Metabolism of tissue cells in suspension. Synthesis of ribonucleic acid by liver cells in suspension

1. Liver cells in suspension are shown to incorporate several RNA precursors into their RNA. 2. The incorporation of [³²P]phosphate and [¹⁴C]adenine into the RNA of the cell suspension is usually of the same order as that in the perfused (or unperfused) liver slices. However, the initial lag in the incorporation of adenine into the RNA of the cell suspensions is much longer than that obtained for the tissue slices, and the optimum incorporation of adenine in the former, unlike that in the latter, needs exogenous glucose and probably a high concentration of phosphate. 3. The cell suspensions also differ from the tissue slices in being unable to incorporate [¹⁴C]orotic acid into their RNA, and resemble tumour tissues in incorporating uracil into their RNA at a rate significantly higher than that obtained with the tissue slices. 4. The above differences in the metabolic behaviour of liver-cell suspensions and tissue slices are considered to be due to the different levels of organization of the liver cells in the two tissue preparations.     

Effect of chloramphenicol on ribonucleic acid synthesis in liver cells in suspension

1. Chloramphenicol has a stimulatory effect on the incorporation of radioactive phosphate into the RNA of perfused rat-liver slices, whole liver homogenates or the liver-cell suspensions, and no effect on the incorporation of [(14)C]adenine and [(14)C]uracil into the RNA of the tissue slices. 2. Chloramphenicol completely inhibits the incorporation of labelled adenine and uracil into the RNA of the cell suspensions, or into the RNA of homogenates derived from the whole liver tissues. 3. Chloramphenicol has at most a slight inhibitory effect on the transport of labelled adenine or uracil in the hepatic cells in suspension; in the slices, the transport of these bases is not inhibited at all. 4. The above observations indicate that: (a) unlike the tissue slices, hepatic cells in suspension are permeable to chloramphenicol; (b) in the presence of chloramphenicol, for reasons that are not clear, the conversion of the base into the appropriate nucleotide does not proceed.     

The uptake of homologous ribonucleic acid by rat-liver parenchymal cells in suspension

1. Native or partially degraded RNA derived from intact rat liver, or from the parenchymal-cell or the non-parenchymatous fraction of liver, has been shown to be transported into rat parenchymal cells in suspension, without prior degradation to acid-soluble components, when the cell suspension is incubated with the RNA at 37 degrees . The amount of RNA of exogenous origin present in the parenchymal cells in an acid-precipitable form increased rapidly up to 30-60min., after which it gradually decreased, indicating intracellular degradation to acid-soluble components of the RNA taken up by the cells. 2. The RNA taken up by the parenchymal cells from the medium, and the acid-soluble products of its degradation within the cells, could be released back into the medium. 3. The RNA of exogenous origin present in acid-precipitable form in the parenchymal cells represented up to 5% of the RNA of the cells after 60min. of incubation. 4. When the concentration of RNA in the medium was less than 200mug./ml., over 10% of the RNA was transported in an acid-precipitable form in 60min. into the parenchymal cells incubated at a concentration of 2.3x10(6)/ml. 5. Ribonuclease inhibited the uptake of exogenous RNA by the parenchymal cells, whereas 2,4-dinitrophenol, sodium azide, protamine sulphate and polyvinyl sulphate had no significant effect. 6. The uptake of exogenous RNA by liver slices proceeded at a rate which was 4-20% of that obtained in the parenchymal-cell suspensions; the RNA taken up did not appear to become degraded, unlike that taken up by the cell suspensions. 7. It is concluded that dispersion of liver tissue to a suspension of single cells increases the permeability of the parenchymal cells to macromolecular RNA and creates conditions that lead to a rapid degradation of the RNA taken up.     

