scholarly journals Role of Ca2+ for protein turnover in isolated rat hepatocytes

1983 ◽  
Vol 216 (3) ◽  
pp. 529-536 ◽  
Author(s):  
B Grinde
Keyword(s):  
Proteinase Inhibitors ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Inhibitory Effect ◽  
Ionophore A23187 ◽  
Lysosomal Degradation ◽  
Bivalent Cation ◽  
Isolated Rat Hepatocytes ◽  
Autophagic Vacuoles ◽  
Endogenous Protein

Experiments with bivalent-cation chelators (EGTA and EDTA), a Ca2+ ionophore (A23187) and a Ca2+-channel blocker (verapamil) indicate that Ca2+ is required for the lysosomal degradation of endogenous protein in hepatocytes. A distinction is made between lysosomal and non-lysosomal degradation by using the lysosomotropic agent methylamine. As Ca2+ does not appear to be required for the lysosomal degradation of endocytosed asialo-fetuin, the Ca2+-dependence for the degradation of endogenous protein is probably connected with the formation of autophagic vacuoles or the fusion of autophagic vacuoles with lysosomes. EGTA and EDTA had a slight inhibitory effect on the non-lysosomal degradation. This effect could be due to the activity of non-lysosomal Ca2+-dependent thiol proteinases. Together with previous experiments with thiol-proteinase inhibitors, the present experiments indicate that these proteinases have a very limited impact on the bulk protein degradation in the isolated hepatocytes.

scholarly journals Effects of protein-degradation inhibitors on the inactivation of tyrosine aminotransferase, tryptophan oxygenase and benzopyrene hydroxylase in isolated rat hepatocytes

1982 ◽  
Vol 202 (1) ◽  
pp. 191-196 ◽  
Author(s):  
B Grinde ◽  
R Jahnsen
Keyword(s):  
Proteinase Inhibitors ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Tyrosine Aminotransferase ◽  
Weak Base ◽  
Isolated Rat Hepatocytes ◽  
Tryptophan Oxygenase ◽  
The Third ◽  
Lysosomal Proteinase ◽  
Isolated Rat

The following three potent inhibitors of hepatocytic proteolysis were investigated to see if they would inhibit the intracellular inactivation of enzymes: chymostatin and leupeptin (proteinase inhibitors) and methylamine (a lysosomotropic weak base). Chymostatin inhibited the inactivation of two of the three enzymes tested: tyrosine aminotransferase (EC 2.6.1.5) and tryptophan oxygenase (tryptophan 2,3-dioxygenase, EC 1.13.11.11). Leupeptin had no effect on any of the enzymes, whereas methylamine had only a weak inhibitory effect on tyrosine aminotransferase inactivation. Apparently proteolytic cleavage (probably by a non-lysosomal proteinase, since only chymostatin is effective) is involved in the inactivation of tyrosine aminotransferase and tryptophan oxygenase. The third enzyme, benzopyrene hydroxylase (flavoprotein-linked mono-oxygenase, EC 1.14.14.1), is probably inactivated by a non-proteolytic mechanism.

Morphological Changes and Cytotoxicity in Isolated Hepatocytes

1991 ◽  
Vol 19 (2) ◽  
pp. 181-186
Author(s):  
Diana Toivola ◽  
John E. Eriksson
Keyword(s):  
Molecular Mechanisms ◽  
Morphological Changes ◽  
Light And Electron Microscopy ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Ionophore A23187 ◽  
Isolated Rat Hepatocytes ◽  
Morphological Alterations ◽  
Cell Blebbing ◽  
Protein Phosphatase Inhibition

Cells exposed to different types of stress usually exhibit morphological changes. These changes are often referred to as cell blebbing. The aim of this study was to compare a few different cytotoxins acting on freshly isolated rat hepatocytes through different molecular mechanisms. The toxins used were phalloidin and cytochalasin D (for cytoskeletal alterations), menadione (for oxidative stress), the Ca2+-ionophore A23187 (for intracellular Ca2+-elevation) and microcystin-LR (for protein phosphatase inhibition). Light and electron microscopy were used when comparing the morphological effects of the toxins. The morphological effects on microfilaments were studied by labelling F-actin with rhodamine-conjugated phalloidin. The results indicate that, although all the toxins caused cell blebbing, the blebs were morphologically markedly different and the redistribution of actin varied considerably, depending on the toxin used. The modification of the microfilamental architecture seemed to correlate with the morphological changes. Cell blebbing is inevitably a rather vague term for morphological alterations, which may look very different, depending on the subcellular cytotoxic mechanism involved.

