scholarly journals Maintenance of glutathione content is isolated hepatocyctes

1978 ◽  
Vol 170 (3) ◽  
pp. 627-630 ◽  
Author(s):  
J Viña ◽  
R Hems ◽  
H A Krebs
Keyword(s):  
Reduced Glutathione ◽  
Standard Procedure ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Perfusion Medium ◽  
In Cells

1. During the standard procedure for the preparation of rat hepatocytes, about half of the cellular GSH (reduced glutathione) is lost. 2. This loss is prevented by the addition of 0.1 mM-EGTA (but no EDTA) to the perfusion medium. 3. On incubation with and without EGTA, isolated hepatocytes prepared in the presence of EGTA lose GSH. This loss is prevented by near-physiological concentrations of methionine or homocysteine, but not of cysteine. 4. Cysteine, at concentrations above 0.2 mM, causes a loss of GSH probably by non-enzymic formation of a mixed disulphide. 5. Serine together with methionine or homocystein increases GSH above the value in cells from starved rats in vivo. This is taken to suggest that cystathionine may be a cysteine donor in the synthesis of gamma-glutamylcysteine, the precursor of GSH.

scholarly journals Preferable Transplant Site for Hepatocyte Transplantation in a Rat Model

2021 ◽  
Vol 30 ◽  
pp. 096368972110400
Author(s):  
Hiroyuki Ogasawara ◽  
Akiko Inagaki ◽  
Ibrahim Fathi ◽  
Takehiro Imura ◽  
Hiroki Yamana ◽  
...  
Keyword(s):  
Serum Albumin ◽  
In Vivo Imaging ◽  
Standard Procedure ◽  
Graft Function ◽  
Rat Hepatocytes ◽  
Hepatocyte Transplantation ◽  
Current Standard ◽  
Imaging Evaluation ◽  
Transplant Site

Intraportal injection is regarded as the current standard procedure of hepatocyte transplantation (HTx). In islet transplantation, which shares many aspects with HTx, recent studies have clarified that instant blood-mediated inflammatory reaction (IBMIR), characterized by strong innate immune responses, can cause poor engraftment, so other transplant sites to avoid such a reaction have been established. Although IBMIR was reported to occur in HTx, few reports have evaluated alternative transplant sites for HTx. In this study, we sought to determine the optimum transplant site for HTx. Rat hepatocytes (1.0 × 107) were transplanted at the 9 transplant sites (intraportal (IPO), intrasplenic (IS), liver parenchyma, subcutaneous, intraperitoneal, renal subcapsular, muscle, inguinal subcutaneous white adipose tissue, and omentum) of analbuminemic rats. The serum albumin levels, immunohistochemical staining (albumin, TUNEL, and BrdU), and in vivo imaging of the grafts were evaluated. The serum albumin levels of the IPO group were significantly higher than those of the other groups ( p < .0001). The BrdU-positive hepatocyte ratio of liver in the IS group (0.9% ± 0.2%) was comparable to that of the IPO group (0.9% ± 0.3%) and tended to be higher than that of the spleen in the IS group (0.5% ± 0.1%, p = .16). Considering the in vivo imaging evaluation and the influence of splenectomy, the graft function in the IS group may be almost entirely achieved by hepatocytes that have migrated to the liver. The present study clearly showed that the intraportal injection procedure is more efficient than other procedures for performing HTx

Prophylactic Effect Of Aqueous Propolis Extract Against Acute Experimental Hepatotoxicity In Vivo

2002 ◽  
Vol 57 (3-4) ◽  
pp. 379-385 ◽  
Author(s):  
Aiman S. El-Khatib ◽  
Azza M. Agha ◽  
Laila G. Mahran ◽  
Mohamed T. Khayyal
Keyword(s):  
Reduced Glutathione ◽  
Wide Spectrum ◽  
Folk Medicine ◽  
Lipid Peroxide ◽  
Isolated Hepatocytes ◽  
Propolis Extract ◽  
Single Intraperitoneal Injection ◽  
Liver Homogenates

