No description available.

Na lesão renal aguda, os mecanismos de defesa atuam como genes protetores, como a proteína heat shock 32 (HSP 32), também conhecida como heme oxigenase-1 (HO-1). A polimixina B (PmxB) é um antimicrobiano nefrotóxico. O objetivo deste estudo foi caracterizar a participação da enzima HO-1 na toxicidade da PmxB em células LLC-PK1. As células foram submetidas aos seguintes tratamentos: Controle (CTL- 0µM); Hemin (indutor de HO-1, 25µM); Hemin II (250M), Protoporfirina de zinco (ZnPP - inibidor de HO-1, 10M,); Nitro-L-arginina-metilester (L-NAME - inibidor de iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM de Hemin uma hora antes da PmxB); PmxB + ZnPP (10M de ZnPP uma hora antes da PmxB); PmxB + Hemin + L-NAME (25M de Hemin e 0,1mM de L-NAME uma hora antes da PmxB). Os grupos foram avaliados em 24 e 72 horas. Foram analisados os seguintes parâmetros: desidrogenase láctica (DHL), peroxidação lipídica (MDA), expressão gênica da HO-1 por RT-PCR, síntese protéica da HO-1 por imunofluorescência, óxido nítrico (NO) pelo método de Griess e expressão protéica da HO-1 e da iNOS por western blotting. Os resultados mostraram que a PmxB elevou o DHL com aumento dos níveis de MDA. O Hemin e a ZnPP elevaram as variáveis DHL, MDA e óxido nítrico (NO). O indutor de HO-1 incrementou a expressão protéica da HO-1 e da iNOS. A PmxB se confirmou como citotóxica e a HO-1 intensificou a lesão por mecanismos oxidativos. O efeito da HO-1 na lesão celular parece ser mediado pelo NO / In the acute kidney injury, the mechanisms of defense act as protector genes, as the protein heat shock 32 (HSP 32), also known as heme oxygenase-1 (HO-1). The polymyxin B (PmxB) is a nephrotoxic antimicrobial. The aim of this study was to distinguish the role of the HO-1 enzyme in the PmxB toxicity in LLC-PK1 cells. The cells were submitted to the following treatments: Control (CTL- 0µM); Hemin (inhibitor of HO-1, 25µM); Hemin II (250M), Zinc protoporphyrin (ZnPP - inhibitor of HO-1, 10M,); NG-nitro-L-arginine methyl ester (L-NAME - inhibitor of iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM of Hemin one hour before the PmxB); PmxB + ZnPP (10M of ZnPP one hour before the PmxB); PmxB + Hemin + L-NAME (25M of Hemin and 0,1mM of L-NAME one hour before the PmxB). All groups were evaluated in 24 and 72 hours. The following parameters were analysed: lactate dehydrogenase (LDH), lipid peroxidation (MDA), genic expression of HO-1 by RT-PCR, protein syntesis of HO-1 by immunofluorescence, nitric oxide (NO) by Griess method and protein expression of HO-1 and of iNOS by western blotting. The results showed that PmxB increased the LDH and the levels of MDA. Hemin and ZnPP also increased the LDH variables, MDA and nitric oxide (NO). The inducer of HO-1 improved the protein expression of HO-1 and of iNOS. The PmxB was confirmed as a cytotoxic and the HO-1 intensified the failure by oxidative mechanisms. The effect of HO-1 in the cell injury seemed to be mediated by NO