Effect of hepatocyte swelling on microtubule stability and tubulin mRNA levels

1994 ◽  
Vol 72 (1-2) ◽  
pp. 12-19 ◽  
Author(s):  
Dieter Häussinger ◽  
Barbara Stoll ◽  
Stephan vom Dahl ◽  
Panayiotis A. Theodoropoulos ◽  
Emmanuel Markogiannakis ◽  
...  
Keyword(s):  
Cell Function ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Inhibitory Effect ◽  
Cell Swelling ◽  
Mrna Levels ◽  
Microtubule Network ◽  
Isolated Rat Hepatocytes ◽  
Tubulin Mrna ◽  
Induced Changes

Incubation of isolated rat hepatocytes under conditions known to induce cell swelling caused several alterations in microtubule physiology. As shown by immunofluorescence microscopy experiments in the absence and presence of triethyllead or colchicine (two well-established microtubule inhibitors), an apparent stabilization of the microtubule network became evident in hepatocytes exposed to hypotonic (190 mosmol/L) conditions. A similar stabilizing effect was also observed upon cell swelling induced by addition of insulin (100 nmol/L) or glutamine (10 mmol/L). The differential microtubule stabilities were not attributed to a differential incorporation of the antimicrotubular agents into hepatocytes as shown by [3H]colchicine-uptake experiments. The swelling-induced alterations of microtubules may contribute to the swelling-induced changes of liver cell function: in perfused rat liver it was found that the established inhibitory effect of hypotonic cell swelling on hepatic proteolysis was largely abolished in presence of colchicine. Tubulin mRNA levels increased by 1.9-, 2.1- and 2.7-fold in isolated hepatocytes being exposed for 120 min to hypotonic medium, insulin, or glutamine, respectively. The results suggest an involvement of microtubular structures in the regulation of liver metabolism in response to alterations of the cellular hydration state.Key words: microtubules, cell swelling, glutamine, gene expression, proteolysis.

scholarly journals Inhibition of protein degradation in isolated rat hepatocytes

1977 ◽  
Vol 164 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Melvyn F. Hopgood ◽  
Michael G. Clark ◽  
F. John Ballard
Keyword(s):  
Protein Degradation ◽  
Cathepsin B ◽  
Proteinase Inhibitors ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Cell Protein ◽  
Isolated Rat Hepatocytes ◽  
Cell Energy ◽  
High Concentrations ◽  
Inhibitory Agents

1. Isolated parenchymal cells were prepared by collagenase perfusion of livers from fed rats that had been previously injected with [3H]leucine to label liver proteins. When these cells were incubated in a salts medium containing glucose, gelatin and EDTA, cellular integrity was maintained over a period of 6h. 2. Cells incubated in the presence of 2mm-leucine to minimize radioactive isotope reincorporation released [3H]leucine into the medium at a rate accounting for the degradation of 4.5% of the labelled cell protein per h. 3. Degradation of [3H]protein in these cells was inhibited by insulin and by certain amino acids, of which tryptophan and phenylalanine were the most effective. 4. Protein degradation was decreased by several proteinase inhibitors, particularly those that are known to inhibit lysosomal cathepsin B, and by inhibitors of cell-energy production. 5. Ammonia inhibited degradation, but only at concentrations above 1.8mm. Aliphatic analogues of ammonia were effective at lower concentrations than was ammonia. 6. High concentrations of ammonia inhibited degradation by 50%. The extent of this inhibition could not be increased further by the addition of the cathepsin B inhibitor leupeptin, which by itself inhibited degradation by approx. 30%. 7. The sensitivity of proteolysis in isolated hepatocytes to these various inhibitory agents is discussed in relation to their possible modes of action.