Propolis has been extensively used in folk medicine for the management of a wide spectrum of disorders. In a previous study, we demonstrated the protective effect of the aqueous propolis extract (APE) against the injurious effects of carbon tetrachloride (CCl4) on hepatocytes in vitro. The present investigation was carried out to show whether the hepatoprotective effect of the extract could also be manifested in vivo. Rats were given APE orally for 14 consecutive days, before being subjected to a single intraperitoneal injection of CCl4. One day after the CCl4 injection, the animals were sacrificed, hepatocytes were isolated and liver homogenates were prepared for the assessment of liver injury. In isolated hepatocytes, APE afforded protection against CCl4-induced injury as manifested by a decrease in the leakage of the cytosolic enzyme lactate dehydrogenase (LDH), decreased generation of lipid peroxide and maintenance of cellular reduced glutathione (GSH) content. In principle, similar findings were observed in liver homogenates. The present findings show that APE has in vivo hepatoprotective potential which could be attributed at least in part to the maintenance of cellular GSH content. The latter effect seems to play an important role in conserving the integrity of biomembranes as it was associated with a decrease in lipid peroxidation and reduced leakage of cytosolic LDH

Inhibition of GSH efflux from rat liver by methionine: effects of GSH synthesis in cells and perfused organ

1990 ◽  
Vol 258 (6) ◽  
pp. G967-G973 ◽  
Author(s):  
J. C. Fernandez-Checa ◽  
T. Maddatu ◽  
M. Ookhtens ◽  
N. Kaplowitz
Keyword(s):  
Rat Liver ◽  
Reduced Glutathione ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Perfused Liver ◽  
Isolated Rat Hepatocytes ◽  
Dependent Inhibition ◽  
Krebs Henseleit Buffer ◽  
Isolated Rat ◽  
In Cells

The inhibition of efflux of intracellular reduced glutathione (GSH) by methionine was determined in isolated rat hepatocytes suspended either in Krebs-Henseleit buffer or in modified Fisher's medium. Methionine (1 mM) added to Krebs-Henseleit suspensions of isolated rat hepatocytes inhibited GSH efflux, with greater retention of GSH in the cells compared with control. Results were similar with methionine and 0.3 mM propargylglycine cystathionase inhibitor), suggesting no net synthesis of GSH from methionine. In Fisher's medium, the inhibitory effect of methionine on GSH efflux was masked due to increasing cellular GSH; however, the inhibitory effect of methionine was unmasked by propargylglycine, which prevented the utilization of methionine for GSH synthesis. The addition of serine (0.1 mM) to methionine in Krebs-Henseleit buffer raised cellular GSH, overcoming the inhibition of GSH efflux. In the perfused liver, infusion of 1 and 5 mM methionine initially inhibited GSH efflux, but the inhibition was reversed with continued methionine infusion. After removal of methionine, GSH efflux increased immediately. The reversal and rebound were blocked by propargylglycine, revealing concentration-dependent inhibition of sinusoidal GSH efflux by methionine. Thus, when methionine is utilized to promote GSH synthesis, its inhibitory effect on GSH efflux tends to be overcome.

Hormonal induction of malic enzyme in rat hepatocytes cultured on laminin-rich gels

1992 ◽  
Vol 8 (3) ◽  
pp. 235-242 ◽  
Author(s):  
D. J. Mann ◽  
A. J. Strain ◽  
E. Bailey
Keyword(s):  
Malic Enzyme ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Specific Expression ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Species Being ◽  
Rat Tail

ABSTRACT The levels of malic-enzyme mRNA and activity were determined in primary cultures of adult rat hepatocytes maintained on either rat-tail collagen or a laminin-rich substratum. Cells plated on laminin-rich gels exhibited substantially improved patterns of albumin and malic-enzyme expression when compared with cells maintained on rat-tail collagen. Moreover, hepatocytes plated on the laminin-rich matrix displayed marked malic-enzyme inducibility in response to tri-iodothyronine and dichloroacetate, especially in the presence of insulin. However, Northern blot analysis revealed that the ratio of the amounts of the two major malic-enzyme mRNA species (2.0 and 3.1 kb) was reversed when compared with that found in the liver in vivo, the altered levels of these two species being closer to those found in non-hepatic tissues. These findings indicate that, although the hormonal responsiveness of isolated hepatocytes maintained on laminin-rich gels is markedly improved, and approaches the degree of induction demonstrated in the liver in vivo, the mechanisms of control differ, indicating a loss of liver-specific expression.

