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Biomedical subjects

P O Seglen

Publications and source records attributed to P O Seglen.

At least 109 records · Page 6Linked to original sources
PubMed1984

Amino acid control of autophagic sequestration and protein degradation in isolated rat hepatocytes.

Sequestration of the inert cytosolic marker [14C]sucrose by sedimentable organelles was measured in isolated rat hepatocytes made transiently permeable to sucrose by means of electropermeabilization. Lysosomal integrity, protein degradation, autophagic sequestration, and other cellular functions were not significantly impaired by the electric treatment. Hepatocytes sequestered sucrose at an initial rate of approximately 10%/h, which is threefold higher than the estimated rate of autophagic-lysosomal protein degradation. Almost one-third would appear to represent mitochondrial fluid uptake; the rest was nearly completely and specifically inhibited by 3-methyladenine (3MA) and can be regarded as autophagic sequestration. A complete amino acid mixture was somewhat less inhibitory than 3MA, and partially antagonized the effect of the latter. This paradoxical effect, taken together with the high sequestration rate, may suggest heterogeneity as well as selectivity in autophagic sequestration. There was no detectable recycling of sequestered [14C]sucrose between organelles and cytosol. Studies of individual amino acids revealed histidine as the most effective sequestration inhibitor. Leucine may have a regulatory function, as indicated by its unique additive/synergistic effect, and a combination of Leu + His was as effective as the complete amino acid mixture. Asparagine inhibited sequestration only 20%, i.e., its very strong effect on overall (long-lived) protein degradation must partially be due to post-sequestrational inhibition. The lysosomal (amine-sensitive) degradation of short-lived protein was incompletely inhibited by 3MA, indicating a contribution from nonautophagic processes like crinophagy and endocytic membrane influx. The ability of an amino acid mixture to specifically antagonize the inhibition of short-lived protein degradation by AsN + GIN (but not by 3MA) may suggest complex amino acid interactions at the level of fusion between lysosomes and other vesicles in addition to the equally complex interactions at the level of autophagic sequestration.

Acid Phosphatase↗
PubMed1984

Emergence of a population of small, diploid hepatocytes during hepatocarcinogenesis.

The sequential treatment of young Wistar rats with two different carcinogens (diethylnitrosamine - plus partial hepatectomy - as an initiator, and 2-acetylaminofluorene as a cytotoxic selection pressure) induces the appearance of foci and nodules of liver cells which are phenotypically altered. By means of an algorithm which takes into account binuclearity as well as cell-to-cell aggregation it is possible to compute cellular ploidy distributions from flow-cytometric analysis of either hepatocyte suspensions or suspensions of hepatocytic nuclei. Cell suspensions isolated from carcinogen-treated rats can be shown to contain, already after 8 weeks, approximately 70% small, diploid hepatocytes, whereas suspensions from normal or partially hepatectomized control livers contain only approximately 10% diploid cells (the remainder being mostly tetraploid). Isolated nodules, i.e., expanding clones of proliferating cells, believed to be neoplastic precursor lesions, contained almost only diploid cells. These observations suggest that the selective outgrowth of a population of small, diploid hepatocytes may be a significant early step in the development of liver cancer.

2-Acetylaminofluorene↗
PubMed1983

Paradoxical stimulation by amino acids of the degradation of [35S]methionine-labelled, short-lived protein in isolated rat hepatocytes.

A complete amino acid mixture inhibited the degradation of long-lived and [14C]valine-labelled short-lived protein in isolated rat hepatocytes, but paradoxically stimulated the degradation of [35S]methionine-labelled short-lived protein. The stimulation persisted in the presence of autophagiclysosomal pathway inhibitors like 3-methyladenine and propylamine, indicating the existence of an hitherto unrecognized non-lysosomal degradation mechanism with selectivity towards methionine-rich proteins or peptide regions.

Adenine↗
PubMed1983

Structural and physical changes in lysosomes from isolated rat hepatocytes treated with methylamine.

Incubation of isolated rat hepatocytes with 10 mM methylamine resulted in an inhibition of endogenous protein degradation and a microscopically visible enlargement of the lysosomes. Lysosomes from methylamine-treated cells exhibited increased buoyancy in metrizamide gradients and increased fragility as measured by the release of acid phosphatase activity in vitro, despite the fact that no methylamine remained in the gradient-isolated organelles. When methylamine was extracted from intact cells, the inhibition of protein degradation was immediately relieved, whereas the lysosomal enlargement (and to a certain extent also the increased fragility) persisted for some time. The methylamine-induced osmotic swelling of the lysosomes would thus seem to involve not merely a passive stretching of the lysosomal membrane, but rather some structural change (e.g., an increased amount of membrane material) which is relatively slowly reversible, but without effect on lysosomal function.

Acid Phosphatase↗
PubMed1983

Cellular and lysosomal uptake of methylamine in isolated rat hepatocytes.