scholarly journals The roles of different protein kinases and of calmodulin in the effects of Ca2+ mobilization on 3-hydroxy-3-methylglutaryl-CoA reductase activity in isolated rat hepatocytes

1991 ◽  
Vol 273 (2) ◽  
pp. 485-488 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell
Keyword(s):  
Protein Kinase ◽  
Reductase Activity ◽  
Rat Hepatocytes ◽  
Total Activity ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Hmg Coa Reductase ◽  
Lysosomal Proteolysis ◽  
Dependent Protein ◽  
Coa Reductase

The roles of protein kinase C, Ca2+/calmodulin-dependent protein kinase and AMP-activated protein kinase in the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase induced by Ca2(+)-mobilizing conditions in isolated hepatocytes were investigated. Only partial evidence for the involvement of AMP-activated kinase was found. Antagonism of calmodulin action prolonged the decrease in expressed/total activity ratio induced by vasopressin plus glucagon. Protease inhibitors active against Ca2(+)-dependent cytosolic proteases or lysosomal proteolysis did not attenuate the loss of total HMG-CoA reductase induced by glucagon plus vasopressin, but calmodulin antagonists largely prevented this effect.

Regulation of ANG II and AVP receptors in isolated hepatocytes of pregnant rats

1990 ◽  
Vol 258 (4) ◽  
pp. E597-E605
Author(s):  
G. Massicotte ◽  
L. Coderre ◽  
J. L. Chiasson ◽  
G. Thibault ◽  
E. L. Schiffrin ◽  
...  
Keyword(s):  
Binding Sites ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Maximum Response ◽  
Receptor Subtype ◽  
Ang Ii ◽  
Single Class ◽  
Pregnant Rats ◽  
Isolated Rat Hepatocytes ◽  
Biological Responses

Recent evidence suggests that angiotensin II (ANG II) and vasopressin (AVP) act on the liver via specific receptors. We have examined the binding properties of these receptors in isolated rat hepatocytes and studied the regulation of the biological responses to ANG II and AVP during pregnancy in the rat. In contrast to [3H]ANG II, 125I-labeled-[Sar1-Ile8]ANG II was markedly resistant to degradation by isolated liver cells. Displacement and saturation experiments with this iodinated antagonist revealed the presence of a single class of binding sites [2 x 10(5) sites/cell, dissociation constant (KD) = 1.0 nM]. The potency of ANG II analogues to displace 125I-[Sar1-Ile8]-ANG II agrees closely with data reported for vascular smooth muscle cells. Isolated hepatocytes have approximately 8 x 10(4) [3H]AVP binding sites/cell (KD = 1.0 nM) based on saturation experiments. AVP analogues selectively displaced [3H]AVP, suggesting the presence of V1-AVP receptor subtype. The maximum response of [Sar1]ANG II-induced glycogenolysis in the cells was decreased during gestation, whereas the effective concentration producing 50% of maximum response (EC50) was significantly increased (0.15-0.28 nM) when compared with cells from nonpregnant animals. In pregnancy, receptors for 125I-[Sar1-Ile8]ANG II were not changed in affinity (KD) or in density (Bmax). The maximum response and EC50 of AVP on liver glycogenolysis were not significantly decreased during pregnancy, whereas an increased number of AVP binding sites (from 5.0 +/- 0.5 x 10(4) to 11.0 +/- 1.7 x 10(4)) with similar KD was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Evidence that Ca2+-release-activated Ca2+ channels in rat hepatocytes are required for the maintenance of hormone-induced Ca2+ oscillations

2003 ◽  
Vol 370 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Roland B. GREGORY ◽  
Gregory J. BARRITT
Keyword(s):  
High Selectivity ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Cation Channels ◽  
Isolated Rat Hepatocytes ◽  
Pharmacological Agents ◽  
Crac Channels ◽  
Kinetics Of ◽  
Ca2 Channels

