scholarly journals Effects of insulin, glucagon, dexamethasone, cyclic GMP and spermine on the stability of phosphatidate phosphohydrolase activity in cultured rat hepatocytes

1986 ◽  
Vol 240 (1) ◽  
pp. 253-257 ◽  
Author(s):  
R A Pittner ◽  
R Fears ◽  
D N Brindley
Keyword(s):  
Cyclic Gmp ◽  
Half Life ◽  
Rat Hepatocytes ◽  
Incubation Mixture ◽  
Phosphatidate Phosphohydrolase ◽  
Cultured Rat Hepatocytes ◽  
The Stability ◽  
Specific Control

Hepatocytes were preincubated with 10mM-glucagon and 100 microM-corticosterone to increase phosphatidate phosphohydrolase activity. Addition of 10 nM-glucagon or 100 microM-8-bromo cyclic GMP to a second incubation mixture that contained cycloheximide increased the half-life of the phosphohydrolase activity. Dexamethasone (100 nM) had no significant effect, but insulin (500 pM) or spermine (1 mM) decreased the half-life. None of these compounds altered the general rate of degradation of proteins labelled with [3H]leucine. There appears to be a specific control of the half-life of phosphatidate phosphohydrolase activity, which could contribute to its long-term regulation in the liver.

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 Autoregulation of tubulin synthesis in hepatocytes and fibroblasts.

1985 ◽  
Vol 101 (5) ◽  
pp. 1763-1772 ◽  
Author(s):  
J M Caron ◽  
A L Jones ◽  
M W Kirschner
Keyword(s):  
Half Life ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Low Doses ◽  
Specific Level ◽  
The Stability ◽  
Tubulin Protein ◽  
Uniform Population

Microtubule polymer levels in mouse 3T6 fibroblasts and primary cultures of rat hepatocytes can be manipulated by treatment of cells with long term, low doses of colcemid. Such treatment produces a rather uniform population of cells with microtubules of reduced lengths. Using this system, we demonstrate (a) that the rate of tubulin synthesis is sensitive to small changes (10%) in microtubule polymer mass and (b) that the percent of inhibition of synthesis is proportional to the level of soluble tubulin. Experiments with hepatocytes indicate that not only synthesis but the stability of tubulin protein was also regulated to maintain a specific level of tubulin. Treatment of hepatocytes with colcemid or other microtubule-depolymerizing drugs reduced the half-life of tubulin from 50 to 2 h, whereas taxol, which stabilizes microtubules, increased the half-life. To assess the consequences of altering microtubule polymer mass, we have analyzed the effect of controlled depolymerization of microtubules in rat hepatocytes on the processing of endocytosed ligands and found it sensitive to small changes in microtubule polymer levels.

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.

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

scholarly journals Dexamethasone- and osmolarity-dependent expression of the multidrug-resistance protein 2 in cultured rat hepatocytes1

1999 ◽  
Vol 340 (3) ◽  
pp. 585-591 ◽  
Author(s):  
Ralf KUBITZ ◽  
Ulrich WARSKULAT ◽  
Marcus SCHMITT ◽  
Dieter HÄUSSINGER
Keyword(s):  
Multidrug Resistance ◽  
Culture Medium ◽  
Rat Hepatocytes ◽  
Multidrug Resistance Protein ◽  
Short Term ◽  
Multidrug Resistance Protein 2 ◽  
Long Term Effect ◽  
Cultured Rat Hepatocytes ◽  
Resistance Protein

Expression of the conjugate export pump multidrug-resistance protein 2 (MRP2) in liver is regulated by endotoxin and anti-tumour agents. This paper reports on the effects of dexamethasone and osmolarity on MRP2 expression. MRP2 expression was studied at the protein, mRNA, immunocytochemical and functional levels in cultured rat hepatocytes. Protein and mRNA expression of MRP2 in rat hepatocytes 24 and 48 h after isolation were largely dependent on the presence of dexamethasone (100 nmol/l) in the culture medium. MRP2 was localized at the pseudocanalicular membrane and increased expression of MRP2 was accompanied by a widening of the pseudocanaliculi. In presence of dexamethasone, hypo-osmolarity (205 mosmol/l) led to a strong induction of MRP2 mRNA and protein, whereas expression was decreased by hyperosmolarity (405 mosmol/l). Also, a decay of MRP2 protein and mRNA following dexamethasone withdrawal was osmosensitive. Expression of dipeptidylpeptidase IV, another canalicular protein, was unaffected by dexamethasone and osmolarity. It is concluded that glucocorticoids are strong inducers of MRP2 in liver. Besides short-term carrier insertion/retrieval, osmoregulation of MRP2 also involves a long-term effect on MRP2 expression.

Influence of the isolation method on the stability of differentiated phenotype in cultured rat hepatocytes

1991 ◽  
Vol 25 (1) ◽  
pp. 85-94 ◽  
Author(s):  
J. Bayad ◽  
N. Sabolovic ◽  
D. Bagrel ◽  
J. Magdalou ◽  
G. Siest
Keyword(s):  
Rat Hepatocytes ◽  
Isolation Method ◽  
Cultured Rat Hepatocytes ◽  
The Stability
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