Selective inhibition of smooth muscle plasma membrane transport Са2+,Mg2+-АТРase by calixarene C-90 and its activation by IPT-35 compound

We investigated the influence of calixarene C-90 and IPT-35 on plasma membrane Ca2+pumping АТРase (PMCA), intracellular calcium homeostasis and myometrium smooth muscle strain contractions. It has been shown that both effectors (100 μM) affect PMCA enzymatic activity: calixarene C-90 inhibits it by 75% and IPT-35 activates it by 40%. These compounds don’t affect the Mg2+-АТРase, Mg2+-independent Са2+-АТРase and Na+,K+-АТРase enzymatic activities. C-90 inhibition coefficient I0.5 magnitude was approximately 20 μM and the Hill coefficient nH was 0.55. For IPT-35 activation, constant А0.5 was 6.4 and nH was 0.7. Mathematical modeling demonstrated the implication of calixarene C-90 on unexcited myocytes, which allows for a precise change in cytoplasm Ca2+ concentration and an influence on basal muscle tonus. By the same method, we determined that IPT-35 has a little influence on Ca2+ concentration in unexcited myocytes. It was also shown that calixarene C-90 in vitro can increase velocity of oxytocin-initiated contractions, whereas IPT-35 can suppress this aforementioned parameter. These results are promising for the design of new pharmacological compounds as better regulators of uterine contractions. Calixarene C-90 can be used in obstetric cases for the simultaneous use of oxytocin for enhancing uterine contractions, and IPT-35 for its antispasmodic effect on uterine tone.


Introduction
Smooth muscles (SM) promote functioning of internal organs and their systems (Barany 1996;Shmidt and Tevsa 2005;Guibert et al. 2011). SM activity depends on one of the key events in cell life -changing of intracellular calcium ion concentration (Aguilar and Mitchell 2010;Iino 2010;Santo-Domingo and Demaurex 2010). The uterus is the largest SM organ, as it performs special functions in gestation and labor. Uterine contractility can be changed by influence of environmental factors (sex and hypophysis hormones, mediators, Correspondence to: Oleksandr Shkrabak, Palladin Institute of Biochemistry of NAS of Ukraine, Leontovicha str. 9, Kyiv 01601, Ukraine E-mail: sashashkrabak32@gmail.com ions, mechanical stretching, and others) (Buxton 2004(Buxton , 2007Aguilar 2010). Investigation of cellular mechanisms involved in the control of myometrium contractility is important for a better understanding of obstetric pathology; for example, this would include premature and complicated labor, as well as post-labor bleeding etc, and for improving of treatment methods for these pathologies.
Spontaneous action potentials that spread on entire myometrium provoke periodical synchronous intracellular Ca 2+ concentration elevations, which in turn control myometrium contractility and tone. Ca 2+ concentration changes induce contraction/relaxation in response to some agents. The Mg 2+ -АТР-dependent plasma membrane (PM) calcium pump (transporting Са 2+ ,Mg 2+ -АТРase or PMCA, the Ca 2+ activation constant К Са ≈ 0.3-0.4 µM) participate in intracellular Ca 2+ concentration regulation, which influences myometrium contractions. It is important to thoroughly investigate peculiarities of PMCA activity regulation by compounds that can selectively change them. Moreover, direct changes in PMCA activity can elucidate the role of this protein in intracellular Ca 2+ concentration regulation in normal and pathological conditions. It will likely be possible to correct myometrium activity dysfunctions by targeting changes in PMCA activity.
Nowadays there are no well-known low molecular weight effectors which selectively regulate PMCA activity. In our previous work, we found that two compounds -calixarene C-90 was the selective inhibitor of PMCA activity , while another compound, IPT-35, activated the Ca 2+ -transport activity of Са 2+ , Mg 2+ -АТРase of PM. Therefore, in this work, we will continue to investigate calixarene C-90 and IPT-35 effects on SM cell PMCA and then attempt to compare them. The above compounds comprise a promising platform for the design of selective modifiers, which can be used for targeting PMCA activity regulation, and pharmacological drugs in medical practice (Consoli et al. 2006;Demchenko et al. 2009;Kononevych et al. 2011).
Cyclopantane [d]pirimidine derivate IPT-35 was synthesized in Department of Synthesis of Physiologically Active Compounds (in GA "Institute of Pharmacology and Toxicology of NAMS of Ukraine"). Its structure is also shown at Fig. 1 (Demchenko et al. 2009;Kononevych et al. 2011).

