Effects of amino acids, ammonia and leupeptin on protein synthesis and degradation in isolated rat hepatocytes

Seglen, Per Ottar

doi:10.1042/bj1740469

Cited by 100 publications

(35 citation statements)

References 42 publications

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“…Two injections were given, 18 and 4 h before the cells were isolated; the total dose of [14C]valine was 50 gCi/rat (Grinde & Seglen, 1981). The rate of protein degradation was measured as described by Seglen (1978) and Grinde & Seglen (1981) in Krebs-Henseleit bicarbonate buffer. The rates of proteolysis were measured from 60 to 120 min to avoid interference by short-lived protein degradation (Woodside & Mortimore, 1972).…”

Section: Isolation Of Hepatocytesmentioning

confidence: 99%

“…The rates of proteolysis were measured from 60 to 120 min to avoid interference by short-lived protein degradation (Woodside & Mortimore, 1972). The samples were treated and the radioactivity was measured as described by Seglen (1978). Hepatocyte primary culture Cells were inoculated on collagen-gel-coated culture dishes (8.5 cm diameter; Nunc) at a density of 0.6 x 106 cells/ml (6.5 ml per dish).…”

Section: Isolation Of Hepatocytesmentioning

confidence: 99%

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Urea synthesis in freshly isolated and in cultured periportal and perivenous hepatocytes

Pösö¹,

Penttilä²,

Suolinna³

et al. 1986

Biochemical Journal

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Periportal hepatocytes isolated by digitonin/collagenase perfusion produced urea faster than did similarly prepared perivenous hepatocytes, in both the presence and the absence of amino acids and various urea precursors. There was no difference between the two cell types in rates of intracellular proteolysis. The initial difference in urea synthesis persisted for 5 days during primary culture, but then gradually disappeared. Our results demonstrate that the periportal dominance of urea formation is unrelated to the currently existing acinar microenvironment in the intact liver, but probably reflects differences in acinar key enzyme activities only slowly converging during culture.

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Section: Isolation Of Hepatocytesmentioning

confidence: 99%

Section: Isolation Of Hepatocytesmentioning

confidence: 99%

Urea synthesis in freshly isolated and in cultured periportal and perivenous hepatocytes

Pösö¹,

Penttilä²,

Suolinna³

et al. 1986

Biochemical Journal

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“…Protein synthesis was measured, in the presence of amino acids, as the incorporation of [14C]valine into acid-precipitable material [31] and protein degradation was measured as and H-89 from Calbiochem (San Diego, CA, USA). After the completion of these experiments Sigma has informed us that the H-7 preparation used in our study is not l-(5-isoquinolinylsulfonyl)-2-methylpiperazine as originally described by Hidaka et al [33], but rather 1 -(5-isoquinolinylsulfonyl)-3-methylpiperazine, which inhibits protein kinases A and C with a somewhat lower potency than the original H-7 [34, 351.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors

Holen

Gordon

Seglen

1993

European Journal of Biochemistry

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Autophagy, measured as the sequestration of electroinjected [3H]raffinose or endogenous lactate dehydrogenase, was inhibited in isolated rat hepatocytes by the protein phosphatase inhibitors okadaic acid, calyculin A and microcystin-LR. Okadaic acid, the most potent inhibitor, suppressed autophagy almost completely at 15 nM, suggesting inhibition of a protein phosphatase of type 2A. Okadaic acid had no effect on ATP levels, protein synthesis or cellular viability at this concentration, but caused a disruption of the hepatocytic cytoskeleton and a consequent reduction in organelle sedimentability, potentially interfering with the autophagy assay unless the necessary precautions are taken. Lysosomal (propylamine-sensitive) degradation of endogenous protein was inhibited by okadaic acid, whereas non-lysosomal (propylamine-resistant) degradation was unaffected. The autophagy-inhibitory effect of okadaic acid was not affected by inhibitors of CAMP-dependent protein kinase or protein kinase C (H-7, H-89, calphostin C) but eliminated by the non-specific inhibitor K-252a and its analogues (KT-5720, KT-5823, KT-5926) and by KN-62, a specific inhibitor of Ca2+/ calmodulin-dependent protein lunase 11. Protein phosphorylation by this kinase would thus seem to play a role in regulation of the autophagic-lysosomal degradation pathway.Autophagy, an intracellular mechanism for the bulk degradation of cytoplasm, plays an important role in protein metabolism and growth control [ 1 -31. The stepwise nature of the process has been suggested by ultrastructural and biochemical studies. In the first step, autophagic sequestration, a portion of the cytoplasm is wrapped up by a membraneous organelle which eventually forms a closed autophagic vacuole, an autophagosome [4]. This vacuole then matures into, or delivers the autophagocytosed cytoplasm to a second autophagic vacuole, an amphisome, so called because it can also receive material from endocytosis [ 5 ] . The amphisome in turn delivers its contents to a lysosome for degradation [61.The biochemistry of autophagy is largely uncharted. Regulation of the process occurs mainly at the initial sequestration step, which is sensitive to various hormones, growth factors and metabolites [3, 71. In rat hepatocytes the rate of autophagic sequestration and the formation of autophago- somes is influenced by several agents known to transmit their effects through protein kinases, such as glucagon [8, 91, insulin [lo, 111, adrenergic agonists [12, 131 and cyclic nucleotides [14]. An involvement of protein phosphorylation in the biological control of autophagy would, therefore, seem likeIn the present study we have examined this possibility by treating isolated rat hepatocytes with okadaic acid and other protein phosphatase inhibitors. Okadaic acid is a toxin associated with diarrhetic seafood poisoning, first isolated from the marine sponge Hulichondria okudui [15]. Its mechanism of action is a specific inhibition of protein phosphatases 1 (PP1) and 2A (PP2A), two of the major phosphatases responsi...

