Ammonia inhibition of protein degradation in isolated rat hepatocytes

Seglen, Per Ottar; Reith, Albrecht

doi:10.1016/0014-4827(76)90148-8

Cited by 163 publications

(48 citation statements)

References 6 publications

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“…The present study confirmed the ability of asparagine to induce accumulation of autophagocytosed LDH and of LDH-containing vacuoles. Although the latter could not be resolved from lysosomes in sucrose density gradients, they were extensively density-shifted by endocytosed AOM-gold, unlike the LDH-containing lysosomes from ammonia-or propylamine-treated cells, thus discounting the possibility that the asparagine effect could be mediated by its deamination to ammonia, a lysosomotropic agent (43,44). Asparagine (but not ammonia or propylamine) likewise allowed density shifting of endosomes and lysosomes, supporting the notion that the latter were not detectably affected by asparagine-generated ammonia.…”

Section: Discussionmentioning

confidence: 99%

“…Density Shifts-Ammonia, propylamine, and other lysosomotropic, pH-elevating amines inhibit intralysosomal protein degradation and cause lysosomal swelling (42)(43)(44)(45), possibly also accumulation of prelysosomal autophagic vacuoles (41). Both propylamine and ammonia (NH 4 Cl) prevented the formation of acid-soluble degradation products from endocytosed 125 I-TC-AOM (Fig.…”

Section: Effect Of Lysosomotropic Amines On Aom-gold-inducedmentioning

confidence: 99%

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Isolation and Characterization of Rat Liver Amphisomes

Fengsrud¹,

Strømhaug²,

Berg³

et al. 1998

Journal of Biological Chemistry

339

151

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Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes). Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.

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Section: Discussionmentioning

confidence: 99%

Section: Effect Of Lysosomotropic Amines On Aom-gold-inducedmentioning

confidence: 99%

Isolation and Characterization of Rat Liver Amphisomes

Fengsrud¹,

Strømhaug²,

Berg³

et al. 1998

Journal of Biological Chemistry

339

151

View full text Add to dashboard Cite

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“…1 and table 1, of the tested amines, all with weak-base properties inhibited the spreading of hepatocytes on adsorbed collagen, a process which is normally completed within 3 h. These amines are also potent inhibitors of protein degradation (table 1) by virtue of their ability to accumulate in lysosomes [2], whereas they have little effect on protein synthesis under the experimental conditions Isolated rat hepatocytes were incubated under the conditions in section 2, and the effects of various amines (at 10 mM) on cell spreading, protein degradation and protein synthesis were tested. The amine effects are expressed in % of control values (+ means stimulation, -means inhibition).…”

Section: Resultsmentioning

confidence: 99%

“…The osmotic swelling of vacuoles and the neutralization of intravacuolar acidity caused by the weak-base amines may conceivably result in a functional impairment and a retardation of membrane flow through the vacuolar compartments. There is some evidence for continued influx and accumulation of material in lysosomes during ammonia blockage [9] and the degree of lysosomal swelling observed [2] is hardly possible without the acquisition of new membrane material. A relative accumulation of membrane material in intracellular vacuoles, as suggested in fig.2, would seem to be a more likely mechanism for the 'surface' effects of weak-base amines than, e.g., direct binding to cell surface components, particularly since amines with structures as dissimilar as, e.g., methylamine and imidazole both inhibit cell spreading (table 1).…”

Section: Resultsmentioning

confidence: 99%

“…The NaCl and NaOH concentrations were so adjusted that the final osmolarity and pH were 290 mosM and 7.6, respectively, at 37'C. The dishes were incubated for 3 h at 37°C fmed in 1% glutaraldehyde [2] and the percentage of spread cells (cells which had lost their rounded outline) was counted microscopically. In control cultures (no amines or other inhibitors added), 90% of the cells had spread in 3 h.…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of cell spreading by lysosomotropic amines

Seglen

Gordon

1979

FEBS Letters

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Measurement of Autophagic Activity in Mammalian Cells

Menzies¹,

Moreau²,

Puri³

et al. 2012

CP Cell Biology

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Macroautophagy (referred to herein as autophagy) is a process in which cells engulf portions of cytoplasm in double‐membrane vesicles called autophagosomes. These autophagosomes can also capture protein oligomers associated with neurodegenerative diseases, infectious agents (like bacteria), and even organelles (like mitochondria). Autophagosomes are transported along microtubules towards the microtubule organizing center of cells, where the lysosomes are clustered. After fusion and content exchange with lysosomes, the autophagosome cargo is degraded by lysosomal hydrolases. This unit describes some of the core autophagy assays that are in common use, including LC3 immunoblotting, light microscopy analyses of different stages of autophagy, electron microscopy, and an assay of autophagy substrate accumulation. Autophagy assays should generally not be performed alone, but should be accompanied by complementary assays to enable robust interpretations. Curr. Protoc. Cell Biol. 54:15.16.1‐15.16.25. © 2012 by John Wiley & Sons, Inc.

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Ammonia inhibition of protein degradation in isolated rat hepatocytes

Cited by 163 publications

References 6 publications

Isolation and Characterization of Rat Liver Amphisomes

Isolation and Characterization of Rat Liver Amphisomes

Inhibition of cell spreading by lysosomotropic amines

Measurement of Autophagic Activity in Mammalian Cells

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