Modelling of Mg2+, ATP-dependent mitochondrial Ca ions transport in smooth muscle cells using protonophore CCCP-sensitive fluorescent tetracycline

Mg2+, ATP-dependent Ca2+ accumulation in the rat myometrial mitochondria was investigated in complex experiment using Ca2+ isotope (45Ca2+) and Ca(2+)-sensitive label tetracycline. Monotonous increase of the fluorescence signal, insensitive to thapsigargin (100 nM) was observed with following establishing the stationary state of incubation at 2 min. which correlates with results obtained using isotope technique. Experiments with isotope label signify, that protonophore CCCP, ruthenium red and sodium azide, in concentration 1 microM, 10 microM and 10 mM respectively, totally inhibits the accumulation of the Ca ions in mitochondria. At the same time, in conditions of Mg2+, ATP-dependent Ca2+ accumulation modeling in these cellular structures, CCCP and sodium azide, used in the same concentration, diminished tetracycline fluorescence signal increase. In the same conditions, the introduction of the CCCP (1 mM) into the incubation medium at 75 sec. after initiation of the transport process induced reversible quenching of the tetracycline fluorescence signal to the level, observed in case of initial CCCP presence in the medium. According to data obtained in the experiment, using Ca2+ isotope, Ca(2+)-ionophore A-23187 induces both the reversible release of previously accumulated Ca ions, and cause reversible quenching of the tetracycline fluorescence signal to the level, observed in case of initial CCCP (1 mM) and sodium azide (10 mM) presence in the incubation medium. Conclusion was drawn that the thapsigargin-insensitive and CCCP, sodium azide and A-23187-sensitive tetracycline fluorescence increasing in case of modeling of Mg2+, ATP-dependent Ca2+ accumulation in myometrial mitochondria reflect the Ca2+ uniporter functioning in those subcellular structures.