Activity and secretion of sialyltransferase in primary monolayer cultures of rat hepatocytes cultured with and without dexamethasone

1986 ◽  
Vol 64 (2) ◽  
pp. 79-84 ◽  
Author(s):  
Willem van Dijk ◽  
Willem Boers ◽  
Mieke Sala ◽  
Anne-Marie Lasthuis ◽  
Sailen Mookerjea
Keyword(s):  
Enzyme Activity ◽  
Culture Media ◽  
Rat Hepatocytes ◽  
Progressive Increase ◽  
Isolated Rat Hepatocytes ◽  
Sialyltransferase Activity ◽  
The Media ◽  
Primary Monolayer ◽  
Triton X ◽  
In Cells

Monolayers of hepatocytes attached on collagen-coated dishes were cultured for 20–24 h and were found suitable to study the activity and secretion of CMP-N-acetylneuraminate:asialo-α1-acid glycoprotein sialyltransferase. A progressive increase of sialyltransferase activity in the culture medium was observed during incubation of the hepatocytes. After 24 h 34–48% of the total sialyltransferase activity of the hepatocyte incubation system was present in the medium. The enzyme activity present in the medium was soluble in nature and could not be stimulated by Triton X-100. The secretion of the enzyme was stimulated about twofold by dexamethasone. The activity of sialyltransferase in the hepatocytes was also increased by dexamethanasone. The Km of either hepatocyte or medium sialyltransferase for CMP-sialic acid was only slightly changed by dexamethasone, whereas the Vmax was increased about twofold. The secretion of sialyltransferase could be inhibited partially by the anti-microtubular agent colchicine. The dexamethasone-induced increase of the sialyltransferase activity in cells and media could be eliminated by inclusion of α-amanitin in the culture media at 0 h. The inhibiting effect of α-amanitin was only partially expressed when the drug was added 4 h after the addition of dexamethasone to the media. The results suggest that isolated rat hepatocytes actively secrete sialyltransferase and that the increase in the sialyltransferase activity in cells and media owing to the synthetic glucocorticosteroid dexamethasone results from increased synthesis of the enzyme molecule. It is supposed that in the intact rat the increased levels of the enzyme activity in serum observed in inflammation may originate from an induction of the synthesis of sialyltransferase in the hepatocytes of rat liver by the increased levels of circulating corticosteroids.

scholarly journals Exposure of cultured hepatocytes to cyclic AMP enhances the vasopressin-mediated stimulation of inositol phosphate production

1989 ◽  
Vol 257 (2) ◽  
pp. 455-460 ◽  
Author(s):  
R A Pittner ◽  
J N Fain
Keyword(s):  
Cyclic Amp ◽  
Inositol Phosphate ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Phosphate Accumulation ◽  
Primary Monolayer Culture ◽  
Inositol Phosphate Production ◽  
Inositol Phosphate Accumulation ◽  
Primary Monolayer ◽  
Stimulation Of

Isolated rat hepatocytes in primary monolayer culture were maintained for 18-24 h in the presence of 10% (v/v) serum and [3H]inositol. Vasopressin (100 nM) stimulated the production of inositol mono-, bis- and tris-phosphates (IP1, IP2, and IP3). Prior exposure of hepatocytes to 8-bromo cyclic AMP (8Br-cAMP; 100 microM), but not 8-bromo cyclic GMP, enhanced the vasopressin-mediated stimulation of inositol phosphate accumulation, but had no significant effect on their formation in the absence of vasopressin. The effect of the cyclic AMP analogue was mimicked by glucagon (10 nM), and was seen whether cyclic AMP or glucagon was added 5 min or 12 h before the addition of vasopressin. An 8 h incubation with dexamethasone (100 nM) enhanced the accumulation of IP3, but not that of IP2 or IP1, in the presence of 8Br-cAMP and vasopressin. Cycloheximide or actinomycin D had little effect on the vasopressin stimulation of inositol phosphate accumulation, after an 8 h incubation in the presence or absence of 8Br-cAMP.

