A new Affine inhibitor of sodium pump thiacalix[4]arene С-1193 increases the intrAcellulAr concentrAtion of ca ions And modifies myometrium contrActility

The methods of enzymatic and kinetic analysis were used to demonstrate that thiacalix[4]arene-bis-hydroxymethylphosphonic acid С-1193 had the inhibitory effect (І 0.5 = 42.1 ± 0.6 nm) on na + ,k + -aTPase activity in the plasma membrane of myometrium cells with no effect on the relative activity of other aTPases localized in this subcellular structure. The method of confocal microscopy and Са 2+ -sensitive fluorescent probe fluo-4 were used to demonstrate that thiacalix[4]arene С-1193 increased the intracellular concentration of ca ions in the immobilized uterine myocytes. The tenzometric studies proved that С-1193 (10 and 100 μМ) increased the isometric phasic contractions, induced via the paths of both electromechanical (depolarization with high-potassium solution) and pharmacomechanical (application of uterotonic hormone oxytocin, neu - rotransmitter acetylcholine or selective agonist of muscarinic acetylcholine receptors cevimeline) coupling. application of thiacalix[4]arene С-1193 as a selective and effective inhibitor of nа + ,k + -aTPase may be useful both for studyng the regulation of ion homeostasis in smooth muscle cells and creation of new uterotonics based on the calixarene core

The methods of enzymatic and kinetic analysis were used to demonstrate that thiacalix [4]arene-bishydroxymethylphosphonic acid С-1193 had the inhibitory effect (І 0.5 = 42.1 ± 0.6 nm) on na + ,k + -aTPase activity in the plasma membrane of myometrium cells with no effect on the relative activity of other aTPases localized in this subcellular structure.The method of confocal microscopy and Са 2+ -sensitive fluorescent probe fluo-4 were used to demonstrate that thiacalix [4]arene С-1193 increased the intracellular concentration of ca ions in the immobilized uterine myocytes.The tenzometric studies proved that С-1193 (10 and 100 μМ) increased the isometric phasic contractions, induced via the paths of both electromechanical (depolarization with high-potassium solution) and pharmacomechanical (application of uterotonic hormone oxytocin, neurotransmitter acetylcholine or selective agonist of muscarinic acetylcholine receptors cevimeline) coupling.application of thiacalix [4]arene С-1193 as a selective and effective inhibitor of nа + ,k + -aTPase may be useful both for studyng the regulation of ion homeostasis in smooth muscle cells and creation of new uterotonics based on the calixarene core.k e y w o r d s: na + ,k + -АТРase, myometrium, thiacalix [4]arene, contractility mechanokinetics.N owadays the search for novel low-molecular regulators of membrane-bound ion transport systems, including active transport, is required for further studies on biochemical mechanisms of electro-and pharmacomechanical coupling in muscles, including smooth muscles (SM).In addition, the findings of this search could have many practical prospects, as selective highly-affine reverse effectors, capable of modifying the activity of specific ion-transporting systems, can serve as a basis for the elaboration of new-generation pharmacological preparations to correct the contractile function of SM, if it is impaired due to pathological states.Among SM organs, the uterus has an exclusive role, explained by its unique function in the organism -child-bearing and delivery.The impaired contractile activity of myometrium is the foundation for various pathologies: slow labor, spontaneous abortions, early labor, miscarriages, atony, hypo-and hypertonicity of the uterus [1][2][3].Thus, there is an urgent need to elaborate novel efficient medications capable of preventing and counteracting the impairments in myometrium functioning.
A number of cation-dependent ATPases are present in the plasma membrane (PM) of smooth muscle cells.For instance, fundamental significance is attributed to electroenzyme Na + ,K + -АТРase, supporting high K + concentrations and low Na + concentrations in the cytoplasm, ensuring the excitability of the cell and other biochemical and biophysical pro-doi: https://doi.org/10.15407/ubj95.05.005

e x p e r i m e n t A l w o r k s e x p e r i m e n t A l w o r k s
cesses.This enzyme is present in all the excitable tissues, and its activity is very sensitive to the energy state of the cell; thus, to some extent, it may be assumed that the catalytic and transporting activi ty of Na + ,K + -ATPase characterizes the energy potential of the cell.Na + ,K + -ATPase has a key role in the regulation of many biochemical processes; it is a target for both physiological regulatory factors and pharmacological agents [4][5][6][7][8].
