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P O Seglen

Publications and source records attributed to P O Seglen.

At least 19 recordsLinked to original sources
PubMed1992

Protein kinase-dependent effects of okadaic acid on hepatocytic autophagy and cytoskeletal integrity.

The protein phosphatase inhibitor okadaic acid suppressed autophagy completely in isolated rat hepatocytes, as measured by the sequestration of electroinjected [3H]raffinose into sedimentable autophagic vacuoles. Okadaic acid was effectively antagonized by the general protein kinase inhibitors K-252a and KT-5926, the calmodulin antagonist W-7, and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK-II). These inhibitors also antagonized a cytoskeleton-disruptive effect of okadaic acid, manifested as the disintegration of cell corpses after breakage of the plasma membrane. CaMK-II, or a closely related enzyme, would thus seem to play a role in the control of autophagy as well as in the control of cytoskeletal organization.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Prelysosomal and lysosomal connections between autophagy and endocytosis.

In isolated rat hepatocytes electroloaded with [14C]sucrose, autophaged sugar accumulated in lysosomes under control conditions, and in prelysosomal autophagic vacuoles (amphisomes) in the presence of asparagine, an inhibitor of autophagic-lysosomal fusion. Endocytic uptake of the sucrose-cleaving enzyme invertase resulted in rapid and complete degradation of autophaged sucrose in both amphisomes and lysosomes. Pre-accumulated sucrose was degraded equally well in both compartments, regardless of amphisomal-lysosomal flux inhibition by asparagine, suggesting that endocytic entry into the autophagic pathway can take place both at the lysosomal and at the amphisomal level. The completeness of sucrose degradation by endocytosed invertase furthermore indicates that all lysosomes involved in autophagy can also engage in endocytosis. Endocytosed invertase reached the amphisomes even when autophagy was blocked by 3-methyladenine, and autophaged sucrose reached this compartment even when endocytic influx was blocked by vinblastine, suggesting that amphisomes may exhibit some degree of permanence independently of either pathway.

Animals↗
PubMed1992

Proteases and proteolysis in the lysosome.

Proteins sequestered by a non-selective bulk process within the lysosomes turn over with an apparent half-life of about 8 minutes and this rapid lysosomal proteolysis is initiated by endopeptidases, in particular by the cathepsins D and L. We describe also the cathepsins B and H which show mainly exopeptidase and only low endopeptidase activity. Especially cathepsin H is most probably the only lysosomal aminopeptidase in many cell types. Additionally, the properties of other mammalian lysosomal endo- and exopeptidases are compared. Finally, we discuss some of the conditions for the action of lysosomal proteases as the low intralysosomal pH, the high part of lysosomal thiol groups and the absence of intralysosomal proteinase inhibitors.

Animals↗
PubMed1992

Autophagy and other vacuolar protein degradation mechanisms.

Autophagic degradation of cytoplasm (including protein, RNA etc.) is a non-selective bulk process, as indicated by ultrastructural evidence and by the similarity in autophagic sequestration rates of various cytosolic enzymes with different half-lives. The initial autophagic sequestration step, performed by a poorly-characterized organelle called a phagophore, is subject to feedback inhibition by purines and amino acids, the effect of the latter being potentiated by insulin and antagonized by glucagon. Epinephrine and other adrenergic agonists inhibit autophagic sequestration through a prazosin-sensitive alpha 1-adrenergic mechanism. The sequestration is also inhibited by cAMP and by protein phosphorylation as indicated by the effects of cyclic nucleotide analogues, phosphodiesterase inhibitors and okadaic acid. Asparagine specifically inhibits autophagic-lysosomal fusion without having any significant effects on autophagic sequestration, on intralysosomal degradation or on the endocytic pathway. Autophaged material that accumulates in prelysosomal vacuoles in the presence of asparagine is accessible to endocytosed enzymes, revealing the existence of an amphifunctional organelle, the amphisome. Evidence from several cell types suggests that endocytosis may be coupled to autophagy to a variable extent, and that the amphisome may play a central role as a collecting station for material destined for lysosomal degradation. Protein degradation can also take place in a 'salvage compartment' closely associated with the endoplasmic reticulum (ER). In this compartment unassembled protein chains are degraded by uncharacterized proteinases, while resident proteins return to the ER and assembled secretory and membrane proteins proceed through the Golgi apparatus. In the trans-Golgi network some proteins are proteolytically processed by Ca(2+)-dependent proteinases; furthermore, this compartment sorts proteins to lysosomes, various membrane domains, endosomes or secretory vesicles/granules. Processing of both endogenous and exogenous proteins can occur in endosomes, which may play a particularly important role in antigen processing and presentation. Proteins in endosomes or secretory compartments can either be exocytosed, or channeled to lysosomes for degradation. The switch mechanisms which decide between these options are subject to bioregulation by external agents (hormones and growth factors), and may play an important role in the control of protein uptake and secretion.

