Analysis of discontinuous variation in albumin production by hepatoma cells at the cellular level

The clonal variation in the rate of albumin production in cultured rat hepatoma cells has been studied on a cellular basis by immunoperoxidase techniques using specific antisera against rat serum albumin. Previously, it has been shown that an array of clonal hepatoma cell populations that produce serum albumin at different rates can be isolated simply by subcloning a single clonal hepatoma cell line (Fu5). The present study demonstrates conclusively that this phenotypic variation is the result of quantal shifts in the rate of albumin production in the individual cells and is not due to changes in the percentage of albumin-producing cells. Also, by analyzing individual colonies as they develop from single cells, it was possible to establish that the rate of variation in albumin content in several hepatoma cell clones is on the order of 0.5-1.4 10(-2) per cell per generation. This variation in albumin content probably reflects shifts in the rate of albumin synthesis. Even after several sequential subclonings, the same clonal variation persists. The variants are not the result of fluctuations in albumin synthesis with different phases of the cell cycle.     

Variability in the intensity of albumin synthesis in mouse hepatocytes

Using immunoenzyme method followed by the cytophotometrical determination of individual protein content in single cells, a comparative analysis was made of albumin contents in hepatocytes of inbred mouse stocks significantly differing in their albumin contents in the blood. Some reasons of heterogeneity of parenchymal liver cells in respect to albumin synthesis were identified. The content of albumin in hepatocytes was shown to correlate with the cell ploidy. A probable contribution of the tissue level to gene expression control is discussed.     

Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)⁺ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.     

Plasma volume expansion in humans after a single intense exercise protocol.

We used intense intermittent exercise to produce a 10% expansion of plasma volume (PV) within 24 h and tested the hypothesis that PV expansion is associated with an increase in plasma albumin content. The protocol consisted of eight 4-min bouts of exercise at 85% maximal O2 uptake with 5-min recovery periods between bouts. PV, plasma concentrations of albumin and total protein (TP), and plasma osmolality were measured before and during exercise and at 1, 2, and 24 h of recovery from exercise. During exercise, PV decreased by 15%, while plasma TP and albumin content remained at control levels. At 1 h of recovery, plasma albumin content was elevated by 0.17 +/- 0.04 g/kg body wt, accounting for the entire increase in plasma TP content. PV returned to control level at 1 h of recovery without fluid intake by the subjects, despite a 820 +/- 120-g reduction in body weight. At 2 h of recovery, plasma TP content remained significantly elevated, and plasma TP and albumin concentration were significantly elevated. At 24 h of recovery, PV was expanded by 4.5 +/- 0.7 ml/kg body wt (10 +/- 1%), estimated from hematocrit and hemoglobin changes, and by 3.8 +/- 1.3 ml/kg body wt (8 +/- 3%), measured by Evans blue dye dilution. Plasma albumin content was increased by 0.19 +/- 0.05 g/kg body wt at 24 h of recovery. If 1 g of albumin holds 18 ml of water, this increase in plasma albumin content can account for a 3.4-ml/kg body wt expansion of the PV. No significant changes in plasma osmolality occurred during recovery, but total plasma osmotic content increased in proportion to PV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute suppression of albumin synthesis in systemic inflammatory disease: an individually graded response of rat hepatocytes.

The effects of an inflammatory insult on albumin of the rat liver were investigated at the cellular level and were correlated with serum albumin concentration. After SC injection of turpentine, the livers were perfused and fixed in vivo; serial liver sections were stained using a streptavidin-ABC-immunoperoxidase technique with an antibody to rat albumin. Albumin and total protein were measured at intervals after turpentine injection in whole livers and in serum. Fibrinogen was determined in plasma only. Twenty-four hours after turpentine injection serum albumin had dropped by 25% and was at 50% of its initial value at Day 3. Serum fibrinogen increased 2.4-fold within 24 hr and decreased thereafter. Liver homogenates showed no significant changes in albumin concentration. Immunohistochemically, all hepatocytes stained positive for albumin in normal animals. During inflammation, the immunostainable albumin content vanished entirely in a majority of all hepatocytes while remaining unchanged in other cells, thus producing a strikingly patchy staining pattern. No signs of resumption of albumin accumulation in depleted hepatocytes were seen after 8 days, despite a clear trend towards normalization of serum albumin concentration. These results suggest that individual hepatocytes differ widely in their response to agents that suppress albumin synthesis in an acute-phase reaction.     