The targeted synthesis and the study of the properties of effectors capable of penetrating via PM and changing the enzymatic or transporting activity of some intracellular membrane-bound and cytoplasmic proteins in a reversible way and with high affinity and selectivity is one of the most important tasks of modern biological, biophysical, and bioorganic chemistry.Indeed, the elaboration of novel, highly effective low-toxic selective inhibitors and activators of enzymes is extremely necessary, in particular, for the purpose of further research on ion, molecular, and membrane mechanisms of intracellular signalling.In this context, one should consider calixarenes, "cup"-like macrocyclic compounds, whose specifics of three-dimensional molecular structure and practically unlimited synthetic potential allow for rational designing of targeted effectors, to be rather promising agents [9][10][11].
The unique structure and properties of calixarene molecules allow for the modification of their structure (involving different functional groups on the upper and lower rims of the molecule); thus, the search for new calixarene effectors of enzymes has only begun.It should be noted that calixarenes are remarkable for their low toxicity [12] and immunogenicity [9,11,12], as well as affordable and low-cost synthesis.
This article presents new findings obtained by us while studying the regularities in the effect of thiacalix [4]arene С-1193, the inhibitor of Na + ,K + -dependent ATP-hydrolase of the plasma membrane, on the intracellular concentration of Ca ions in uterine myocytes and the mechanokinetics of contractility.In the enzymological part of this study, we compare the effects of the inhibitory activity of a novel thiacalix [4]arene С-1193 with a bigger size, containing four sulfur atoms in the macrocyclic platform, and the effects of calix [4]arene С-99, the impact of which on Na + ,K + -ATPase was studied by us before [16,17].materials and methods a. Synthesis of (thia)calix [4]arene-bis-hydroxymethylphosphonic acids С-99 and С-1193.The NMR spectra were registered on a Varian VXR-400 spectrometer operating at 399.987 MHz ( 1 Н), 1050.8MHz ( 13 С), using TMS as a reference.The 31 P NMR spectra were recorded on a Varian VXR-400 spectrometer operating at 162 MHz using 85% H 3 PO 4 as reference.The melting points were measured on a Boёtius heating block and not corrected.The reactions were carried out in anhydrous solvents .
The general method of synthesizing С-99 and С-1193.Tris-trimethylsilylphosphite (5 mmol) was added to a solution of diformylthiacalixarene 1 (1 mmol) in dry methylene chloride (20 ml) at room temperature.The mixture was stirred for 15 h.Methy lene chloride and silyl phosphite residues were evaporated under reduced pressure and kept in a vacuum of 0.1 mm Hg at 50°C for 2 h.Wet methanol (30 ml) was added to the obtained silyl derivative of thiacalixarene 2 and the reaction mixture was stirred for 2 h at 40°C.The solvent was evaporated under reduced pressure; the residue was kept in a vacuum of 0.1 mm Hg at 50°C for 2 h.C-99 and C-1193 acids were dissolved in methanol (10 ml) and diluted with water (10 ml).The precipitate formed was filtered, dried in a vacuum of 0.1 mm Hg at room temperature for 5 h.
The protein content in the membrane fraction was determined by the method of M. Bradford [23] using the reaction with Coomassie reagent -G250.
The myocytes were isolated from the uterus of non-pregnant laboratory rats using collagenase and soy inhibitor trypsin by Mollard's method [24].
The work was conducted in accordance with the Declaration of Helsinki (World Medical Assembly, 1964), the International principles of the European Convention for the Protection of Vertebrate Animals, Used for Experimental and Other Scientific Purposes (Strasbourg, 1986), the Declaration of Principles on Tolerance (28 th UNESCO Assembly, 1995), the Universal Declaration on Bioethics and Human Rights (UNO, 1997), the norms of the Convention for the Protection of Human Rights, adopted in 1997 due to the introduction of new biomedical technologies, in Oviedo (Spain) and ratified by the Verkhovna Rada of Ukraine in 2002, the Law of Ukraine No. 3447 IV "On Protection of Animals from Cruelty".enzymological studies.The total ATPase activity was determined in the PM fraction of myometrium cells, as described before [21], at 37°С in the standard medium (volume -0.4 ml), containing (mM): 1 АТР, 3 MgCl 2 , 125 NaCl, 25 KCl, 1 EDTA, 20 Hepes-tris-buffer (рН 7.4), 1 NaN 3 , 0.1 µM thapsigargin and 0.1 % digitonin.The presence of Са 2+chelating agent of EDTA in the incubation medium ensured the binding of endogenous ions of Ca therein.The amount of membrane fraction protein in the probe was 20-30 µg.The incubation time at 37°С was 4 min.The enzymatic reaction was initiated by the introduction of the aliquot (50 µl) of PM fragment suspension (8°C) to the incubation medium, and terminated by the introduction of 1 ml of "stop"solution to the incubation mixture as follows: 1.5 M sodium acetate, 3.7% formaldehyde, 14% ethanol, 5% TCA, рН 4.3 (at 8°С).