scholarly journals Glutathione analogues as novel inhibitors of rat and human glutathione S-transferase isoenzymes, as well as of glutathione conjugation in isolated rat hepatocytes and in the rat in vivo

1995 ◽  
Vol 308 (1) ◽  
pp. 283-290 ◽  
Author(s):  
S Ouwerkerk-Mahadevan ◽  
J H van Boom ◽  
M C Dreef-Tromp ◽  
J H T M Ploemen ◽  
D J Meyer ◽  
...  
Keyword(s):  
Ethyl Ester ◽  
Rat Liver ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Glutathione S Transferase ◽  
Isolated Rat Hepatocytes ◽  
Glutathione S Transferases ◽  
Gamma S ◽  
Isolated Rat

Inhibitors of rat and human Alpha- and Mu-class glutathione S-transferases that effectively inhibit the glutathione (GSH) conjugation of bromosulphophthalein in the rat liver cytosolic fraction, isolated rat hepatocytes and in the rat liver in vivo have been developed. The GSH analogue (R)-5-carboxy-2-gamma-(S)-glutamylamino-N-hexylpentamide [Adang, Brussee, van der Gen and Mulder (1991) J. Biol. Chem. 266, 830-836] was used as the lead compound. To obtain more potent inhibitors, it was modified by replacement of the N-hexyl moiety by N-2-heptyl and by esterification of the 5-carboxy group with ethyl and dodecyl groups. In isolated hepatocytes, the branched N-2-heptyl derivatives were stronger inhibitors of GSH conjugation of bromosulphophthalein than the N-hexyl derivatives. The ethyl ester compounds were more efficient than the corresponding unesterified derivatives. The dodecyl ester of the N-2-heptyl analogue was the most effective inhibitor in isolated hepatocytes, but was relatively toxic in vivo. However, the corresponding ethyl ester was a potent in vivo inhibitor: GSH conjugation of bromosulphophthalein (as assessed by biliary excretion of the conjugate) was decreased by 70% after administration of a dose of 200 mumol/kg. The isoenzyme specificity of the inhibitors towards purified rat and human glutathione S-transferases was also examined. The unesterified compounds were more potent than the esterified analogues, and inhibited Alpha- and Mu-class isoenzymes of both rat and human glutathione S-transferase (Ki range 1-40 microM). Other GSH-dependent enzymes, i.e. GSH peroxidase, GSH reductase and gamma-glutamyltranspeptide, were not inhibited. Thus (R)-5-ethyloxycarbonyl-2-gamma-(S)-glutamylamino-N-2-hept ylpentamide, the in vivo inhibitor of GSH conjugation, may be useful in helping to assess the role of the Alpha and Mu classes of glutathione S-transferases in cellular biochemistry, physiology and pathology.

scholarly journals Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance

2009 ◽  
Vol 106 (37) ◽  
pp. 15714-15719 ◽  
Author(s):  
Srivatsan Kidambi ◽  
Rubin S. Yarmush ◽  
Eric Novik ◽  
Piyun Chao ◽  
Martin L. Yarmush ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Drug Clearance ◽  
Adaptation Period ◽  
Culture Methods ◽  
Cytochrome P450 Activity