The effects of dexamethasone on α-fetoprotein and albumin synthesis in cultured hepatoma 7777 cells

Dexamethasone inhibits -fetoprotein (AFP) synthesis, and stimulates albumin synthesis, in cultured hepatoma 7777 cells. These changes are due to a decrease in AFT-mRNA, and an increase in albumin-mRNA, in cells.     

Effects of bilirubin on potassium (86Rb+) influx and ionic content in Ehrlich ascites cells.

Potassium influx, intracellular potassium and sodium content and cellular volume were determined in vitro in Ehrlich ascites cells in the presence of up to 0.8 mM bilirubin in the incubation medium. Bilirubin uptake into cells as a function of bilirubin concentration in the incubation medium increased linearly with a molar bilirubin/albumin ratio of 20 : 1. Potassium influx and intracellular content decreased while cellular volume increased after 180 min of incubation of cells in bilirubin at a molar bilirubin/albumin ratio of 20 : 1. At a bilirubin/albumin ratio 2 : 1, potassium influx decreased, cellular volume remained unchanged, and bilirubin uptake into cells became saturated at bilirubin concentrations greater than 0.3 mM. It is suggested that bilirubin-induced alterations in potassium gradients across cell membranes may play a role in toxic effects of bilirubin on cells.     

Effects of bilirubin on potassium (86Rb+) influx and ionic content in Ehrlich ascites cells

Potassium influx, intracellular potassium and sodium content and cellular volume were determined in vitro in Ehrlich ascites cells in the presence of up to 0.8 mM bilirubin in the incubation medium. Bilirubin uptake into cells as a function of bilirubin concentration in the incubation medium increased linearly with a molar bilirubin/albumin ratio of 20 : 1. Potassium influx and intracellular content decreased while cellular volume increased after 180 min of incubation of cells in bilirubin at a molar bilirubin/albumin ratio of 20 : 1. At a bilirubin/albumin ratio 2 : 1, potassium influx decreased, cellular volume remained unchanged, and bilirubin uptake into cells became saturated at bilirubin concentrations greater than 0.3 mM. It is suggested that bilirubin-induced alterations in potassium gradients across cell membranes may play a role in toxic effects of bilirubin on cells.     

Regulation of albumin expression in fetal rat hepatocytes cultured under proliferative conditions: role of epidermal growth factor and hormones.

Sustained production of plasma proteins, notably albumin, is a reliable indicator of the differentiated state of hepatocytes. In this work, we have developed a fetal hepatocyte culture system where studying the regulation of albumin expression in proliferating liver cells. Our results show that under proliferative conditions (i.e., in the presence of EGF) fetal hepatocytes maintain albumin production above control quiescent non-treated cells. Glucagon and noradrenaline have no effect on the proliferation induced by EGF in cultured fetal hepatocytes; however, they act synergistically with the growth factor, increasing intracellular albumin levels. The maximum response is obtained by treatment of cells with EGF and noradrenaline. The stimulatory noradrenergic effect is mimicked by agents that increase cyclic AMP levels (forskolin plus IBMX). However, vasopressin or phorbol esters have no effect on albumin production, neither alone nor in combination with EGF. Dexamethasone, which does not alter the proliferative induction of EGF, increases albumin content. This effect is independent of the proliferative status of the cells and is not enhanced by glucagon, noradrenaline, or cyclic AMP increasing agents. The hormonal changes observed in albumin production partially correlate with changes in mRNA levels. This is the first time that cyclic AMP increasing agents are shown to act synergistically with EGF, increasing the expression of this liver specific gene.     

Identification of albumin-bound fatty acids as the major factor in serum-induced lipid accumulation by cultured cells