Upon addition of methylamine to intact cells, this lysosomotropic weak base accumulates intracellularly as the result of at least two different mechanisms: (1) facilitated diffusion across the plasma membrane, i.e. a process which is carrier-mediated and subject to both trans-stimulation (accelerative exchange) and cis-inhibition (competition) by other amines (e.g. ammonia, methylamine and triethylamine); this transport process is furthermore non-concentrative, energy-independent, and (although moderately temperature-sensitive) operative even at 0 degrees C; (2) active uptake, i.e. an energy-dependent concentrative process which is inhibited by anoxia and energy inhibitors. With time, methylamine accumulates in lysosomes and gives rise to a lysosomal swelling which is easily visible by optical microscopy, and which causes the cells to appear coarsely granular. After a 1h incubation with 10mM-methylamine, the total cell volume is increased by about 12%. Under anoxic conditions or in the presence of energy inhibitors, lysosomal swelling is abolished regardless of there being a high concentration of methylamine intracellularly (taken up by facilitated diffusion). The continuous accumulation of methylamine in lysosomes therefore seems to depend on an energy-requiring process (such as continuous proton pumping), and not only on trapping by Donnan-equilibrium-generated protons.

Amines↗
PubMed1982

The relation between protein accumulation and cell cycle traverse of human NHIK 3025 cells in unbalanced growth.

Human NHIK 3025 cells, synchronized by mitotic selection, were given 2 mM thymidine, which inhibited DNA synthesis without reducing the rate of protein accumulation. After removal of the thymidine the cells proceeded towards mitosis and cell division, with an S duration 2 hours shorter than, but a G2 and M duration nearly identical to that of the control cells. If cycloheximide (1.25 muM) was present together with thymidine, no net protein accumulation took place during the treatment, and the subsequent duration of S, G2, and M was similar to that of untreated cells. The shortening of S seen after treatment with thymidine alone would therefore indicate that the rate of DNA synthesis depended on the amount of some preaccumulated protein. The postreplicative period in thymidine-treated cells was lengthened by cycloheximide treatment although the protein content had already been doubled. This suggests that proteins required for the traverse of this part of the cell cycle might have to be synthesized after completion of DNA replication. Shortly after removal of thymidine, the rate of protein accumulation declined markedly, indicating the existence of some mechanism for negative control of cell mass. In addition, the daughters of thymidine-treated cells had their cell cycle shortened by 2 hours. As a result, the cells had returned to balanced growth already in the first cell cycle following the induction of unbalanced growth. In conclusion, our experiments suggest that NHIK 3025 cells might require a minimum time in order to traverse the cell cycle, which is independent of cell mass.

Cell Division↗
PubMed1982

Amino acid and energy requirements for rat hepatocytes in primary culture.

The amino acid and energy requirements of rat hepatocytes in suspension and early culture were investigated. Among a number of potential energy substrates tested, pyruvate (20 mM) was found to be most effective in stimulating hepatocytic protein synthesis. Amino acids stimulated protein synthesis both as energy substrates and as protein precursors. An amino acid mixture was designed to provide maximal inhibition of protein degradation as well as maximal stimulation of protein synthesis. In a defined medium containing amino acids at these concentrations, and supplemented with glucocorticoid hormone and insulin, hepatocytes could be maintained--on a collagen substratum--for at least a week without any significant net loss of cells or cellular protein.

Amino Acids↗
PubMed1982

Autophagic sequestration of [14C]sucrose, introduced into rat hepatocytes by reversible electro-permeabilization.

Isolated rat hepatocytes could be made permeable to small molecules such as [14C]sucrose (but not to proteins) by subjecting the cells to repeated electric discharges in a high-voltage field. During subsequent incubation at 37 degrees C, the permeability changes were reversed within 15 min, the electro-injected [14C]sucrose remaining trapped inside the re-sealed plasma membrane. Autophagic sequestration of [14C]sucrose, i.e., the transfer of radioactivity from cytosol to sedimentable vesicles (autophagosomes and lysosomes), could be followed by incubating the [14C]sucrose-loaded hepatocytes for up to 2 h at 37 degrees C. After incubation, the cells were disrupted by a single high-voltage discharge in electrolyte-free medium (sucrose), and sedimentable cell components were separated from the cytosol by centrifugation through metrizamide. By the use of these methods, which are particularly suitable for the analysis of many small cell samples, it could be shown that [14C]sucrose was autophagically sequestered in the hepatocytes at a rate of 4-5%/h. The sequestration was nearly completely inhibited by the specific autophagy inhibitor 3-methyladenine.

Animals↗
PubMed1982

3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes.

3-Methyladenine (5 mM) inhibits endogenous protein degradation in isolated rat hepatocytes by about 60%, while having no adverse effect on the degradation of an exogenous protein (asialofetuin), on protein synthesis, or on intracellular ATP levels. 3-Methyladenine appears to act specifically upon the autophagic/lysosomal pathway of degradation, as judged from its lack of effect in the presence of amino acids or a lysosomotropic amine (propylamine). The effect of the purine is not mediated by amino acids because the inhibition of protein degradation is accompanied by a significant depression of intracellular amino acid levels. The ability of 3-methyladenine to suppress the formation of electron microscopically visible autophagosomes suggests that it may be regarded as a specific inhibitor of autophagy.