The effect of actinomycin D on the synthesis of ribonucleic acid and protein in rat liver parenchymal cells in suspension and liver slices

1. Rat liver parenchymal cells in suspension are shown to require a higher concentration of actinomycin D than liver slices for equivalent inhibition of the incorporation of [(14)C]adenine, [(14)C]uracil and [(32)P]phosphate into RNA, and of (14)C-labelled amino acids into protein; protein synthesis is much less susceptible to actinomycin D inhibition than RNA synthesis in both the tissue preparations. Possible causes for these differences are discussed. 2. The uptake of [(3)H]actinomycin D in the first few minutes was much greater in the cell suspensions than in the tissue slices; that in the next 1-4hr. was about the same in both the cases. The uptake by both the tissue preparations was at all times proportional to the concentration of the drug within the range 0.5-2.0mug./ml. 3. In the slices actinomycin D taken up initially was concentrated almost exclusively in the nuclei; with time the concentration of the drug in the mitochondria and the supernatant increased more rapidly than in the nuclei though at no stage did it exceed that in the nuclei. In the cell suspension the largest concentration of the drug taken up initially was found in the supernatant; most of the drug taken up subsequently also stayed in the supernatant. 4. When the drug concentration in the incubation medium was 1mug./ml., its concentration within the parenchymal cells in suspension and the parenchymal cells in the slices reached 2.2 and 1.6mug./cm.(3) of cellular volume respectively. On average, 7% of the drug was removed from the medium by the cells in suspension and 23% by the cells in the slices; the average ratio of intracellular to extracellular concentration was 2.4 in the former and 2.1 in the latter case.     

Activation of a ribonuclease on dispersion of rat liver to a single cell suspension and incubation of the cells at 37 degrees

Incubation of rat liver parenchymal cell suspensions at 37° results in degradation, to acid-soluble material, of 15% of cellular RNA at 30 minutes and 40% at three hours, beyond which there is little, if any, further degradation. The RNA which remains in the acid-insoluble form in the cells up to 30 minutes appears to exist largely in the native state. However, after 30 minutes, the acidinsoluble RNA of the cells is found to be partially depolym-erised. These observations suggest the activation of an intracellular nuclease on dispersion of the liver tissue to a single cell suspension and incubation at 37°. This nuclease appears to be responsible also for the degradation, reported earlier, of exogenous RNA taken up by the cells. Activation of the nuclease is not due to depletion of pool of ATP or of other ribonucleotides from the cells, during either dispersion of the tissue or incubation at 37°. Incubation of the cells at 28°, or of liver slices at 37°, does not lead to any significant degradation to acid-soluble material, or to partial depolymerisation, of RNA. Analysis of RNA obtained from cells incubated at 37° for various periods showed that chromatography on methylated albumin-kiesulguhr (MAK) and Sephadex columns is not suitable for detecting partial depolymerisation of cellular RNA; RNA shown to be partially depolymerised by analysis on sucrose density gradient, in an analytical ultracentrifuge, and on a cellulose column, gave the normal pattern in MAK or Sephadex runs.     

Rat choroid plexus specializes in the synthesis and the secretion of transthyretin (prealbumin). Regulation of transthyretin synthesis in choroid plexus is independent from that in liver

Synthesis of total protein and of transthyretin in rat choroid plexus was studied by measuring the incorporation of radioactive leucine into proteins in choroid plexus tissue incubated in vitro. About 20% of the protein newly synthesized in choroid plexus and about 50% of the newly synthesized protein secreted into the medium was transthyretin. Evidently, the choroid plexus is very active in the biosynthesis of this carrier protein for thyroid hormones and could be an important link in the chemical communication between the body and the central nervous system. Acute inflammation, which leads to a profound rearrangement of the pattern of plasma protein synthesis rates in the liver, produced distinct changes in the levels for plasma protein mRNAs in the liver. The levels of the mRNAs for alpha 1-acid glycoprotein and major acute phase alpha 1-protein increased more than 30-fold, those for transthyretin and albumin decreased to 27 and 57% of normal, respectively. The pattern of the observed changes in the levels of mRNAs for plasma proteins in the liver was independent of whether the acute inflammation was produced by subcutaneous injection of turpentine or intraperitoneal injection of a suspension of talcum. However, levels of transthyretin mRNA in choroid plexus were affected only very slightly, or not at all. Apparently, transthyretin synthesis in liver and choroid plexus is regulated independently during the acute phase response. No mRNA was detected in choroid plexus for albumin, alpha 1-acid glycoprotein, and major acute phase alpha 1-protein under any conditions.     