Nа + ,K + -ATPase activity was estimated by the difference between the values of the total ATPase activity and the basal Mg 2+ -ATPase activity.The basal Mg 2+ -АТРase activity was determined in the same medium but in the presence of 1 mM ouabain (selective inhibitor of Nа + ,K + -ATPases of PM [25,26]).
The incubation medium, with a composition, similar to the above described one but lacking fragments of plasma membranes, served as a control for non-enzymatic hydrolysis of ATP.The control of the amount of endogenous non-organic phosphorus (Р і ) in the membrane preparation was the medium, containing only the suspension of the membrane preparation in the aqueous solution.The basal АТРase enzymatic activity was estimated as a difference between the amount of Р і , which was formed in the incubation medium in the presence and in the absence of PM fragments with the consideration of the amendment for non-enzymatic hydrolysis of ATP and the content of endogenous Р і in the membrane preparation.The amount of P i reaction product was determined by the method [27].
Са 2+ ,Mg 2+ -АТРase activity was estimated by the difference between the values of ATPase activities in the presence and in the absence of exogenous Ca 2+ (against the background of 1 mM EDTAspecific chelating agent for Ca 2+ ) in the incubation medium .In the sarcolemma of the porcine myometrium, the relative enzymatic activity of Са 2+ ,Mg 2+ -ATPase was 3.4 ± 0.3 µmol Р і /mg of protein per one hour, respectively (n = 7).
It should be noted that the PM of uterine myocytes was found also to contain Са 2+ -АТРase, the properties of which differ from Са 2+ ,Mg 2+ -АТРase, since its activity was manifested in the presence of millimolar concentrations of Са 2+ and АТР in the incubation medium against the background of Mg 2+ [28,29].Са 2+ -АТРase was of low affinity to the activating cation -the constant of activation with Ca 2+ for K Са was 1 mM [29].The low-affinity Mg 2+ -independent Ca 2+ -ATPase activity was determined in the PM fraction of myometrium cells at 37°С in the medium (volume -0.4 ml), containing (mM): 1 АТР, 3 CaCl 2 , 125 NaCl, 25 KCl, 1 EDTA, 20 Hepes-tris-buffer (рН 7.4), 1 NaN 3 , 1 ouabain, 0.1 µM thapsigargin, and 0.1 % digitonin.The mentioned Ca 2+ -АТРase activity was estimated as a difference between the amount of Р і , which was formed in the incubation medium in the presence and in the absence of PM fraction with the consideration of the amendment for the content of endogenous Р і in the membrane preparation.In the sarcolemma of the porcine myometrium, the relative enzymatic activity of low-affinity Mg 2+ -independent Ca 2+ -ATPase of PM was 12.7 ± 2.0 µmol Р і /mg of protein per hour, respectively (n = 7).
In the experiments on the impact of different concentrations of calixarene compounds С-99 and C-1193 (1-100 µM) on AТР-hydrolase activity, the above-described standard incubation medium was used with the addition of the aliquot of the solution of thiacalix [4]arene in the relevant concentration.The experiments involved the use of the concentrated (20 mM) solutions of С-99 and C-1193 in DMSO, which were further diluted with water.
Determining the changes in the concentration of intracellular Са 2+ with the addition of confocal microscopy.To register the changes in the concentration of Са 2+ in SM cells, the suspension of myocytes, obtained by [24], was loaded with Са 2+ -sensitive probe fluo-4 AM at room temperature for 20 min, then the cells were precipitated by centrifugation for 15 min at 1,000 g, diluted in the isotonic storage medium (containing 25 mM HEPES-KOH (рН = 7.4; 8°С, 150 mM NaCl) and applied to a glass surface with poly-L-lysine.The obtained preparation of the immobilized cells was investigated with the laser scanning confocal microscope LSM 510 META.For analysis, we selected the spindle-shaped cells with a clearly defined nucleus, stained with DNA-sensitive fluorescent probe Hoechst, (applied 10 min before the registration).To register the relative changes in the concentration of Са 2+ in the cytoplasm, a series of consecutive photographs were taken, during which the aliquot of the C-1193 solution in the concentration of 20 µM (5 µl) was added.
kinetic estimates.To study the concentration dependence of the effect of calix [4]arenes on the enzymatic activity, the values of inhibition coefficients І 0.5 and Hill coefficients n H were estimated using the linearized charts of Hill according to the equation lg[(a max -a)/a] = -n H lgІ 0.5 + n H lg[С-1193], where a max and a -relative enzymatic activities in the absence ("zero point") and in the presence of calix [4]arene in the incubation medium in the concentration [С-1193].
