scholarly journals Synchronized synthesis and intracellular transport of serum albumin and apolipoprotein B in cultured rat hepatocytes as studied by double immunofluorescence.

1986 ◽  
Vol 34 (9) ◽  
pp. 1223-1230 ◽  
Author(s):  
G A Keller ◽  
C Glass ◽  
D Louvard ◽  
D Steinberg ◽  
S J Singer
Keyword(s):  
Serum Albumin ◽  
Apolipoprotein B ◽  
Intracellular Transport ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Cell System ◽  
Secretory Proteins ◽  
Transport Pathway ◽  
Apo B ◽  
Normal Rat

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.

scholarly journals Insulin-mediated inhibition of apolipoprotein B secretion requires an intracellular trafficking event and phosphatidylinositol 3-kinase activation: studies with brefeldin A and wortmannin in primary cultures of rat hepatocytes

1996 ◽  
Vol 313 (2) ◽  
pp. 567-574 ◽  
Author(s):  
Janet D. SPARKS ◽  
Thuy L. PHUNG ◽  
Mary BOLOGNINO ◽  
Charles E. SPARKS
Keyword(s):  
Apolipoprotein B ◽  
Enzyme Inhibitor ◽  
Primary Cultures ◽  
Brefeldin A ◽  
Rat Hepatocytes ◽  
Phosphatidylinositol 3 Kinase ◽  
Apo B ◽  
Insulin Effect ◽  
Insulin Inhibition ◽  
Phosphatidylinositol 3

Insulin inhibition of the secretion of apolipoprotein B (apo B) was studied in primary cultures of rat hepatocytes by using brefeldin A (BFA), an inhibitor of protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and by using the phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin. Incubation of hepatocytes with BFA (10 μg/ml) for 1 h inhibited the subsequent secretion of apo B, albumin and transferrin for up to 3 h. BFA treatment resulted in the time-dependent accumulation in cells of [14C]leucine-labelled proteins and apo B. Under conditions where insulin decreased total apo B (cell plus secreted), BFA blocked the insulin-dependent effect. These results suggest that export of apo B from the ER is a prerequisite for the observed insulin effect. Treatment of hepatocytes with wortmannin for 20 min abolished insulin inhibition of apo B secretion, suggesting that the insulin effect on the apo B pathway involves activation of PI 3-K. Enzyme inhibitor studies indicate that chymostatin and (+)-(2S,3S)-3-[(S)-methyl-1-(3-m e t h y l b u t y l c a r b a m o y l) - b u t y l c a r b a m o y l] - 2-oxiranecarboxylate (E-64-c) partially block insulin effects on apo B compared with leupeptin, which had no discernible effect. The cell-permeable derivative of E-64-c, EST, and N-Ac-Leu-Leu-norleucinal (ALLN) were most effective in blocking insulin effects on apo B. These results suggest that insulin action on apo B in primary rat hepatocytes involves (1) vesicular movement of apo B from the ER; (2) activation of PI 3-K and (3) a cellular protease that is either a cysteine- or calcium-activated neutral protease.

scholarly journals Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes.

1986 ◽  
Vol 261 (24) ◽  
pp. 11398-11403 ◽  
Author(s):  
Y Misumi ◽  
Y Misumi ◽  
K Miki ◽  
A Takatsuki ◽  
G Tamura ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Brefeldin A ◽  
Rat Hepatocytes ◽  
Secretory Proteins ◽  
Cultured Rat Hepatocytes

scholarly journals Effects of nonketotic streptozotocin diabetes on apolipoprotein B synthesis and secretion by primary cultures of rat hepatocytes.

1988 ◽  
Vol 82 (1) ◽  
pp. 37-43 ◽  
Author(s):  
J D Sparks ◽  
C E Sparks ◽  
M Bolognino ◽  
A M Roncone ◽  
T K Jackson ◽  
...  
Keyword(s):  
Apolipoprotein B ◽  
Primary Cultures ◽  
Streptozotocin Diabetes ◽  
Rat Hepatocytes

scholarly journals Inhibition of apolipoprotein B net synthesis and secretion from cultured rat hepatocytes by the calcium-channel blocker diltiazem

1989 ◽  
Vol 263 (2) ◽  
pp. 411-415 ◽  
Author(s):  
T C Kwong ◽  
J D Sparks ◽  
D J Pryce ◽  
J F Cianci ◽  
C E Sparks
Keyword(s):  
Molecular Mass ◽  
Apolipoprotein B ◽  
Channel Blocker ◽  
Rat Hepatocytes ◽  
Acetate Incorporation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Apo B ◽  
Cellular Lipids ◽  
Mass Form

1. The effect of the Ca2+-channel blocker diltiazem on hepatic apolipoprotein B (apo B) synthesis and secretion was studied in 12-18 h cultures of collagenase-dispersed rat hepatocytes. 2. The presence of diltiazem in the medium decreased apo B secretion by hepatocytes in a concentration-dependent manner. At 25 microM, diltiazem inhibited apo B secretion by approx. 36%, but there was no evidence of intracellular accumulation of apo B. 3. The inhibition of apo B secretion by hepatocytes was significantly correlated with cell-associated diltiazem (r = 0.72, P less than 0.01). 4. The rate of apo B secretion remained linear over 16 h even in the presence of 50 microM-diltiazem. 5. At diltiazem concentrations in the medium which were inhibitory for apo B secretion, [14C]acetate incorporation into cellular lipids and [35S]methionine incorporation into protein were enhanced. 6. Diltiazem inhibited the secretion of the apo B variants with a preferential inhibition of the higher-molecular-mass form of apo B (apo BH) over the lower-molecular-mass form (apo BL) at diltiazem concentrations in the medium greater than 25 microM. 7. Together, these results suggest that Ca2+ may play an important role in the synthesis and secretion of apo B-containing lipoproteins.

