A critical evaluation of in situ measurement of mitochondrial electrical potentials in single hepatocytes

AbstractThe range of applicability and the critical parameters involved in the assessment of mitochondrial electrical potential (ΔΨmit) 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 ΔΨmit 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 ΔΨmit 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 ΔΨmit was not altered in a measurable manner during Ca2+ oscillations consecutive to α1-agonist stimulation