Physiological methods
Physiological investigations were performed in Department of Cardiovascular Agent Pharmacology (in GA "Institute of pharmacology and toxicology of NAMS of Ukraine").
Longitudinal muscle strips were cut out from uterine horns after rat decapitation. Animals were narcotized by diethyl ether regarding to International guiding principles for biomedical research involving animals (1985 year).
Obtained strips were fixed in flow chamber on two steel hooks with load of 10 mN. Chamber volume was 0.5 ml and it was perfused by Ringer's solution (velocity 1.7 ml per min). Temperature was constant: 37°С.
Contraction power was measured in isometric regime by capacitive strain sensor (FTK-0.1; PLC «Miosensor», Russian Federation). Contraction registration was on PC using programs DataTrax2 and LabScribe2,.
Contraction records of longitudinal muscle strips from isolated rat uterine horns were performed after stabilization period -about 40 min in flow chamber. Input data were recorded for 10 min after obtaining stable phase contractions of isolated SM preparations. Then investigated compounds (100 µM) were added for 15 min, whereafter oxytocin was added (0.1 IU/ml). Normalized maximal oxytocin-initiated contraction velocity was determined on contraction records by the method described in (Burdyga and Kosterin 1991). Group with intact myometrium was used as a control.

Biochemical investigations
Biochemical investigations were carried out in Department of Muscle Biochemistry in Palladin Institute of Biochemistry of NAS of Ukraine.

Preparative chemistry
Experiments were carried out in accordance with the European Guidelines and International Laws and Policies (86/609/EEC). All procedures conformed to the guidelines of the Palladin Institute of Biochemistry. Before starting the experiments, the protocols were approved by the Animal Care and Use Committee of the Palladin Institute of Biochemistry (Protocol No.1 from 21/04/2014).
Uterus smooth muscle cell PM fraction was isolated from swine myometrium as was previously described (Kondratuk et al. 1986;Veklich and Kosterin 2005). Protein concentration in membrane fraction was determined by method (Bradford 1976) using reaction with Coomassie dye G250.
Са 2+ ,Mg 2+ -АТРase activity was estimated as a difference between ATPase activity in case of Ca 2+ presence and absence in the incubation medium.
Nа + ,К + -АТРase activity was determined in the same medium as a difference between ATPase activity in presence and absence of 1 mM ouabain.
In order to study the influence of calixarene C-90 and IPT-35 in different concentrations on ATPase activities of PM fraction we used incubation mediums described above. Solutions (20 mM) of calixarene and IPT-35 in DMSO were diluted by water to obtain required concentration of agents.

Mathematical modeling of intracellular calcium concentration in unexcited myocytes
For this purpose program ("Groovy") was composed, which made calculations with presented precision.

Kinetic analysis
Inhibition coefficient І 0.5 and Hill coefficient n H were calculated from kinetic curves of calixarene concentration depend-ency influence on enzyme activity. For this purpose linearized Hill graphs were used according to equation: lg[(A о -A)/A] = -n H lgІ 0.5 + n H lg [С-90], where A о and A are specific enzyme activity in case of absence ("zero point") and presence of calixarene C-90 in incubation medium in concentration [C-90].
Likewise, activation coefficient А 0.5 and Hill coefficient n H were calculated from linearized Hill graphs according to equation: lg[(А max -А)/(А-А 0 )] = n H ·lgA 0.5 -n H ·lg , where A о and A are specific enzyme activities in case of absence ("zero point") and presence of IPT-35 in incubation medium in concentration , А max is maximal specific enzyme activity in case of maximal IPT-35 concentration (100 µM).

Statistical analysis
Obtained data were statistically analyzed with Student's t-criterion. All the experiments were repeated minimum five times (n = 5) and the results are presented as mean ± standard error. Kinetic and statistic calculations were made with MS Excel.

Results
In this work, we studied the effect of calixarene C-90 and IPT-35 on ATPase activities, and free Ca 2+ concentration changes in smooth muscle cells and myometrium contractions.

A B
relative to control. Both of these compounds do not influence other PM ATPase activities.
Calixarene C-90, compared to IPT-35, is a more efficient effector of PMCA enzyme activity when both compounds are examined in the same maximal concentration. Change of ATPase activity by C-90 action was 5-fold (inhibition), while for IPT-35, it was only 1.4 (activation) (Fig. 3).

