A CRITICAL-EVALUATION OF IN-SITU MEASUREMENT OF MITOCHONDRIAL ELECTRICAL POTENTIALS IN SINGLE HEPATOCYTES

The range of applicability and the critical parameters involved in the assessment of mitochondrial electrical potential (Delta Psi(mit)) usi ng 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 heterog eneity 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 th e cell membrane for monovalent cations using nystatin and gramicidin, and equilibrating the cell with a K+-free bath solution. A voltage-cla mp at defined Delta Psi(mit) was then achieved after addition of valin omycin in the presence of different K+ concentrations in the bath. The oretical analysis indicated that the ratio of fluorescence intensity m easured in mitochondria-rich and mitochondria-poor regions of cell was related with Delta Psi(mit) and yielded quantitative estimates of ele ctrical potential with an accuracy of 10-20 mV. The ratio tended to pl ateau at potentials more negative than -140 mV, showing a limitation o f the technique. Manoeuvres such as imposing a heavy ATP demand or int erfering 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.