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Isolated Rat Hepatocytes (isolated + rat_hepatocyte)
Selected AbstractsAllosteric activation of pyruvate kinase via NAD+ in rat liver cellsFEBS JOURNAL, Issue 14 2001Anne Devin In isolated rat hepatocytes, it has previously been reported that a rise in the ATP content induces a proportional increase in cytosolic NAD+ concentration [Devin, A., Guérin, B. & Rigoulet, M. (1997) FEBS Lett.410, 329,332]. This occurs under physiological conditions such as various substrates or different energetic states. To investigate the effect of a physiological rise in cytosolic [NAD+] per se on glycolysis and gluconeogenesis, an increase in [NAD+] induced by exogenous nicotinamide addition was obtained without a change in redox potential, ATP/ADP ratio and ATP concentration. Using dihydroxyacetone as substrate, we found that an increase in cytosolic [NAD+] decreases gluconeogenesis and enhances glycolysis without significant alteration of dihydroxyacetone consumption rate. These modifications are the consequence of an allosteric activation of pyruvate kinase via cytosolic NAD+ content. Thus, in addition to the well-known thermodynamic control of glycolysis by pyridine-nucleotide redox status, our study points to a new mechanism of glycolytic flux regulation by NAD+ concentration at the level of pyruvate kinase activity. [source] Glucagon induces the plasma membrane insertion of functional aquaporin-8 water channels in isolated rat hepatocytesHEPATOLOGY, Issue 6 2003Sergio A. Gradilone Although glucagon is known to stimulate the cyclic adenosine monophosphate (cAMP)-mediated hepatocyte bile secretion, the precise mechanisms accounting for this choleretic effect are unknown. We recently reported that hepatocytes express the water channel aquaporin-8 (AQP8), which is located primarily in intracellular vesicles, and its relocalization to plasma membranes can be induced with dibutyryl cAMP. In this study, we tested the hypothesis that glucagon induces the trafficking of AQP8 to the hepatocyte plasma membrane and thus increases membrane water permeability. Immunoblotting analysis in subcellular fractions from isolated rat hepatocytes indicated that glucagon caused a significant, dose-dependent increase in the amount of AQP8 in plasma membranes (e.g., 102% with 1 ,mol/L glucagon) and a simultaneous decrease in intracellular membranes (e.g., 38% with 1 ,mol/L glucagon). Confocal immunofluorescence microscopy in cultured hepatocytes confirmed the glucagon-induced redistribution of AQP8 from intracellular vesicles to plasma membrane. Polarized hepatocyte couplets showed that this redistribution was specifically to the canalicular domain. Glucagon also significantly increased hepatocyte membrane water permeability by about 70%, which was inhibited by the water channel blocker dimethyl sulfoxide (DMSO). The inhibitors of protein kinase A, H-89, and PKI, as well as the microtubule blocker colchicine, prevented the glucagon effect on both AQP8 redistribution to hepatocyte surface and cell membrane water permeability. In conclusion, our data suggest that glucagon induces the protein kinase A and microtubule-dependent translocation of AQP8 water channels to the hepatocyte canalicular plasma membrane, which in turn leads to an increase in membrane water permeability. These findings provide evidence supporting the molecular mechanisms of glucagon-induced hepatocyte bile secretion. [source] Role of mitogen-activated protein kinases in tauroursodeoxycholic acid-induced bile formation in cholestatic rat liverHEPATOLOGY RESEARCH, Issue 7 2008Gerald Ulrich Denk Aim:, Ursodeoxycholic acid exerts anticholestatic effects in various cholestatic disorders and experimental models of cholestasis. Its taurine conjugate (TUDCA) stimulates bile salt secretion in isolated perfused rat livers (IPRL) under physiological, non-cholestatic conditions, in part by mitogen-activated protein kinase (MAPK)-dependent mechanisms. The role of MAPK in the anticholestatic effect of TUDCA, however, is unclear. Therefore, we studied the role of MAPK in the anticholestatic effect of TUDCA in IPRL and isolated rat hepatocytes (IRH) in taurolithocholic acid (TLCA)-induced cholestasis. Methods:, Bile flow, biliary levels of 2,4-dinitrophenyl-S-glutathione (GS-DNP) as a marker of hepatobiliary organic anion secretion and activity of lactate dehydrogenase (LDH) in hepatovenous effluate as a marker of hepatocellular damage in IPRL perfused with TUDCA and/or TLCA were determined in the presence or absence of MAPK inhibitors. In addition, phosphorylation of Erk 1/2 and p38MAPK induced by TUDCA and/or TLCA was studied by Western immunoblot in IPRL and IRH. Results:, TUDCA-induced bile flow was impaired by the Erk 1/2 inhibitor PD98059 in normal livers (,28%), but not in