Simulation of calixarene C-90 (selective PMCA inhibitor) affected equilibrium intracellular Ca 2+ concentration in unexcited myocytes
We have previously shown that calixarene C-90 causes a decrease in enzyme turnover, or the apparent maximal velocity <V max > of PMCA reaction in control (without calixarene in incubation medium), which was 6.7 µmol Р і /hour per mg of protein, then dropping off in the case of calixarene action. The PMCA apparent activation constant <K Сa > without calixarene in incubation medium was 190 ± 1 nM, with the Hill coefficient n H = 2.1. Application of calixarene C-90 in a concentration of 0.1-10 µM had no influence on PMCA's apparent activation constant by Ca 2+ <K Сa > and the Hill coefficient n H for Ca 2+ . Further increase of C-90 concentration (> 50 µM) caused significant changes in kinetic parameters: <K Сa > increased to 312 nM, and the Hill coefficient n Са decreased to 1.5. Thus, only a high calixarene concentration (50-100 µM) could induce reduction in PMCA affinity for Ca 2+ and decrease the positive cooperative effect of PMCA activation by Ca ions (Veklich et al. 2013). Considering all determined kinetic parameters of calixarene C-90 inhibitory action on PMCA enzyme activity, we have elaborated a quantitative permanent model of intracellular calcium concentration which changes in uterine myocytes in the case of effector application.  74.9 ± 0.5 -Activation (%) -40.9 ± 0.4 Data are presented as mean ± standard error (n = 5). І 0.5 , inhibition coefficient; А 0.5 , activation coefficient; n H , Hill coefficient.
It was shown (Veklich et al. 2013) that calixarene C-90' s influence on PMCA velocity can be described by the Hill equation: induce reduction in PMCA affinity for Ca 2+ and decrease the positive cooperative effect of PMCA activation by Ca ions (Veklich et al. 2013). Considering all determined kinetic parameters of calixarene C-90 inhibitory action on PMCA enzyme activity, we have elaborated a quantitative permanent model of intracellular calcium concentration which changes in uterine myocites in the case of effector application.
It was shown (Veklich et al. 2013) that calixarene C-90's influence on PMCA velocity can be described by the Hill equation: where <V max > is apparent maximum velocity of Ca 2+ extrusion from the cell across the membrane; i Ca ] [ 2 is concentration of intracellular Са 2+ , < K Са > is apparent activation constant for Ca 2+ , n is Hill coefficient for Ca 2+ .
According to recent data, PMCA is responsible for maintaining Ca 2+ homeostasis and preventing its increase. It counteracts input of Ca 2+ flow, which is caused by the Ca 2+ transmembrane gradient between cytoplasm and an outside medium (Strehler et al. 2007;Oloizia and Paul 2008). Taking into account that the Hill coefficient n Са of PMCA is approximately equal to 2 (Veklich et al. 2013), changing the cytoplasm Ca 2+ concentration in unexcited uterine myocites can be described by the following differential equation: Here, the first part of the right-hand side describes input flow of Ca where <V max > is apparent maximum velocity of Ca 2+ extrusion from the cell across the membrane, [Ca 2+ ] i is concentration of intracellular Са 2+ , <K Са > is apparent activation constant for Ca 2+ , n is Hill coefficient for Ca 2+ . According to recent data, PMCA is responsible for maintaining Ca 2+ homeostasis and preventing its increase. It counteracts input of Ca 2+ flow, which is caused by the Ca 2+ transmembrane gradient between cytoplasm and an outside medium (Strehler et al. 2007;Oloizia and Paul 2008). Taking into account that the Hill coefficient n Са of PMCA is approximately equal to 2 (Veklich et al. 2013), changing the cytoplasm Ca 2+ concentration in unexcited uterine myocytes can be described by the following differential equation: coefficient n Са decreased to 1.5. Thus, only a high calixarene concentration (50-100 µM) could induce reduction in PMCA affinity for Ca 2+ and decrease the positive cooperative effect of PMCA activation by Ca ions (Veklich et al. 2013). Considering all determined kinetic parameters of calixarene C-90 inhibitory action on PMCA enzyme activity, we have elaborated a quantitative permanent model of intracellular calcium concentration which changes in uterine myocites in the case of effector application.
It was shown (Veklich et al. 2013) that calixarene C-90's influence on PMCA velocity can be described by the Hill equation: where <V max > is apparent maximum velocity of Ca 2+ extrusion from the cell across the membrane; i Ca ] [ 2 is concentration of intracellular Са 2+ , < K Са > is apparent activation constant for Ca 2+ , n is Hill coefficient for Ca 2+ .
According to recent data, PMCA is responsible for maintaining Ca 2+ homeostasis and preventing its increase. It counteracts input of Ca 2+ flow, which is caused by the Ca 2+ transmembrane gradient between cytoplasm and an outside medium (Strehler et al. 2007;Oloizia and Paul 2008). Taking into account that the Hill coefficient n Са of PMCA is approximately equal to 2 (Veklich et al. 2013), changing the cytoplasm Ca 2+ concentration in unexcited uterine myocites can be described by the following differential equation: Here, the first part of the right-hand side describes input flow of Ca The fixed values of γ = 10 -3 sm -1 (Oloizia and Paul 2008), i Ca ] [ 2 = 10 -3 М (Kosterin 1990), K Са = 1.8×10 -7 М and І 0.5 = 2×10 -5 М were used, while V max was simulated by a model, with an initial baseline of 4.6×10 -6 М/min (Kosterin 1990). As a result, according to Eqs. 3 and 4, dependence of basal equilibrium Ca 2+ concentration in unexcited uterus cells on calixarene C-90 (selective PMCA inhibitor) was obtained (Fig. 4A).
From this result, we may assume that the action of calixarene C-90 on [Ca 2+ ] і has two phases: C-90 concentration 1-25 µM provokes the slow increase of intracellular Ca 2+ concentration.
Application of this compound in a specific concentration allows us to precisely change the Ca 2+ . As such, Eq. 2 can be rewritten as: (particularly for Ca 2+ ) γ. The second part involves the velocity of PM 1.8×10 -7 М and І 0.5 = 2×10 -5 М were used, while V max was simulated baseline of 4.6×10 -6 М/min (Kosterin 1990). As a result, according basal equilibrium Ca 2+ concentration in unexcited uterus cells on cal inhibitor) was obtained (Fig. 4A).
From this result, we may assume that the action of calixarene phases: C-90 concentration 1-25 µM provokes the slow increase of Application of this compound in a specific concentration allows us t concentration and gently influence its basal muscle tonus. Applicati concentrations higher than 25 µM causes an exponential increase of which can yield SM contracture.