Na lesão renal aguda, os mecanismos de defesa atuam como genes protetores, como a proteína heat shock 32 (HSP 32), também conhecida como heme oxigenase-1 (HO-1). A polimixina B (PmxB) é um antimicrobiano nefrotóxico. O objetivo deste estudo foi caracterizar a participação da enzima HO-1 na toxicidade da PmxB em células LLC-PK1. As células foram submetidas aos seguintes tratamentos: Controle (CTL- 0µM); Hemin (indutor de HO-1, 25µM); Hemin II (250M), Protoporfirina de zinco (ZnPP - inibidor de HO-1, 10M,); Nitro-L-arginina-metilester (L-NAME - inibidor de iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM de Hemin uma hora antes da PmxB); PmxB + ZnPP (10M de ZnPP uma hora antes da PmxB); PmxB + Hemin + L-NAME (25M de Hemin e 0,1mM de L-NAME uma hora antes da PmxB). Os grupos foram avaliados em 24 e 72 horas. Foram analisados os seguintes parâmetros: desidrogenase láctica (DHL), peroxidação lipídica (MDA), expressão gênica da HO-1 por RT-PCR, síntese protéica da HO-1 por imunofluorescência, óxido nítrico (NO) pelo método de Griess e expressão protéica da HO-1 e da iNOS por western blotting. Os resultados mostraram que a PmxB elevou o DHL com aumento dos níveis de MDA. O Hemin e a ZnPP elevaram as variáveis DHL, MDA e óxido nítrico (NO). O indutor de HO-1 incrementou a expressão protéica da HO-1 e da iNOS. A PmxB se confirmou como citotóxica e a HO-1 intensificou a lesão por mecanismos oxidativos. O efeito da HO-1 na lesão celular parece ser mediado pelo NO / In the acute kidney injury, the mechanisms of defense act as protector genes, as the protein heat shock 32 (HSP 32), also known as heme oxygenase-1 (HO-1). The polymyxin B (PmxB) is a nephrotoxic antimicrobial. The aim of this study was to distinguish the role of the HO-1 enzyme in the PmxB toxicity in LLC-PK1 cells. The cells were submitted to the following treatments: Control (CTL- 0µM); Hemin (inhibitor of HO-1, 25µM); Hemin II (250M), Zinc protoporphyrin (ZnPP - inhibitor of HO-1, 10M,); NG-nitro-L-arginine methyl ester (L-NAME - inhibitor of iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM of Hemin one hour before the PmxB); PmxB + ZnPP (10M of ZnPP one hour before the PmxB); PmxB + Hemin + L-NAME (25M of Hemin and 0,1mM of L-NAME one hour before the PmxB). All groups were evaluated in 24 and 72 hours. The following parameters were analysed: lactate dehydrogenase (LDH), lipid peroxidation (MDA), genic expression of HO-1 by RT-PCR, protein syntesis of HO-1 by immunofluorescence, nitric oxide (NO) by Griess method and protein expression of HO-1 and of iNOS by western blotting. The results showed that PmxB increased the LDH and the levels of MDA. Hemin and ZnPP also increased the LDH variables, MDA and nitric oxide (NO). The inducer of HO-1 improved the protein expression of HO-1 and of iNOS. The PmxB was confirmed as a cytotoxic and the HO-1 intensified the failure by oxidative mechanisms. The effect of HO-1 in the cell injury seemed to be mediated by NO

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

In the past, hyperlipidemia (HL) has been shown to affect the pharmacokinetic and pharmacodynamic properties of lipophilic drugs extensively bound to lipoproteins, including halofantrine (HF). The present thesis examined the effect of HL on the biodistribution, metabolism and electrocardiographic (ECG) effects of HF in the poloxamer 407 rat model of HL. The HL state caused unexpected changes in the distribution of HF enantiomers. In contrast to plasma, concentrations of desbutyl-HF (DHF) were much higher in highly perfused tissues. Following in vitro incubation of racemic HF and DHF, HF and DHF enantiomers shifted from the lipoprotein deficient fraction to triglyceride-rich fractions in HL plasma. No significant differences were noted in HF metabolism between NL and HL liver microsomes. It appears that both reduced plasma unbound fraction and lipoprotein associated directed uptake of lipoprotein-bound drug by tissues play roles in enantiomer biodistribution. In everted gut metabolism, formation of DHF enantiomers was inversely proportional to bile concentration whereas addition of lipids in the presence of bile caused a significant decrease in DHF:HF ratio of (-)-enantiomers. Pre-treatment of rats with peanut oil had no significant effect on DHF formation in the incubated sacs or microsomal preparations, nor did it affect the expression of intestinal CYP450. The above results were consistent with previous in vivo evaluations showing that the DHF to HF ratio was decreased by the ingestion of a high fat meal. In the ECG study, HL by itself had no effect on the ECG. In HL rats, plasma but not heart concentrations of the HF enantiomers were significantly higher compared to NL rats. DHF did not impart significant ECG prolonging effects after HF administration. The unbound fraction of HF was the controlling factor for drug uptake by the heart. Despite the lack of difference in HF heart concentrations, the QT and QTc intervals were significantly higher in HL compared to NL rats, suggesting a greater vulnerability towards HF induced QT interval prolongation in the HL state. The HL serum resulted in decreased metabolism of HF enantiomers in the isolated primary rat hepatocytes. Studies with LLC PK1 and NRK 52E cells showed that HF is not a substrate of P-glycoprotein transporters. / Pharmaceutical Sciences

