Calix[4]arenes C-136 and C-137 hyperpolarize myometrium mitochondria membranes

Calixarenes are supramolecular compounds interacting with bioactive molecules and ions, causing changes in biochemical and biophysical processes. The aim of this work was to study the effects of calix4]arenes C-136, C-137, and C-138 at the level of polarization of the rat myometrium mitochondria membrane. The structure of synthesized calix4]arene molecules was confirmed by the methods of ¹H NMR and infrared spectroscopy. Calix4]arenes C-136 and C-137 each possess two chalcone amide moieties at the lower rim, while calix4]arene C-138, only one. Calix4]arenes C-136 and C-137 differ by the presence of ether or hydroxyl groups, respectively, at the lower rim of calix4]arene skeleton, as well as the length of alkyl spacer between chalcone amide group and the macrocycle. It was shown that calix4]arenes C-136, C-137, and C-138 form micelles in aqueous medium and in dimethylformamide (DMF). Irradiation of micelles with an argon laser on the flow cytometer results in the rise of autofluorescence. In an aqueous medium, calix4]arene micelles interact with a positively charged voltage-sensitive fluorescent probe TMRM, which can testify to the presence of negative charge in these structures. However, calix4]arene micelles do not interact with TMRM in DMF solution. The mitochondrial membrane potential was measured using fluorescent dyes MTG and TMRM with confocal microscopy and fluorescent dye TMRM with flow cytometry. Experiments were conducted on myometrium cells in culture and on suspension of digitonin-permeabilized uterus myocytes. It was shown that the fluorescent signal was stable during the time of experiment. Calix4]arenes C-136 and C-137 (10 μM) hyperpolarize mitochondria membranes. At maximum, the effect was 173% relative to the control. At the same time, calix4]arene C-138 did not influence the mitochondria membrane potential. The relationship between the structural organization of investigated calix4]arene molecules and their effect on polarization of the mitochondria membrane is discussed.