The liver is a major site for the metabolism of xenobiotic compounds due to its abundant level of phase I/II metabolic enzymes. With the cost of drug development escalating to over $400 million/drug there is an urgent need for the development of rigorous models of hepatic metabolism for preclinical screening of drug clearance and hepatotoxicity. Here, we present a microenvironment in which primary human and rat hepatocytes maintain a high level of metabolic competence without a long adaptation period. We demonstrate that co-cultures of hepatocytes and endothelial cells in serum-free media seeded under 95% oxygen maintain functional apical and basal polarity, high levels of cytochrome P450 activity, and gene expression profiles on par with freshly isolated hepatocytes. These oxygenated co-cultures demonstrate a remarkable ability to predict in vivo drug clearance rates of both rapid and slow clearing drugs with an R2 of 0.92. Moreover, as the metabolic function of oxygenated co-cultures stabilizes overnight, preclinical testing can be carried out days or even weeks before other culture methods, significantly reducing associated labor and cost. These results are readily extendable to other culture configurations including three-dimensional culture, bioreactor studies, as well as microfabricated co-cultures.

Purpurogallin—a natural and effective hepatoprotector in vitro and in vivo

1991 ◽  
Vol 69 (10-11) ◽  
pp. 747-750 ◽  
Author(s):  
Tai-Wing Wu ◽  
Ling-Hua Zeng ◽  
Jun Wu ◽  
Doug Carey
Keyword(s):  
Xanthine Oxidase ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Oxidase Inhibition ◽  
The Mean ◽  
Xanthine Oxidase Inhibition ◽  
Oxidase Reaction ◽  
Dose Dependent

Purpurogallin is a plant phenol that is sometimes added as an oxidation retardant to fats–oils or to certain fuels or lubricants. However, it was unknown if purpurogallin is cytoprotective. Here we examined this issue, both in isolated hepatocytes and in vivo. From 0.5 to 2.0 mM, purpurogallin prolongs survival of rat hepatocytes substantially against oxyradicals generated with xanthine oxidase and hypoxanthine. The protection was dose dependent and surpassed that given by such antioxidants as ascorbate, mannitol, superoxide dismutase, catalase, and Trolox, when each was examined at or near its optimal concentration in the same system. When 1.5,3, and 6 μmol of purpurogallin in saline were infused into rats with postischemic livers shortly before reperfusion, the mean hepatic salvages were 42, 76, and 86%, respectively. Such salvage effects would rank purpurogallin highly among the hepatoprotectors known. Over the range of 31 to 500 μM, purpurogallin inhibited the rate of O2 consumption in the xanthine oxidase reaction by ~90%, which was 2- to several-fold higher than the inhibition elicited by allopurinol over the same concentrations. Thus, purpurogallin is an effective natural hepatoprotector that may operate partly or principally as an inhibitor of xanthine oxidase.Key words: purpurogallin, hepato-protection, xanthine oxidase inhibition.

scholarly journals The effect of acute ethanol treatment on rates of oxygen uptake, ethanol oxidation and gluconeogenesis in isolated rat hepatocytes

1985 ◽  
Vol 230 (3) ◽  
pp. 595-602 ◽  
Author(s):  
K M Stowell ◽  
K E Crow
Keyword(s):  
Ethanol Oxidation ◽  
Physiological State ◽  
Rat Hepatocytes ◽  
O2 Uptake ◽  
Isolated Rat Hepatocytes ◽  
Liver Preparation ◽  
Acute Ethanol ◽  
Ethanol Pretreatment ◽  
In Cells

In hepatocytes isolated from fed rats, acute ethanol pretreatment (at a dose of 5.0 g/kg body wt.) did not change rates of O2 uptake. In cells from starved animals, acute ethanol pretreatment increased O2 uptake by 17-29%. The increased O2 uptake in hepatocytes from starved rats was not accompanied by increased rates of ethanol oxidation, but was accompanied by increased rates of gluconeogenesis under some conditions. The provision of ethanol (10 mM) as a substrate to cells from fed or starved rats decreased O2 uptake in the absence of other substrates or in the presence of lactate, and increased it in the presence of pyruvate or lactate and pyruvate. The results of this study show that the acute effects of ethanol on liver O2 uptake are dependent on the physiological state of the liver. Previously reported large (2-fold) increases in O2 uptake after acute ethanol pretreatment may have been an artefact owing to low control uptake rates (approximately 1.8 micromol/min per g wet wt. of cells) in the liver preparation used. The ATP contents (2.4-2.6 micromol/g wet wt. of cells) and rates of O2 uptake (2.5-5.0 micromol/min per g wet wt. of cells) of cells used in the present study were the same as values reported under conditions close to those in vivo. Therefore the increase in O2 uptake in cells from starved rats after acute ethanol pretreatment is likely to be of physiological significance.

