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.

Keywords: calix[4]arenes, mitochondria, photon correlation spectroscopy, plasma membrane, smooth muscle, transmembrane potential, uterus