Inhibition of GSH efflux from rat liver by methionine: effects of GSH synthesis in cells and perfused organ

1990 ◽  
Vol 258 (6) ◽  
pp. G967-G973 ◽  
Author(s):  
J. C. Fernandez-Checa ◽  
T. Maddatu ◽  
M. Ookhtens ◽  
N. Kaplowitz
Keyword(s):  
Rat Liver ◽  
Reduced Glutathione ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Perfused Liver ◽  
Isolated Rat Hepatocytes ◽  
Dependent Inhibition ◽  
Krebs Henseleit Buffer ◽  
Isolated Rat ◽  
In Cells

The inhibition of efflux of intracellular reduced glutathione (GSH) by methionine was determined in isolated rat hepatocytes suspended either in Krebs-Henseleit buffer or in modified Fisher's medium. Methionine (1 mM) added to Krebs-Henseleit suspensions of isolated rat hepatocytes inhibited GSH efflux, with greater retention of GSH in the cells compared with control. Results were similar with methionine and 0.3 mM propargylglycine cystathionase inhibitor), suggesting no net synthesis of GSH from methionine. In Fisher's medium, the inhibitory effect of methionine on GSH efflux was masked due to increasing cellular GSH; however, the inhibitory effect of methionine was unmasked by propargylglycine, which prevented the utilization of methionine for GSH synthesis. The addition of serine (0.1 mM) to methionine in Krebs-Henseleit buffer raised cellular GSH, overcoming the inhibition of GSH efflux. In the perfused liver, infusion of 1 and 5 mM methionine initially inhibited GSH efflux, but the inhibition was reversed with continued methionine infusion. After removal of methionine, GSH efflux increased immediately. The reversal and rebound were blocked by propargylglycine, revealing concentration-dependent inhibition of sinusoidal GSH efflux by methionine. Thus, when methionine is utilized to promote GSH synthesis, its inhibitory effect on GSH efflux tends to be overcome.

scholarly journals Differential feedback regulation of cholesterol 7α-hydroxylase mRNA and transcriptional activity by rat bile acids in primary monolayer cultures of rat hepatocytes

1993 ◽  
Vol 290 (3) ◽  
pp. 685-691 ◽  
Author(s):  
J Twisk ◽  
E M Lehmann ◽  
H M G Princen
Keyword(s):  
Enzyme Activity ◽  
Bile Acid ◽  
Bile Acids ◽  
Transcriptional Activity ◽  
Rat Hepatocytes ◽  
Taurocholic Acid ◽  
Monolayer Cultures ◽  
Primary Monolayer ◽  
Primary Monolayer Cultures ◽  
Rat Bile

We have used primary monolayer cultures of rat hepatocytes to study the effects of physiological concentrations of various bile acids, commonly found in bile of normal rats, on the mechanism of regulation of cholesterol 7 alpha-hydroxylase and bile acid synthesis. Addition of taurocholic acid, the most predominant bile acid in rat bile, to the culture medium suppressed cholesterol 7 alpha-hydroxylase activity and mRNA time- and dose-dependently. The decrease in enzyme activity paralleled the changes in mRNA. Maximal suppression of cholesterol 7 alpha-hydroxylase mRNA (-91%) and enzyme activity (-89%) was observed after a 16 h incubation period with 50 microM taurocholic acid. The declines in mRNA and enzyme caused by taurocholic acid were tightly coupled and followed first-order kinetics with a half-life of 4 h. Transcriptional activity, as assessed with nuclear run-on assays, was decreased by 44% at 50 microM taurocholic acid. Mass production of bile acids (chenodeoxycholic acid and beta-muricholic acid) was inhibited to a similar extent as the cholesterol 7 alpha-hydroxylase when different concentrations of taurocholic acid were used, giving maximal inhibition (-81%) at 50 microM taurocholic acid. Glycocholic acid and unconjugated cholic acid were equally effective as taurocholic acid in suppressing cholesterol 7 alpha-hydroxylase mRNA. The more hydrophobic bile acids (chenodeoxycholic acid and deoxycholic acid) showed profound suppression of the cholesterol 7 alpha-hydroxylase mRNA by 85% and 75% respectively, whereas the other trihydroxy bile acids in rat bile, alpha- and beta-muricholic acid, were not or only marginally active. We conclude that rat bile acids, in particular the more hydrophobic ones, in concentrations commonly observed in portal blood, exert negative feedback control at the level of cholesterol 7 alpha-hydroxylase mRNA in cultured rat hepatocytes through a direct effect on the hepatocytes, and that down-regulation of transcription is only one of the mechanisms involved in this regulation.

