos trocadores NHE1 e NHE3, que pode ser secundária à inibição da Na+, K+-ATPase por ET-1. / The kidney is both a target organ and the main source of ET-1 production, a peptide that regulates the excretion of Na+ and water by this organ that expresses its respective receptors, ETA and ETB, in addition to the NHE1 and NHE3 exchanger which are Essential for the basic acid and electrolyte balance of cells. Thus, the objective of this study was to investigate, in IRPTC cells, the role of ET-1 in the activity of the NHE1 and NHE3 exchanger. Our results indicate that the acute treatment with ET-1 (10-9 M) increases the rate of recovery of pHi (dpHi/dt) in the first two minutes after the acid pulse, suggesting an increase in the activity of the NHE1 and NHE3 exchanger, which occurs via activation Of ETA and ETB receptors and appears to be secondary to the activity of p90RSk and p38MAPK. Chronic treatment with ET-1 (10-9 M) reduces dpHi/dt in the first two minutes after the acid pulse, suggesting a reduction in NHE1 and NHE3 exchanger activity, which may be secondary to inhibition of Na+, K+- ATPase by ET-1.

In the yellow fever mosquito, Aedes aegypti (L.), hemolymph-circulating diuretic hormones act upon the renal organs (Malpighian tubules) to regulate primary urine composition and secretion rate; however, the molecular endocrine mechanisms underlying rapid water elimination upon adult eclosion and blood feeding are not fully understood. Bioinformatic analysis of the current Aedes aegypti genome assembly reveals only a single predicted corticotropin releasing factor (CRF)-like diuretic hormone 44 (DH44) gene, but two DH44 receptor genes. The tissue expression profiles of the DH44 receptor(s), and specifically the identity of the DH44 receptor(s) in the Malpighian tubule, are undetermined in any mosquito species. This dissertation shows that Vectorbase gene ID AAEL008292 encodes a DH44 receptor (AaegGPRdih1) transcribed in Malpighian tubules. Sequence analysis and transcript localization indicate that AaegGPRdih1 is the co-ortholog of the Drosophila melanogaster DH44 receptor (CG12370-PA). The presence of conserved amino acid residues between AaegGPRdih1 and vertebrate CRF receptors suggests this mosquito receptor modulates multiple G protein-dependent intracellular signaling pathways. Quantitative PCR analysis of a time course of Malpighian tubule cDNA reveals AaegGPRdih1 abundance increases paralleling periods of observed urination. This suggests that target tissue receptor biology is linked to the known periods of release of diuretic hormones from the nervous system, pointing to a common up-stream regulatory mechanism. Higher relative abundance of AaegGPRdih1 transcript in female Malpighian tubules 24 hours after blood feeding suggests a role for AaegGPRdih1 in the excretion of nitrogen waste. RNA-mediated silencing to establish the significance of AaegGPRdih1 to mosquito Malpighian tubule physiology was inconclusive.