livers made cholestatic by TLCA. GS-DNP secretion was unaffected by PD98059 under both conditions. TUDCA-induced bile formation and organic anion secretion both in the presence and absence of TLCA were unaffected by the p38MAPK inhibitor SB202190. Erk 1/2 phosphorylation in liver tissue was unchanged after bile salt exposure for 70 min, but was transiently enhanced by TUDCA in IRH. Conclusion:, MAPK do not mediate the anticholestatic effects of TUDCA in TLCA-induced cholestasis. [source] Cytotoxic effects of polychlorinated biphenyl hydroquinone metabolites in rat hepatocytesJOURNAL OF APPLIED TOXICOLOGY, Issue 2 2010Katie Chan Abstract Polychlorinated biphenyls (PCBs) are persistent organic pollutants that exhibit various toxic effects in animals and exposed human populations. The molecular mechanisms of PCB toxicity have been attributed to the toxicological properties of its metabolites, such as hydroquinones, formed by cytochrome-P-450 oxidation. The effects of PCB hydroquinone metabolites towards freshly isolated rat hepatocytes were investigated. Hydroquinones can be oxidized to semiquinones and/or quinone metabolites. These metabolites can conjugate glutathione or can oxidize glutathione as a result of redox cycling. This depletes hepatocyte glutathione, which can inhibit cellular defence mechanisms, causing cell death and an increased susceptibility to oxidative stress. However in the following, glutathione-depleted hepatocytes became more resistant to the hydroquinone metabolites of PCBs. This suggested that their glutathione conjugates were toxic and that there was a third type of quinone toxicity mechanism which involved a hydrogen peroxide-accelerated autoxidation of the hydroquinones to form toxic electrophilic quinone and semiquinone,glutathione conjugates. Copyright © 2009 John Wiley & Sons, Ltd. [source] Metabolites of an orally active antimicrobial prodrug, 2,5-bis(4-amidinophenyl)furan-bis- O -methylamidoxime, identified by liquid chromatography/tandem mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2004Lian Zhou Abstract DB75 (2,5-bis(4-amidinophenyl)furan) is a promising antimicrobial agent against African trypanosomiasis and Pneumocystis carinii pneumonia. However, it suffers from poor oral activity in rodent models for both infections. In contrast, a novel prodrug of DB75, 2,5-bis(4-amidinophenyl)furan-bis- O -methylamidoxime (DB289), has excellent oral activity. DB289 is currently undergoing clinical investigation as a candidate drug to treat primary stage African trypanosomiasis and Pneumocystis carinii pneumonia. In this study, metabolites of DB289 formed after incubation with freshly isolated rat hepatocytes were characterized using liquid chromatography/ion trap mass spectrometry. Administration of DB289 and octadeuterated DB289 in a 1 : 1 mixture greatly facilitated metabolite identification by providing isotope patterns with twin ions separated by 8 m/z units in the ratio 1 : 1, in the extracted ion chromatograms of molecular ions and in the product ion mass spectra of metabolites. Ten metabolites were identified. Series of O -demethylations and N -dehydroxylations led to the metabolic activation of DB289 to DB75 with the production of four intermediate phase I metabolites. Phase II glucuronidation and sulfation led to the formation of four glucuronide and one sulfate metabolites. Copyright © 2004 John Wiley & Sons, Ltd. [source] Metabolism of isometamidium in hepatocytes isolated from control and inducer-treated ratsJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2006I. BOIBESSOT Little is known about the metabolism and mechanism of action of the trypanocide, isometamidium (ISM), the major drug used for prophylaxis of trypanosomiasis. We have investigated its metabolism and distribution in isolated rat hepatocytes using liquid chromatography-mass spectrometry and confocal laser scanning microscopy (CLSM). Two putative metabolites were formed, which were proposed to be a mono-acetyl derivative and an oxidized metabolite (SII). This is the first demonstration of the hepatic metabolism of ISM, as previous in vivo studies were hampered by dose-limiting toxicity and insensitive analytical methods. The intrinsic fluorescence of the drug enabled its intracellular uptake to be followed by CLSM. It is taken up rapidly into the nucleolus, nuclear membrane and endoplasmic reticulum within 5 min, and retained in the nucleus for at least 24 h. Persistent binding of ISM to cellular macromolecules may contribute to its prophylactic effect in vivo. Pretreatment of rats with 3-methylcholanthrene, phenobarbitone (PB) or the widely used pyrethroid pesticide, deltamethrin, resulted in an increase in metabolism of ISM to the proposed SII after 1 h incubation with hepatocytes. 