Effects of insulin, dexamethasone and cytokines on ?1-acid glycoprotein gene expression in primary cultures of normal rat hepatocytes

Inflammation ◽  
1996 ◽  
Vol 20 (2) ◽  
pp. 191-202 ◽  
Author(s):  
B�atrice Barraud ◽  
Sophie Balavoine ◽  
G�rard Feldmann ◽  
Bernard Lardeux
Keyword(s):  
Gene Expression ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Acid Glycoprotein ◽  
Glycoprotein Gene ◽  
Normal Rat

scholarly journals Collagen measured in primary cultures of normal rat hepatocytes derives from lipocytes within the monolayer.

1988 ◽  
Vol 82 (2) ◽  
pp. 450-459 ◽  
Author(s):  
J J Maher ◽  
D M Bissell ◽  
S L Friedman ◽  
F J Roll
Keyword(s):  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Normal Rat

scholarly journals Differential arrest of secretory protein transport in cultured rat hepatocytes by azide treatment.

1988 ◽  
Vol 107 (6) ◽  
pp. 2503-2510 ◽  
Author(s):  
R Persson ◽  
E Ahlström ◽  
E Fries
Keyword(s):  
Intracellular Transport ◽  
Protein Transport ◽  
Rat Hepatocytes ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Atp Content ◽  
Isolated Rat Hepatocytes ◽  
Atp Level ◽  
Atp Levels ◽  
Endoglycosidase H

The effect of reduced cellular ATP content on intracellular transport of two secretory proteins, albumin and haptoglobin, in isolated rat hepatocytes was studied. The cells were labeled with [35S]methionine and the cellular ATP content was then rapidly reduced to different stable levels by incubation with azide at different concentrations (2.0-10 mM). The amount of the radioactively labeled secretory proteins in the cells and in the medium after 150 min of incubation was determined by immunoprecipitation followed by gel electrophoresis, fluorography, and densitometry. At progressively lower ATP levels, down to 50% of normal, the protein secretion was unaffected, whereas at even lower levels an increasing portion of the proteins remained in the cells; at 30 and 10% of normal ATP level, 25 and 75% of albumin, respectively, was arrested intracellularly. Analysis of the carbohydrate structure of intracellularly arrested haptoglobin showed that in cells with an ATP level of approximately 30% of normal, the majority of haptoglobin molecules (55%) were fully or partially resistant to endoglycosidase H. This result indicates that exit from the medial and/or the trans part of the Golgi complex (GC) was inhibited under these conditions. It also shows that the protein had accumulated in the GC, since under normal conditions the fraction of the intracellular haptoglobin that is endoglycosidase H resistant is approximately 10%. By similar criteria it was found that at ATP levels below 10% of normal transport of haptoglobin from the endoplasmic reticulum to the medial GC (and possibly also to the cis GC) as well as from the trans GC to the medium were blocked.

scholarly journals Effect of serum proteins on haem uptake and metabolism in primary cultures of liver cells

1988 ◽  
Vol 256 (1) ◽  
pp. 159-165 ◽  
Author(s):  
P R Sinclair ◽  
W J Bement ◽  
N Gorman ◽  
H H Liem ◽  
A W Wolkoff ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Chick Embryo ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Serum Proteins ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Liver Cells ◽  
Metabolic Effects ◽  
High Affinity

A role of haemopexin in transporting haem to hepatocytes for degradation has been inferred from the high affinity of haemopexin for haem. We have examined this question in primary cultures of chick-embryo and adult rat liver cells. We present here the results of four sets of experiments which indicate that haemopexin retarded haem uptake by hepatocytes in culture. (1) Haem bound to bovine serum albumin is known to repress the activity of delta-aminolaevulinate synthase in chick cultures as indicated by decreased porphyrin accumulation. When haem-albumin was added in the presence of excess purified or freshly secreted chicken haemopexin, no haem-mediated repression of porphyrin production was observed. The haem-mediated repression of porphyrin accumulation was partially prevented when human, but not chicken, albumin was added to cultures. This finding reflected the higher affinity of human albumin for haem compared with that of chicken albumin. (2) Haemopexin inhibited the ability of haem to be incorporated into cytochrome P-450 induced in the chick cultures in the presence of the iron chelator desferrioxamine. (3) The rate of association of [55Fe]haem with cultured rat hepatocytes when [55Fe]haem-haemopexin was added was one-eighth of the rate observed when [55Fe]haem-bovine serum albumin was used as the haem donor. (4) The presence of haemopexin also diminished the catabolism of haem by both rat and chick-embryo liver cell cultures. It is concluded that the uptake and subsequent metabolic effects of haem are inhibited in cultured hepatocytes by proteins such as haemopexin which have a high affinity for haem.

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