EXPERIMENTAL SUBSTANTIATION OF PERMEABILIZED HEPATOCYTES MODEL FOR INVESTIGATION OF MITOCHONDRIA IN SITU RESPIRATION
| Autor: | V. V. Manko, B. O. Manko, V. M. Merlavsky, O. V. Ikkert |
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| Rok vydání: | 2016 |
| Předmět: |
Male
Cell Membrane Permeability Cellular respiration Liver cytology Cell Respiration Detergents Primary Cell Culture Succinic Acid Cell Count Digitonin Mitochondria Liver Biology Biochemistry Oxidative Phosphorylation lcsh:Biochemistry chemistry.chemical_compound Respiration polycyclic compounds medicine Animals lcsh:QD415-436 permeabilized hepatocytes Dose-Response Relationship Drug Biological Transport respiratory system succinate Rats mitochondria carbohydrates (lipids) Adenosine Diphosphate Adenosine diphosphate α-ketoglutarate medicine.anatomical_structure chemistry Liver Hepatocyte Biophysics Hepatocytes Ketoglutaric Acids Trypan blue Respiration rate respiration |
| Zdroj: | Ukrainian Biochemical Journal, Vol 87, Iss 6, Pp 113-121 (2015) |
| ISSN: | 2409-4943 |
| Popis: | To verify experimentally the model of permeabilized hepatocytes, the degree of cell permeability was assessed using trypan blue and polarographycally determined cell respiration rate upon succinate (0.35 mM) and α-ketoglutarate (1 mM) oxidation. Oxidative phosphorylation was stimulated by ADP (750 μM). Hepatocyte permeabilization depends on digitonin concentraion in medium and on the number of cells in suspension. Thus, the permeabilization of 0.9-1.7 million cells/ml was completed by 25 μg/ml of digitonin, permeabilization of 2.0-3.0 million cells/ml – by 50 μg/ml of digitonin and permeabilization of 4.0-5.6 million cells/ml – by 100 μg/ml. Thus, the higher is the suspension density, the higher digitonin concentration is required. Treatment of hepatocytes with digitonin resulted in a decrease of endogenous respiration rate to a minimum upon 20-22 μg of digitonin per 1 million cells. Supplementation of permeabilized hepatocytes with α-ketoglutarate maintained stable respiration rate on the level higher than endogenous respiration at the corresponding digitonin concentration, unlike the intact cells. Respiration rate of permeabilized hepatocytes at the simultaneous addition of α-ketoglutarate and ADP increased to the level of intact cell respiration, irrespective of digitonin concentration. Addition of solely succinate and especially succinate plus ADP markedly intensified the respiration of permeabilized hepatocytes to the level higher than that of intact cells. The dependence of succinate-stimulated respiration on digitonin concentration reached maximum at 20-22 μg of digitonin per 1 million cells. Optimal ratio of digitonin amount and the cell number in suspension is expected to be different in various tissues. |
| Databáze: | OpenAIRE |
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