Store-operated Ca2+ channels in liver cells have been shown previously to exhibit a high selectivity for Ca2+ and to have properties indistinguishable from those of Ca2+-release-activated Ca2+ (CRAC) channels in mast cells and lymphocytes [Rychkov, Brereton, Harland and Barritt (2001) Hepatology 33, 938—947]. The role of CRAC channels in the maintenance of hormone-induced oscillations in the cytoplasmic free Ca2+ concentration ([Ca2+]cyt) in isolated rat hepatocytes was investigated using several inhibitors of CRAC channels. 2-Aminoethyl diphenylborate (2-APB; 75μM), Gd3+ (1μM) and 1-{β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SK&F 96365; 50μM) each inhibited vasopressin- and adrenaline (epinephrine)-induced Ca2+ oscillations (measured using fura-2). The characteristics of this inhibition were similar to those of inhibition caused by decreasing the extracellular Ca2+ concentration to zero by addition of EGTA. The effect of 2-APB was reversible. In contrast, LOE-908 {(R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamidemesylate}(30μM), used commonly to block Ca2+ inflow through intracellular-messenger-activated, non-selective cation channels, did not inhibit the Ca2+ oscillations. In the absence of added extracellular Ca2+, 2-APB, Gd3+ and SK&F 96365 did not alter the kinetics of the increase in [Ca2+]cyt induced by a concentration of adrenaline or vasopressin that induces continuous Ca2+ oscillations at the physiological extracellular Ca2+ concentration. Ca2+ inflow through non-selective cation channels activated by maitotoxin could not restore Ca2+ oscillations in cells treated with 2-APB to block Ca2+ inflow through CRAC channels. Evidence for the specificity of the pharmacological agents for inhibition of CRAC channels under the conditions of the present experiments with hepatocytes is discussed. It is concluded that Ca2+ inflow through CRAC channels is required for the maintenance of hormone-induced Ca2+ oscillations in isolated hepatocytes.

scholarly journals Inhibition of oxidative metabolism by propionic acid and its reversal by carnitine in isolated rat hepatocytes

1986 ◽  
Vol 236 (1) ◽  
pp. 131-136 ◽  
Author(s):  
E P Brass ◽  
P V Fennessey ◽  
L V Miller
Keyword(s):  
Palmitic Acid ◽  
Propionic Acid ◽  
Oxidative Metabolism ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Acid Oxidation ◽  
Isolated Rat Hepatocytes ◽  
Pyruvate Oxidation ◽  
Metabolic Basis ◽  
Isolated Rat

The present study was designed to study the interaction of propionic acid and carnitine on oxidative metabolism by isolated rat hepatocytes. Propionic acid (10 mM) inhibited hepatocyte oxidation of [1-14C]-pyruvate (10 mM) by 60%. This inhibition was not the result of substrate competition, as butyric acid had minimal effects on pyruvate oxidation. Carnitine had a small inhibitory effect on pyruvate oxidation in the hepatocyte system (210 +/- 19 and 184 +/- 18 nmol of pyruvate/60 min per mg of protein in the absence and presence of 10 mM-carnitine respectively; means +/- S.E.M., n = 10). However, in the presence of propionic acid (10 mM), carnitine (10 mM) increased the rate of pyruvate oxidation by 19%. Under conditions where carnitine partially reversed the inhibitory effect of propionic acid on pyruvate oxidation, formation of propionylcarnitine was documented by using fast-atom-bombardment mass spectroscopy. Propionic acid also inhibited oxidation of [1-14C]palmitic acid (0.8 mM) by hepatocytes isolated from fed rats. The degree of inhibition caused by propionic acid was decreased in the presence of 10 mM-carnitine (41% inhibition in the absence of carnitine, 22% inhibition in the presence of carnitine). Propionic acid did not inhibit [1-14C]palmitic acid oxidation by hepatocytes isolated from 48 h-starved rats. These results demonstrate that propionic acid interferes with oxidative metabolism in intact hepatocytes. Carnitine partially reverses the inhibition of pyruvate and palmitic acid oxidation by propionic acid, and this reversal is associated with increased propionylcarnitine formation. The present study provides a metabolic basis for the efficacy of carnitine in patients with abnormal organic acid accumulation, and the observation that such patients appear to have increased carnitine requirements (‘carnitine insufficiency’).

