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A critical evaluation of in situ measurement of mitochondrial electrical potentials in single hepatocytes
- Source :
- Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1276(2):124-132
- Publication Year :
- 1996
- Publisher :
- Elsevier BV, 1996.
-
Abstract
- The range of applicability and the critical parameters involved in the assessment of mitochondrial electrical potential (delta psi mit) using epifluorescence microscopy were evaluated based on both theoretical and experimental analysis. Rat hepatocytes loaded with the potential-dependent fluorescent dye rhodamine 123 exhibited the expected heterogeneity of fluorescence distribution with dark regions corresponding to the nucleus and bright regions corresponding to the mitochondria-rich cytosol. Calibration of the signal was performed by permeabilizing the cell membrane for monovalent cations using nystatin and gramicidin, and equilibrating the cell with a K(+)-free bath solution. A voltage-clamp at defined delta psi mit was then achieved after addition of valinomycin in the presence of different K+ concentrations in the bath. Theoretical analysis indicated that the ratio of fluorescence intensity measured in mitochondria-rich and mitochondria-poor regions of cell was related with delta psi mit and yielded quantitative estimates of electrical potential with an accuracy of 10-20 mV. The ratio tended to plateau at potentials more negative than-140 mV, showing a limitation of the technique. Manoeuvres such as imposing a heavy ATP demand or interfering with the mitochondrial respiration depolarized mitochondria, while delta psi mit was not altered in a measurable manner during Ca2+ oscillations consecutive to alpha 1-agonist stimulation.
- Subjects :
- Analytical chemistry
Biophysics
Mitochondria, Liver
Mitochondrion
Rhodamine 123
Biochemistry
Membrane Potentials
Cell membrane
Phenylephrine
Valinomycin
chemistry.chemical_compound
Phenformin
Mitochondrial potential
medicine
Fluorescence microscope
Animals
Hypoglycemic Agents
Fluorescent Dyes
Rhodamines
Reproducibility of Results
Ca2+ oscillation
Cell Biology
Models, Theoretical
Fluorescence
Metformin
Rats
Cytosol
medicine.anatomical_structure
Liver
chemistry
Evaluation Studies as Topic
Gramicidin
Biguanide
Cytophotometry
Adrenergic alpha-Agonists
Subjects
Details
- ISSN :
- 00052728
- Volume :
- 1276
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- Biochimica et Biophysica Acta (BBA) - Bioenergetics
- Accession number :
- edsair.doi.dedup.....533ccf0843b20aca1f0c6139a6523f7c
- Full Text :
- https://doi.org/10.1016/0005-2728(96)00067-9