Characterization and cell cycle kinetics of hepatocyte populations isolated from adult liver tissue by a nonenzymatic procedure

Suspensions of liver cells enriched in lobular parenchymal hepatocytes were isolated from adult mouse hepatic tissue by nonenzymatic dispersion in chelating buffer and sedimentation of the released cells at unit g. Single cell suspensions so obtained were suitable for flow cytometric measurements of hepatic ploidy class distributions. The more quickly sedimenting cell population consisted of 88% albumin/transferrin-positive epithelial hepatocytes, the nuclei of which were bimodally distributed with respect to RNA content. This dual G1 population was observed in 2C DNA content liver nuclei prepared by several methods and appears to be a general cytochemical characteristic of adult liver parenchymal cells.     

Flow cytometric evaluation of cell dispersion from human head and neck tumors

The preparation of single-cell suspensions from 25 human head and neck tumors is described. Dispersal was performed overnight at 4 degrees C under slight agitation of the tissue suspensions using various combinations of enzymes and additives. The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM). It was shown that both trypsin/dithioerythritol (TD) and collagenase/D Nase (CDse) were of value in dispersing single cells from tumor tissue. In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes. In a number of cases, DNA-FCM revealed ploidy abnormalities in a TD-suspension, which were not discernible in the concurrent CDse-suspension. Cell culture of primary cell suspensions corroborated the reliability of the DNA-FCM measurements. Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity. Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension.     

Effect of cell concentration on the uptake of amino acids by rat liver parenchymal cells in suspension.

The accumulation of several amino acids in the acid-soluble fraction and their incorporation into protein in rat liver parenchymal cell suspensions, has been shown to depend on the concentration of cells in the incubation medium; the uptake, both in the acid-soluble and the acid-insoluble fractions, decreased as the cell concentration increased from 0.03 X 10(6) cells/ml upwards, reaching a plateau at high cell concentrations (3-5 X 10(6) cells/ml). The uptake values at high cell concentrations were the same as those obtained in liver slices in which a similar effect was not observed. Evidence is presented which suggests that this phenomenon is mediated by a material released from the cells in suspension, which is inhibitory to enhancement of the uptake of amino acids by these cells over and above the value obtained in normal, adult liver slices.     

Effects of prolonged incubation and cell concentration on lipogenesis from glucose in isolated human omental fat cells

The behavior of human omental fat cells in vitro has been examined in order to define conditions under which glucose is converted to glyceride-glycerol and glyceride fatty acids. Synthesis of glyceride fatty acids from glucose reached maximal rates only after several hours of incubation in Krebs-Henseleit bicarbonate buffer, with or without added bovine albumin. Conversion of glucose to glyceride fatty acids was readily demonstrable with concentrated cell suspensions and was stimulated 3- to 8-fold by insulin. With dilute cell suspensions, little fatty acid was synthesized even after prolonged incubation in the presence of insulin. Conversion of glucose to glyceride-glycerol was linear during 6-hr incubations in buffer and unaffected by the concentration of the cell suspension. In the presence of bovine albumin, glyceride-glycerol synthesis was readily demonstrable at all cell concentrations used, although synthesis was faster in dilute suspensions. Thus, different incubation conditions produce widely divergent patterns of glucose metabolism in human omental fat cells.     

Interferon suppresses glutamine synthetase induction in chick embryonic neural retina.

