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.     

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.     

Effect of hypophysectomy on liver nuclear ribonucleic acid synthesis in aging rats.

Changes in RNA synthesis in liver nuclei were observed at different ages and after hypophysectomy and hormone replacement in female Sprague-Dawley rats. As determined by the incorporation of [3H]UMP into an acid-insoluble product, RNA synthesis decreased by about 75% in intact rats from 6 months to 24 months of age. This decline with age was not observed in liver nuclei from 24-month-old rats that had been hypophysectomized at 12 months and maintained on a minimal hormone-replacement therapy. Thyroid hormones and somatotropin (growth hormone) had an additive effect on RNA synthesis in liver nuclei from these hypophysectomized rats. The same hormones had no significant effect on intact, age-matched rats. With advancing age, nuclei of intact rats had an increase in the pool of free RNA polymerase and an apparent decrease in the enzyme activity bound to nuclear chromatin. There was no change in total enzyme with age. In hypophysectomized, hormone-treated rats, free RNA polymerase activity decreased and chromatin-bound activity increased. There was no difference in total nuclear RNA polymerase activity between operated or intact rats. However, the ratio of the bound to the free activity was different. These results suggest that the ability of RNA polymerase to bind to chromatin may be involved in the age-related decrease in liver nuclear RNA synthesis of intact rats.     

Effect of biotin on ribonucleic acid synthesis.

A single injection of biotin to biotin-deficient rats produces a two-fold increase in the incorporation, both in vivo and in vitro of precursors into nucleic acids as early as 2 h after the biotin treatment. The specific activity of the precursor pool is not affected by biotin. Analysis of the polysome profile at various times following biotin treatment and a kinetic study of the effect of excess poly(U) on the incorporation of phenylalanine by cell-free amino acid incorporation experiments indicate a marked decrease in messenger-free ribosomes in rat liver after biotin administration.     

Effect of ethionine on synthesis and methylation of ribosomal ribonucleic acid in regenerating rat liver

Ethionine, a hepatocarcinogen, was administered into rats 24 h before partial hepatectomy and immediately thereafter. Hepatic precursor ribosomal RNA (pre-rRNA) obtained 20 h after the operation of rats injected with ethionine and adenine resulted in methyl deficiency as judged by the incorporation of [3H]methyl group of S-adenosylmethionine into nuclear rRNA by partially purified rRNA methylase. The ethionine and adenine treatment causes methyl deficiency of nuclear rRNA at 2'-hydroxyribose sites of cytidine and uridine, but not at base sites. Although the ethionine and adenine treatment produced no significant change in total hepatic RNA synthesis in vivo assayed by the incorporation of labeled orotate, a one-third increase in nuclear rRNA synthesis as well as a one-third decrease in microsomal rRNA synthesis was found under the treatment. These results suggest that the undermethylation at 2'-hydroxyribose of pre-rRNA in liver nucleus, which is caused by ethionine and adenine administration into rats, causes an inhibition of the processing of nuclear pre-rRNA to cytoplasmic rRNA.     

The effect of ethionine on ribonucleic acid synthesis in rat liver.

1. By 1h after administration of ethionine to the female rat the appearance of newly synthesized 18SrRNA in the cytoplasm is completely inhibited. This is not caused by inhibition of RNA synthesis, for the synthesis of the large ribosomal precursor RNA (45S) and of tRNA continues. Cleavage of 45S RNA to 32S RNA also occurs, but there was no evidence for the accumulation of mature or immature rRNA in the nucleus. 2. The effect of ethionine on the maturation of rRNA was not mimicked by an inhibitor of protein synthesis (cycloheximide) or an inhibitor of polyamine synthesis [methylglyoxal bis(guanylhydrazone)]. 3. Unlike the ethionine-induced inhibition of protein synthesis, this effect was not prevented by concurrent administration of inosine. A similar effect could be induced in HeLa cells by incubation for 1h in a medium lacking methionine. The ATP concentration in these cells was normal. From these two observations it was concluded that the effect of etionine on rRNA maturation is not caused by an ethionine-induced lack of ATP. It is suggested that ethionine, by lowering the hepatic concentration of S-adenosylmethionine, prevents methylation of the ribosomal precursor. The methylation is essential for the correct maturation of the molecule; without methylation complete degradation occurs.     

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.     

Sequence complexity of polyadenylated ribonucleic acid from soybean suspension culture cells

The sequenc complexity of total poly(A) RNA from a higher plant system, soybean cultured cells, was determined. Labeled cDNA synthesized from the poly(A) RNA hybridized exclusively with the unique sequence component of total soybean DNA. Analysis of the hybridization reaction between cDNA and the poly(A) RNA template revealed three abundance classes in the poly(A) RNA. These classes represent 18, 44, and 38% of the poly(A) RNA and contain information for approximately 60, 1900 and 30,000 different 1400-nucleotide RNA molecules. From these results, the total sequence complexity of poly(A) RNA was estimated to be 4.5 X 10(7) nucleotides. Saturation hybridization of labeled unique DNA with RNA showed that the total cell RNA represents 12.4% of the unique DNA sequence complexity, or 6.4 X 10(7) nucleotides, while poly(A) RNA respresent 8.7% of the unique DNA sequence complexity, or 3.3 X 10(7) nucleotides. Thus, it is estimated that 50--70% of total RNA sequence complexity is contained in poly(A) RNA in these cells.