scholarly journals Evaluation of oxidative status in acetaminophen treated rat hepatocytes in culture

2009 ◽  
pp. 239-246
Author(s):  
T Roušar ◽  
O Kučera ◽  
P Křiváková ◽  
H Lotková ◽  
R Kanďár ◽  
...  
Keyword(s):  
Functional Capacity ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Oxidative Status ◽  
Ros Production ◽  
Cellular Viability ◽  
Time Intervals ◽  
Leakage Test ◽  
Cultured Rat Hepatocytes ◽  
In Cells

The present study describes the estimation of acetaminophen (AAP) toxicity in cultured rat hepatocytes. We used different concentrations of AAP – 1, 2.5, 5, 10 and 20 mM, to test influence of AAP on cellular viability, functional capacity and oxidative status at given time intervals. WST-1 test showed decrease of dehydrogenase activity in 5, 10 and 20 mM AAP to 75 % of control values after 1 hour of incubation. At 12 h of treatment, all AAP concentrations decreased WST-1 signal; no enzyme activity was found since 18 h in cells treated with 20 mM AAP according to LDH leakage test performed at 24 h of incubation. Functional capacity was tested by albumin assay where the decrease was strictly related to AAP dose. Intracellular oxidative status was assessed by analysis of GSH/GSSG levels and time course of ROS production and glutathione reductase (GR) activity. Increased ROS production was found already after 3 h of incubation in 2.5, 5, 10 and 20 mM AAP, respectively. The highest ROS production was measured after 12 h treatment. GR activity was decreased already after 3 h of incubation and remained also decreased in cells treated with 2.5, 5, 10 and 20 mM AAP during further incubation.

scholarly journals The turnover of L-type pyruvate kinase in cultured rat hepatocytes

1982 ◽  
Vol 204 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Graeme P. Poole ◽  
David P. Bloxham
Keyword(s):  
Pyruvate Kinase ◽  
Rat Hepatocytes ◽  
Cell Protein ◽  
Rapid Phase ◽  
First Order Kinetics ◽  
Enzyme Degradation ◽  
Fructose 1,6 Bisphosphatase ◽  
Cultured Rat Hepatocytes ◽  
In Cells

Hepatocytes were isolated by collagenase perfusion of livers from rats that had been allowed access to a carbohydrate-rich diet or laboratory chow or had been deprived of food 48h before use. By incubation with l-[4,5-3H]leucine and precipitation with anti-(L-type pyruvate kinase) sera the rates of synthesis and degradation of L-type pyruvate kinase were measured in freshly prepared cells and hepatocytes maintained in monolayer culture for up to 5 days. Hepatocytes from carbohydrate-rich-diet-fed rats synthesized more L-type pyruvate kinase than did cells from chow-fed animals, which in turn synthesized more than cells from 48h-starved rats. Hepatocytes maintained in culture for up to 5 days synthesized L-type pyruvate kinase at similar rates to freshly prepared cells. The degradation of [3H]leucine-labelled L-type pyruvate kinase was shown to be biphasic. A phase with t½ (half-time) 4.9h and a duration of 8–10h was followed by a phase with t½ 79.2h. Cells from chow-fed and carbohydrate-rich-diet-fed rats showed similar patterns of degradation of L-type pyruvate kinase. The addition of 2mm-fructose and 0.1μm-insulin to the culture medium increased the t½ of the rapid phase to 12h in cells isolated from carbohydrate-rich-diet-fed rats, but not in cells from chow-fed rats. The secondary, slower, phase of degradation remained unaffected. The degradation of fructose 1,6-bisphosphatase and total cell protein followed first-order kinetics. The half-life of fructose 1,6-bisphosphatase was 41.0h in cells from chow-fed animals and 48.5h in cells from carbohydrate-rich-diet-fed donors. Fructose and insulin did not affect the rate of enzyme degradation. We propose that there is a role for protein catabolism in the short-term and long-term control of L-type pyruvate kinase concentration.

Selective determination of mitochondrial chelatable iron in viable cells with a new fluorescent sensor

2002 ◽  
Vol 362 (1) ◽  
pp. 137-147 ◽  
Author(s):  
Frank PETRAT ◽  
Daniela WEISHEIT ◽  
Martina LENSEN ◽  
Herbert de GROOT ◽  
Reiner SUSTMANN ◽  
...  
Keyword(s):  
Time Course ◽  
Fluorescent Sensor ◽  
Rat Hepatocytes ◽  
Intact Cells ◽  
Free System ◽  
Selective Determination ◽  
Viable Cells ◽  
Pyridoxal Isonicotinoyl Hydrazone ◽  
Cultured Rat Hepatocytes