3-methylcholanthrene was the most potent inducer, causing a maximal 19.5-fold induction of SII formation after exposure of hepatocytes to ISM for 1 h compared with formation by control hepatocytes. In comparison, at the 1 h timepoint deltamethrin pre-treatment caused a 10.2-fold induction, and PB only 8.2 fold. [source] Maintenance of integrity and function of isolated hepatocytes during extended suspension culture at 25°CLIVER INTERNATIONAL, Issue 3 2003Alan J. Wigg Abstract: Isolated hepatocytes in suspension provide a number of advantages for use in bioartificial liver device, however, poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. We therefore investigated the integrity and differentiated function of isolated rat hepatocytes under conditions of mild hypothermia. Isolated hepatocytes were suspended in a bicarbonate buffered saline medium, supplemented with glucose and bovine serum albumin (BSA), and maintained for 48 h at 25 °C on a rotary shaker under an atmosphere of 95% O2 and 5% CO2. Under these conditions there was no significant decline in cell viability and good preservation of cellular morphology on transmission electron microscopy for at least 24 h. Isolated hepatocytes in suspension at 25 °C were also able to maintain normal Na + and K + ion gradients. The cellular energy status ([ATP], ATP/ADP ratio, cytoplasmic and mitochondrial redox potentials), metabolic function (urea synthesis and ammonia removal), albumin synthesis and phase I and phase II drug detoxification activity of these cells were also maintained for at least 24 h post isolation. These observations demonstrate the robust nature of mildly hypothermic isolated hepatocytes in suspension and encourage further studies re-examining the feasibility of using this cell preparation in bioartificial livers. [source] Copper-catalyzed ascorbate oxidation results in glyoxal/AGE formation and cytotoxicityMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 4 2007Nandita Shangari Abstract Previously we showed that 10 ,M glyoxal compromised hepatocyte resistance to hydrogen peroxide (H2O2) by increasing glutathione (GSH) and NADPH oxidation and decreasing mitochondrial membrane potential (MMP) before cytotoxicity ensued. Since transition metal-catalyzed oxidation of ascorbate (Asc) has been shown to result in the generation of both glyoxal and H2O2, we hypothesized that glyoxal formation during this process compromises hepatocyte resistance to H2O2. We used isolated rat hepatocytes and incubated them with Asc/copper and measured cytotoxicity, glyoxal levels, H2O2, GSH levels, and MMP. To investigate the role of Asc/copper on glyoxal-BSA adducts, we measured the appearance of advanced glycation end-products (AGE) in the presence and absence of catalase or aminoguanidine (AG). Asc/copper increased glyoxal and H2O2 formation. Hepatocyte GSH levels were decreased and cytotoxicity ensued after a collapse of the hepatocyte MMP. Glyoxal traps protected hepatocytes against Asc/copper-induced cytotoxicity. In cell-free studies with BSA, incubation with Asc and copper resulted in glyoxal-hydroimidazolone formation, which was decreased by both AG and catalase. To the best of our knowledge, this is the first study that illustrates the importance of glyoxal production by transition metal-catalyzed Asc autoxidation. Understanding this mechanism of toxicity could lead to the development of novel copper chelating drug therapies to treat diabetic complications. [source] Hepatoprotective and antioxidant activities of Tetracera loureiriPHYTOTHERAPY RESEARCH, Issue 7 2003Veerapol Kukongviriyapan Abstract Tetracera loureiri is one of the most valued herbs in Thai traditional medicine. In this study, we describe its in vitro and in vivo antioxidant and hepatoprotective activities. The ethanol extract of T. loureiri possessed potent antioxidant and strong free radical scavenging properties assayed using ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH), respectively. The cytoprotective effects of T. loureiri were demonstrated in ethanolic extracts of freshly isolated rat hepatocytes against the chemical toxicants paracetamol and tertiary-butylhydroperoxide. The cells pretreated with the extract maintained the GSH/GSSG ratio and suppressed lipid peroxidation in a dose dependent manner. Pretreating rats with the ethanol extract orally, one hour prior to intraperitoneal injection of toxic doses of paracetamol, signi,cantly prevented elevations of plasma ALT and AST. These results suggest that T. loureiri may be of potential therapeutic value in some liver disorders. Copyright © 2003 John Wiley & Sons, Ltd. [source] Effect of macrolide antibiotics on uptake of digoxin into rat liverBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 3 2007Suwako Ito Abstract The objective of this study was to examine the effect of macrolide