Tenzometric experiments.The contractile activi ty in the preparations of longitudinal SM of uterine horns with preserved endothelium was registered in the isometric mode.Muscle stripes (2×10 mm) were placed into the working chamber (the volume of 2 ml) with the flowing Krebs solution (the flow rate of 5 ml/min), thermostated at 37°С.The prepa rations were provided with passive tension at the rate of 10 mN and left for 1 h until achieving stable reproduction of contractions.The signals were registered with an analogue-to-digital transformer.
Thiacalix [4]arene C-1193 was applied in concentrations of 10 and 100 μМ; it was preliminary dissolved in DMSO and added to the working solution to obtain the final aliquot of this organic solvent of 0.1% from the total volume of the solution.The control contractions were registered against the background of 0.1% DMSO.
mechanokinetic analysis of contractions.The study of the spontaneous contractile activity in SM preparations was conducted according to the empirical multiparameter method of the complex mechanokinetic analysis, previously developed by us [30].
To analyze the complete profile of single spontaneous contractions, they were linearized in the coordinates [ln( f r /f c ) vs ln(1+Δt/t)], where f and t -instant values of force and time at the level of the contraction cycle (С and r -symbols for the phases of contraction and relaxation, respectively), F C and F R -the values of the force at the inflexion points of the mechanogram at the level of the phases of contraction (from the beginning of the increase in the force to its maximal value F max ) and relaxation (from the maximal value of the force F max at the time moment τ 0 and until its return to the basal level), Δt -arbitrary fixed time interval (which varied within 15-50 s).The lineariza-tion charts were used to determine the characteristic constants k and n, which were further used to calculate the parameters: time (τ 0 , τ C and τ R ), force (F max , F C and F R ), velocity (V C and V R ), and impulse (І 0 , І C and І R ) parameters.Here, V C and V R -maximal velocities of the phases of contraction and relaxation, respectively, І 0 , І C and І R -force impulses at the level of amplitude and maximal velocities of contraction and relaxation, respectively.Spontaneous contraction, registered within 10-30 min from the start of C-1193 application were used in the analysis.
The analysis of kinetic properties of the induced contractions was made according to the method [31].The analysis involved the estimation of the indices, independent from the amplitude of contractile responses, the maximal velocities of the contraction phase (V nc ) and relaxation phase (V nr ), normalized in terms of the amplitude.The induced contractions registered within 20-30 min from the start of C-1193 application were used in the analysis.
Statistical analysis.The statistical analysis of the data obtained was conducted by the methods of variation statistics.Data were normally distributed.The Student's t-test was used to determine the reliable differences between the mean values of samplings.In all cases, the results were considered reliable on condition of P < 0.05.The validation analysis of data approximation by the linear function was performed using Fisher's F-test; the value of the determination coefficient (R 2 ) was at least 0.96 in all cases.The results were presented as the mean ± standard error of mean value, n -number of experiments.The kinetic and statistical calculations were done using the programs MS Excel and Origin 2018.

results and discussion
(Thia)calixarenes С-99, С-1193 were obtained by the one-pot synthesis, envisaging two chemical stages (Scheme).In the first stage, the interaction between diformyl(thia)calixarenes 1а,b and tris-trimethylsilylphosphite was used to obtain silyl derivatives of (thia)calixarene-bis-hydroxymethylphospho-nic acids 2a,b.During the processing of the obtained silyl derivatives 2a,b with methanol, the bonds of Р-О-Si and C-O-Si get bifurcated, and the target (thia)calixarene-hydroxymethylphosphonic acids С-99, С-1193 are formed.The yields of reaction products in both stages are practically quantitative.