No description available.

In eukaryotischen Zellen sind die Lipidspezies häufig asymmetrisch zwischen den Hälften der Plasmamembran verteilt. Insbesondere Phosphatidylserin (PS) weist oft eine ausgeprägte transversale Asymmetrie auf, da es fast ausschliesslich auf die innere Hälfte der Plasmamembran beschränkt ist. In den letzten Jahren wurden mehrere Proteine diskutiert, die Lipide zwischen den Membranhälften transportieren und möglicherweise die transversale Lipidasymmetrie sowie damit verbundene Zelleigenschaften beeinflussen. Im Mittelpunkt der vorliegenden Promotion steht der Auswärtstransport fluoreszierender (C6-NBD-) Lipid-Analoga und endogener Lipide durch das Multidrug Resistance 1 P-Glycoprotein (MDR1 Pgp), das der ATP Binding Cassette (ABC) Transporter Superfamilie angehört. Interessanter Weise wird für MDR1 Pgp eine ungewöhnlich breite Substratspezifität angenommen. Das anionische Lipid PS war hier von besonderem Interesse, obgleich es in vorhergehenden Arbeiten nicht als MDR1 Pgp Substrat betrachtet wurde. Der Auswärtstransport von Phosphatidylcholin-, Phosphatidylethanolamin-, Glucosylceramid- und Sphingomyelin-Analoga durch MDR1 Pgp konnte in einer humanen Magenkarzinomlinie (EPG85-257), die MDR1 überexprimiert, mittels Fluoreszenzspektroskopie bestätigt werden. Zudem legt die verringerte Akkumulation von Diacylglycerol- und Ceramid-Analoga den Transport dieser Lipidspezies durch MDR1 Pgp nahe. Im Anschluß an die intrazelluläre Markierung mit C6-NBD-PS mittels eines neuen Verfahrens konnte der signifikant erhöhte Auswärtstransport dieses Analogons in MDR1 überexprimierenden Zellen durch Verwendung spezifischer Inhibitoren MDR1 Pgp zugeschrieben werden. In flusscytometrischen Versuchen war die Exponierung von endogenem PS auf der äusseren Membranhälfte von MDR1 überexprimierenden Zellen signifikant höher als in Kontrollzellen. Verringerung der PS-Exponierung durch einen Inhibitor von MDR1 Pgp deutet auf den Transport von endogenem PS durch MDR1 Pgp hin. Zusätzlich wurde hier der Transport von C6-NBD-PS in vier weiteren Zellinien mit verschiedener Spezies- und Gewebezugehörigkeit charakterisiert, die unterschiedliche Mengen an MDR1 Pgp synthetisieren. Wie Experimente in einer BCRP überexprimierenden EPG85-257-Sublinie nahelegen, ist ausser MDR1 Pgp möglicherweise ebenfalls der ABC Halb-Transporter Breast Cancer Resistance Protein (BCRP) am Transport von C6-NBD-PS und an der verstärkten Exponierung von endogenem PS beteiligt. / In eukaryotic cells, the lipid species are frequently distributed asymmetrically between the plasma membrane leaflets. Phosphatidylserine (PS), in particular, often exhibits a distinct transverse asymmetry, being restricted almost exclusively to the inner leaflet. In the past years, several proteins were suggested to transport lipids between the leaflets of a membrane, and to potentially influence transverse lipid asymmetry and related cell properties. This thesis focuses on outward transport of fluorescent (C6-NBD-) lipid analogs and endogenous lipids by the Multidrug Resistance 1 P-Glycoprotein (MDR1 Pgp), a member of the ATP binding cassette (ABC) transporter superfamily. Interestingly, MDR1 Pgp has been suggested to exhibit an unusually broad substrate specificity. Here, the anionic PS was of particular concern, although previously reported not to be an MDR1 Pgp substrate. In a human gastric carcinoma cell line (EPG85-257) overexpressing MDR1, outward transport of phosphatidylcholine, phosphatidylethanolamine, glucosylceramide and sphingomyelin analogs via MDR1 Pgp was confirmed using fluorescence spectroscopy. In addition, decreased accumulation of analogs of diacylglycerol and ceramide suggest MDR1 Pgp mediated transport of these lipid species. Upon intracellular labelling with C6-NBD-PS using a novel approach, significantly increased outward transport of this analog in MDR1 overexpressing cells could be attributed to MDR1 Pgp by employing specific inhibitors. In a flow cytometry setup, the exposure of endogenous PS on the outer plasma membrane leaflet was significantly elevated in MDR1 overexpressing cells compared to controls. Reduction of PS exposure by an MDR1 Pgp inhibitor suggests transport of endogenous PS by MDR1 Pgp. Transport of C6-NBD-PS was furthermore characterized here in four additional cell lines of different species and tissue origin with varying synthesis levels of MDR1 Pgp. Besides MDR1 Pgp, the ABC half-size transporter Breast Cancer Resistance Protein (BCRP) is possibly also involved in transport of C6-NBD-PS and in increased exposure of endogenous PS, as found in a BCRP overexpressing EPG85-257 subline.