Factors responsible for the high lipogenic activity of rabbit serum were investigated using an assay procedure based on the gravimetric determination of the 24 hr increase in cell lipid. Cellular synthesis of fatty acids was inhibited by the presence of serum in the assay medium. Approximately 90% of the increase in cell lipid produced by serum fractions was due to triglyceride accumulation. Fractionation of rabbit serum by precipitation with ammonium sulfate or by ultracentrifugation in high density medium, both indicated that three-quarters of its lipogenic activity was associated with albumin. The lipoproteins prepared by ultracentrifugation also exhibited about one-half the activity of whole serum. The lipogenic activity of albumin was confirmed by the high potency of the albumin isolated in a nearly pure form from proteins of d>1.21 by precipitation with trichloroacetic acid and extraction with ethanol. As judged from chemical and isotopic analysis, neither the lipid content nor the lipid composition of the albumin was appreciably altered during its isolation. Of the albumin-bound lipids, only the free fatty acids, as determined by DEAE column chromatography, were present in an amount sufficient to account for the observed increase in cell triglycerides. In control experiments with horse serum of low lipogenic activity, the proteins of d>1.21 also possessed low activity in conjunction with a low content of free fatty acid. However, the albumin isolated from the latter preparation exhibited the high lipogenic activity of rabbit serum albumin. Chemical and isotopic analysis of the recovered horse serum albumin revealed that its free fatty acid content was the same as that of rabbit serum albumin. These results indicated that the isolation of horse serum albumin was attended by a substantial increase in its free fatty acid content. When the rabbit serum and horse serum content of media were adjusted to provide equivalent concentrations of albumin-bound fatty acids, the rabbit liver cells grown on the former media accumulated more lipid than cells grown on the latter media. This difference was shown to be due to the higher concentration of albumin per micro mole of fatty acid in horse serum as compared with rabbit serum. Consequently, the albumin to fatty acid ratio also controls the lipogenic activity of a serum. A linear relationship is presented which relates the cell lipid content to the molar ratio of albumin to free fatty acids and to the absolute concentration of free fatty acids in the medium.     

Neoplastic progression of rat tracheal epithelial cells is associated with a reduction in the number of growth factors required for clonal proliferation in culture

Summary Factors regulating the proliferation of normal, preneoplastic, and neoplastic rat tracheal epithelial (RTE) cells were investigated to identify changes taking place during the progression of RTE cells to neoplasia. Normal RTE cells exhibit clonal proliferation in a serum-free medium containing pituitary extract, serum albumin, cholera toxin, epidermal growth factor, hydrocortisone, and insulin. All combinations of these six factors were examined for their abilities to support clonal proliferation of normal, preneoplastic, and neoplastic RTE cells. In general, preneoplastic RTE cells required fewer factors for proliferation than normal RTE cells, and neoplastic cells required fewer factors than preneoplastic cells. A common pattern of reductions has been identified in the growth factors required for the clonal proliferation of preneoplastic vs. normal RTE cells and for neoplastic vs. preneoplastic and normal RTE cells. Normal RTE cells exhibit clonal proliferation in a serum-free medium supplemented with a minimum of six factors: bovine serum albumin, bovine pituitary extract, cholera toxin, epidermal growth factor, hydrocortisone, and insulin. Preneoplastic RTE cells exhibit clonal proliferation in a serum-free medium supplemented with four factors: bovine serum albumin, bovine pituitary extract, hydrocortisone, and insulin. Finally, neoplastic RTE cells exhibit clonal proliferation in a serum-free medium supplemented with two factors: bovine serum albumin and bovine pituitary extract. These results suggest that the progression of RTE cells to neoplasia is associated with a series of changes in regulatory pathways that control cell proliferation.     

Biochemical and physico-chemical changes in albumin in dynamics of experimental allergic encephalomyelitis

The experiments on guinea pig with experimental allergic encephalomyelitis (EAE) have shown that the most significant changes in the content, binding ability and thermodynamic characteristics of serum albumin are found at the early stages preceding the appearance of neurological characters. The plasma perfusion through granulated carbon-activated sorbents increases the binding ability and decreases the temperature denaturation of albumin down to the normal level.     

Uptake of immunoglobulin and albumin by granulated metrial gland cells in vitro

Single cell suspensions of metrial gland tissue from rats at Day 14 of pregnancy were prepared for maintenance in vitro. During the first 2 days of culture IgG was detected in glycoprotein granule-containing granulated metrial gland (GMG) cells. Albumin was also detected in GMG cells at the same stages. The IgG and albumin were not detected during the next 4 days in culture. When metrial gland cells, maintained in vitro for 5 days, were incubated with rat serum for a further 24 h, IgG and albumin were detected in GMG cells. When similar cultures were incubated for 24 h with purified rat IgG or purified rat albumin, GMG cells were positive for IgG and albumin respectively. Albumin was not detected in GMG cells in wax sections of metrial gland tissue, although IgG has previously been demonstrated. The uptake of serum proteins by GMG cells in vitro has been clearly shown but the difference in IgG and albumin content of these cells in paraffin-wax sections indicates that the means by which IgG accumulates intracellularly may be different in vitro and in vivo.     

