Tag Archives: transmembrane potential
Comparative investigation by spectrofluorimetry and flow cytometry of plasma and inner mitochondrial membranes polarisation in smooth muscle cell using potential-sensitive probe DIOC(6)(3)
G. V. Danylovych, Yu. V. Danylovych, V. F. Gorchev
Palladine Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua
Possibility of the use of flow cytometry and spectrofluorimetry analysis for investigation mitochondria and plasma membrane polarization in myometrium cell suspension using potential-sensitive probe 3,3′-dihexyloxacarbocyanine [DiOC6(3)] has been demonstrated. The obtained results confirm the use of DiOC6(3) for studying the influence of effectors on transmembrane potentials of intact cell compartments.
Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na(+), K(+)-ATPase
G. V. Danylovych1, Yu. V. Danylovych1, O. V. Kolomiets1,
S. O. Kosterin1, R. V. Rodik2, S. O. Cherenok2, V. I. Kalchenko2,
A. Ju. Сhunikhin1, V. F. Gorchev1, S. A. Karakhim1
1Palladine Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv
2Institute of Organic Chemistry, National Academy of Science of Ukraine, Kyiv
e-mail: danylovych@biochem.kiev.ua; vik@bpci.kiev.ua
The influence of supramolecular macrocyclic compounds – calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K+-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied.
By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM).
Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide.
Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too.
It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.
Investigation of nitrosactive compounds influence on polarization of the mitochondrial inner membrane in the rat uterus myocytes using potential sensitive fluorescent probe DіOC(6)(3)
Yu. V. Danylovych, H. V. Danylovych, O. V. Kolomiets, S. O. Kosterin, S. A. Karakhim, A. Yu. Chunikhin
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua
The effect of nitrosactive compounds (sodium nitroprusside and sodium nitrite) on the polarization level of the uterus myocytes inner mitochondrial membrane using the confocal laser microscopy and fluorescent probe potentialsensitive DiOC6(3) (3,3′-dihexyloxacarbocyanine) was ivestigated. Colocalisation of mitochondrial membranes specific fluorescent probes (MitoTracker Orange CM H2TMRos, 10 – nonyl acridine orange and DiOC6(3)) was demonstrated. It was shown that sodium nitroprusside at 0.1 mM concentration caused a moderate decrease in mitochondrial transmembrane potential. That observation was confirmed by flow cytometry. Action efficiency of sodium nitrite in a similar concentration was significantly lower than that of sodium nitroprusside. It is shown that it was sodium nitroprusside which caused a slight swelling of the mitochondria. A possible protecting role of nitric oxide as to mitochondria was discussed.