Animals↗
PubMed1992

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.

Animals↗
PubMed1992

Reduced proliferative activity of polyploid cells in primary hepatocellular carcinoma.

The proliferative activity of tumor cells differing in DNA content (ploidy) and nuclearity was investigated in primary hepatocellular carcinomas of the rat by flow cytometric analysis of collagenase-isolated cells immunostained after labelling with bromodeoxyuridine (BrdU) in vivo. The diploid cell fraction in these euploid tumours was higher than in normal liver, and the rate of binucleation as well as the proliferative activity of the binuclear cells was very low. The highest proliferative activity (BrdU labelling index) was found among the diploid tumour cells. The activity in mononuclear tetraploid and octoploid cells was reduced in inverse proportion to their increasing DNA content, possibly suggesting a loss of proliferative potential associated with polyploidization. There was a significant correlation between the proliferative activity of hepatocellular carcinoma cells and nonparenchymal liver cells in the different tumours, indicating that different cell types within a tumour may respond to common growth stimuli. Treatment of tumour-bearing rats with a promoting carcinogen (2-acetylaminofluorene) resulted in significant stimulation of tumour cell proliferation (all ploidy classes), whereas the proliferation of non-parenchymal (stromal) cells in the tumour was slightly inhibited.

2-Acetylaminofluorene↗
PubMed1991

Inhibition of autophagic-lysosomal delivery and autophagic lactolysis by asparagine.

Overall autophagy was measured in isolated hepatocytes as the sequestration and lysosomal hydrolysis of electroinjected [14C]lactose, using HPLC to separate the degradation product [14C]glucose from undegraded lactose. In addition, the sequestration step was measured separately as the transfer from cytosol to sedimentable cell structures of electroinjected [3H]raffinose or endogenous lactate dehydrogenase (LDH; in the presence of leupeptin to inhibit lysosomal proteolysis). Inhibitor effects at postsequestrational steps could be detected as the accumulation of autophaged lactose (which otherwise is degraded intralysosomally), or of LDH in the absence of leupeptin. Asparagine, previously shown to inhibit autophagic but not endocytic protein breakdown, strongly suppressed the autophagic hydrolysis of electroinjected lactose. Vinblastine, which inhibits both types of degradation, likewise suppressed lactose hydrolysis. Asparagine had little or no effect on sequestration, but caused an accumulation of autophaged LDH and lactose, indicating inhibition at a postsequestrational step. Neither asparagine nor vinblastine affected the degradation of intralysosomal lactose preaccumulated in the presence of the reversible lysosome inhibitor propylamine. However, if lactose was preaccumulated in the presence of asparagine, both asparagine and vinblastine suppressed its subsequent degradation. The data thus indicate that autophagic-lysosomal delivery, i.e., the transfer of autophaged material from prelysosomal vacuoles to lysosomes, is inhibited selectively by asparagine and non-selectively by vinblastine.

Animals↗
PubMed1991

Diploid growth pattern of hepatocellular tumours induced by various carcinogenic treatments.