The binding of tris-trimethylsilylphosphite to C=O bonds of formyl groups of calixarenes 1a,b occurs in a diastereoselective way with the formation of only mezo-form of silyl derivatives 2a,b with R and S configuration of chiral carbon atoms of phosphonomethylol fragments of СН(ОН)Р.This is evident in the same set of signals in 1 Н, 13 С and 31 Р NMR spectra of the acids С-99 and С-1193 (SI5 -SI10), obtained on their basis.Here, the spectra and physical-chemical properties of acid С-99 coincide with the corresponding characteristics of the mezoform of this compound, previously obtained by diastereoselective binding of dialkyl esters of phosphorous acid to diformylcalixarene 1а [21].
The obtained acids С-99, С-1193 have a coneshaped conformation of the macrocyclic frame.As for acid С-99, the cone-shaped conformation is clearly confirmed by the presence of two duplets of the spin system АВ of axial and equatorial protons of ArCH 2 Ar groups with chemical shifts of 3.37 and 4.24 ppm respectively, and the constants of spin-spin interaction 2 J HН J 13.0 Hz.As for thiacalixarene-bishydroxymethylphosphonic acid С-1193, the coneshaped conformation is demonstrated in the values of chemical shifts of the protons in ОСН 2 groups at 4.40 ppm.In the case of conformation 1,3-alternate, the signals of these groups should be considerably shifted to the strong field of δ 3.81-3.86ppm due to the occurrence of ОСН 2 groups into the cones of screening for two sin-oriented phenyl rings of the macrocyclic frame [32,33].
The cone-shaped conformation of С-99 and С-1193 is also confirmed by the data of molecular modelling using HyperChem program (Fig. 1).
In the obtained models, the phosphorylated benzene rings have coplanar orientation and paranon-substituted benzene rings -perpendicular orientation regarding the main plane of the macrocycle, formed by linker atoms of carbon or sulfur.
Therefore, the replacement of methylene linkers in the macrocyclic platform С-99 with sulfur atoms (molecule С-1193) changes the geometric parameters of the macrocycle and impacts the biological activity of the molecule.
In our previous studies, we showed that calixarene С-99 in the concentration of 100 µM inhibited the activity of Nа + ,K + -ATPase in PM of uterine myocytes as compared to the control [16].At the same time, it practically did not impact the basal Mg 2+ -ATPase activity (the decrease in the activity was only down to 91% as compared to the control value) (Fig. 2).

arene С-1193 (in blue) (hyperchem) T a b l e. The dihedral angles (°), formed by benzene rings i, ii, iii, iV with the main plane of the macrocycle in calix
We tried to determine the selectivity of the effect of thiacalix [4]arene С-1193 on АТР-hydrolase activities of PM in myocytes and demonstrated that thiacalix [4]arene C-1193 in the concentration of 100 µM effectively inhibited Na + ,K + -АТРase activity of PM in myometrium cells to the level of 5.2 ± 0.8% as compared to the control value (accepted as 100%) (n = 5) (Fig. 2).

Fig. 2. The results of the study on the comparative effect of calix[4]arene С-99 and thiacalix[4]arene c-1193 (100 µm) on aТР-hydrolase activities of plasma membranes of the myometrium cells (n = 5). The values of enzymatic activities in the absence of calix[4]arenes in the incubation medium are accepted as 100%
Relative activity, % effectively (І 0.5 = 42.1 ± 0.6 nM) inhibits the enzymatic activity of Na + ,K + -АТРase of PM, not affecting the activities of Mg 2+ -ATPase, Са 2+ -ATPase and Са 2+ ,Mg 2+ -ATPase of PM (Fig. 2, 3).It is known that PM of uterine myocytes has a functioning energy-dependent system of Na + -Ca 2+exchange, this antiport cation exchange is ensured with the energy of transmembrane sodium gradient, created due to the functioning of the sodium pump [34][35][36].Since thiacalix [4]arene С-1193 inhibited the activity of Nа + ,K + -ATPase of plasma membrane effectively, it should have been expected that the mentioned inhibitory effect would be accompanied with the increase in the concentration of Ca ions in the myoplasm due to the inhibition of Na + -dependent release of Са 2+ from myocytes.Thus, in our further experiments, we tried to find out whether thiacalix [4]arene С-1193 would affect the intracellular concentration of Са 2+ in smooth muscle cells.In these experiments, we estimated the changes in the concentration of Са 2+ in myocytes under the effect of thiacalix [4]arene С-1193, using the method of confocal microscopy and Са 2+ -sensitive probe fluo-4.It was demonstrated that under the effect of thiacalix [4] arene С-1193 (20 µM), there was a sharp increase in the fluorescent response of Са 2+ -sensitive probe fluo-4 АМ in the cell (Fig. 4 and Fig. 5).Within the next two minutes, the concentration of Са 2+ decreased which demonstrated the involvement of other ener-Т.О. Veklich, S. О. cherenok, О. V. Tsymbalyuk et al.