scholarly journals Interactions of dietary fat and 2,5-anhydro-d-mannitol on energy metabolism in isolated rat hepatocytes

2002 ◽  
Vol 282 (3) ◽  
pp. R715-R720 ◽  
Author(s):  
Hong Ji ◽  
Grazyna Graczyk-Milbrandt ◽  
Mary D. Osbakken ◽  
Mark I. Friedman
Keyword(s):  
Oxygen Consumption ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Metabolic Effects ◽  
Atp Content ◽  
Isolated Rat Hepatocytes ◽  
Palmitate Oxidation ◽  
Low Carbohydrate ◽  
Lower Oxygen ◽  
Hepatocyte Metabolism

The fructose analog 2,5-anhydro-d-mannitol (2,5-AM) stimulates feeding in rats by reducing ATP content in the liver. These behavioral and metabolic effects occur with rats fed a high-carbohydrate/low-fat (HC/LF) diet, but they are prevented or attenuated when the animals eat high-fat/low-carbohydrate (HF/LC) food. To examine the metabolic bases for this effect of diet, we assessed the actions of 2,5-AM on ATP content, oxygen consumption, and substrate oxidation in isolated hepatocytes from rats fed one of the two diets. Compared with cells from rats fed the HC/LF diet (“HC/LF” cells), cells from rats fed the HF/LC diet (“HF/LC” cells) had similar ATP contents but lower oxygen consumption, decreased fructose, and increased palmitate oxidation. 2,5-AM did not decrease ATP content or oxygen consumption in HF/LC cells as much as it did in HC/LF hepatocytes, and it only affected fructose and palmitate oxidation in HC/LF cells.31P-NMR spectroscopy indicated that differences in phosphate trapping accounted for differences in depletion of ATP by 2,5-AM. These results suggest that intake of the HF/LC diet prevents the eating response and attenuates the decline in liver ATP by shifting hepatocyte metabolism to favor fat over carbohydrate as an energy-yielding substrate.

scholarly journals Taurolithocholate-induced Ca2+ release is inhibited by phorbol esters in isolated hepatocytes

1992 ◽  
Vol 287 (3) ◽  
pp. 891-896 ◽  
Author(s):  
L Combettes ◽  
B Berthon ◽  
M Claret
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Inhibitory Effect ◽  
Cell Uptake ◽  
Intracellular Binding ◽  
Pkc Activation

The monohydroxy bile acid taurolithocholate (TLC) causes a rapid and transient increase in free cytosolic Ca2+ concentration ([Ca2+]i) in suspensions of rat hepatocytes similar to that elicited by the InsP3-dependent hormone vasopressin. The effect of the bile acid is due to a mobilization of Ca2+, independent of InsP3, from the endoplasmic reticulum (ER). Short-term preincubation of cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), which activates protein kinase C (PKC), blocked the increase in [Ca2+]i induced by TLC, but did not alter that mediated by vasopressin. We obtained the following results, indicating that the effect of PMA is mediated by the activation of PKC. (1) Phorbol esters were effective over a concentration range where they activate PKC (IC50 = 0.5 nM); (2) phorbol esters that do not activate PKC did not inhibit the effects of TLC; (3) the permeant analogue oleoylacetylglycerol mimicked the inhibitory effect of PMA; (4) lastly, the inhibition of the TLC-induced Ca2+ mobilization by phorbol esters was partially prevented by preincubating the cells with the PKC inhibitors H7 and AMG-C16. Preincubating hepatocytes with PMA had no effect on the cell uptake of labelled TLC, indicating that the phorbol ester does not interfere with the transport system responsible for the accumulation of bile acids. In saponin-treated liver cells, PMA added before or after permeabilization failed to abolish TLC-induced Ca2+ release from the ER. The possibility is discussed that PMA, via PKC activation, may alter the intracellular binding or the transfer of bile acids in the liver.

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