Two different proteins precipitable with antiserum to albumin exist in liver. One is albumin, the other is precursor albumin. Liver cells in suspension contain mainly precursor, but secrete only albumin. In subcellular fractions isolated from liver homogenate, 95.3% of anti-albumin precipitable protein in the rough endoplasmic reticulum, 51.4% in the smooth endoplasmic reticulum, 33.5% in the Golgi apparatus and 0% in the supernatant fraction was precursor albumin. The results suggest that albumin precursor is synthesized in the rough endoplasmic reticulum and converted into albumin in the smooth endoplasmic reticulum and the Golgi apparatus.     

Apolipoprotein B production and very low density lipoprotein secretion by calf liver slices.

Secretion of triglycerides by the liver in ruminants as components of very low density lipoproteins particles is low as compared with that in primates or rodents. The rate-limiting steps for the hepatic export of very low density lipoproteins have been studied in liver slices to determine the origin of the low lipotropic capacity of calf liver compared to that of rat liver. The rates of production of apolipoprotein B (apo B) and albumin as well as the rate of secretion of VLDL-apolipoproteins were measured during 12-h incubation of liver slices in organo-culture using [35S]methionine-cysteine labeling. Hepatic apo B production was similar in the two animal species but the VLDL-apolipoprotein secretion rate for calf liver slices amounted to only 20% of that observed for rat liver slices. Although calf and rat liver slices synthesized similar amounts of total protein, the hepatic production of albumin, measured in cells and media, was much higher in calf than rat liver slices (around 2.7-fold), whereas the rate secretion of albumin was similar in the two species. Our results showed that the slow rate of secretion of VLDL by calf liver cells was not consecutive to a low rate of synthesis of apo B but rather to a defect in VLDL assembly and/or secretion.     

Studies on the assembly and secretion of a multicomponent protein. The biosynthesis of vitellogenin, an oestrogen-induced glycolipophosphoprotein

1. Incorporation of [(32)P]P(i) and [(3)H]leucine into vitellogenin secreted in vitro by liver slices from oestrogen-treated Xenopus laevis is accompanied by a 2h lag; no lag is apparent for the incorporation into total tissue protein. 2. The addition of cycloheximide was found immediately to inhibit further incorporation of radioactive leucine into total tissue protein. The incorporation into secreted vitellogenin, however, continued for 2h after the addition of cycloheximide. 3. Pulse-labelling of liver slices with [(3)H]leucine for 30min, followed by a chase with a large excess of unlabelled leucine, resulted in the appearance of radioactivity in secreted vitellogenin from 90min after the end of the pulse period. 4. Evidence is presented which suggests that of the radioactivity from [(3)H]leucine incorporated into proteins by the liver of oestrogen-treated Xenopus some 70% is present in the single protein vitellogenin. 5. The incorporation of [(32)P]P(i) into vitellogenin followed a pattern identical with that found for [(3)H]leucine in the pulse-labelling experiments and this indicates that synthesis of the polypeptide chain and incorporation of P(i) are closely linked processes. 6. The cumulative evidence suggests that the 2h lag phase represents the time required for the assembly and secretion of this multicomponent protein.     

Ultrastructural location of albumin and fibrinogen in primary cultures of new-born rat liver tissue

In primary cultures of new-born rat liver tissue, albumin and frbrinogen, two proteins normally synthesized by the liver and secreted into plasma were demonstrated by specific antibodies labelled with peroxidase in about 50 and 70% of the hepatocytes; these proteins were not demonstrated in the other types of cells, in particular fibroblasts, present in primary cultures. These two proteins were detected on the ribosomes of the rough endoplasmic reticulum and were also present in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus. It is concluded that

1. 1. In primary cultures of liver tissue, only the hepatocytes synthesize albumin and fibrinogen.

2. 2. Proliferating cultured hepatocytes are able to synthesize albumin and fibrinogen.

3. 3. The presence of detectable albumin and fibrinogen in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in hepatocytes of primary cultures and their absence in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in the hepatocytes of adult rat liver might indicate an alteration in the translocation of albumin and fibrinogen through these organelles in cultured hepatocytes.