Inhibition by tranilast of collagen accumulation in hypersensitive granulomatous inflammation in vivo and of morphological changes anf functions of fibroblasts in vitro

We examined the effects of tranilast, an anti-allergic agent, on hypersensitive inflammation and on morphology and functions of fibroblasts. In vivo, tranilast suppressed the content of collagen in granulation tissue of hypersensitive granulomatous inflammation induced by methylated bovine serum albumin (m-BSA) in rats. In culture, tranilast inhibited the TGF-β-independent inflammatory exudate-induced stimulation of morphological changes of fibroblasts to myofibroblast-like cells and their proliferation. Collagen gel contraction by myofibroblast-like cells and fibroblasts was also inhibited by tranilast. Flow cytometric analysis revealed that tranilast suspended the cell cycle of fibroblasts at the G0/G1 phase. These results suggest that tranilast modulates the fibrosis and contraction of granulation tissue by inhibiting the growth of myofibroblast-like cells and fibroblasts.     

The role of serum and serum components in the merocyanine 540-sensitized photoinactivation of K562 leukemia cells.

Serum is known to inhibit the merocyanine 540 (MC540)-sensitized photoinactivation of cells and enveloped viruses in a concentration-dependent manner. In diagnostic applications of MC540, a moderate amount of serum or serum albumin is frequently added to the staining solution because it enhances the contrast between intensely staining cells (e.g., electrically excitable cells or leukemia cells) and cells with a lower affinity for the dye (e.g., nonexcitable cells, red cells, normal leukocytes). In this communication we report on a quantitative analysis of the interactions of MC540 with serum and serum components. Human serum inhibited the MC540-sensitized photoinactivation of K562 leukemia cells most effectively, followed in order of decreasing potency by calf, newborn calf, horse, and fetal bovine serum. The photoprotective capacity of these five sera was directly proportional to their albumin content. Gel filtration experiments and differential spectroscopy showed that MC540 bound to serum albumin and lipoproteins. Both delipidated and lipidated albumin were capable of binding MC540. However, lipidated albumin had a considerably higher binding capacity and affinity for dye molecules.     

The role of arachidonic acid in vasogenic brain edema

Arachidonic acid is released rapidly from cellular membrane phospholipids after pathological insults associated with the delayed development of brain edema. Intracerebral injection of arachidonic acid caused significant increases in brain water and sodium content with decreases in potassium content and Na+,K+-ATPase activity. The 125I-labeled bovine serum albumin spaces in brain (a measure of blood-brain barrier permeability) rose threefold 24 h after arachidonic acid injection. There was gross and microscopic evidence of edema. Saturated fatty acids and monounsaturated fatty acids were not effective. These data indicate that the endothelial cells of the blood-brain barrier are target sites for the action of arachidonic acid. It is hypothesized that the increased permeability of endothelial cells to macromolecules and water results from alterations of membrane phospholipids and increased vesicular transport, changes that are responsible for the delayed development of vasogenic edema.     

The influence of glucocorticoid on albumin synthesis and its messenger RNA in rat in vivo and in hepatocyte suspension culture

Corticosteroids are known to stimulate the synthesis of a number of liver-specific proteins. The reports regarding the effect of glucocorticoid on albumin synthesis in vivo and in vitro are controversial. In an attempt to determine the mechanism by which glucocorticoid exerts its influence on hepatic albumin synthesis and to find an explanation for the conflicting data, we have studied the effect of dexamethasone disodium phosphate on albumin synthesis and albumin messenger RNA as determined by the molecular hybridization technique in hepatocytes in rat in vivo and in suspension culture. In hepatocyte suspension culture, addition of 0.48 μM dexamethasone in medium at zero time led to a significant increase (20%) in incorporation of labeled precursor into albumin as compared to control experiments; this was accompanied by a maintainance of the initial level of full-length albumin mRNA for a 9 h period. In hepatocytes cultured without dexamethasone in the medium there was a progressive loss of albumin mRNA content. Despite this finding, dexamethasone was not able to increase the albumin mRNA content in hepatocyte to a level higher than the initial value. Moreover, administration of this hormone either intraperitoneally or intravenously into rats did not lead to enhanced cell-free albumin synthesis or to an increased level of albumin mRNA. These findings suggest that glucocorticoid does not play an essential role in the regulation of albumin synthesis in vivo. In vitro, however, glucocorticoid leads to a preservation of the initial level of albumin mRNA and thus plays a role in the control of spontaneous dedifferentiation of liver cells in culture.     

