the inhibitory influence of calix [ 4 ] arene of c90 on the activity of ca 2 + , mg 2 +-atpases in plasma membrane and sarcoplasmic reticulum in myometrium сells

Our study on the plasma membrane vesicles and myometrium cells treated with 0.1% digitonin showed
that inhibitory effect of calix[4]arene C-90 (5,11,17,23-tetra(trifluoro)methyl(phenylsulphonylimino)-methylamino-
25,26,27,28-tetrapropoxy-calix[4]arene) on the plasma membrane Ca2+,Mg2+-ATPase was more significant
than on the Ca2+,Mg2+-ATPase in sarcoplasmic reticulum (the inhibition coefficient I0.5 values were
20.2 ± 0.5 μM and 57.0 ± 1.4 μM for the plasma membrane Ca2+,Mg2+-ATPase and Ca2+,Mg2+-ATPase in
sarcoplasmic reticulum, respectively (n = 5)). Inhibition kinetics of calix[4]arene C-90 effect on the Ca2+,Mg2+-
ATPase activities in plasma membrane and sarcoplasmic reticulum were studied. This substance inhibited
both pumps as complete noncompetitive inhibitor. Calix[4]arene C-90 caused the increase of intracellular
Ca2+ concentration and decrease of hydrodynamic diameter in smooth muscle cells similar to the action of
uterotonic drug oxytocin.

C alciu m t ranspor ters, wh ich i nclude, for exa mple, the calcium pumps of plasma membrane (PM) and sarcoplasmic reticulum (SR), play an essential role in controlling Ca ion concentration in the smooth muscle (SM) cytoplasm [1][2][3][4][5][6] The PM Ca 2+ ,Mg 2+ -ATPase maintains low Ca ion concentration in the relaxed myocytes and reduces Ca 2+ concentration in myoplasm after muscle contraction and hence contributes to muscle relaxation [7,8].The SR Ca 2+ ,Mg 2+ -ATPase reduces intracellular Ca 2+ concentration owing to cation accumulation in reticular pool [6,9].Recent literature data have indicated that SR is one of the largest cellular calcium depots [10].
Disorder in contractile function of myometrium in women often causes various pathologies, namely poor uterine contraction strength, spontaneous abortion, pre-term birth, miscarriage, atony, hypo-or hypertonic uterus [11,12].Typically, these pathologies are caused by abnormalities in the functioning of membrane-mediated cation transport systems.Therefore, the search for compounds which capable modifying the contractile function of myometrium upon these pathological conditions is a current point of interest.
In this context it would be of interest to study calixarenes, macrocyclic compounds synthesized by cyclocondensation of para-substituted phenols and formaldehyde.These compounds exhibit antiviral, antibacterial, antitumor and antithrombotic properties and may serve as effective inhibitors or activators of enzymatic, receptor and transport membranebound proteins [13,14].It should be noted that the majority of calixarenes are low toxic [14,15], that along with their ability to bind to molecules of various ligands [16,17] makes these compounds highly promising agents in biotechnology and medicine.
In our previous studies we showed that calix [4]arene C-90 (the code name) at concentration of 100 µM inhibited significantly (by 75% compared to control) the PM Ca 2+ ,Mg 2+ -ATPase activity in uterus myocytes and practically did not affect the activities of Mg 2+ -independent Ca 2+ -dependent ATPase, Na + ,K + -ATPase and Mg 2+ -ATPase localized in the same membrane structure [18].Ca ion accumulation in myometrium mitochondria, sensitive to the action of CCCP protonophore, was practically resistant to the calixarene action.
The aim of our study was to compare the effect of calix [4]arene C-90 on the activities of the PM and SR Ca 2+ ,Mg 2+ -ATPases in myometrium myocytes and to study kinetics of this effect, as well as influen ce of calix [4]arene C-90 on the intracellular Ca 2+ concentration and hydrodynamic diameter of smooth muscle cells (SMC).

materials and methods
Synthesis and structure of calix [4] Preparative biochemistry.Fraction of uterine PM SMC was isolated from pig myometrium as previously described [20,21].
Protein concentration in the membrane frac tion was determined by Bradford assay with Coomassie G-250 [22].
Myocytes were isolated from the uterus of nonpregnant rats using collagenase and soybean trypsin inhibitor as described [23].