     

Biosynthesis of Rat serum albumin

1. The labelling of intracellular and extracellular serum albumin was studied in liver slices and in whole rats by using new methods for the purification of the protein. 2. The results suggest that a polypeptide precursor is formed that is converted relatively slowly into serum albumin. 3. The effect of liver cell K(+) has been examined by a double-label method and it is shown that K(+) accelerates the rate of conversion of ;precursor' into albumin. The rate of transit of albumin across the cell membrane appears to be unrelated to the concentration of K(+) within the cell. 4. The time-course of incorporation of radioactive amino acid into albumin follows a sigmoidal mode. There is a pronounced time-lag before label starts to appear in intracellular albumin, and a further time-lag before it appears in extracellular albumin. 5. In slices the sum of intra- and extra-cellular label rises steadily from 30min after the start of labelling with a pulse of labelled leucine or valine and continues to rise for at least another 60min. This occurs whether labelling is stopped by addition of excess of carrier amino acid or with cycloheximide (100mum) or both. 6. The intracellular albumin content remains constant whether slices are maintained with low or normal intracellular K(+) concentrations. 7. Specific radioactivities of intracellular albumin (and fractions thereof) and of extracellular albumin were determined in vitro and in vivo. The results show that the intracellular albumin cannot be a precursor of extracellular albumin, unless a very small compartment is turning over much more rapidly than the bulk of the liver albumin or even of the microsomal albumin.     

A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration

Current methods of cryopreservation of hepatocytes in single cell suspensions result in low overall yields of hepatocytes, demonstrating long-term preservation of hepatocellular functions. A novel culture method has recently been developed to culture liver cells in a sandwich configuration of collagen layers in order to stabilize the phenotypic expression of these cells in vitro (J. C. Y. Dunn, M. L. Yarmush, H. G. Koebe, and R. G. Tompkins, FASEB J. 3, 174, 1989). Using this culture system, rat hepatocytes were frozen with 15% (v/v) Me2SO to -70 degrees C, and stored at approximately -100 degrees C. Following rapid thawing, long-term function was assessed by measuring albumin secretion in culture for 7-14 days postfreezing. Comparison was made with cryopreservation of liver cells in single cell suspensions. Cryopreservation of liver cells in suspension resulted in only a 2% yield of cells which could be successfully cultured; albumin secretion rates in these cultured cells over 48 hr were 26-30% of secretion rates for nonfrozen hepatocytes. Freezing cultured liver cells in the sandwich configuration after 3, 7, and 11 days in culture maintained 0, 26, and 19% of the secretion rates of nonfrozen hepatocytes, respectively. Morphology of the cryopreserved cells appeared grossly similar to cells without freezing; however, this morphological result was patchy and represented approximately 30% of the cells in culture. These results represent the first demonstration of any quantitative long-term preservation of hepatocellular function by cryopreservation, suggesting that cultured hepatocytes can survive freezing and maintain function.     

Tissue responses against Cladosporium trichoides and its parasitic forms in congenitally athymic nude mice and their heterozygous littermates

The tissue responses against Cladosporium trichoides and its parasitic forms were studied using nude (nu/nu) mice and their heterozygous (nu/+) littermates of BALB/c background.1.0,0.1 and 0.01% cell suspensions were prepared from a culture broth which had been inoculated with the C. trichoides and cultured with reciprocal shaking at 27 ° C for 7 days. Sixty nu/nu or 60 nu/+ mice were divided into three groups consisting of 20 each which was allotted to one of the three cell suspensions. Each mouse was inoculated intravenously with 0.1 ml of either the cell suspensions. Two mice from each of the six groups were sacrificed at adequate intervals until 30 days after inoculation and histopathologic sections stained with H & E or by PAS were prepared from their visceral organs.There were no characteristic findings in the nu/nu and nu/+ mice inoculated with the 0.01% cell suspension. When inoculated with the 1.0% cell suspension, the brain was the favorite target organ in both groups of mice and the kidney was the second. When inoculated with the 0.1% cell suspension, brain lesions were observed only in the nu/nu mice. The susceptibility of the nu/nu mice was higher than that of the nu/+ mice.The parasitic forms in the brain of the nu/nu and nu/+ mice were slender septate true hyphae with or without polymorphonuclear leucocyte infiltrate, while in the liver, spleen and lung of both groups of mice the parasitic forms were short thick hyphae, moniliform hyphae, chlamydospores or round cells (sclerotic cells). Many giant cells containing fungal elements appeared in the liver of the nu/nu mice.     