Hepatocellular carcinomas from rats of different strains, subjected to a variety of carcinogenic treatment regimens in different laboratories (initiation by diethylnitrosamine or dimethylhydrazine, promotion by phenobarbital, 2-acetylaminofluorene, nafenopin, orotic acid or deoxycholic acid, growth stimulation by partial hepatectomy or necrogenic CCl4 treatment), were all found to be predominantly diploid by flow cytometric analysis, in contrast to normal liver tissue in which polyploid nuclei were predominant. A switch from polyploidization to diploid growth would thus seem to be a common property of malignant liver tumours. Benign neoplastic liver nodules were likewise predominantly diploid, with the exception of nodules induced by long-term deoxycholic acid treatment in Fischer rats. In addition to containing a majority of polyploid cells, the latter nodules failed to progress to the carcinoma stage.

Animals↗
PubMed1991

Hepatocytic autophagy.

Autophagy is a non-selective bulk process for degradation of cytoplasm, as indicated by ultrastructural evidence and by the similarity in autophagic sequestration rates of various cytosolic enzymes with different half-lifes. The initial autophagic sequestration step is subject to feedback inhibition by amino acids, an effect which is potentiated by insulin and antagonized by glucagon. Epinephrine and other adrenergic agonists inhibit autophagic sequestration through a prazosin-sensitive, alpha 1-adrenergic mechanism. The sequestration is also inhibited by cAMP and by protein phosphorylation as indicated by the effects of cyclic nucleotide analogues, phosphodiesterase inhibitors and okadaic acid. Asparagine specifically inhibits autophagic-lysosomal fusion without having any significant effects on autophagic sequestration, intralysosomal degradation or on the endocytic pathway. Autophaged material that accumulates in prelysosomal vacuoles in the presence of asparagine is accessible to endocytosed enzymes, revealing the existence of an amphifunctional organelle, the amphisome. Evidence from several cell types suggests that endocytosis may be coupled to autophagy in a differential (ligand-dependent) manner, and that amphisomes may play a central role as collecting stations for material destined for lysosomal degradation.

Amino Acids↗
PubMed1991

Role of cyclic nucleotides in the control of hepatic autophagy.

Using isolated hepatocytes as a model system we have investigated whether the cyclic nucleotides cAMP and cGMP are involved in the regulation of the autophagic process. The dibutyryl-cyclic nucleotide analogues db-cAMP and db-cGMP both inhibited autophagic sequestration, suggesting that cAMP and cGMP may be of significance for this step. The adenylate cyclase stimulator deacetyl-forskolin both raised the level of intracellular cAMP and reduced sequestration markedly. In contrast, the guanylate cyclase stimulating agent atriopeptin did not affect sequestration although, it effectively elevated, the level of cGMP. Several inhibitors of cyclic nucleotide phosphodiesterases strongly suppressed autophagy and elevated the level of both cAMP and cGMP. However, one inhibitor, milrinone, raised the cAMP level 3-4 x while having no significant effect on cGMP. These results suggest that cAMP may be involved in the control of hepatic autophagy, whereas the role of cGMP, if any, remains unclear.

Animals↗
PubMed1991

Effects of adrenergic agonists and antagonists on autophagic activity in isolated rat liver cells.

The effect of various adrenergic agonists on autophagic sequestration--measured as the transfer of electroinjected [3H]raffinose from cytosol to vacuoles of the autophagic pathway--was investigated. Epinephrine and other agonists with alpha-effects inhibited sequestration through a specific alpha 1-adrenergic, i.e. prazosin-sensitive, mechanism. The beta-adrenergic agonist isoproterenol also inhibited sequestration, but by a non-beta-specific (propranolol-insensitive) mechanism. All sequestration-inhibitory agents suppressed overall autophagic-lysosomal proteolysis. The inhibitory action of the adrenergic agonists on protein metabolism was not specific to the autophagic pathway since protein synthesis was suppressed as well. However, intracellular levels of ATP were not adversely affected, ruling out the possibility that the agonists might be generally cytotoxic.

Adenosine Triphosphate↗
PubMed1990

Both endocytic and endogenous protein degradation in fibroblasts is stimulated by serum/amino acid deprivation and inhibited by 3-methyladenine.