scholarly journals Inhibition of hormonal induction of tyrosine aminotransferase by polyamines in freshly isolated rat hepatocytes

1983 ◽  
Vol 214 (3) ◽  
pp. 679-685 ◽  
Author(s):  
P Auberger ◽  
M Samson ◽  
A Le Cam
Keyword(s):  
Enzyme Activity ◽  
Rat Hepatocytes ◽  
Fold Increase ◽  
Dose Dependence ◽  
Tyrosine Aminotransferase ◽  
Enzyme Inactivation ◽  
Post Translational Modification ◽  
Isolated Rat Hepatocytes ◽  
Hormonal Induction ◽  
High Concentrations

We have analysed the effects of natural aliphatic polyamines on hormonal induction of tyrosine aminotransferase (TAT) in suspensions of hepatocytes isolated from adult fed rats. Glucagon or cyclic AMP derivatives (dibutyryl and 8-bromo) used alone caused a 4-5 fold increase in enzyme activity within 4h. This effect was independent of glucocorticoids, which also increased TAT activity (2.5-fold); when combined, the effects of the two inducers were additive. Spermine and putrescine totally inhibited the hormonally-mediated increase in enzyme activity when added at the onset of incubation with the inducers. Furthermore, polyamines could block the hormonal effect at any time during the course of TAT induction, with, however, a 30 min lag period, suggesting that they must enter the cells. Hepatocytes were indeed shown to take up spermine. At low external concentrations (less than 50 microM), an Na+-dependent, saturable and concentrative mechanism was predominant; at high concentrations (greater than 0.5 mM) transport occurred mainly through a non-saturable, Na+-independent mechanism, building up intracellular concentrations slightly lower than those in the medium. Dose-dependence analysis of the polyamine effect on enzyme induction indicated that half-maximal and maximal inhibition occurred with 0.75 mM- and 2.5 mM-spermine respectively, whereas 2.5mM- and 7.5 mM-putrescine were required respectively to obtain similar effects. Spermidine was much less effective and cadaverine had virtually no effect. None of the polyamines affected the rate of decay of TAT, nor did they directly or indirectly cause enzyme inactivation, indicating that a post-translational modification was unlikely to account for the polyamine effects. Similarly, these effects could not be ascribed to a non-specific inhibition of overall protein synthesis. We conclude that, in hepatocytes, polyamines (or their metabolites) directly interfere with one or several steps controlled by hormones in the synthesis of tyrosine aminotransferase.

scholarly journals Studies on the assembly of cytochrome oxidase in isolated rat hepatocytes

1982 ◽  
Vol 204 (1) ◽  
pp. 239-245 ◽  
Author(s):  
A Wielburski ◽  
S Kuźela ◽  
B D Nelson
Keyword(s):  
Cytochrome Oxidase ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Liver Mitochondria ◽  
Small Scale ◽  
Pulse Label ◽  
Isolated Rat Hepatocytes ◽  
Sequential Event ◽  
Triton X ◽  
Isolated Liver