Fig. 3. The concentration dependencies of the effect of calix[4]arene С-99 and thiacalix[4]arene c-1193 on the activity of na + ,k + -aТРases in plasma membranes of the myometrium cells (n = 5). The values of specific enzymatic activity in the absence of calix[4]arenes in the incubation medium are accepted as 100%
Relative activity, % gy-dependent mechanisms, regulating the homeostasis of Са 2+ concentration (Са 2+ ,Mg 2+ -ATPases of PM and sarcoplasmic reticulum, Са 2+ -uniporter of mitochondria).There remained a stable fluorescence level of the DNA-sensitive probe, Hoechst, which was mainly localized in the nucleus of smooth muscle cells.So thiacalix [4]arene С-1193 is a selective inhibitor of Na + ,K + -ATPase of PM increased cytosolic Са 2+ concentration in smooth muscle cells.The previous series of experiments clearly demonstrated that thiacalix [4]arene С-1193 inhibited Nа + ,K + -ATPases in PM and induced considerable changes in the homeostasis of Са 2+ ions in myocytes, so one could assume that it was also capable of changing the functional properties of the integral SM tissue.So, our subsequent studies were related to the investigation of spontaneous and induced contractile activity of pluricellular smooth muscle preparations of uterine horns of non-pregnant rats.
Thiacalix [4]arene С-1193 induced dose-dependent inhibition of spontaneous contractions in the myometrium.For instance, under the effect of this compound in the concentration of 100 μМ, there was a decrease in the amplitude of contractions on average down to 72.3 ± 9.1% (n = 6, P < 0.05); against the background of the increase in the concentration of С-1193 up to 100 μМ, the amplitude was 63.1 ± 8.4% on average (n = 6, P < 0.05) (Fig. 6).Also, against the background of C-1193, there were some changes in single spontaneous contractionsan enhanced manifestation of complex contractions with the increase in the concentration of the mentioned compound.

Fig. 4. A. The fluorescence intensity of probes of ca 2+ -sensitive fluo-4 am (1) and Dna-sensitive hoechst (2) in Sm under the effect of thiacalix[4]arene С-1193. B. The image of a smooth muscle cell in the uterus, obtained using the scanning laser confocal microscope. The introduction of the aliquot of the thiacalix[4]arene С-1193 solution (the final concentration -20 µm) is indicated with the asterisks. The results of the typical experiment are presented
The method of complex mechanokinetic analysis was applied to specific spontaneous contractions (in control and under the effect of С-1193) [30].It was found that against the background of both investigated concentrations of thiacalix [4]arene С-1193, the indices of temporal parameters of amplitude (τ max ) and time of the mechanogram, when the maximal velocity of the relaxation was observed (τ R ), remained at the level of the control (Fig. 7, a), whereas the temporal parameter of achieving the maximal velocity of the contraction (τ C ) decreased reliably.
It was also found that under the effect of С-1193, the force (F max , F C and F R ) parameters decreased considerably and to the same degree (Fig. 7,  B): for instance, if C-1193 was present in the washing solution in the concentration of 10 μМ, on average these amounted to 72.3, 68.0 and 69.4% regarding the corresponding control parameters, accepted as 100%.When the concentration of С-1193 increased by one order (up to 100 μМ), these indices tended to decrease further, amounting to 63.1, 49.0, and 60.2%, respectively.
The mechanokinetic analysis demonstrated that under the effect of thiacalix [4]arene C-1193, the impulse parameters decreased considerably, tending towards dose dependence.For instance, against the background of 10 μМ С-1193, the values of the parameters І C , І R , and І 0 were 54.1, 65.5, and 63.3%, respectively, regarding the control values, whereas at the effect of 100 μМ of this compound, the average values of these indices were 47.7, 60.4, and 57.1%, respectively.Also, under the effect of С-1193, there was a considerable decrease in the velocities of the phases of contraction (V C ) and relaxation (V R ) with the tendency towards dose-dependence (Fig. 7, D).For instance, against the background of 10 μМ of this compound, the values of parameters V C and V R on average were 72.1 and 70.0%, respectively, whereas under the effect of 100 μМ С-1193 the corresponding indices were on average 69.3 and 55.4% regarding the control.It should be noted that the decrease in the velocity parameters of spontaneous contractions cannot be considered specific in terms of Са 2+ -transporting systems of myocytes, since the norm-setting of V C and V R regarding the contraction amplitude does not have statistically significant differences from the normalized maximal velocities of the phases of contraction and relaxation in control.
