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.

scholarly journals Interactions of insulin, glucagon and dexamethasone in controlling the activity of glycerol phosphate acyltransferase and the activity and subcellular distribution of phosphatidate phosphohydrolase in cultured rat hepatocytes

1985 ◽  
Vol 230 (2) ◽  
pp. 525-534 ◽  
Author(s):  
R A Pittner ◽  
R Fears ◽  
D N Brindley
Keyword(s):  
Neutral Lipids ◽  
Relative Proportion ◽  
Rat Hepatocytes ◽  
Glycerol Phosphate ◽  
Acyltransferase Activity ◽  
Phosphatidate Phosphohydrolase ◽  
Cultured Rat Hepatocytes ◽  
Total Glycerol

Rat hepatocytes were incubated in monolayer culture for 8 h. Glucagon (10nM) increased the total phosphatidate phosphohydrolase activity by 1.7-fold. This effect was abolished by adding cycloheximide, actinomycin D or 500 pM-insulin to the incubations. The glucagon-induced increase was synergistic with that produced by an optimum concentration of 100 nM-dexamethasone. Theophylline (1mM) potentiated the effect of glucagon, but it did not affect the dexamethasone-induced increase in the phosphohydrolase activity. The relative proportion of the phosphohydrolase activity associated with membranes was decreased by glucagon when 0.15 mM-oleate was added 15 min before the end of the incubations to translocate the phosphohydrolase from the cytosol. This glucagon effect was not seen at 0.5 mM-oleate. Since glucagon also increased the total phosphohydrolase activity, the membrane-associated activity was maintained at 0.15 mM-oleate and was increased at 0.5 mM-oleate. This activity at both oleate concentrations was also increased in incubations that contained dexamethasone, particularly in the presence of glucagon. Insulin increased the relative proportion of phosphatidate phosphohydrolase that was associated with membranes at 0.15 mM-oleate, but not at 0.5 mM-oleate. It also decreased the absolute phosphohydrolase activity on the membranes at both oleate concentrations in incubations that also contained glucagon and dexamethasone. None of the hormonal combinations significantly altered the total glycerol phosphate acyltransferase activity. However, glucagon significantly increased the microsomal activities, and insulin had the opposite effect. Glucagon also decreased the mitochondrial acyltransferase activity. There was a highly significant correlation between the total phosphatidate phosphohydrolase activity and the synthesis of neutral lipids from glycerol phosphate and 0.5 mM-oleate in homogenates of cells from all of the hormonal combinations. Phosphatidate phosphohydrolase activity is increased in the long term by glucocorticoids and also by glucagon through cyclic AMP. In the short term, glucagon increases the concentration of fatty acid required to translocate the cytosolic reservoir of activity to the membranes on which phosphatidate is synthesized. Insulin opposes the combined actions of glucagon and glucocorticoids. The long-term events explain the large increases in the phosphohydrolase activity that occur in vivo in a variety of stress conditions. The expression of this activity depends on increases in the net availability of fatty acids and their CoA esters in the liver.

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.

Pyruvate kinase in cultured rat hepatocytes

1981 ◽  
Vol 9 (5) ◽  
pp. 399-399
Author(s):  
GRAEME P. POOLE ◽  
ANTHONY D. POSTLE ◽  
DAVID P. BLOXHAM
Keyword(s):  
Pyruvate Kinase ◽  
Rat Hepatocytes ◽  
Cultured Rat Hepatocytes

Long-term cytotoxicities of various pesticides evaluated by albumin secretion of primary cultured rat hepatocytes

1996 ◽  
Vol 10 (2) ◽  
pp. 99-102 ◽  
Author(s):  
Kazuhiro Ichikawa ◽  
Yasuyuki Sakai ◽  
Akiyoshi Sakoda ◽  
Motoyuki Suzuki
Keyword(s):  
Rat Hepatocytes ◽  
Albumin Secretion ◽  
Primary Cultured Rat Hepatocytes ◽  
Cultured Rat Hepatocytes

Long-term preservation and induction of drug metabolizing enzymes in co-cultured rat hepatocytes

1994 ◽  
Vol 22 (2) ◽  
pp. 124S-124S ◽  
Author(s):  
MAY AKRAWI ◽  
VERA ROGIERS ◽  
ANTOINNE VERCRUYSSE ◽  
IAN R. PHILLIPS ◽  
ELIZABETH A. SHEPHARD
Keyword(s):  
Rat Hepatocytes ◽  
Drug Metabolizing Enzymes ◽  
Metabolizing Enzymes ◽  
Cultured Rat Hepatocytes ◽  
Long Term Preservation

Uptake of L-triiodothyronine into human cultured lymphocytes

1980 ◽  
Vol 95 (3) ◽  
pp. 350-358 ◽  
Author(s):  
Ann-Charlotte Holm ◽  
K. Y. Wong ◽  
Nathan B. Pliam ◽  
Eugene C. Jorgensen ◽  
Ira D. Goldfine
Keyword(s):  
Thyroid Hormone ◽  
Incubation Temperature ◽  
Potassium Cyanide ◽  
Rat Hepatocytes ◽  
Side Chain ◽  
Affinity Constant ◽  
Uptake System ◽  
Mitochondrial Inhibitors ◽  
First Order Kinetics ◽  
Cultured Rat Hepatocytes

Abstract. The cellular uptake of [125I]L-triiodothyronine and its analogues was investigated in IM-9 human cultured lymphocytes. Uptake of L-triiodothyronine was one half maximal within 15 min of incubation and maximal within 45 min. The efflux of the hormone followed first order kinetics having a one half time of 15 min. Treating the cells with either the mitochondrial inhibitors antimycin-A and potassium cyanide, or lowering the incubation temperature to 12°C, markedly reduced uptake. The uptake of [125I]L-triiodothyronine was saturable having an affinity constant (Kd) of 110 nm. When thyroid hormone analogues were studied D-triiodothyronine and triiodothyroacetic acid (analogues known to bind avidly to nuclear receptors) competed only weakly with [125I]L-triiodothyronine for uptake. These findings indicated the importance of the intact L-alanine side chain of the thyroid hormone molecule for its uptake into lymphocytes. Other studies, with cultured rat hepatocytes, demonstrated a similar saturable uptake system for [125I]L-triiodothyronine. These studies suggest, therefore, that the uptake of thyroid hormones and their analogues into cells may be an important step in the biological actions of these hormones.

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