1. The assembly of rat liver cytochrome oxidase was studied in isolated hepatocytes and isolated liver mitochondria labelled with L-[35S]methionine. 2. Labelled subunits II and III appeared in the immunoabsorbed holoenzyme within minutes after the initiation of a pulse label. In contrast, labelled subunit I appeared in immunoabsorbed holoenzyme only after a subsequent 2 h chase or after an additional 2 h of labelling. Subunit I was heavily labelled, however, in intact mitochondria after 10 min. 3. A similar pattern of labelling was observed in holo-cytochrome oxidase which was chemically isolated by a small scale procedure adapted for this purpose. The appearance of subunit I in the holoenzyme was delayed for 1.5-2 h after a 60 min pulse with labelled methionine. 4. Incubation of hepatocytes for 4 h in the presence of cycloheximide had no effect on the labelling pattern described above. 5. Methods were developed in which newly translated, presumably unassembled, subunits of cytochrome oxidase could be separated from the holoenzyme by fractionation in Triton X-114. Short-term pulse experiments indicate that subunits II and III are associated with the holoenzyme fraction immediately after their completion, whereas subunit I is not. 6. The data are consistent with a model in which cytochrome oxidase assembly is viewed as an ordered and sequential event.

scholarly journals The effect of acute ethanol treatment on rates of oxygen uptake, ethanol oxidation and gluconeogenesis in isolated rat hepatocytes

1985 ◽  
Vol 230 (3) ◽  
pp. 595-602 ◽  
Author(s):  
K M Stowell ◽  
K E Crow
Keyword(s):  
Ethanol Oxidation ◽  
Physiological State ◽  
Rat Hepatocytes ◽  
O2 Uptake ◽  
Isolated Rat Hepatocytes ◽  
Liver Preparation ◽  
Acute Ethanol ◽  
Ethanol Pretreatment ◽  
In Cells

In hepatocytes isolated from fed rats, acute ethanol pretreatment (at a dose of 5.0 g/kg body wt.) did not change rates of O2 uptake. In cells from starved animals, acute ethanol pretreatment increased O2 uptake by 17-29%. The increased O2 uptake in hepatocytes from starved rats was not accompanied by increased rates of ethanol oxidation, but was accompanied by increased rates of gluconeogenesis under some conditions. The provision of ethanol (10 mM) as a substrate to cells from fed or starved rats decreased O2 uptake in the absence of other substrates or in the presence of lactate, and increased it in the presence of pyruvate or lactate and pyruvate. The results of this study show that the acute effects of ethanol on liver O2 uptake are dependent on the physiological state of the liver. Previously reported large (2-fold) increases in O2 uptake after acute ethanol pretreatment may have been an artefact owing to low control uptake rates (approximately 1.8 micromol/min per g wet wt. of cells) in the liver preparation used. The ATP contents (2.4-2.6 micromol/g wet wt. of cells) and rates of O2 uptake (2.5-5.0 micromol/min per g wet wt. of cells) of cells used in the present study were the same as values reported under conditions close to those in vivo. Therefore the increase in O2 uptake in cells from starved rats after acute ethanol pretreatment is likely to be of physiological significance.

scholarly journals Mechanisms of elevation of adenosine levels in anoxic hepatocytes

1993 ◽  
Vol 290 (3) ◽  
pp. 671-677 ◽  
Author(s):  
F Bontemps ◽  
M F Vincent ◽  
G Van den Berghe
Keyword(s):  
Adenine Nucleotides ◽  
Rat Hepatocytes ◽  
Adenosine Kinase ◽  
Slight Effect ◽  
Isolated Rat Hepatocytes ◽  
Adenosylhomocysteine Hydrolase ◽  
Adenosine Concentration ◽  
Isolated Rat ◽  
Rate Of Accumulation ◽  
In Cells

