scholarly journals Studies on the assembly of cytochrome oxidase in isolated rat hepatocytes

1982 ◽  
Vol 204 (1) ◽  
pp. 239-245 ◽  
Author(s):  
A Wielburski ◽  
S Kuźela ◽  
B D Nelson
Keyword(s):  
Cytochrome Oxidase ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Liver Mitochondria ◽  
Small Scale ◽  
Pulse Label ◽  
Isolated Rat Hepatocytes ◽  
Sequential Event ◽  
Triton X ◽  
Isolated Liver

1. The assembly of rat liver cytochrome oxidase was studied in isolated hepatocytes and isolated liver mitochondria labelled with L-[35S]methionine. 2. Labelled subunits II and III appeared in the immunoabsorbed holoenzyme within minutes after the initiation of a pulse label. In contrast, labelled subunit I appeared in immunoabsorbed holoenzyme only after a subsequent 2 h chase or after an additional 2 h of labelling. Subunit I was heavily labelled, however, in intact mitochondria after 10 min. 3. A similar pattern of labelling was observed in holo-cytochrome oxidase which was chemically isolated by a small scale procedure adapted for this purpose. The appearance of subunit I in the holoenzyme was delayed for 1.5-2 h after a 60 min pulse with labelled methionine. 4. Incubation of hepatocytes for 4 h in the presence of cycloheximide had no effect on the labelling pattern described above. 5. Methods were developed in which newly translated, presumably unassembled, subunits of cytochrome oxidase could be separated from the holoenzyme by fractionation in Triton X-114. Short-term pulse experiments indicate that subunits II and III are associated with the holoenzyme fraction immediately after their completion, whereas subunit I is not. 6. The data are consistent with a model in which cytochrome oxidase assembly is viewed as an ordered and sequential event.

Flux through glycine cleavage system in isolated hepatocytes: effects of glucagon, cAMP, and calcium

1990 ◽  
Vol 68 (2) ◽  
pp. 543-546 ◽  
Author(s):  
Markandeya Jois ◽  
Beatrice Hall ◽  
Vaughn M. Collett ◽  
John T. Brosnan
Keyword(s):  
Acid Metabolism ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Liver Mitochondria ◽  
Glycine Cleavage System ◽  
Isolated Rat Hepatocytes ◽  
Maximal Stimulation ◽  
Glycine Cleavage ◽  
Camp Levels ◽  
Stimulation Of

The hepatic glycine cleavage system (GCS) is the principal route for the metabolism of glycine in mammals. Flux through the GCS in isolated rat hepatocytes was stimulated by about 100% by glucagon (10−7 M), forskolin (10−4 M), and dibutyryl cAMP (10−4 M). The stimulation of flux through the GCS by these agents was accompanied by marked elevation of cellular cAMP levels. A significant correlation was observed between increased cellular cAMP levels induced by glucagon and stimulation of flux through the GCS by glucagon. Exclusion of calcium from the incubation medium reduced the basal flux by 38%, but did not affect the degree of stimulation of flux through the GCS by glucagon. A single intraperitoneal injection of glucagon to rats prior to isolation of hepatocytes resulted in a 76% stimulation of flux through the GCS. These hepatocytes with stimulated flux through the GCS showed reduced sensitivity for further stimulation by glucagon. Half-maximal stimulation of flux through the GCS occurred at 3.8 ± 1.1 and 8.5 ± 1.4 nM glucagon in hepatocytes isolated from control and glucagon-injected rats, respectively. We conclude that cAMP is involved in the regulation of flux through the GCS by gluagon.Key words: amino acid, metabolism, liver, mitochondria, hormones.

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.

Propranolol metabolism by isolated hepatocytes from normal and cirrhotic rat livers: the effect of albumin

1990 ◽  
Vol 68 (6) ◽  
pp. 657-662 ◽  
Author(s):  
Louise Gariepy ◽  
Daphna Fenyves ◽  
Jean-Luc Petit ◽  
Ginette Raymond ◽  
Jean-Pierre Villeneuve
Keyword(s):  
Free Fraction ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Hepatic Uptake ◽  
Albumin Concentration ◽  
Isolated Rat Hepatocytes ◽  
Subsequent Elimination ◽  
Rat Livers ◽  
Mediated Catalysis ◽  
Isolated Rat

Several recent reports have shown that the hepatic uptake and subsequent elimination of some substrates is faster in the presence of albumin than in its absence, as if some of the substrate bound to albumin was also available for uptake. In the present study, we examined the effect of albumin on the clearance of propranolol by isolated rat hepatocyte suspensions. The clearance of total drug decreased progressively as albumin concentration increased. There was also a progressive decrease in the free fraction of propranolol and the net result was an increase in the clearance of unbound drug (+50% at 40 g/L albumin). This increase was not due to an oncotic pressure effect of albumin, nor to the presence of fatty acids bound to albumin. The clearance of propranolol by isolated hepatocytes from cirrhotic rats was decreased compared with controls (−50%), and albumin also increased propranolol free clearance, albeit to a lesser extent than in control animals. Our results indicate that albumin facilitates the elimination of propranolol by hepatocytes, possibly because of surface-mediated catalysis of the albumin–propranolol complexes.Key words: propranolol clearance, albumin, isolated rat hepatocytes, cirrhosis.