Simulation of IPT-35 (selective PMCA activator) effect on equilibriu concentration in unexcited myocites
As seen in our results, IPT-35 had no influence on the Ca 2+ activatio increased enzyme hydrolysis velocity maximum (data not shown); t of enzyme kinetics, IPT-35 was accepted as a non-competitive PMC Ca 2+ and IPT-35 interacts with PMCA independently, creating a tern velocity of ATP hydrolysis. Maximal apparent PMCA velocity V m following expression: (3) (particularly for Ca 2+ ) γ. The second part involves the velocity of PMCA Ca 2+ -transport activity. In the case of a permanent condition, in which Ca 2+  Given that C-90 caused the decrease of PMCA V max , it was assumed that in concentrations of 0.1-50 µM, calixarene acted as a full non-competitive inhibitor of PMCA. Using principles of enzyme kinetics, the following equation is obtained: The fixed values of γ = 10 -3 sm -1 (Oloizia and Paul 2008), i Ca ] [ 2 = 10 -3 М (Kosterin 1990), K Са = 1.8×10 -7 М and І 0.5 = 2×10 -5 М were used, while V max was simulated by a model, with an initial baseline of 4.6×10 -6 М/min (Kosterin 1990). As a result, according to Eqs. 3 and 4, dependence of basal equilibrium Ca 2+ concentration in unexcited uterus cells on calixarene C-90 (selective PMCA inhibitor) was obtained (Fig. 4A).
From this result, we may assume that the action of calixarene C-90 on [Ca 2+ ] і has two phases: C-90 concentration 1-25 µM provokes the slow increase of intracellular Ca 2+ concentration.
Application of this compound in a specific concentration allows us to precisely change the Ca 2+ concentration and gently influence its basal muscle tonus. Application of calixarene C-90 in concentrations higher than 25 µM causes an exponential increase of basal Ca 2+ concentration, which can yield SM contracture.

