scholarly journals Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes

2006 ◽  
Vol 395 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Yongmin Ma ◽  
Herbert de Groot ◽  
Zudong Liu ◽  
Robert C. Hider ◽  
Frank Petrat
Keyword(s):  
Time Course ◽  
Iron Chelation ◽  
Rat Hepatocytes ◽  
Calibration Method ◽  
Ex Situ ◽  
Iron Chelators ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Iron Pool ◽  
Intracellular Fluorescence

A series of fluorescent iron chelators has been synthesized such that a fluorescent function is covalently linked to a 3-hydroxypyridin-4-one. In the present study, the fluorescent iron chelators were loaded into isolated rat hepatocytes. The intracellular fluorescence was not only quenched by an addition of a highly lipophilic 8-hydroxyquinoline–iron(III) complex but also was dequenched by the addition of an excess of the membrane-permeable iron chelator CP94 (1,2-diethyl-3-hydroxypyridin-4-one). The time course of uptake of iron and iron chelation in single, intact cells was recorded on-line by using digital fluorescence microscopy. Intracellular concentrations of various fluorescent iron chelators were determined by using a spectrofluorophotometer subsequent to lysis of probe-loaded cells and were found to depend on their partition coefficients; the more hydrophobic the compound, the higher the intracellular concentration. An ex situ calibration method was used to determine the chelatable iron pool of cultured rat hepatocytes. CP655 (7-diethylamino-N-[(5-hydroxy-6-methyl-4-oxo-1,4-dihydropyridin-3-yl)methyl]-N-methyl-2-oxo-2H-chromen-3-carboxamide), which is a moderately lipophilic fluorescent chelator, was found to be the most sensitive probe for monitoring chelatable iron, as determined by the intracellular fluorescence increase induced by the addition of CP94. The concentration of the intracellular chelatable iron pool in hepatocytes was determined by this probe to be 5.4±1.3 μM.

scholarly journals The glucagon-induced activation of pyruvate dehydrogenase in hepatocytes is diminished by 4β-phorbol 12-myristate 13-acetate. A role for cytoplasmic Ca2+ in dehydrogenase regulation

1987 ◽  
Vol 241 (3) ◽  
pp. 729-735 ◽  
Author(s):  
J M Staddon ◽  
R G Hansford
Keyword(s):  
Dehydrogenase Activity ◽  
Phorbol Ester ◽  
Pyruvate Dehydrogenase ◽  
Kinase Activity ◽  
Rat Hepatocytes ◽  
Pyruvate Kinase Activity ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Subsequent Addition ◽  
Isolated Rat

Phenylephrine, vasopressin and glucagon each increased the amount of active (dephospho) pyruvate dehydrogenase (PDHa) in isolated rat hepatocytes. Treatment with 4 beta-phorbol 12-myristate 13-acetate (PMA) opposed the increase in PDHa caused by both phenylephrine and glucagon, but had no effect on the response to vasopressin: PMA alone had no effect on PDHa. As PMA is known to prevent the phenylephrine-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and to diminish the increase [Ca2+]c caused by glucagon, while having no effect on the ability of vasopressin to increase [Ca2+]c, these data are consistent with the notion that in intact cells an increase in [Ca2+]c results in an increase in the mitochondrial free Ca2+ concentration, which in turn leads to the activation of PDH. In the presence of 2.5 mM-Ca2+, glucagon caused an increase in NAD(P)H fluorescence in hepatocytes. This increase is taken to reflect an enhanced activity of mitochondrial dehydrogenases. PMA alone had no effect on NAD(P)H fluorescence; it did, however, compromise the increase produced by glucagon. When the extracellular free [Ca2+] was decreased to 0.2 microM, glucagon could still increase NAD(P)H fluorescence. Vasopressin also increased fluorescence under these conditions; however, if vasopressin was added after glucagon, no further increase in fluorescence was observed. Treatment of the cells with PMA resulted in a smaller increase in NAD(P)H fluorescence on addition of glucagon: the subsequent addition of vasopressin now caused a further increase in fluorescence. Changes in [Ca2+]c corresponding to the changes in NAD(P)H fluorescence were observed, again supporting the idea that [Ca2+]c indirectly regulates intramitochondrial dehydrogenase activity in intact cells. PMA alone had no effect on pyruvate kinase activity, and the phorbol ester did not prevent the inactivation caused by glucagon. The latter emphasizes the different mechanisms by which the hormone influences mitochondrial and cytoplasmic metabolism.

Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes

1989 ◽  
Vol 256 (1) ◽  
pp. C28-C34 ◽  
Author(s):  
S. R. Kimball ◽  
W. V. Everson ◽  
K. E. Flaim ◽  
L. S. Jefferson
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Hepatocytes ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell

A cell-free system, which maintained a linear rate of protein synthesis for up to 20 min of incubation, was prepared from isolated rat hepatocytes. The rate of protein synthesis in the cell-free system was approximately 20% of the rate obtained in isolated hepatocytes or perfused liver. More than 70% of total protein synthesis in the cell-free system was due to reinitiation, as indicated by addition of inhibitors of initiation, i.e., edeine or polyvinyl sulfate. The rate of protein synthesis and formation of 43S initiation complexes in the cell-free system were reduced to 60 and 30% of the control values, respectively, after incubation of hepatocytes in medium deprived of an essential amino acid. Therefore, the cell-free system maintained the defect in initiation induced in the intact cells by amino acid deprivation. The defect in initiation was corrected by addition of either rat liver eukaryotic initiation factor 2 or the guanine nucleotide exchange factor (GEF) to the cell-free system. A role for GEF in the defect in initiation was further implicated by experiments that showed that the activity of the factor was decreased in extracts from livers perfused with medium deficient in amino acids. The cell-free system should provide a valuable tool for investigation of mechanisms involved in the regulation of initiation of protein synthesis.

Time course of ethanol-induced impairment in fluid-phase endocytosis in isolated rat hepatocytes

Hepatology ◽  
1993 ◽  
Vol 17 (4) ◽  
pp. 661-667 ◽  
Author(s):  
Kenneth B. Camacho ◽  
Carol A. Casey ◽  
Robert L. Wiegert ◽  
Michael F. Sorrell ◽  
Dean J. Tuma
Keyword(s):  
Time Course ◽  
Fluid Phase ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Fluid Phase Endocytosis ◽  
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.

scholarly journals Hormonal control of fructose 2,6-bisphosphate concentration in isolated rat hepatocytes

1983 ◽  
Vol 214 (3) ◽  
pp. 829-837 ◽  
Author(s):  
R Bartrons ◽  
L Hue ◽  
E Van Schaftingen ◽  
H G Hers
Keyword(s):  
Cyclic Amp ◽  
Pyruvate Kinase ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Hormonal Control ◽  
Isolated Rat Hepatocytes ◽  
Free System ◽  
Significant Difference ◽  
Dependent Protein

The ability of glucagon and of adrenaline to affect the concentration of fructose 2,6-bisphosphate in isolated hepatocytes was re-investigated because of important discrepancies existing in the literature. We were unable to detect a significant difference in the sensitivity of the hepatocytes with regard to the effect of glucagon to initiate the interconversion of phosphorylase, pyruvate kinase, 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase, and also to cause the disappearance of fructose 2,6-bisphosphate. In contrast, we have observed differences in the time-course of these various changes, since the interconversions of phosphorylase and of pyruvate kinase were at least twice as fast as those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. When measured in a cell-free system in the presence of MgATP, the cyclic AMP-dependent interconversion of pyruvate kinase was 5-10-fold more rapid than those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. These data indicate that 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase are relatively poor substrates for cyclic AMP-dependent protein kinase; they also support the hypothesis that the two catalytic activities belong to a single protein. Adrenaline had only a slight effect on the several parameters under investigation, except for the activation of phosphorylase. In the absence of Ca2+ ions from the incubation medium, however, adrenaline had an effect similar to that of glucagon.

scholarly journals Glucosamine-sensitive and -insensitive detritiation of [2-3H]glucose in isolated rat hepatocytes: a study of the contributions of glucokinase and glucose-6-phosphatase

1995 ◽  
Vol 308 (1) ◽  
pp. 23-29 ◽  
Author(s):  
E Van Schaftigen
Keyword(s):  
Exchange Reaction ◽  
Regulatory Protein ◽  
Liver Microsomes ◽  
Rat Hepatocytes ◽  
Amino Sugar ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
In Situ Experiments ◽  
Glucose Phosphorylation ◽  
Isolated Rat

Glucosamine, a potent inhibitor of glucokinase (hexokinase IV or D), was used to estimate the contribution of this enzyme to glucose phosphorylation in freshly isolated rat hepatocytes and its sensitivity to fructose 6-phosphate in situ. Experiments with radiolabelled glucosamine indicated that this amino sugar, at concentrations of 5 or 40 mM, readily penetrated hepatocytes to reach in 1 min a total (i.e., glucosamine+metabolites) intracellular concentration equal to 0.8-1.2-fold its extracellular concentration. In marked contrast, N-acetylglucosamine barely penetrated the cells. The detritiation of [2-3H]glucose, used to estimate glucose phosphorylation in intact cells, was inhibited by glucosamine much more potently than by N-acetylglucosamine, half-maximal effects being reached at about 2.5 and 30 mM respectively. Extrapolation of the data indicated that about 12% of the detritiation was resistant to glucosamine. Dihydroxyacetone (10 mM), lactate (10 mM) + pyruvate (1 mM), and glucagon (1 microM) increased up to 8-fold the concentration of hexose 6-phosphates (glucose 6-phosphate+fructose 6-phosphate) and, against expectations, modestly decreased the detritiation rate measured in the absence of glucosamine. In the presence of 40 mM glucosamine, these agents increased the detritiation rate, which then positively correlated with the concentration of hexose 6-phosphates. This hexose 6-phosphates-dependent detritiation was sensitive to inhibition by vanadate, and was also catalysed by gel-filtered cell-free extracts, as well as by liver microsomes in the presence of phosphoglucoisomerase; it can be explained by an exchange reaction catalysed by glucose-6-phosphatase. When this exchange reaction is taken into account, it appears that the rate of glucose detritiation attributable to glucokinase decreases when the concentration of hexose 6-phosphates increases. This is in agreement with the known effect of fructose 6-phosphate to potentiate the inhibition of glucokinase by its regulatory protein.