Adenine↗
PubMed1982

Inhibition of hepatocytic protein degradation by inducers of autophagosome accumulation.

Out of nine compounds known to induce accumulation of autophagosomes, seven were found to inhibit degradation of endogenous protein and all of them to inhibit degradation of an exogenous protein (asialofetuin) in isolated rat hepatocytes. On the basis of these findings we propose two possible common mechanisms by which the drugs may cause autophagosome accumulation: 1) The inhibition of protein degradation may result in a decrease in the intracellular amino acid levels, a change which in turn serves as a stimulus for increased autophagic sequestration. 2) Disturbance of the function of the lysosomes may reduce their ability to fuse with newly formed autophagosomes, thereby causing accumulation of the latter.

Animals↗
PubMed1981

Inhibition of hepatocytic protein degradation by methylaminopurines and inhibitors of protein synthesis.

Nutritional control of protein degradation in isolated rat hepatocytes can take place in the absence of protein synthesis. Suppression of degradation by amino acids (step-up) is unaffected and the enhanced degradation seen upon amino acid deprivation (step-down) is only partially inhibited by cycloheximide at a concentration (10(-3) M) which inhibits protein synthesis virtually completely. Protein degradation per se is, however, inhibited by cycloheximide as well as by puromycin, apparently at least in part by mechanisms additional or unrelated to their effect on protein synthesis. Several puromycin analogues (methylaminopurines) are stronger inhibitors of protein degradation than of protein synthesis, most notably puromycin aminonucleoside and 6-dimethylaminopurine riboside (N6,N6-dimethyladenosine). The latter compounds appear to specifically inhibit cellular autophagy, since neither the degradation of endocytosed protein (asialofetuin) nor the extralysosomal (amino acid-, propylamine- and leupeptin-resistant) degradation are affected.

Adenosine↗
PubMed1981

Vanadate inhibits protein degradation in isolated rat hepatocytes.

Vanadate (10 mM) strongly inhibited endogenous protein degradation as well as the degradation of an exogenous, endocytosed protein (asialofetuin) in isolated rat hepatocytes. Protein synthesis and cellular viability were unaffected, but changes in cell morphology suggested some interference with cytoskeletal elements. The effect of vanadate was comparable to the effects of several other degradation inhibitors (lysosomotropic amines, leupeptin, vinblastine, amino acids, dimethylaminopurine riboside) known to inhibit the autophagic/lysosomal pathway of protein degradation. Vanadate inhibited proteolysis in a liver homogenate at pH 5, suggesting a direct effect upon the lysosomal proteinases.

Animals↗
PubMed1981

Effects of amino acid analogues on protein degradation in isolated rat hepatocytes.

Analogues and derivatives of six of the amino acids which most effectively inhibit protein degradation in isolated rat hepatocytes (leucine, asparagine, glutamine, histidine, phenylalanine and tryptophan) were investigated to see if they could antagonize or mimic the effect of the parent compound. No antagonists were found. Amino alcohols and amino acid amides tended to inhibit protein degradation strongly, apparently by direct lysosomotropic effect as indicated by their ability to cause lysosomal vacuolation. Amino acid alkyl esters and dipeptides inhibited degradation to approximately the same extent as did their parent amino acids, possibly by being converted to free amino acids intracellularly. Of several leucine analogues tested, four (L-norleucine, L-norvaline, D-norleucine and L-allo-isoleucine) were found to be as effective as leucine in inhibiting protein degradation. None of the analogues had any effect on protein synthesis. Since leucine appears to play a unique role as a regulator of bulk autophagy in hepatocytes, the availability of active leucine agonists may help to elucidate the biochemical mechanisms for control of this important process.

Amino Acids↗
PubMed1981

Effect of serum step-down on protein metabolism and proliferation kinetics of NHIK 3025 cells.

Human NHIK 3025 cells growing exponentially in 30% or 3% serum had population doubling times of 19.1 and 27.6 hours, respectively. These values were equal to the calculated protein doubling times (17.6 and 26.5 hours, respectively), showing that the cells were in balanced growth at both serum concentrations. Stepdown from 30% to 3% serum reduced the rate of protein synthesis within 1--2 hours, from 5.7%/hour to 4.3%/hour, while the rate of protein degradation was unchanged (1.7%/hour). In cells synchronized by mitotic selection from an exponentially growing population, the median cell cycle durations in 30% and 3% serum were 17.2 and 23.6 hours, respectively, which were also in good agreement with the protein doubling times. The median G1 durations were 7.1 and 9.6 hours, respectively. Thus the duration of G1 relative to the total cell cycle duration was the same in the two cases. Complete removal of serum for a period of 3 hours resulted in a 3-hour prolongation of the cell cycle regardless of the time after mitotic selection at which the serum was removed. For synchronized cells, the rate of entry into both the S phase and into the subsequent cell cycle were reduced in 3% serum as compared to 30% serum, the former rate being significantly greater than the latter at both serum concentrations. Our results thus indicate that these cells are continuously dependent upon serum throughout the entire cell cycle.

Blood↗