Simulation of IPT-35 (selective PMCA activator) effect on equilibrium intracellular Ca 2+ concentration in unexcited myocites
As seen in our results, IPT-35 had no influence on the Ca 2+ activation constant K Саwhile it increased enzyme hydrolysis velocity maximum (data not shown); therefore, according to principles of enzyme kinetics, IPT-35 was accepted as a non-competitive PMCA activator. This indicates that Ca 2+ and IPT-35 interacts with PMCA independently, creating a ternary complex and increasing the velocity of ATP hydrolysis. Maximal apparent PMCA velocity V max  change was described by the following expression: Solving Eq. 3 for [Ca 2+ ] i we obtain dependence of basal [Ca 2+ ] i in unexcited myocytes on constant of PM permeability γ, Ca 2+ concentration in extracellular medium [Ca 2+ ] e , the apparent activation constant for Ca 2+ <K Са > and apparent pump velocity <V max >. This equation has three roots: one has a negative value, while the others are coupled, with the value of positive one corresponding to the expected intracellular Ca 2+ concentration (≈100 nM).
Given that C-90 caused the decrease of PMCA V max , it was assumed that in concentrations of 0.1-50 µM, calixarene acted as a full non-competitive inhibitor of PMCA. Using principles of enzyme kinetics, the following equation is obtained: (particularly for Ca 2+ ) γ. The second part involves the velocity of PMCA Ca the case of a permanent condition, in which Ca 2+ concentration in the cell is As such, Eq. 2 can be rewritten as: Given that C-90 caused the decrease of PMCA V max , it was assumed of 0.1-50 µM, calixarene acted as a full non-competitive inhibitor of PMC enzyme kinetics, the following equation is obtained: The fixed values of γ = 10 -3 sm -1 (Oloizia and Paul 2008), i Ca ] [ 2 = 10 -3 М 1.8×10 -7 М and І 0.5 = 2×10 -5 М were used, while V max was simulated by a m baseline of 4.6×10 -6 М/min (Kosterin 1990). As a result, according to Eqs. basal equilibrium Ca 2+ concentration in unexcited uterus cells on calixarene inhibitor) was obtained (Fig. 4A).
From this result, we may assume that the action of calixarene C-90 o phases: C-90 concentration 1-25 µM provokes the slow increase of intracel Application of this compound in a specific concentration allows us to precis concentration and gently influence its basal muscle tonus. Application of ca concentrations higher than 25 µM causes an exponential increase of basal C which can yield SM contracture.
From this result, we may assume that the action of calixarene C-90 on [Ca 2+ ] і has two phases: C-90 concentration 1-25 µM provokes the slow increase of intracellular Ca 2+ concentration. Application of this compound in a specific concentration allows us to change precisely the Ca 2+ concen-

A B
tration and gently influence its basal muscle tonus. Application of calixarene C-90 in concentrations higher than 25 µM causes an exponential increase of basal Ca 2+ concentration, which can yield SM contracture.

Simulation of IPT-35 (selective PMCA activator) effect on equilibrium intracellular Ca 2+ concentration in unexcited myocytes
As seen in our results, IPT-35 had no influence on the Ca 2+ activation constant K Са -while it increased enzyme maximum hydrolysis velocity (data not shown); therefore, according to principles of enzyme kinetics, IPT-35 was accepted as a non-competitive PMCA activator. This indicates that Ca 2+ and IPT-35 interacts with PMCA independently, creating a ternary complex and increasing the velocity of ATP hydrolysis. Maximal apparent PMCA velocity <V max > change was described by the following expression: where K A is the activation constant (А 0.5 = 6.4 µM, Table 1), and β (β = 1.31) is the ratio of k 2 /k 2 ' in the Botts and Morales mechanism.
Likewise, using Eq. 3 in the case of the calixarene C-90 model and Eq. 5, we obtained the dependence of Ca 2+ concentration in unexcited myometrium cells on IPT-35 concentration (Fig.   4B). According to this model, IPT-35 causes the decrease of Ca 2+ concentration by 35%, relative to its initial level, as it is the plateau level which is insensitive to the additional effector concentration increase.
Compared to calixarene C-90, an activator of PMCA -IPT-35 is a mild effector, and its application does not cause significant change of Ca 2+ concentration in myocites (Fig. 4B). The action of IPT-35 on [Ca 2+ ] і can be described as a sigmoid curve, with the most effective concentration being 0.3-3 µM.
These results confirm the antispasmodic effect of IPT-35 in smooth muscles in vitro and in vivo (Pupysheva 2013).

Discussion
PMCA is one of the high affinity Ca 2+ -extrusion systems of cells (Kosterin 1990). This enzyme is (5) where K A is the activation constant (А 0.5 = 6.4 µM, Table 1), and β (β = 1.31) is the ratio of k 2 /k 2 ' in the Botts and Morales mechanism. Likewise, using Eq. 3 in the case of the calixarene C-90 model and Eq. 5, we obtained the dependence of Ca 2+ concentration in unexcited myometrium cells on IPT-35 concentration (Fig. 4B). According to this model, IPT-35 causes the decrease of Ca 2+ concentration by 35%, relative to its initial level, as it is the plateau level which is insensitive to the additional effector concentration increase.
Compared to calixarene C-90, an activator of PMCA -IPT-35 is a mild effector, and its application does not cause significant change of Ca 2+ concentration in myocytes (Fig.  4B). The action of IPT-35 on [Ca 2+ ] і can be described as a sigmoid curve, with the most effective concentration being 0.3-3 µM.