Organische-Anionen-Transporter (OATs) sind maßgeblich an der Ausscheidung von körpereigenen und körperfremden Substanzen über die Niere beteiligt. In Ratten, einem häufig verwendeten Tiermodell in präklinischen Studien, ist bekannt, dass die basolateral lokalisierten Organischen-Anionen-Transporter 1 (Oat1) und 3 (Oat3) in männlichen Tieren stärker und darüber hinaus Testosteron abhängig exprimiert werden. Beide Transporter sind an der Ausscheidung von organischen Anionen, einschließlich negativ geladener Medikamente wie zum Beispiel Adefovir, Furosemid oder Penicillin, beteiligt. In den menschlichen Nieren zählen der OAT1 und der OAT3 zu den klinisch relevanten Transportern, deren Funktionen im Laufe neuer Medikamentenentwicklung berücksichtigt werden sollten. Für das Antibiotikum Penicillin wurde bei Frauen ein vermehrtes Auftreten von Nebenwirkungen im Vergleich zu Männern gezeigt. Dieses erhöhte Risiko könnte möglicher Weise auf einer geschlechtsabhängigen Expression des OAT1 und OAT3 zurückzuführen sein. Ziel der vorliegenden Arbeit war es, den molekularen Mechanismus, der für die höhere Oat1 und Oat3 Expression in den männlichen Rattennieren verantwortlich ist, zu identifizieren. Mit Hilfe von Luciferase assays wurde die Aktivierung von Ratten und menschlichen Oat1/OAT1 und Oat3/OAT3 Promotoren untersucht. Hierzu wurden zunächst Oat1/OAT1 und Oat3/OAT3 Promotorkonstrukte generiert, welche unterschiedlich lange Promotorregionen enthielten, und diese anschließend transient in OK oder LLC-PK1 Zellen transfiziert. Mittels Co-Transfektion potentieller transkriptioneller Regulatoren konnte deren Einfluss auf die Promotoraktivität von Oat1/OAT1 und Oat3/OAT3 untersucht werden. Zur Identifikation geschlechtsabhängig exprimierter Gene in der proximalen Tubuluszelle der Rattennieren wurden von vier männlichen und vier weiblichen Tieren je eine Niere präpariert und deren RNA mit Hilfe eines microarrays und real-time PCR analysiert. Im Rahmen dieser Arbeit konnte gezeigt werden, dass die bereits bekannte männlich dominierende Expression von Oat1 und Oat3 in Rattennieren nicht durch den klassischen Testosteron/Androgenrezeptor vermittelten, transkriptionellen Mechanismus reguliert wird. Vergleichbar zu den Ratten Oat1 und Oat3, zeigten auch die menschlichen OAT1 und OAT3 Promotoren keine Aktivierung durch den Testosteron/Androgenrezeptor-Komplex. Während der Suche nach geschlechtsabhängig exprimierter transkriptioneller Regulatoren in der Rattenniere, konnte die Expression des Transkriptionsfaktors B-cell CLL/ lymphoma 6 (BCL6) erstmalig als männlich dominierend identifiziert werden. Die bereits bekannten Aktivatoren der Oats/OATs Expression, hepatocyte nuclear factor 1α (HNF1α), HNF1β und HNF4α zeigten keine geschlechtsabhängige Expression. Zudem konnte gezeigt werden, dass BCL6 die Promotoren der Ratten und menschlichen Oat1/OAT1 und Oat3/OAT3 aktiviert. Die BCL6-vermittelte Aktivierung von Oat1/OAT1 und Oat3/OAT3 erfolgt nicht über die bislang vorhergesagten BCL6-Bindungsstellen, aber möglicher Weise über Protein-Protein Interaktionen mit den Transkriptionsfaktoren HNF1 oder cAMP response element binding protein (CREB). Zusammenfassend konnte gezeigt werden, dass der Transkriptionsfaktor BCL6 einen vielversprechenden Regulator der geschlechtsabhängigen Expression von Oat1 und Oat3 in Ratten darstellt. Es ist anzunehmen, dass BCL6 ebenso die humane OAT1 und OAT3 Expression reguliert.

No description available.

The inwardly-rectifying K+ current (IK1) is important for heart cell function because it sets the resting potential, influences cell excitability, and promotes repolarization of the action potential. My objective was to investigate the modulation of IK1 by extracellular K+ (K+o) and intracellular K+ (K+i). IK1 was recorded from whole-cell-configured guinea-pig ventricular myocytes that were dialyzed with Na+-free EGTA-buffered pipette-filling solution and bathed with Na+ or NMDG+ solution that contained agents that inhibit non-IK1 currents. Lowering K+o from standard 5.4 to 2 and 1 mM shifted the reversal potential (Erev) of IK1 in accord with calculated K+ equilibrium potential (EK), and altered IK1 amplitude in accord with conductance (GK1)? ?K+o. Surprisingly, myocytes bathed with 0-mM K+ solution had a small outward IK1 at holding potential (Vhold) ?85 mV. This IK1 was attributed to channel-activation by T-tubular K+ (K+T) whose concentration is sensitive to the flow of T-tubular IK1. K+T in myocytes bathed with 0-mM K+ solution was ? 3.2 mM at Vhold ?85 mV, but ? 0.3 mM following large K+T-depleting flows of inward IK1 at ?160 mV. Results consistent with interplay of IK1 and K+T were also obtained in experiments on myocytes bathed with 2-, 5.4-, and 15-mM K+ solution. Myocytes were dialyzed with pipette solutions that contained 0-140 mM K+ to investigate modulation by K+i. When IK1 at Vhold was kept small, Erev varied with pipette K+ in a near-Nernstian manner (i.e., Erev ? EK); however, when IK1 (Vhold) was large and inward, Erev was markedly negative to nominal EK. Findings in experiments that involved shifting Vhold, changing K+o, and application of Ba2+ and Cs+ suggest that the magnitude/direction of IK1 strongly affects the concentration of K+ in submembrane cytoplasm. Classical GK1-voltage parameters GK1max and V0.5 (but not slope factor) were affected by decreases in K+i: GK1max declined by ? 25% per decade decrease in K+i, and V0.5 shifted approximately as 0.5 ? EK-shift. The latter findings are discussed and compared with those of earlier studies on the dependence of inwardly-rectifying K+ conductance on K+i.