Therefore, in the following series of experiments, we investigated the effect of thiacalix [4]arene C-1193 on the contractile reactions of rat myometrium, induced via the pathways of electro-and phar-macomechanical coupling of excitation-contraction.The depolarization of PM in smooth muscle cells by high-potassium solution (80 mM) was used as an adequate model of electromechanical coupling; the pharmacomechanical coupling was studied on the example of contractions, induced by the uterotonic hormone oxytocin (0.1 IU), the neurotransmitter of parasympathetic nervous system acetylcholine (10 µM) and the selective agonist of muscarinic acetylcholine receptors of M3-type, cevimeline (100 µM).
Against the background of calix [4]arene C-1193 (10 µM), there was an increase in the amplitude of contractions, activated by hyperkalemic depolarization of PM in smooth muscle cells (Fig. 8).In these conditions, the value of the phasic component increased up to 126.1% on average (P < 0.05, n = 5), there were also tendencies towards the increase in the tonic component of contractions.
The method of kinetic analysis [31] was used to determine that thiacalix [4]arene С-1193 activated Fig. 7.The parameters of the spontaneous contractile activity of rat myometrium in the control and under the effect of thiacalix [4]arene c-1193 (10 and 100 μМ): А -time parameters (τ 0 , τ c and τ r ); B -force parameters (F max , F c and F r ); C -impulse parameters (І 0 , І c and І r ); D -velocity parameters (V c and V r ); n = 6; *P < 0.05 and **P < 0.01 -the difference is reliable as compared to the control the process of accele rating the force of contractions, induced by the application of the high-potassium solution, in a considerable way: the parameter V nc was 165.9% on average (P < 0.01, n = 6).Also, regardless of the increased tonic component, there was a considerab le increase in the normalized maximal velocity of the relaxation phase V nr (on average up to 155.9% regarding the control, P < 0.01, n = 6) (Fig. 8, А and Fig. 9).Also, under preliminary incubation of smooth muscle preparations of rat uterus with thiacalix [4]arene C-1193, there were changes in the oxytocin-induced contractions: the amplitude of phasic contractions tended to increase, and their tonic components increased considerably on average up to 121.8% (P < 0.05, n = 6) (Fig. 8, B and 9).
Noteworthy are considerable changes in the kinetics of oxytocin-induced contractions, caused by thiacalix [4]arene С-1193 (10 μМ): similar to the HPS-induced contractions, in this case, there was a considerable increase in the normalized maximal velocity of the contraction phase V nc (on average up to 177.5%, P < 0.01, n = 6).As for the normalized maximal velocity of the relaxation phase V nr , there was a decrease down to 88.6% on average regarding the control, P < 0.05, n = 6) (Fig. 8, А and Fig. 9).Also, against the background of thiacalix [4]arene C-1193 (10 µM), there was an increase in the amplitude of contractions, activated by the exogenous application of the neurotransmitter acetylcholine (Fig. 8, c).Against the background of C-1193, these contractions were characterized by the increase in the phasic component up to 130.2% on average (P < 0.05, n = 6); there were tendencies towards the increase in the tonic component.
The method of kinetic analysis [31] was used to determine that thiacalix [4]arene С-1193 activated the process of accelerating the force of acetylcholine-induced contractions: the parameter V nc was 119.5% on average (P < 0.05, n = 6).Also, in these conditions,  there was a conside rable decrease in the normalized maximal velocity of the relaxation phase V nr (on average down to 72.4% regarding the control, P < 0.05, n = 6).So, under the acetylcholine-induced activation of myometrium contractions against the background of C-1193, there generally were the effects, similar to those for oxytocin-activated contractions.
In the following stage, we studied the contractile activity of myometrium, activated by the selective agonist of muscarinic М3-cholinoreceptors, cevimeline (100 µM).Under the preliminary incubation of smooth muscle preparations of rat uterus with thiacalix [4]arene C-1193, there was a considerab le increase in the phasic contractions (on average up to 190.2%, P < 0.01, n = 6) (Fig. 8, D).In these conditions, contrary to acetylcholine-induced contractions, there was a decrease in the normalized maximal velocity of the contraction phase V nc (on average down to 76.3%, P < 0.05, n = 6), whereas the normalized maximal velocity of the relaxation phase V nr remained on the level of the control (104.2%regarding the control, P > 0.05, n = 6).