Mitochondrial chelatable ('redox-active') iron is considered to contribute to several human diseases, but has not yet been characterized in viable cells. In order to determine this iron pool, we synthesized a new fluorescent indicator, rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA). In a cell-free system, RPA fluorescence was strongly and stoichiometrically quenched by Fe2+. RPA selectively accumulated in the mitochondria of cultured rat hepatocytes. The intramitochondrial RPA fluorescence was quenched when iron was added to the cells in a membrane-permeant form. It increased when the mitochondrial chelatable iron available to the probe was experimentally decreased by the membrane-permeant transition metal chelators pyridoxal isonicotinoyl hydrazone and 1,10-phenanthroline. The concentration of mitochondrial chelatable iron in cultured rat hepatocytes, quantified from the increase in RPA fluorescence after addition of pyridoxal isonicotinoyl hydrazone, was found to be 12.2±4.9μM. Inhibition of haem synthesis with succinylacetone did not alter the signal obtained in hepatocytes, but a rapid increase in the concentration of mitochondrial chelatable iron was observed in human erythroleukaemia K562 cells. In conclusion, RPA enables the selective determination of the highly physiologically and pathophysiologically interesting mitochondrial pool of chelatable iron in intact cells and to record the time course of alterations of this pool.

Glucagon inhibits phosphatidylcholine biosynthesis via the CDP-choline and transmethylation pathways in cultured rat hepatocytes

1984 ◽  
Vol 62 (4) ◽  
pp. 196-202 ◽  
Author(s):  
Steven L. Pelech ◽  
P. Haydn Pritchard ◽  
Eric F. Sommerman ◽  
Anthony Percival-Smith ◽  
Dennis E. Vance
Keyword(s):  
Fatty Acid ◽  
Rat Hepatocytes ◽  
Biosynthetic Enzymes ◽  
Choline Uptake ◽  
Acetate Incorporation ◽  
Short Term ◽  
Monolayer Cultures ◽  
Phosphatidylcholine Biosynthesis ◽  
Cultured Rat Hepatocytes ◽  
In Cells

The short-term influence of insulin and glucagon on phosphatidylcholine biosynthesis in monolayer cultures of rat hepatocytes was investigated. Under conditions in which insulin (100 nM) stimulated [3H]acetate incorporation into fatty acid almost twofold, synthesis of phosphatidylcholine via CDP-choline and from phosphatidylethanolamine were unaffected. By contrast, glucagon (100 nM), even in the presence of insulin (100 nM), reduced the rate of phosphatidylcholine formation from [Me-3H]phosphocholine by approximately 25% (p < 0.05) within 1 h. Similarly, [3H]phosphatidylethanolamine incorporation into phosphatidylcholine was inhibited in cells exposed to glucagon. Insulin and glucagon had little or no effect on [Me-3H] choline uptake by the hepatocytes. No changes in the activities of the phosphatidylcholine biosynthetic enzymes in cytosol and microsomes from glucagon-treated cells could be detected.

scholarly journals Fasting enhances glycogen synthase activation in hepatocytes from insulin-resistant genetically obese (fa/fa) rats

1990 ◽  
Vol 269 (3) ◽  
pp. 789-794 ◽  
Author(s):  
G van de Werve
Keyword(s):  
Dose Response ◽  
Time Course ◽  
Glycogen Synthase ◽  
Rat Hepatocytes ◽  
Insulin Resistant ◽  
Nutritional Conditions ◽  
Obese Rats ◽  
Elevated Glucose ◽  
Lean Group ◽  
In Cells

Glycogen synthase activation and phosphorylase inactivation by glucose were studied in hepatocytes isolated from fed or overnight-fasted lean or genetically obese (fa/fa) rats. In cells from fed animals, both the time course and dose-response to glucose of synthase activation were the same in both groups, despite higher levels of phosphorylase a in hepatocytes from obese animals. In contrast, in cells from fasted obese animals synthase activation with or without glucose was enhanced severalfold over that of lean controls, despite similar levels of phosphorylase a and of total (a + b) synthase activities. In both nutritional conditions glucose 6-phosphate concentrations were 2-3-fold higher in obese-rat hepatocytes than in lean-rat cells. In addition, synthase activation was transient in the fasted lean group, but was sustained in obese-rat hepatocytes. The rate of synthase activation was, however, comparable in lean- and obese-rat liver Sephadex G-25 filtrates, irrespective of the nutritional state of the donor rats. It is concluded that enhanced synthase activation in hepatocytes from starved obese rats might be due to an unbalanced synthase interconversion brought about by elevated glucose 6-phosphate concentrations and impaired kinase [van de Werve & Massillon (1990) Biochem. J. 269, 795-799], rather than to an intrinsic change in synthase phosphatase.

Influence of bile acids with respect to the time course against cultured rat hepatocytes

1995 ◽  
Vol 108 (4) ◽  
pp. A1128 ◽  
Author(s):  
H Nagai ◽  
K Ishii ◽  
T Hatori ◽  
Y Sumino ◽  
M Sigimoto
Keyword(s):  
Bile Acids ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Cultured Rat Hepatocytes

Nutrition and somatomedin. XXVI. Molecular regulation of IGF-I by insulin in cultured rat hepatocytes

Diabetes ◽  
1991 ◽  
Vol 40 (11) ◽  
pp. 1525-1530 ◽  
Author(s):  
L. S. Phillips ◽  
S. Goldstein ◽  
C. I. Pao
Keyword(s):  
Rat Hepatocytes ◽  
Molecular Regulation ◽  
Igf I ◽  
Cultured Rat Hepatocytes
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