Intracellular accumulation of free fatty acids in isolated white adipose cells

A simple, rapid, and accurate method was developed for measuring intracellular FFA levels in isolated white adipose cells using sucrose-(14)C or inulin carboxyl-(14)C as nontransportable, nonutilizable markers of the extracellular space. Following incubation, medium and cells were separated by centrifugation and the infranatant medium was removed by aspiration. The volume of medium trapped between cells was determined by measuring the amount of sucrose-(14)C or inulin carboxyl-(14)C retained in the floating packed adipose cells. In this way the FFA content of the adipose cells could be corrected for contamination by FFA bound to extracellular albumin. With this technique the initial events in hormone-activated lipolysis were studied under conditions of maximal and constant rates of triglyceride hydrolysis. The FFA content of isolated adipocytes of fed rats was 0.5 micro mole/g cell lipid. On addition of norepinephrine in the presence of medium albumin, the concentration of intracellular FFA rapidly increased and reached a plateau at a concentration of 2-2.5 micro moles/g cell lipid. In the presence of medium albumin an initial lag in glycerol release occurred and this was attributed to partial hydrolysis of triglyceride with retention of lower glycerides. After 5 min of incubation FFA and glycerol output was constant. In the absence of medium albumin norepinephrine-stimulated lipolysis was reduced more than 90% and extracellular FFA release was not detected. Nevertheless, intracellular FFA accumulation was identical to that seen in the presence of albumin. The data suggest that most of this intracellular pool of FFA is bound to cytoplasmic constituents.     

Regulation of albumin gene expression in a series of rat hepatocyte cell lines immortalized by simian virus 40 and maintained in chemically defined medium.

A series of simian virus 40 (SV40)-immortalized hepatocyte cell lines were characterized for albumin production, the regulation of albumin production, and the expression of other liver-specific genes. This series of cell lines is particularly useful for studying the regulation of hepatocyte gene expression because the cell lines express liverlike levels of a number of liver-specific functions and do so while growing in a chemically defined medium. SV40-immortalized hepatocyte cell lines were derived from colonies of albumin-producing epithelial cells that arose after primary hepatocytes maintained in chemically defined medium were transfected with SV40 DNA. Some cell lines secreted albumin at levels equal to or greater than those secreted by freshly plated primary hepatocytes, and all but one line continued to produce albumin for more than 20 passages. The variation in albumin secretion among cell lines reflected differences in the amount of albumin produced per cell and not in the percentage of albumin-producing cells in each line. The characterization of selected cell lines showed that albumin production was regulated by cell density during the growth cycle. Albumin production in most cell lines was also regulated by dexamethasone; however, one cell line continued to produce high levels of albumin when the cells were grown in medium lacking dexamethasone, demonstrating that although glucocorticoid can induce albumin production in some cell lines, it is not required for high levels of albumin production by all cells in culture. Regulation of albumin production measured at the level of protein secretion was paralleled by changes in steady-state levels of a 2.3-kilobase albumin RNA. Albumin-producing SV40-immortalized hepatocytes secreted a variety of other plasma proteins, including transferrin, hemopexin, and the third component of complement. These cells also expressed tyrosine aminotransferase activity that was inducible by dexamethasone. Alpha-fetoprotein production was not detected in any of the cell lines examined.     

Distribution of albumin in normal and regenerating livers of mice. A light microscopic immunohistochemical and autoradiographic study

The conditions affecting the immunohistochemical identification of albumin in livers of male NMRI-mice were investigated by light microscopy. In normal livers albumin is randomly distributed, revealing a pancytoplasmic nearly homogen reaction in groups of hepatocytes or single parenchymal cells. However, combined autoradiographic studies after pulse labelling with 3H-valine and perfusion experiments with human albumin indicate that this distribution is caused by albumin from blood plasma and does not reflect true protein synthesis. After perfusion of the livers followed by immunohistochemical amplification techniques which allowed to dilute the primary antibody up to 1:30,000, albumin could be detected nearly in all liver parenchymal cells as granular deposits decreasing in its density from periportal fields towards the terminal hepatic venules. In regenerating livers due to partial hepatectomy no remarkable differences in granular albumin deposits between G1- and S-phase of the cell cycle could be detected as was demonstrated by combined immunohistochemistry and 3H-dThd-autoradiography. However, during mitosis the content of albumin was often considerably reduced.