The study was performed 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) enzyme assays.Total ATPase activity was determined in fractions of myometrial PM at 37 °C in a standard medium (0.4 ml) containing (mM): 3 ATP, 3 MgCl 2 , 0.95 CaCl 2 , 25 NaCl, 125 KCl, 1 EGTA, 20 Hepes-tris-buffer (pH 7.4), 1 NaN 3 (mitochondrial ATPase inhibitor [24]), 1 ouabain (selective Na + ,K + -ATPase inhibitor [25,26]), 0.1 µM thapsigargin (selective inhibitor of Ca 2+ ,Mg 2+ -ATPase of endo(sarco)-plasmic reticulum [24]) and 0.1% digitonin (factor of PM perforation [27]).Concentration of free Ca 2+ was calculated using Maxchel software and found to be 1 µM upon the given physicalchemical and concentration conditions of incubation medium.When studying the effect of various Ca ion concentrations on the Ca 2+ ,Mg 2+ -ATPase activity, required cation concentrations were calculated using mentioned above software.The protein concentration in the sample was 20-30 µg.Incubation time was 5 min.The enzyme reaction was initiated by adding aliquots (50 µl) of the PM suspension to the incubation medium and terminated by adding 1 ml of "stop"-solution comprising of 1.5 M sodium ace tate, 3.7% formaldehyde, 14% ethanol, 5% TCA (pH 4.3 at 8 °C).The amount of the reaction product (P i ) was determined by the method of W. Rathbun et V. Betlach [28].
Plasma membrane Ca 2+ ,Mg 2+ -ATPase activity was calculated as the difference between the values of ATPase activity in the presence and absence of exogenous Ca ions in the incubation medium (in the Calix [4]arene C-90 presence of 1 mM EGTA, a specific chelator of Ca ions).
The specific Ca 2+ ,Mg 2+ -ATPase activity in the fraction of myometrium PM was found to be 3.63 ± 0.21 µM P i /mg protein per 1 h (M ± m; n = 7).
For the assessment of the ATPase activity in SR, cells (protein concentration in a sample of 20 µg) were permeabilized by digitonin (0.1%).Total ATPase activity was measured in a standard medium (0.4 ml) containing (mM): 3 ATP, 3 MgCl 2 , 0.95 CaCl 2 , 25 NaCl, 125 KCl, 1 EGTA, 20 Hepestris-buffer (pH 7.4), 1 NaN 3 , 1 ouabain.Ca 2+ ,Mg 2+ -ATPase activity in SR was calculated as the difference between the values of ATPase activity in the presence and absence of 0.1 µM thapsigargin in the incubation medium.
The Ca 2+ ,Mg 2+ -ATPase specific activity in rat myometrial SR was found to be 2.45 ± 0.14 µM P i / mg protein per 1 h (M ± m; n = 7).
In the experiments on the effect of various concentrations of calix [4]arene C-90 (1-100 µM) on Ca 2+ ,Mg 2+ -ATPase activity we used a standard incubation medium (as described above) to which aliquot of calix [4]arene solution at the corresponding concentration was added.Concentrated (20 mM) calix [4]arene C-90 solution in DMSO (diluted with water for further experiments) was used.
kinetics study.For the study of the effect of different concentrations of calix [4]arene C-90 on the enzyme activities, inhibition coefficients I 0.5 and Hill coefficients nH were calculated using linear Hill plots according to equation lg[(A 0 -A)/A] = -n H lg I 0.5 + +n H lg[I], where A 0 and A -specific enzyme activities in the absence of calix [4]arene C-90 ("zero point") and in the presence of calix [4]arene C-90 at a concentration [I] in the incubation medium.
The values of the activation constant for Ca 2+ and Mg 2+ (k Ca , k Mg ), the activation constant for ATP (k ATP ) and the corresponding Hill coefficient n H (n H ,Ca, n H,Mg , n H,ATP ) were calculated using linear Hill plots according to equation lg , where A 0 ("zero point") and A are specific enzyme activities at actual concentration of compound S (Ca 2+ , Mg 2+ and ATP) in the incubation medium.