Previous work has shown that normoxic isolated rat hepatocytes continuously produce adenosine from AMP and that the nucleoside is not catabolized further but immediately rephosphorylated by adenosine kinase [Bontemps, Van den Berghe and Hers (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2829-2833]. We now report the effect of anoxia on adenosine production and on the AMP/adenosine substrate cycle. In cell suspensions incubated in O2/CO2, the adenosine concentration was about 0.4 microM. It increased 30-fold in cells incubated in N2/CO2 or with 5 mM KCN, and 20-fold in cells incubated with 2 mM amytal. Adenosine production, measured in hepatocytes in which adenosine kinase and adenosine deaminase were inhibited by 5-iodotubercidin and deoxycoformycin respectively, was about 18 nmol/min per g of cells in normoxia; it increased about 2-fold in anoxia, although AMP increased 8-16-fold in this condition. From studies with inhibitors of membrane 5′-nucleotidase and of S-adenosylhomocysteine hydrolase, it was deduced that adenosine is produced by the latter enzyme and by cytosolic 5′-nucleotidase in normoxia, and by cytosolic and membrane 5′-nucleotidases in anoxia. Unlike in normoxic hepatocytes, inhibition of adenosine kinase by 5-iodotubercidin neither elevated the adenosine concentration nor enhanced total purine release from adenine nucleotides in cells treated with N2/CO2 or KCN; it had only a slight effect in cells treated with amytal. This indicates that recycling of adenosine is suppressed or profoundly inhibited in anoxia. The rate of accumulation of adenosine in anoxia was several-fold lower than the rate of its rephosphorylation upon reoxygenation. It is concluded that the elevation of adenosine in anoxic hepatocytes is much more dependent on decreased recycling of adenosine by adenosine kinase than on increased production by dephosphorylation of AMP.

Morphological and biochemical analyses of changes in rat hepatocytes related to wine and ethanol consumption

1984 ◽  
Vol 42 ◽  
pp. 232-233
Author(s):  
S.M. Geyer ◽  
C.L. Mendenhall ◽  
J.T. Hung ◽  
E.L. Cardell ◽  
R.L. Drake ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Malic Enzyme ◽  
Smooth Endoplasmic Reticulum ◽  
Rat Hepatocytes ◽  
Mature Male ◽  
Treatment Groups ◽  
Malic Enzyme Activity ◽  
Biochemical Analyses

Thirty-three mature male Holtzman rats were randomly placed in 3 treatment groups: Controls (C); Ethanolics (E); and Wine drinkers (W). The animals were fed synthetic diets (Lieber type) with ethanol or wine substituted isocalorically for carbohydrates in the diet of E and W groups, respectively. W received a volume of wine which provided the same gram quantity of alcohol consumed by E. The animals were sacrificed by decapitation after 6 weeks and the livers processed for quantitative triglycerides (T3), proteins, malic enzyme activity (MEA), light microscopy (LM) and electron microscopy (EM). Morphometric analysis of randomly selected LM and EM micrographs was performed to determine organellar changes in centrilobular (CV) and periportal (PV) regions of the liver. This analysis (Table 1) showed that hepatocytes from E were larger than those in C and W groups. Smooth endoplasmic reticulum decreased in E and increased in W compared to C values.

Macrophage Inhibition of Collagen-Induced Platelet Aggregation

1979 ◽  
Vol 42 (04) ◽  
pp. 1207-1216 ◽  
Author(s):  
Berit Mørland
Keyword(s):  
Enzyme Activity ◽  
Platelet Aggregation ◽  
Peritoneal Macrophage ◽  
Cultured Cells ◽  
Culture Media ◽  
Human Platelets ◽  
Mouse Peritoneal Macrophage ◽  
Collagenase Activity ◽  
Partial Inhibition ◽  
Macrophage Phagocytosis

SummaryCollagen was incubated with cells or media fractions of mouse peritoneal macrophage cultures, and its aggregating effect on human platelets was tested. Incubation with lysates of cultured cells completely abolished the normal collagen-induced platelet aggregation, while incubation with media fractions only caused partial inhibition. The latter inhibition was more pronounced after macrophage phagocytosis of latex particles, while endocytosis of endotoxin had no effect.Corresponding macrophage cultures were also tested for specific collagenase activity, using 14C-glycine labelled collagen as substrate. Collagenase activity was found in the culture media fractions only, and the enzyme activity could be enhanced by endocytosis of latex as well as endotoxin.It appears that the effect of macrophage lysates and media on collagen-platelet interaction cannot be ascribed only to secretion of collagenase from macrophages.

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