Mitochondria play an important role in the cell aging process. Changes in calcium homeostasis and/or increased reactive oxygen species (ROS) production lead to the opening of mitochondrial permeability transition pore (MPTP), depolarization of the inner mitochondrial membrane, and decrease of ATP production. Our work aimed to monitor age-related changes in the Ca2+ ion effect on MPTP and the ability of isolated rat liver mitochondria to accumulate calcium. The mitochondrial calcium retention capacity (CRC) was found to be significantly affected by the age of rats. Measurement of CRC values of the rat liver mitochondria showed two periods when 3 to 17-week old rats were tested. 3-week and 17-week old rats showed lower CRC values than 7-week old animals. Similar changes were observed while testing calcium-induced swelling of rat liver mitochondria. These findings indicate that the mitochondrial energy production system is more resistant to calcium-induced MPTP opening accompanied by the damaging effect of ROS in adult rats than in young and aged animals.
Values of the calcium retention capacity (CRC) of rat liver mitochondria are highly dependent on the experimental conditions used. When increasing amounts of added calcium chloride are used (1.25-10 nmol), the values of the CRC increase 3-fold. When calcium is added in 75 s intervals, the CRC values increase by 30 % compared with 150 s interval additions. CRC values are not dependent on the calcium/protein ratio in the measured sample in our experimental design. We also show that a more detailed evaluation of the fluorescence curves can provide new information about mitochondrial permeability transition pore opening after calcium is added.
By determining the calcium retention capacity (CRC) of rat liver
mitochondria, we confirmed and extended previous observations
describing the activation of mitochondrial swelling by phosphate
and tert-butyl hydroperoxide (t-BHP). Using CRC measurements,
we showed that both phosphate and t-BHP decrease the extent
of calcium accumulation required for the full mitochondrial
permeability transition pore (MPTP) opening to 35 % of control
values and to only 15 % when both phosphate and t-BHP are
present in the medium. When changes in fluorescence were
evaluated at higher resolution, we observed that in the presence
of cyclosporine A fluorescence values return after each
Ca2+ addition to basal values obtained before the Ca2+ addition.
This indicates that the MPTP remains closed. However, in the
absence of cyclosporine A, the basal fluorescence after each
Ca2+ addition continuously increased. This increase was
potentiated both by phosphate and t-BHP until the moment when
the concentration of intramitochondrial calcium required for the
full opening of the MPTP was reached. We conclude that in the
absence of cyclosporine A, the MPTP is slowly opened after each
Ca2+ addition and that this rate of opening can be modified by
various factors such as the composition of the media and the
experimental protocol used.