Dynamic light scattering.The characteristic size of myocytes (hydrodynamic diameter) was measured by DLS spectrometer "ZetaSizer-3" equipped with a computing correlator type 7032 (Malvern Instruments, UK).Helium-neon laser LGN-111 (P = 25 mW, λ = 633 nm) was used.The measu re range was from 1 nm to 20 µm.Registration and statistical processing of the autocorrelation function of light scattering in cell suspension was carried out for 1 min, 5 times at a scattering angle of 90 °.Autocorrelation function was processed using a standard software PCS-Size mode v1.61.
confocal microscopy.The SMC suspension (100 µl) was immobilized on a microscope slide treated with poly-L-lysine (200 µl) for 2 h at 25 °C.Unattached myocytes were washed with solution B ((mM): 136.9 NaCl, 5.36 KCl, 0.44 KH 2 PO 4 , 0.26 NaHCO 3 , 0.26 Na 2 HPO 4 , 0.03 CaCl 2 , 5.5 glucose), and then 100 µl of solution B was added to the immobilized cells.To determine the changes in [Ca 2+ ] i , myocytes were treated with probes Hoechst 33342 (specific to cell nucleus) and fluo-4 AM (specific to changes in the intracellular Ca 2+ concentration), 0.2% Pluronic was also added to the solution to facili tate the loading.Measurements were carried out in the Multi Track mode using laser scanning confocal microscope LSM 510 META (Carl Zeiss, Germany).
Cells images (time series) were captured for 5 min at 15-20 s each, fluorescence was induced by diode laser at 405 nm for Hoechst 33342, recorded using filter BP 420-480 nm, the fluorescence of fluo-4 AM was induced by an argon laser at 488 nm and recorded in the range of 505-530 nm (filter BP 505-530).For quantitative analysis, ROI (Region of Interest) function was used.It enabled to obtain plots of the fluorescence intensity dependence on time averaged in a selected area.
Spindle-shaped cells with well-defined nucleus stained by DNA-sensitive fluorescent probe Hoechst were selected for analysis.Changes in the cytoplasm Ca 2+ concentration were registered by a series of consecutive images during which 2 µl of 20 µM C-90 (sample) or 2 µl of 20 µM C-150 (control) were added.
Statistical analysis.Statistical processing of the obtained data was performed by standard methods using Student t-test.Kinetic and statistical calculations were performed using software MS Excel.
Slight difference in the calix [4]arene C-90 inhibitory effect on the PM and SR Ca 2+ ,Mg 2+ -ATPase activities may be caused by the different structure of Ca 2+ ,Mg 2+ -ATPase in PM and SR, namely the presen ce of the regulatory COOH-terminal tail in PM Ca 2+ ,Mg 2+ -ATPase [29].
To understand the kinetic mechanism of calix [4]arene C-90 inhibitory effect, it was examined whether the affinity of Ca 2+ ,Mg 2+ -ATPases in PM and SR for their substrates would change under the influence of inhibitor.
Increasing the ATP concentration in the incubation medium from 0.01 to 3 µM (Fig. 2, a and b, control) resulted in an increase in the Ca 2+ ,Mg 2+ -ATPase activities in PM (Fig. 2, a) and SR (Fig. 2, b) at fixed MgCl 2 concentration (3 mM) in the incubation medium.Apparent half-activation constant k ATP and Hill coefficient n H,ATP were calculated and found to be 56.3 ± 4.3 µM and 132.1 ± 5.1 µM in the case of Ca 2+ ,Mg 2+ -ATPase activity in PM and 1.32 ± 0.14 and 0.96 ± 0.01 in the case of Ca 2+ ,Mg 2+ -ATPase activity in SR (n = 5).
In further experiments, the effect of calix [4]arene C-90 on the enzyme affinity for ATP was studied.The influence of calix [4]arene C-90 (50 µM) on the concentration dependence of both ATPases activities on ATP was examined.In both cases of the C-90 action, the decrease in the Ca 2+ ,Mg 2+ -ATPases activities in PM and SR was observed, but the dependences of the enzyme activity on ATP were similar to the corresponding dependences in control (in the absence of calix [4]arene C-90), however a decrease in V max occurred (Fig. 2, a, b).
