Abstract
We demonstrated using Ca(2+)-sensitive fluorescent probe, mitochondria binding dyes, and confocal laser scanning microscopy, that elimination of electrochemical potential of uterus myocytes' inner mitochondrial membrane by aprotonophore carbonyl cyanide m-chlorophenyl hydrazone (10 μM), and by a respiratory chain complex IV inhibitor sodium azide (1 mM) is associated with substantial increase of Ca2+ concentration in myoplasm in the case of the protonophore effect only, but not in the case of the azide effect. In particular, with the use of nonyl acridine orange, a mitochondria-specific dye, and 9-aminoacridine, an agent that binds to membrane compartments in the presence of proton gradient, we showed that both the protonophore and the respiratory chain inhibitor cause the proton gradient on mitochondrial inner membrane to dissipate when introduced into incubation medium. We also proved with the help of 3,3'-dihexyloxacarbocyanine, a potential-sensitive carbocyanine-derived fluorescent probe, that the application of these substances results in dissipation of the membrane's electrical potential. The elimination of mitochondrial electrochemical potential by carbonyl cyanide m-chlorophenyl hydrazone causes substantial increase in fluorescence of Ca(2+)-sensitive Fluo-4 AM dye in myoplasm of smooth muscle cells. The results obtained were qualitatively confirmed with flow cytometry of mitochondria isolated through differential centrifugation and loaded with Fluo-4 AM. Particularly, Ca2+ matrix influx induced by addition of the exogenous cation is totally inhibited by carbonyl cyanide m-chlorophenyl hydrazone. Therefore, using two independent fluorometric methods, namely confocal laser scanning microscopy and flow cytometry, with Ca(2+)-sensitive Fluo-4 AM fluorescent probe, we proved on the models of freshly isolated myocytes and uterus smooth muscle mitochondria isolated by differential centrifugation sedimentation that the electrochemical gradient of inner membrane is an important component of mechanisms that regulate Ca2+ homeostasis in myometrium cells.
Highlights
M itochondrial Ca2+ transport plays a major role in maintaining Ca2+ homeostasis in smooth muscle cells, as it provides for post-transient energy-dependent Ca2+ uptake from myoplasm
We prove in this study the identical subcellular localization of 9-AA and nonyl acridine orange (NAO) fluorescent probes in myocytes (Fig. 1)
As NAO interacts with cardiolipin, which is abundant in mitochondrial membrane [13], and 9-AA interacts with subcellular membrane structures bearing ΔpH [14], we can assert that we have positively identified energized mitochondria with proton gradient on their inner membrane
Summary
M itochondrial Ca2+ transport plays a major role in maintaining Ca2+ homeostasis in smooth muscle cells, as it provides for post-transient energy-dependent Ca2+ uptake from myoplasm. It has been proven that in unexcitable tissues and smooth muscle cells the Ca2+/H+ exchanger plays the main role in the maintaining of the optimal matrix concentrations of Ca2+ [6, 7]. The aim of the present work was to investigate the effect of sodium azide (NaN3), a conventional inhibitor of complex IV of mitochondrial electron transport chain, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a protonophore, on the electrochemical potential of the inner mitochondrial membrane and myoplasm Ca2+ concentration in ute rus smooth muscle (myometrium) cells
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