Protein synthesis with membrane-bound polysomes and albumin messenger RNA from livers of mutant mice

Summary Investigation of deficiencies in serum protein synthesis resulting from deletion-mutations at the albino locus in mice was continued usingin vitro conditions. Previous work showed that although total protein synthesis was only slightly lower in livers from albinos, newly synthesized protein appearing in plasma was 22% of that in controls. It was thought that the disorganized endoplasmic reticulum and Golgi apparatus, characteristic for the liver (and kidney) of these mutants, might be responsible for the observed deficiencies. In the present study mebrane-bound polysomes isolated from the livers of newborn albinos were 55% (c^3H/c^3H strain) and 62% (c^14CoS/c^14CoS strain) as efficient as those from normal littermates in incorporating radioactive leucine into protein in a cell-free system. These differences could not be eliminated by the addition of excess liver mRNA, exogenous soluble factors or by the exchange of cell sap between albino and control polysomes. In an earlier study albino liver slices synthesized only 13% (or 17% per mg of total protein synthesized) as much albumin as controls. We have now found that the level of albumin poly(A)⁺-RNA isolated from albino livers and assayed with a reticulocyte lysate, was almost as high (85%) as in controls. It was concluded that the very low level of albumin synthesis in albino livers did not result from a deficiency of albumin mRNA. Whether the rate-limiting step in synthesis of albumin in mutant livers is at the level of translation or processing for secretion requires further investigation.     

The separation of intracellular serum albumin from rat liver

1. Antibody precipitation of serum albumin from rat liver extracts yields impure preparations of the protein. 2. When rat liver is labelled with l-[1-(14)C]leucine, antibody precipitation of albumin leads to material that is contaminated with a protein or proteins of very high specific radioactivity. Only 10-25% of the radioactivity of the antibody precipitate is associated with serum albumin. 3. A chromatographic procedure is described that can be used to separate radiochemically pure serum albumin from antibody precipitates obtained from extracts of rat liver. 4. Extracellular albumin secreted by liver slices yields a precipitate with antibody which contains much less radioactive impurity. About 70-90% of the radioactivity is associated with serum albumin. Serum albumin separated by antibody precipitation from rat serum labelled in vivo was not contaminated with the radiochemical impurities associated with intracellular albumin. 5. A simple method is described of obtaining the content of serum albumin in rat liver extracts by the technique of isotope dilution and ion-exchange chromatography.     

Synthesis and secretion of some plasma proteins by tissue slices of Morris hepatoma 7777 and liver from control and turpentine-injected rats

Slices of Morris hepatoma 7777 or rat liver isolated from control or turpentine-injected rats were incubated for 2 h with 14C-leucine. Radioactivities incorporated into albumin, alpha-fetoprotein, fibrinogen, alpha 1-AP-globulin, haptoglobin and alpha 1-acid glycoprotein were determined after the proteins had been isolated from the incubation medium or tissue homogenate by immunoprecipitation with monospecific antisera. It was found that hepatoma synthesizes fibrinogen, alpha 1-AP-globulin and alpha 1-acid glycoprotein in the amounts comparable to rat liver, whereas formation of albumin and haptoglobin is reduced 5- to 10-fold. Local inflammation elicited by injection of turpentine to tissue donors increased formation of acute-phase protein in liver slices but had no effect on synthesis of these proteins in preparations of Morris hepatoma, although certain ultrastructural changes in the Golgi complex were observed not only in the liver but also in the tumour.