Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence

The occurrence and the mode of opening of the mitochondrial permeability tr ansition pore (MTP) were investigated directly in intact cells by monitorin g the fluorescence of mitochondrial entrapped calcein, When MH1C1 cells and hepatocytes were loaded with calcein AM, calcein was also present within m itochondria, because (i) its mitochondrial signal was quenched by the addit ion of tetramethylrhodamine methyl ester and (ii) calcein-loaded mitochondr ia could be visualized after digitonin permeabilization. Under the latter c ondition, the addition of Ca2+ induced a prompt and massive release of the accumulated calcein, which was prevented by CsA, indicating that calcein re lease could, in principle, probe MTP opening in intact cells as well. To st udy this process, we developed a procedure by which the cytosolic calcein s ignal was quenched by Co2+. In hepatocytes and MH1C1 cells coloaded with Co 2+ and calcein AM, treatment with MTP inducers caused a rapid, though limit ed, decrease in mitochondrial calcein fluorescence, which was significantly reduced by CsA. We also observed a constant and spontaneous decrease in mi tochondrial calcein fluorescence, which was completely prevented by CsA. Th us MTP likely fluctuates rapidly between open and closed states in intact c ells.