Discussion
PMCA is one of the high affinity Ca 2+ -extrusion systems of cells (Kosterin 1990). This enzyme is part of the systems of calcium homeostasis, signal transduction, relaxation/ contraction and their regulation the case of smooth muscle cells. It is important to study the properties of PMCA to understand the specific physiological value of this enzyme, its regulation and interaction with other proteins. Today the most widely-used methods are genetic manipulations with the expression of PMCA. However, as PMCA content in PM is low, there is a methodological problem in obtaining high levels of its expression and the "correct" (physiologic) integration in PM (Adamo et al. 1992;Brini and Carafoli 2009). Due to a tight functional connection between different Ca 2+ -regulation systems, Ca 2+ -transporters, and Ca 2+sensors, they can also change their expression or activity in the case of additional PMCA transcript appearance (Liu et al. 1996). Thus, it is important to use alternative methods of PMCA investigation with agents that can selectively change their activity. It may also help avoid the compensatory effect connected with genetic manipulations.
SM cells should be regarded as a complex receptor tensoelectrochemical system, which is characterized with nonlinear, cooperative, non-additional, and synergistic properties of Ca 2+ homeostasis. Interpretation of the obtained experimental data on a mechanical level is too Figure 5. Application of calixarene C-90 (100 µM) and IPT-35 (100 µM) changes normalized velocity contractions V nc of rat myometrium in vitro initiated by oxytocin (0.1 IU/ml). Data are presented as mean ± standard error (n = 7). Mechanokinetical parameters were calculated by method (Burdyga and Kosterin 1991). * p < 0.05 compared to oxytocin. difficult in this context. However, some observations can be made.
It was established that two compounds with low molecular weight -calixarene C-90 and IPT-35 may selectively (on the PM level) modify PMCA enzyme activity -to inhibit and to activate, respectively (Fig. 2). Investigated compounds are opposite in their effect on PMCA activity, so we also expect that results of their actions on smooth muscle Ca 2+ concentration and contraction pattern can be inverse. Our obtained data confirm our assumption about the action of specific PMCA inhibitors and activators on SM cells.
Calixarene C-90 is a sufficient PMCA inhibitor (І 0.5 = 20.2 ± 0.5 µM, Fig. 3A) which must suppress Mg 2+ and ATP-dependent Ca 2+ transport across myometrium PM. Our results confirm this assumption according to the mathematical model (Fig. 4A) of how calixarene C-90 evokes an increase of Ca 2+ concentration in unexcited myocytes, and increased oxytocin-induced myometrium contractions in vitro (Fig. 5). Calixarene C-90 is assumed to be a prospective agent for application simultaneously with oxytocin to increase uterine contractions.
IPT-35 is a PMCA activator (А 0.5 = 6.40 ± 0.45 µM, Fig.  3B), which according to the mathematical model weakly influences Ca 2+ concentration in unexcited myocytes (Fig.  4B). Nevertheless, it suppresses oxytocin-induced myometrium contraction velocity in vitro (Fig. 5) indicating that IPT-35 may cause an antispasmodic effect on uterine contractions with the simultaneous use with oxytocin. Thus, IPT-35 is also perspective for a pharmacological application, because pathological conditions connected with improper uterine SM activity often require muscle relaxant application, and mild PMCA activation after influence of low effector concentration can curing muscle tone. The role of PMCA in smooth muscles is not limited to the control of muscle basal tonus by maintaining stable Ca 2+ concentration. PMCA participates in the regulation of SM contraction parameters of oxytocin response that are indirectly confirmed by our results (Fig. 5). This may be controlled by changing local calcium signaling waves or the pattern of calcium oscillation. Nevertheless, these assumptions must be confirmed by additional experimental data.
There are no selective inhibitors or activators of PMCA besides the so-called caloxins (Pande et al. 2011) that complicate the investigation of physiological functions of this enzyme. We can conclude that obtained results may be useful for further design of new selective effectors of PMCA -on the basis of calixarene C-90 and IPT-35 for investigation of PMCA's physiological functions and pharmacological regulation of uterine contractility in pathological conditions.