The purpose of this study was to establish whether curcumin protects renal proximal tubule cells against ischemic injury, determine whether this postulated cytoprotective effect is mediated through the upregulation of HSP70, and investigate whether the mechanism by which curcumin induces HSP70 expression and confers its protective effect is through activation of the Unfolded Protein Response. LLC-PK1 cells were cultured on collagen-coated filters to mimic conditions of in vivo renal proximal tubule cells and induce cell polarization. Injury with and without curcumin treatment was studied by using chemically-induced ATP-depletion which mimics renal ischemic injury. Cell injury was assessed using a TUNEL assay in order to evaluate DNA cleavage associated with ischemia-induced apoptosis and actin staining used to assess cytoskeletal disruption. Renal ischemic damage was further investigated by determining detachment of the Na-K ATPase from the basolateral membrane, which represents loss of cell polarity. Cells were incubated with curcumin in a dose- and time-response fashion and subsequent levels of HSP70 expression were assessed. Cells were then incubated with AEBSF, an inhibitor of the Unfolded Protein Response (UPR) and HSP70 and BiP/GRP78 (an ER resident chaperone that is upregulated by the UPR) expression levels were evaluated. Results demonstrated that treatment with curcumin during two hours of injury results in significantly less injury-related apoptosis and cytoskeletal disruption compared to control injured cells. It was demonstrated that curcumin induces HSP70 in both a dose- and time-response fashion. Moreover, curcumin treatment resulted in profound stabilization of Na-K ATPase on the basolateral membranes as there was significantly less Na-K ATPase detachment in cells treated with curcumin during two hours of injury compared to control injured cells. Finally, treatment with AEBSF inhibited HSP70 upregulation in curcumin-treated cells as well as inhibiting the GRP78 over-expression otherwise demonstrated in curcumin-treated cells. Protection of proximal tubule cells against renal ischemic injury by curcumin was therefore indicated to be mediated by the activation of the UPR through which HSP70 is upregulated. Curcumins activation of the UPR and induction of HSP70 explains the stabilization of Na-K ATPase on the cytoskeleton and also provides a potential mechanism explaining many of curcumins therapeutic and protective qualities.

Thesis (Ph.D.)--University of Toledo, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Zi-Jian Xie. Includes abstract. Document formatted into pages: iii, 156 p. Title from title page of PDF document. Bibliography: pages 64-67, 97-100, 116-117, 125-155.