scholarly journals Continuous bioluminescent monitoring of cytoplasmic ATP in single isolated rat hepatocytes during metabolic poisoning

1993 ◽  
Vol 295 (1) ◽  
pp. 165-170 ◽  
Author(s):  
A Koop ◽  
P H Cobbold
Keyword(s):  
Time Course ◽  
Rat Hepatocytes ◽  
Firefly Luciferase ◽  
Beta Oxidation ◽  
Isolated Rat Hepatocytes ◽  
Chemical Hypoxia ◽  
Luminescent Signal ◽  
Bioluminescent Monitoring ◽  
Cell To Cell Variability ◽  
Isolated Rat

We have devised a technique for monitoring cytoplasmic ATP continuously in single hepatocytes. Single isolated rat hepatocytes were injected with the ATP-dependent luminescent protein firefly luciferase, and then superfused with 45 microM luciferin in air-equilibrated medium. Signals of approx. 10-200 photoelectron counts per second could be recorded from individual healthy cells for up to 3 h. The response of the luminescent signal to chemical hypoxia (2-5 mM CN- and 5-10 mM 2-deoxyglucose) was monitored. We found a great cell-to-cell variability in the time course of the ATP decline in response to CN-, 2-deoxyglucose or to their combination; the time for the signal to fall to 10% of the original (corresponding to approx. 100 microM ATP) ranged from approx. 20 to 75 min. This resistance of the cytoplasmic ATP concentration to depletion after blockade of oxidative phosphorylation and glycolysis could be abolished by pretreatment of the cells with etomoxir, which blocks mitochondrial beta-oxidation. Etomoxir alone had no effect on the luciferase signal, but etomoxir-pre-treated cells showed a prompt fall in the luciferase signal starting within 1-2 min of application of cyanide and 2-deoxyglucose and falling to 10% of the original signal in approx. 6-10 min. The technique allows cytoplasmic ATP changes to be monitored in single hepatocytes at concentrations of 1 mM or lower, but more precise calibration of the signal will require correction for the effects of cytoplasmic pH changes.

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

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.

scholarly journals Exogenous phospholipase enzymes mimic effects of phenylephrine on Ca2+ transport in hepatocytes

1983 ◽  
Vol 210 (1) ◽  
pp. 115-119 ◽  
Author(s):  
G J Barritt ◽  
J A Whiting
Keyword(s):  
Plasma Membrane ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Time Course ◽  
Initial Rate ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Glucose Release ◽  
Significant Damage ◽  
Isolated Rat

Phospholipase C from Clostridium perfringens induced the release of 45Ca2+ from isolated rat hepatocytes incubated at 0.1 mM extracellular Ca2+ with a time course similar to that for the action of phenylephrine. Under the conditions of these experiments, no significant damage to the plasma membrane was detected in the presence of phospholipase C. Little 45Ca2+ release was induced by bee venom phospholipase A2. At 1.3 mM extracellular Ca2+, both phospholipase enzymes stimulated the initial rate of 45Ca2+ exchange. Concentrations of phospholipase C comparable with those that stimulated 45Ca2+ release increased the rates of glucose release and O2 utilization by 70 and 20% respectively. An increase in the rate of O2 utilization but not glucose release was observed after the addition of phospholipase A2 to hepatocytes. The possible role for a cellular phospholipase C in the mechanism by which phenylephrine stimulates glycogenolysis in the liver cell is briefly discussed.

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