Fatty acid (FA) transport proteins are important regulators of FA uptake at the cell surface and the mitochondria where they are oxidized. Tight regulation of this process is necessary in order to meet metabolic requirements, while preventing excess lipid accumulation. In an obese state, there is an increase in FA uptake and increased storage of lipids in skeletal muscle, including diacylglycerol (DAG) and ceramides, which interfere with insulin-stimulated glucose uptake. Leptin administration has been shown to reduce muscle triacylglycerol accumulation and restore insulin response in obese rodents. However, it is not known whether this is mediated through a redistribution of the FA transport proteins to the cell surface and mitochondria. In addition to hyperglycemia, post-prandial lipidemia is also observed in the obese state, suggesting a resistance to insulin-stimulated FA uptake. The possibility that insulin-stimulated FA transporter translocation is impaired has received little attention. Lastly, while recent studies have demonstrated that the transverse (t)-tubules may be an important site for glucose uptake in muscle, this has not yet been examined with regards to the FA transporters. In the first study of this thesis, the recovery of insulin response with short-term (2 week) chronic leptin administration in high-fat fed rats was associated with a decrease in muscle reactive lipid species (DAG, ceramide) and an increase in markers of oxidative capacity. Contrary to our expectations, this was not mirrored by an alteration in the distribution of FA transport proteins (FAT/CD36 or FABPpm) at the sarcolemma or the two major mitochondrial populations. To gain further insight into FA transporters and their localization at the cell surface, the second study of this thesis analyzed both the sarcolemma and t-tubules (constitute 40 and 60% of the cell surface, respectively). The novel observation was made that the t-tubules contain FA transport proteins (FAT/CD36, FABPpm, FATP1 and FATP4), and that the distribution and response of these transporters to acute metabolic stimuli (insulin and muscle contraction) was unique from that of the sarcolemma. The third study of this thesis characterized the translocation of FA transport proteins in response to insulin in the obese, insulin resistant Zucker rat. FA transport proteins were chronically increased on both membrane fractions in muscle from the obese rats. Furthermore, a blunting of the insulin-induced translocation of FA transporters to both cell surface domains was observed, demonstrating that insulin resistance extends to the movement of FA as well as glucose transport proteins. The t-tubules appear to play an important role regarding substrate uptake. Together the data from this thesis suggests that a chronic elevation in FA transporters at both cell surface domains contributes to lipid accumulation in obese skeletal muscle, and that reduced sensitivity of both FA and glucose transport proteins to translocate in response to insulin may explain the lipidemia and hyperglycemia that often characterizes post-prandial situations in the obese condition. As the prevalence of obesity reaches epidemic proportions, research into the functional role of FA transport proteins in the progression of obesity related pathologies is warranted as we work to further our knowledge of this significant health issue. / Natural Sciences and Engineering Research Council, Canadian Institute of Health Research

No description available.

Human cells possess multiple nucleoside transporters (NTs) that belong to either the human equilibrative or concentrative NT (hENT: hENT1/2/3/4; hCNT: CNT1/2/3) families. In the kidney, coupling of apical hCNT3 activities to basolateral hENT1/2 activities is hypothesized to mediate renal nucleoside proximal tubular absorption while apical ENT1 may have a role in secretion. The overall aim of this research was to increase understanding of the roles of hENTs and hCNTs in renal handling of physiological nucleosides and anti-cancer nucleoside analog drugs. This was achieved by investigating the distribution of hENTs and hCNTs in human kidney tissue and the function of hENTs and hCNTs in cellular uptake and transepithelial fluxes of nucleosides in cultured human renal proximal tubule cells (hRPTCs). Immunolocalization of hCNT3 and hENT1 in human kidney tissue revealed that hENT and hCNT3 were present in apical membranes of proximal tubules. Production and characterization of adherent hRPTC cultures demonstrated endogenous hCNT3, hENT1, and hENT2 activities. These results provided evidence for the involvement of hCNT3, hENT1, and hENT2 in renal handling of nucleosides. Comparison of adherent hRPTC cultures derived from kidneys from different individuals demonstrated that hCNT3 activities varied between cultures. Also, the extent of cellular uptake of fludarabine, an anti-cancer nucleoside drug, and degree of cytotoxicity was reflected in the different hCNT3 activities observed between cultures. These results suggested that hCNT3 plays an important role in fludarabine renal handling and is a determinant of potential renal toxicities. Production of polarized monolayer cultures of hRPTCs on transwell permeable inserts enabled the functional localization of hCNT3 and hENT1 to apical membranes and hENT2 to basolateral membranes. Transepithelial flux studies demonstrated that (i) apical-to-basolateral fluxes of adenosine were mediated by apical hCNT3 and basolateral hENT2, (ii) basolateral-to-apical fluxes of 2′-deoxyadenosine were mediated, in part, by apical hENT1 and basolateral hOATs, and (iii) apical-to-basolateral fluxes of fludarabine, cladribine, and clofarabine were mediated by apical hCNT3. These studies showed that coupling of apical hCNT3 to basolateral hENT2 mediates proximal tubular nucleoside reabsorption, that coupling of basolateral human organic anion transporters (hOATs) to apical hENT1 mediates proximal tubular nucleoside secretion, and that hCNT3 is a key determinant of fludarabine proximal tubular reabsorption and cytoxicity.