antibiotics, clarithromycin, erythromycin, roxithromycin, josamycin and azithromycin, on the hepatic uptake of digoxin. The uptake of [3H]digoxin was studied in rats in vivo, using the tissue-sampling single-injection technique, and in isolated rat hepatocytes in vitro. The uptake of [3H]digoxin into rat hepatocytes was concentration-dependent with a Michaelis constant (Km) of 445 nM. All the macrolide antibiotics inhibited the uptake of [3H]digoxin into rat hepatocytes in a concentration-dependent manner. However, clarithromycin did not affect the in vivo hepatic uptake of digoxin in rats. The in vivo permeability,surface area product of digoxin for hepatic uptake (PSinf) was estimated to be 12.5 ml/min/g liver from the present in vitro data, which is far larger than the hepatic blood flow rate (1.4 ml/min/g liver). Macrolide antibiotics at clinically relevant concentrations inhibit digoxin uptake by rat hepatocytes in vitro, but not in vivo, probably because hepatic uptake of digoxin in rats is blood flow-limited. Clinically observed digoxin,macrolide interaction in humans could be due to macrolide inhibition of hepatic digoxin uptake, if the uptake is permeation-limited. Copyright © 2007 John Wiley & Sons, Ltd. [source] The sulphonylurea glibenclamide inhibits multidrug resistance protein (MRP1) activity in human lung cancer cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2001Léa Payen Glibenclamide, a sulphonylurea widely used for the treatment of non-insulin-dependent diabetes mellitus, has been shown to inhibit the activities of various ATP-binding cassette (ABC) transporters. In the present study, its effects towards multidrug resistance protein 1 (MRP1), an ABC efflux pump conferring multidrug resistance and handling organic anions, were investigated. Intracellular accumulation of calcein, an anionic dye substrate for MRP1, was strongly increased by glibenclamide in a dose-dependent manner in MRP1-overexpressing lung tumour GLC4/Sb30 cells through inhibition of MRP1-related calcein efflux. By contrast, glibenclamide did not alter calcein levels in parental control GLC4 cells. Another sulphonylurea, tolbutamide, was however without effect on calcein accumulation in both GLC4/Sb30 and GLC4 cells. Glibenclamide used at 12.5 ,M was, moreover, found to strongly enhance the sensitivity of GLC4/Sb30 cells towards vincristine, an anticancer drug handled by MRP1. Efflux of carboxy-2,,7,-dichlorofluorescein, an anionic dye handled by the ABC transporter MRP2 sharing numerous substrates with MRP1 and expressed at high levels in liver, was also strongly inhibited by glibenclamide in isolated rat hepatocytes. In summary, glibenclamide reversed MRP1-mediated drug resistance likely through inhibiting MRP1 activity and blocked organic anion efflux from MRP2-expressing hepatocytes. Such effects associated with the known inhibitory properties of glibenclamide towards various others ABC proteins suggest that this sulphonylurea is a general inhibitor of ABC transporters. British Journal of Pharmacology (2001) 132, 778,784; doi:10.1038/sj.bjp.0703863 [source] Conversion of the Synthetic Catalase Mimic Precursor TAA-1 into the Active Catalase Mimic in Isolated HepatocytesCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2009Ursula Rauen In previous studies we reported on the catalase-like activity and antioxidative properties of a non-heme Fe(III)-tetraaza[14]annulene complex, 5,4-didehydro-5,9,14,18-tetraaza-di(2,2-dimethyl-[5,6]benzo[1,3]dioxolo)[a,h]cyclotetradecene,Fe(III) chloride (TAA-1/Fe). We proposed that intracellular application of the parent, iron-free tetraaza[14]annulene ligand, TAA-1, as precursor would allow antioxidative defense along two lines, i.e. by chelation of potentially toxic cellular iron ions and, subsequently, by catalase-mimic activity. We here set out to establish whether the active catalase mimic is indeed formed intracellularly when cells are loaded with the ligand. When isolated rat hepatocytes were preloaded with TAA-1, they were protected against iron-induced cell injury and oxidative stress elicited by exposure to the membrane-permeable iron complex Fe(III)/8-hydroxyquinoline. After lysis of the cells, followed by ultrafiltration to remove endogenous catalase, the lysate exhibited catalase-like activity, while lysates of control cells not treated with TAA-1 showed no catalase-like activity. By comparison with authentic TAA-1/Fe, an intracellular formation of 2.0 ± 0.3 ,m of the active catalase mimic in native hepatocytes exposed to TAA-1 and of 6.5 ± 1.0 ,m in hepatocytes exposed to both TAA-1 and iron ions was estimated. The intracellular formation of the active catalase mimic thus renders TAA-1 an attractive compound for protection against iron- and/or hydrogen peroxide-dependent cell injuries. [source] | ||||||||||||||||||||||||||||