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 Utilization of endogenous and exogenous sources of substrate for cholesterol biosynthesis by isolated hepatocytes

1979 ◽  
Vol 177 (1) ◽  
pp. 255-263 ◽  
Author(s):  
Geoffrey F. Gibbons ◽  
Clive R. Pullinger
Keyword(s):  
Cholesterol Biosynthesis ◽  
Total Sterol ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Sterol Synthesis ◽  
Isolated Rat Hepatocytes ◽  
Intermediary Metabolites ◽  
Exogenous Glucose ◽  
High Concentrations ◽  
Cholesterol Precursor

The rates of cholesterol biosynthesis in isolated rat hepatocytes were determined by using a method based on measurement of the rate of formation of desmosterol (cholesta-5,24-dien-3β-ol), which accumulates during inhibition of cholesterogenesis by the drug triparanol. Incubation of cells from normal or 24h-starved animals in a medium containing albumin, glucose, amino acids and acetate as the only organic constituents led to an accelerating rate of sterol formation during the earlier stages of a 6h incubation period. The contribution of exogenously added acetate (initial concentration 3.34mm) to sterol synthesis in both types of cells reached an early maximum and then continually declined. Exogenously added pyruvate and lactate were more efficient sources of sterol carbon than was acetate. Exogenous glucose even at relatively high concentrations (11.1mm) was incapable of providing more than 6% of the total sterol carbon. Although the proportion of total sterol carbon supplied from exogenous acetate increased with increasing concentrations of the extracellular substrate, the rates of total sterol synthesis in both types of cell remained unchanged. Similar observations were made when lactate or pyruvate was the cholesterogenic precursor in normal cells. These studies suggest that, although exogenous substrates were capable of expanding an intracellular pool of cholesterol precursor, the normal supply of intermediary metabolites was not rate-limiting for cholesterogenesis.

Inhibition of gluconeogenesis by extracellular ATP in isolated rat hepatocytes

1991 ◽  
Vol 261 (6) ◽  
pp. R1522-R1526 ◽  
Author(s):  
M. Asensi ◽  
A. Lopez-Rodas ◽  
J. Sastre ◽  
J. Vina ◽  
J. M. Estrela
Keyword(s):  
Plasma Membrane ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Extracellular Atp ◽  
Structural Analogue ◽  
Isolated Rat Hepatocytes ◽  
Lactate And Pyruvate ◽  
Alpha Beta ◽  
High Concentrations ◽  
Isolated Rat

The aim of this study was to determine the effect of externally added ATP on gluconeogenesis by isolated hepatocytes from starved rats. High concentrations of extracellular ATP inhibited gluconeogenesis from lactate and pyruvate but not from glycerol or fructose. This inhibition was associated with an increase in intracellular adenosine contents. ADP, AMP, or adenosine but not guanosine 5'triphosphate, inosine 5' triphosphate, or adenine also inhibited gluconeogenesis. alpha, beta-Methylene-ATP, a nonmetabolizable structural analogue of ATP, did not affect the rate of gluconeogenesis. Intracellular ATP levels were increased by externally added ATP or adenosine, but ATP-to-ADP ratios in the cytosolic and mitochondrial compartments were diminished. Malate and phosphoenolpyruvate contents were decreased by extracellular ATP or adenosine. Our results show that inhibition of gluconeogenesis by high levels of extracellular ATP may be mediated by adenosine derived from ATP catabolism at the plasma membrane.

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 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 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.

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