Decreased protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated rat hepatocytes

1992 ◽  
Vol 12 (3) ◽  
pp. 199-206 ◽  
Author(s):  
Victor Sanchez ◽  
Miguel Lucas ◽  
Aureo Sanz ◽  
Raimundo Goberna
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Polymyxin B ◽  
Isolated Rat Hepatocytes ◽  
Protein Kinase C Inhibitors ◽  
Kinase C ◽  
Freshly Isolated Rat Hepatocytes ◽  
Isolated Rat

Apoptosis of freshly isolated rat hepatocytes was induced by either the omission of fetal bovine serum in the culture medium or addition of the protein kinase C inhibitors polymyxin B or staurosporin. The time-course of DNA breakdown into oligonucleosome-sized fragments and the activity of protein kinase C was determined. Hepatocytes were found to be sensitive to bleomycin which induced a high degree of DNA breakdown even within 30 min incubation. Both staurosporin and polymyxin B induced DNA degradation in hepatocytes after three hours incubation, an effect that was partially prevented by phorbol myristate acetate (PMA). After eight hours incubation, PMA failed to counteract this action and itself produced the apoptosis of rat hepatocytes. The results suggest the involvement of protein kinase C in hepatocyte survival.

Characterization of the Binding of Bovine Thrombin to Isolated Rat Hepatocytes

1988 ◽  
Vol 60 (03) ◽  
pp. 419-427 ◽  
Author(s):  
Britta Weyer ◽  
Torben E Petersen ◽  
Ole Sonne
Keyword(s):  
Binding Sites ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Prolonged Incubation ◽  
Receptor Assay ◽  
Isolated Rat

SummaryIsolated rat hepatocytes possess per cell 4,500 high-affinity binding sites for thrombin with a Kd of 30-40 pM, and 2.8 × 105 low-affinity sites with a Kd of 30 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labelled thrombin is achieved after 3 min at 37¸ C and 7 min at 4¸ C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 1—2 × 10−2 s−1 and 3—4 × 10−4 s−1. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radioactivity migrates as intact thrombin upon sodium dodecyl sulphate polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3-treatment. Cell-associated radioactivity dissociated from the cells binds just aswell in a receptor assay as tracer incubated in a conditioned medium under the same conditions, indicating the absence of a quantitatively important receptor-mediated degradation ofthe ligand.

scholarly journals Comparison of the effects of various amino acids on glycogen synthesis, lipogenesis and ketogenesis in isolated rat hepatocytes

1991 ◽  
Vol 273 (1) ◽  
pp. 57-62 ◽  
Author(s):  
A Baquet ◽  
A Lavoinne ◽  
L Hue
Keyword(s):  
Amino Acids ◽  
Time Course ◽  
Glycogen Synthesis ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Aminoisobutyric Acid ◽  
Malonyl Coa ◽  
Lag Period ◽  
Isolated Rat ◽  
Stimulation Of

Several amino acids were found to stimulate glycogen synthesis and lipogenesis, and to inhibit ketogenesis in isolated rat hepatocytes. When hepatocytes were incubated in the presence of 20 mM-glucose, the amino acids could be classified in decreasing order of efficiency as follows: glutamine and proline, alanine, aminoisobutyric acid, asparagine and histidine for stimulation of glycogen synthesis; glutamine, proline and alanine for stimulation of lipogenesis; proline and glutamine for inhibition of ketogenesis. The study of the time course revealed that the rates were not linear and were preceded by a lag period. In all conditions studied, glutamine and proline were found to have similar quantitative effects on glycogen synthesis and lipid metabolism. However, their effects differ qualitatively. Indeed, the effects of proline on glycogen synthesis, lipogenesis and glutamate and aspartate content were faster. Moreover, proline increased the hydroxybutyrate/acetoacetate ratio, whereas glutamine did not change it. Incubation of hepatocytes with aminoisobutyric acid or under hypo-osmotic conditions, which increased cell volume and mimicked the amino acid-induced stimulation of glycogen synthesis, had little effect on lipogenesis. In hepatocytes incubated without glucose, ketogenesis was inhibited, in decreasing order of efficiency, by alanine, asparagine, glutamine and proline. Under these conditions, glutamine increased, alanine decreased and asparagine did not affect the concentration of malonyl-CoA. This indicates that the latter cannot be responsible for the inhibition of ketogenesis by alanine and asparagine.

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