Incubation of BHK-21 hamster fibroblasts in a serum- and amino acid-deficient medium caused a 3-fold increase in the degradation of endogenous protein, a doubling of the degradation of endocytosed epidermal growth factor, and an eightfold increase in the degradation of endocytosed alpha 2-macroglobulin. 3-Methyladenine (3MA) inhibited the deprivation-induced lysosomal degradation of both endogenous and endocytosed protein, but had no effect on basal (non-induced) degradation. 3MA also inhibited deprivation-induced protein degradation in human IMR-90 fibroblasts. Some inhibition of protein synthesis and of endocytic uptake of alpha 2-macroglobulin was observed in 3MA-treated BHK-21 cells, whereas cellular ATP levels were unaffected. These results are different from those obtained with isolated hepatocytes, and suggest that in some cells both endogenous and endocytic protein degradation may be accelerated as part of a general deprivation response.

Adenine↗
PubMed1990

Liver tumor promoters stimulate growth of transplanted hepatocellular carcinomas.

Cell suspensions or tissue fragments from primary hepatocellular carcinomas and benign neoplastic nodules, induced by treating rats with chemical carcinogens, were transplanted by intraportal injection or subcapsular implantation in the livers of syngeneic host rats. Both nodule and carcinoma transplants produced high numbers of hepatocellular carcinomas in the hosts 2 to 5 mo after transplantation. Treatment of the host rats with liver tumor promoters (phenobarbital or 2-acetylaminofluorene) greatly stimulated tumor outgrowth, demonstrating that even established carcinoma cells can be promoter-sensitive. Tumor outgrowth was also stimulated by partial hepatectomy of the hosts, the regenerative stimulus interacting synergistically with the tumor promoters.

2-Acetylaminofluorene↗
PubMed1990

Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes.

Seven cytosolic enzymes with varying half-lives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h; glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organelles (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeptin. Inhibitors of lysosomal fusion processes (vinblastine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolytic inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.

Animals↗
PubMed1990

Studies on the mitoinhibitory effect of orotic acid on hepatocytes in primary culture.

Orotic acid (OA), a promoter of liver carcinogenesis, inhibited proliferation of primary hepatocytes in culture as monitored by labelling index, mitotic index and total DNA content. The mitoinhibitory effect of OA was seen even in the presence of a strong mitogen such as epidermal growth factor (EGF). The growth inhibitory effect of OA was not due to cell killing. Upon exposure to OA the hepatocytes exhibited an increase in the ratio of uridine nucleotides to adenosine nucleotides, and as this ratio increased the response of hepatocytes to proliferate in the presence or absence of EGF decreased. Washing the hepatocytes free of added OA resulted in a gradual decrease in the ratio of uridine nucleotides to adenosine nucleotides, paralleled by an increase in hepatocytic proliferation. Adenine, an agent that inhibits the metabolism of OA to uridine nucleotides, not only inhibited the increase in the ratio of uridine nucleotides to adenosine nucleotides but also counteracted the OA-induced mitoinhibitory effect. These results, together with our earlier observations, suggest that an imbalance in nucleotide pools composed of an increase in uridine nucleotides and a decrease in adenosine nucleotides appears to be important for OA-induced mitoinhibition.

Animals↗
PubMed1990

Growth-related alterations during liver carcinogenesis: effect of promoters.

Bromodeoxyuridine labeling of DNA, binuclearity counting, and flow cytometric analysis of isolated hepatocytes and hepatocyte nuclei has been used to assess hepatocellular growth patterns related to liver carcinogenesis. Three growth patterns can be distinguished. Mononucleating growth is observed during liver regeneration and after treatment with the tumor promoter 2-acetylaminofluorene (2-AAF) and its analogue 4-AAF. In this growth mode binucleation does not occur, resulting in a decrease in the fraction of binucleated cells. Binucleating growth is observed during normal liver development and after treatment with compounds such as phenobarbital, characterized by progressive polyploidization and maintenance of a binucleated cell fraction. Diploid growth is the growth pattern of neoplastic liver hepatocytes. Most of the cells in neoplastic lesions (foci, nodules, and carcinomas) are diploid, in contrast to the normal liver. Diploid tumor cells have a much higher proliferative activity than tetraploid tumor cells, suggesting that the latter may possess a limited growth potential that makes abrogation of binucleation proliferatively advantageous.

2-Acetylaminofluorene↗