scholarly journals A novel method for the study of autophagy: destruction of hepatocytic lysosomes, but not autophagosomes, by the photosensitizing porphyrin tetra(4-sulphonatophenyl)porphine

1997 ◽  
Vol 321 (1) ◽  
pp. 217-225 ◽  
Author(s):  
Per Eivind STRØMHAUG ◽  
Trond Olav BERG ◽  
Kristian BERG ◽  
Per O. SEGLEN
Keyword(s):  
Acid Phosphatase ◽  
Lactate Dehydrogenase ◽  
Proteinase Inhibitor ◽  
Lysosomal Enzymes ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Major Fraction ◽  
Microtubule Inhibitor ◽  
Novel Method

A photoactivatable porphyrin, tetra(4-sulphonatophenyl)porphine (TPPS4), was shown to accumulate in rat hepatocytes as a linear function of dose after intravenous injection, and to localize predominantly in hepatocytic lysosomes. A major fraction of the lysosomal enzymes acid phosphatase and N-acetyl-β-d-glucosaminidase was inactivated by TPPS4 after 20 h of contact with the drug in vivo in the absence of photoactivation. On exposure of isolated hepatocytes to light, photoactivated TPPS4 caused additional inactivation of the lysosomal enzymes as well as inactivation of intralysosomal lactate dehydrogenase (LDH), a cytosolic enzyme that accumulated in lysosomes as a result of autophagy during a 2 h incubation of hepatocytes at 37 °C in the dark (in the presence of the proteinase inhibitor leupeptin to prevent degradation of intralysosomal LDH). Photoactivation of TPPS4 also induced lysosomal rupture, with a loss of lysosomal enzymes, autophagocytosed LDH, endocytosed 125I-tyramine-cellobiose-asialo-orosomucoid and TPPS4 from the lysosomes. However, LDH-containing autophagosomes, accumulated in the presence of vinblastine (a microtubule inhibitor used to prevent the fusion of lysosomes with autophagosomes or endosomes), were not affected by TPPS4. TPPS4 may thus be useful as a selective lysosomal (or endosomal) perturbant in the study of autophagic–endocytic–lysosomal interactions.

scholarly journals Control of glucuronidation during hypoxia. Limitation by UDP-glucose pyrophosphorylase

1984 ◽  
Vol 219 (3) ◽  
pp. 707-712 ◽  
Author(s):  
T Y Aw ◽  
D P Jones
Keyword(s):  
Steady State ◽  
Glucuronic Acid ◽  
Isolated Hepatocytes ◽  
Kinetic Constants ◽  
Regulatory Site ◽  
Exogenous Glucose ◽  
Pathological Conditions ◽  
Intracellular Concentrations ◽  
In Cells

The regulation of glucuronidation during hypoxia was studied in isolated hepatocytes by analysing the dependence of acetaminophen glucuronidation rate on the intracellular concentrations of UTP, glucose 1-phosphate, UDP-glucose and UDP-glucuronic acid. The steady-state concentrations of these metabolites in cells from fed and starved rats were altered by exposure to various hypoxic O2 concentrations and by adding exogenous glucose. Changes in glucuronidation rate under all conditions were explained in terms of the concentrations of the substrates for UDP-glucose pyrophosphorylase, i.e. UTP and glucose 1-phosphate. Steady-state rates for the UDP-glucose pyrophosphorylase reaction, calculated by using published kinetic constants and measured glucose 1-phosphate and UTP concentrations, were in agreement with the measured glucuronidation rates. Thus the UDP-glucose pyrophosphorylase reaction is the key regulatory site for drug glucuronidation during hypoxia. Control at this site indicates that glucuronidation in vivo may be generally depressed in pathological conditions involving hypoxia and energy (calorie) malnutrition.

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