Hepatotoxic effect of (+)usnic acid from Usnea siamensis Wainio in rats, isolated rat hepatocytes and isolated rat liver mitochondria

2004 ◽  
Vol 90 (2-3) ◽  
pp. 381-387 ◽  
Author(s):  
Pornpen Pramyothin ◽  
Withaya Janthasoot ◽  
Nushjira Pongnimitprasert ◽  
Siriwan Phrukudom ◽  
Nijsiri Ruangrungsi
Keyword(s):  
Rat Liver ◽  
Usnic Acid ◽  
Rat Hepatocytes ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat Liver ◽  
Hepatotoxic Effect ◽  
Isolated Rat

scholarly journals Effects of monensin on insulin interactions with isolated hepatocytes. Evidence for inhibition of receptor recycling and insulin degradation

1986 ◽  
Vol 234 (2) ◽  
pp. 463-468 ◽  
Author(s):  
J Whittaker ◽  
V A Hammond ◽  
R Taylor ◽  
K G M M Alberti
Keyword(s):  
Time Course ◽  
Rat Hepatocytes ◽  
Insulin Binding ◽  
Isolated Hepatocytes ◽  
Insulin Degradation ◽  
Receptor Recycling ◽  
Surface Binding ◽  
Binding Studies ◽  
Isolated Rat Hepatocytes ◽  
Receptor Concentration

Recent evidence suggests that, during endocytosis, receptors for many polypeptide ligands are spared degradation and are recycled to the plasma membrane for re-utilization. The univalent ionophore monensin was shown to inhibit membrane recycling. We therefore examined its effects on insulin interactions with isolated rat hepatocytes to characterize further receptor endocytosis and recycling in these cells. At 10 degrees C, in the absence of endocytosis, no change in insulin binding was observed. However, at 37 degrees C a concentration-dependent decrease in 125I-insulin binding was seen in the presence of insulin; this reached a maximum of 60% at 1 nM-insulin. Competitive binding studies showed this to be due to a 50-60% decrease in cell-surface insulin-receptor concentration, although the total cellular receptor concentration remained unchanged, suggesting that monensin causes the intracellular sequestration of receptors. Time-course studies of the processing of 2.5 nM-insulin showed that monensin produced a 50-60% decrease in surface binding, accompanied by a similar decrease in internalization and total inhibition of insulin degradation. When hepatocytes with 125I-insulin prebound to their surface receptors at 10 degrees C were warmed to 37 degrees C, monensin had no effect on internalization, but caused marked impairment of intracellular insulin degradation. It is concluded that monensin inhibits receptor recycling and cellular insulin degradation.

Uptake of unconjugated bilirubin by isolated rat hepatocytes

1979 ◽  
Vol 236 (1) ◽  
pp. C9-C14 ◽  
Author(s):  
T. Iga ◽  
D. L. Eaton ◽  
C. D. Klaassen
Keyword(s):  
Lipid Membrane ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Hepatic Uptake ◽  
Unconjugated Bilirubin ◽  
Metabolic Inhibitors ◽  
Isolated Rat Hepatocytes ◽  
Intracellular Binding ◽  
High Degree ◽  
Isolated Rat

The mechanism responsible for the hepatic uptake of unconjugated bilirubin was examined in isolated rat hepatocytes from control and phenobartital-pretreated rats. The uptake was extremely rapid and the equilibrium between cell and medium was attained within 60 s with a 100-fold higher concentration in the cell than the medium. The initial velocity of uptake (Vo) exhibited a linear relationship to the bilirubin concentration in the medium. Pretreatment of cells with various metabolic inhibitors had no effect on the uptake of unconjugated bilirubin. Ouabain did significantly decrease Vo, but replacement of sodium ion with choline or lithium had no effect on bilirubin uptake. The organic acids sulfobromophthalein (112 muM) and taurocholic acid (50 (muM) and two steroidal compounds, diethylstilbestrol (50 muM) and spironolactone (50 muM), had no effect on the uptake of bilirubin. It is suggested that bilirubin gains access to the hepatocyte interior by passive diffusion into and through the lipid membrane and that intracellular binding may explain the high degree of bilirubin accumulation associated with the isolated hepatocytes.

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