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“…Incubations were terminated by the addition of 0.1 ml perchloric acid (1 0 w/v), and after 15 min at 0 "C and centrifugation for 5 min at 5000 rev./min, duplicate 0.1-ml samples of the supernatant were assayed for acid-soluble radioactivity by liquid scintillation counting [24]. The precipitate was washed three times in 4 ml ice-cold perchloric acid (2'4 w/v), dissolved in 0.5 ml of 0.3 M NaOH (at 37"C), and assayed for total proteinincorporated radioactivity [24]. The net release of [14C]valine during the incubation period was expressed as a percentage of the total initial protein radioactivity of the cell sample.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of the Lysosomal Pathway of Protein Degradation in Isolated Rat Hepatocytes by Ammonia, Methylamine, Chloroquine and Leupeptin

Seglen

Grinde

Solheim

1979

European Journal of Biochemistry

456

252

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1. Protein degradation in isolated rat hepatocytes was measured as the release of [14C]valine from pre-labelled protein. To reduce background radioactivity, the intracellular ['4C]valine pool was depleted by serial extraction at 37 "C, effecting equilibration between the intracellular pool and the valine-free extracellular medium. After extraction, a small, non-equilibrating intracellular [14C]valine pool remained; this pool could only be labelled in the presence of ongoing protein synthesis, and might represent valine and valine-containing oligopeptides derived from protein degradation.2. The ['4C]valine released from degraded protein was not significantly re-utilized for protein synthesis intracellularly (no effect of cycloheximide or high concentrations of unlabelled valine), reflecting the low rate of protein synthesis and the rapid transport of valine into the extracellular medium, both characteristic of isolated hepatocytes.3. From cells pre-labelled for 24 h Ivi vivo, ['4C]valine was released linearly at a rale of 5 %/h, probably representing the true over-all protein degradation rate. The lysosomotropic inhibitor ammonia (10 mM NH4C1) inhibited 70% of the degradation, presumably the contribution by the lysosomal pathway.From 1-h pre-labelled cells, [14C]valine was released at a declining rate, and ammonia inhibited degradation only by 45%, consistent with the view that the majority of short-lived proteins are degraded by the non-lysosomal pathway(s).4. Chloroquine and methylamine, which accumulate in lysosomes by virtue of their weak base properties, inhibited hepatocytic protein degradation to the same extent as ammonia, with no additivity. These compounds therefore seem to block the lysosomal pathway of protein degradation selectively and completely. Leupeptin, which binds to and inhibits the activity of certain lysosomal proteases, also inhibited protein degradation almost to the same extent as ammonia, but with a small part of the effect ( < 20 %) being additive to the NH3 effect and thus probably reflecting a slight inhibition of non-lysosomal protein degradation as well.5. Of the four inhibitors tested, only the effect of ammonia was rapidly reversible within the experimental period (2 h). Leupeptin, on the other hand, was the only degradation inhibitor which did not also affect protein synthesis. Chloroquine caused significant cell death at concentrations above 0.2 mmol/l in this protein-free medium, i.e. in the concentration range needed for maximal inhibition of protein degradation.6. Incubation of hepatocytes under anoxic conditions resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of ammonia, i.e. most of the degradation by the lysosomal pathway and more than one-half of the degradation by the nonlysosomal pathways appears to be energy-dependent.The mechanisms responsible for the general turnover of endogeneous protein in animal cells are not well known. Lysosomes (autophagy) appear to play a major role [I -41 but in addition a numbe...

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Effects of amino acids, ammonia and leupeptin on protein synthesis and degradation in isolated rat hepatocytes

Cited by 100 publications

References 42 publications

Urea synthesis in freshly isolated and in cultured periportal and perivenous hepatocytes

Urea synthesis in freshly isolated and in cultured periportal and perivenous hepatocytes

Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors

Inhibition of the Lysosomal Pathway of Protein Degradation in Isolated Rat Hepatocytes by Ammonia, Methylamine, Chloroquine and Leupeptin

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