We can also foresee the cellular mechanisms, by which the blocking of Nа + ,K + -ATPase under the effect of thiacalix [4]arene С-1193 leads to the increase in the intracellular concentration of Са 2+ There is no doubt that in the excitable tissues, the functioning of Nа + ,K + -ATPase in PM fulfills the function of maintaining the excitability [37].In the myocytes of the uterus, this enzyme creates the gradients of Nа + and K + ions and Са 2+ ions (due to the formation of gradient Nа + , which is a driving force for the system of secondary active ion transport -Nа + , Са 2+ -exchanger) [38][39][40].In the non-pregnant myometrium of rats, the isoforms of all three types of subunits are expressed: α 1 (dominating) and α 2 ; β 1 and β 3 ; FXYD1 [38].It is important that the expression level for some isoforms of α-and β-subunits in the myometrium tissue changes considerably throughout the pregnancy, thus ensuring the optimal level of excitability for fetus bearing and delivery [38,41].
The known inhibitor of Nа + ,K + -ATPAse of PM, ouabain, which binds to α-subunit of the enzyme, induces the effects at the level of the integral tissue of myometrium, changing the contractile function and causing the increase in the frequency, and in some cases, in the force of spontaneous contractions [38,42].To understand the concentration effects of ouabain (and other blockers of Nа + ,K + -ATPase in PM), it is relevant that the sensitivity to inhibiting different isoforms of α-subunits of Nа + ,K + -ATPase differs considerably; in particular, for low-sensitive α 1 -isoform, the effective concentration of ouabain is considered to be 100 μМ, whereas for α 2 -isoform -10 μМ [43,44].It is also relevant that α 2 -isoform performs the signalling function in an ouabain-sensitive way, mediated by Src-kinase, modulating the reaction of SMC on the application of agonists of metabotropic receptors [43][44][45][46].
In our previous studies, we showed that, similarly to ouabain, calix [4]arene C-99 (both in the concentration of 10 µM) induced the activation of spontaneous contractions in the rat myometrium and modulated the mechanokinetics of contractile reactions on HKS-induced depolarization of PM and application of the agonists of metabotropic receptors (oxytocin and acetylcholine) [42].In this study, we revealed that thiacalix [4]arene C-1193 (10 µM) caused the increase in the frequency against the background of the decrease in the amplitude of spontaneous contractions in the myometrium and modulation of induced contractile reactions.It should be noted that in many cases (in terms of the increase in the frequency of spontaneous contractions, the direction of the change in parameters of normalized maximal velocities of the phases of contraction and relaxation, induced by acetylcholine and oxytocin), the effects, previously registered by us against the background of C-99 and C-1193, are in agreement [42].
At the same time, noteworthy is the fact that when thiacalix [4]arene C-1193 (10 and 100 µM) was present in the washing solution, it induced a considerable decrease in the amplitude of spontaneous contractions (Fig. 6).This difference may be explained with the consideration of the data of sustained increase in the level of cytosolic Са 2+ in myocytes under the application of С-1193 (Fig. 4  and 5).It is known that under the increase in the concentration of Са 2+ in the near-membrane space of smooth muscle cells, there is the activation of Са 2+sensitive ion channels, in particular, Са 2+ -sensitive K + -channels of high permeability (K Ca 1.1), which are expressed in the myocytes of the uterus of rats [47][48][49].This probable increase in the initial K + -current may condition the decrease in the amplitude of spontaneous contractions of the myometrium under the effect of С-1193.conclusions.All obtained results are important for understanding and subsequent investigation of mechanisms of Nа + ,K + -ATPase inhibition by calixarene C-1193 and can be a foundation for the creation of new more effective inhibitors of mentioned enzyme and uterotonics for medicine, based on the calixa rene core.

Fig. 8 .
Fig. 8.The induced isometric contractions of longitudinal smooth muscles of rat uterus in the control and under the effect of thiacalix[4]arene c-1193 (10 μМ, the duration of the previous incubation -30 min): A -contractions, activated with high-potassium solution (80 mm); B -contractions, activated with oxytocin (0.1 iu); C -contractions, activated with acetylcholine (10 μМ); D -contractions, activated with cevimeline (100 μМ).The moment of washing muscle preparations with normal krebs solution is shown with asterisks.Typical mechanograms are presented