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 Low Concentration of a Dioxin (2, 3, 7, 8 TCDD) Affects the Glycosidases and Acid Phosphatase Activity in Mice Hepatocytes

Dose-Response ◽  
2014 ◽  
Vol 12 (4) ◽  
pp. dose-response.1
Author(s):  
Jyoti Jigyasi ◽  
Rahul Kundu
Keyword(s):  
Acid Phosphatase ◽  
Lysosomal Enzymes ◽  
Low Dose ◽  
Liver Cells ◽  
Low Doses ◽  
Present Communication ◽  
Cellular Apoptosis ◽  
Mice Liver ◽  
Intracellular Ions

Present communication reports the effects of environmentally available, low doses of tetra chloro di benzo-p-dioxin (2,3,7,8 TCDD) to lysosomal enzymes in mice liver. The study tests the hypothesis, in vivo exposure of low dose TCDD provokes dose and duration dependent toxic effects to key lysosomal enzymes and thereby causes cellular apoptotic changes. Three groups of female Swiss albino mice were subjected to two doses of TCDD (0.004 mg/kg bw/d, 0.04 mg/kg bw/d) for 2, 4 and 6 days of exposure durations. The results indicated significant exposure duration dependent effects of TCDD in mice liver cells. The results suggested that TCDD possibly induced an increase in intracellular ions or ROS which in turn altered different physiological activities by affecting different metabolic pathway of the liver cells. The altered functions of key lysosomal enzymes by TCDD may also evoke the process of cellular apoptosis.

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 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 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.

Interpretation of Hyperamylasemia in Diabetic Coma

1973 ◽  
Vol 19 (4) ◽  
pp. 387-389 ◽  
Author(s):  
Francesco Belfiore ◽  
Elena Napoli
Keyword(s):  
Acid Phosphatase ◽  
Lactate Dehydrogenase ◽  
Enzyme Activities ◽  
Lysosomal Enzymes ◽  
Amylase Activity ◽  
Diabetic Coma ◽  
Serum Lipase

Abstract In sera of patients with diabetic coma, amylase activity was markedly elevated and closely correlated with the activity of some lysosomal enzymes, including β-glucuronidase, N-acetyl-β-glucosaminidase, and acid phosphatase. All these enzyme activities returned to normal with the normalization of glycemia. Activities of serum lipase, aspartate and alanine aminotransferases, aldolase, and lactate dehydrogenase were not changed. Since liver amylase, although primarily contained in microsomes, shows "latency" and is activated by several agents as are lysosomal enzymes, these findings might be regarded as a further evidence of a similarity between amylase and lysosomal enzymes, and make probable the hypothesis that a process of "activation" occurring in liver might be the cause of increased amylase activity in serum as well as of lysosomal enzymes.

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 Proteinase and proteinase-inhibitor activities of rat uterine myometrium during pregnancy and involution

1979 ◽  
Vol 177 (1) ◽  
pp. 99-106 ◽  
Author(s):  
E G Afting ◽  
M L Becker ◽  
J S Elce
Keyword(s):  
Acid Phosphatase ◽  
Proteinase Inhibitor ◽  
Protein Concentration ◽  
Proteinase Activity ◽  
Supernatant Fraction ◽  
Acid Proteinase ◽  
Ph Optimum ◽  
Neutral Proteinase

A supernatant fraction was prepared from rat uterine myometrium by homogenization, sonication and centrifugation. In this supernatant the protein concentration and the activities of an acid proteinase, an acid phosphatase and a proteinase inhibitor were measured. From the fibrous sediment, after washing with 0.5% Triton X-100 and with water, an actomyosin-containing solution was obtained by extraction with 0.6M-NaCl, and in this extract the protein concentration and a neutral proteinase activity were measured. The myometrial wet weight and the activities of the acid proteinase, acid phosphatase and proteinase inhibitor increased by factors of 3-15 during pregnancy and decreased to the same or a greater extent during involution. The amount of protein extracted with 0.6M-NaCl increased by a factor of only 2.3 and the neutral proteinase activity remained essentially constant during pregnancy and involution. The pH optimum of the neutral proteinase, and its pattern of activity compared with those of the lysosomal enzymes, show that the neutral proteinase is not of lysosomal origin. Actomyosin is degraded by the neutral proteinase activity in vitro. Since actomyosin is rapidly broken down only after parturition, the action of the neutral proteinase activity on actomyosin, if this occurs in vivo, must be regulated in some way. The proteinase-inhibitor activity measured in the first supernatant varied in a manner which suggested that it could be involved in this control.

scholarly journals ENZYME LEVELS OF INDIVIDUAL NEURONS IN RELATION TO LIPOFUSCIN CONTENT

1970 ◽  
Vol 18 (4) ◽  
pp. 268-270 ◽  
Author(s):  
HILDE E. HIRSCH
Keyword(s):  
Acid Phosphatase ◽  
Lactate Dehydrogenase ◽  
Nerve Cell ◽  
Lysosomal Enzymes ◽  
Anterior Horn ◽  
Single Neurons ◽  
Histochemical Methods ◽  
Lipofuscin Granules ◽  
Individual Human

By quantitative histochemical methods, activities of (α-naphthyl) acid phosphatase and β-galactosidase, and of malate and lactate dehydrogenase, were measured in individual human anterior horn nerve cell bodies selected for high and low lipofuscin content. No significant differences were found. However, when single neurons were subdivided into pigmented and unpigmented portions, the two lysosomal enzymes were considerably more active, and malate and lactate dehydrogenase less active, in the lipofuscin-rich part of the cell. These results are consistent with the view that lipofuscin granules are derived from lysosomes.

scholarly journals Inhibition of epithelial cell death in the secondary palate in vitro by alteration of lysosome function.

1978 ◽  
Vol 26 (12) ◽  
pp. 1109-1114 ◽  
Author(s):  
R M Greene ◽  
R M Pratt
Keyword(s):  
Cell Death ◽  
Acid Phosphatase ◽  
Epithelial Cell ◽  
Lysosomal Enzymes ◽  
Developmental Age ◽  
Secondary Palate ◽  
Epithelial Cell Death ◽  
Histochemical Examination

The secondary palate in vivo and in vitro exhibits selective cell death at its medialedge epithelium (MEE) at a precise developmental age. This epithelial degeneration is mediated, in part, by MEE lysosomes. Previous studies in vitro (27) showed that the glutamine analogue, diazo-oxo-norleucine (DON), prevented MEE cell death by inhibiting glucosamine synthesis and thereby the glycosylation of proteins without affecting either the synthesis or activity of palatal lysosomal enzymes. In the present study, histochemical examination of MEE from DON treated day-15 rat palates demonstrated that acid phosphatase activity was restricted to Golgi saccules and associated vesicles as well as to lysosomes. Control MEE had reaction product in these structures and distributed diffusely throughout the cytoplasm of degenerating cells. DON treatment therefore appears to alter the intracellular distribution of lysosomal enzymes. Since DON treatment appears to have prevented MEE cell death by inhibiting glycosylation of proteins, glycosylation of lysosomal membranes or lysosomal enzymes may be essential for its role in programmed cell death.

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