It was shown that calix [4]arene C-90 reduces the rate of ATP hydrolysis by both the PM and SR Ca 2+ ,Mg 2+ -ATPases that indicates the reducing of enzyme turnover number during its action.Apparent half-activation constant k ATP and the Hill coefficient n H,ATP were also calculated for both ATPases under the influence of calix [4]arene C-90 and found to be 39.5 ± 9.4 µM and 141.4 ± 8.6 µM for PM Ca 2+ ,Mg 2+ -ATPase and 1.5 ± 0.3 and 0.9 ± 0.1 for SR Ca 2+ ,Mg 2+ -ATPase (n = 5).The obtained results could be interpreted as an absence of C-90 effect on the indicated parameters.That is, the influence of calix [4]arene C-90 on Ca 2+ , Mg 2+ -ATPases in PM and SR is noncompetitive with respect to ATP.Thus, affinity of both ATPases for ATP almost does not depend on the presence of calix [4]arene C-90 in the incubation medium, indicating an absence of competition between the inhibitor and ATP.Therefore, it may be assumed that the substrate sites of Ca 2+ ,Mg 2+ -ATPases in PM and SR and hypothetical interaction site of calix [4]arene C-90 do not overlap on the enzyme surface.
In further experiments, the dependence of the specific activities of Ca 2+ ,Mg 2+ -ATPases in PM and SR on the Ca 2+ concentration in the incubation medium in the presence and absence of calix [4]arene C-90 was studied.The Ca ion concentration was calculated given the ATP and EGTA concentrations and their affinity for Ca 2+ (mentioned in the Materials and Methods).Ca 2+ ,Mg 2+ -ATPase activity in PM and SR of myometrium increased with increasing Ca ion concentration from 100 to 1000 nM (Fig. 3, a, b, control).Apparent activation constant k Ca and Hill coefficient n H,Ca were calculated using Hill method and found to be 190 ± 1 nM and 376 ± 11 nM for PM Ca 2+ ,Mg 2+ -ATPase activity and 2.1 ± 0.1 and 1.90 ± 0.06 for SR Ca 2+ ,Mg 2+ -ATPase activity (n = 5).
The influence of calix [4]arene C-90 (50 µM) on the affinity of Ca 2+ ,Mg 2+ -ATPases of PM and SR for Ca 2+ was also studied.Activity of both studied ATPases decreased, but the dependence of enzyme activity on the Ca 2+ concentration was similar to the corresponding control (without calix [4]arene C-90), though a decrease in V max in the presence of calix [4]arene C-90 was observed (Fig. 3, a the presence of calix [4]arene C-90 were 206 ± 3 nM and 394 ± 29 nM for Ca 2+ ,Mg 2+ -ATPase activity in PM and 1.97 ± 0.08 and 1.90 ± 0.15 for Ca 2+ ,Mg 2+ -ATPase activity in SR (n = 5).Thus, calix [4]arene C-90 (concentration in the incubation medium was 50 µM) had almost no effect on the affinity of Ca 2+ ,Mg 2+ -ATPases of PM and SR for Ca 2+ as well as on the cooperative effect of enzyme activation by this ion.
Mg 2+ plays an essential role in metabolism owing to its ability to modulate macromolecule structure, bind substrate and carry electrons.There are many Mg 2+ -dependent enzymes, where Mg 2+ is involved not only in substrate activation, but also in the formation of active (catalytic) sites.Though, the most significant and well known role of Mg 2+ is the formation of a chelate complex with ATP (substrate in adenosine triphosphate reaction).It is believed that Mg 2+ ions react with ATP phosphate charged groups, polarize them and increase the system's reac tivity, facilitating the nucleophilic attack the ATP terminal phosphate residue [30].
The obtained result showed that the enzyme activities of both Ca 2+ ,Mg 2+ -ATPases (PM and SR) increased with the increasing of MgCl 2 concentration from 0.1 to 3 mM at a fixed concentration of ATP (3 mM) in the incubation medium (Fig. 4, a, b,  control).
The values of the apparent activation constant k Mg of Ca 2+ ,Mg 2+ -ATPases in PM and SR Studying the effect of calix [4]arene C-90 on the enzyme affinity to Mg 2+ , its influence (concentration 50 µM) on dependence of the ATP hydrolase activity on the Mg ion concentration was evaluated.As in previous experiments, a decrease in the Ca 2+ ,Mg 2+ -ATPase activities in PM and SR (regarding control) was observed, however the charac ter of the dependen ce of enzyme activity on the Mg ion concentration remained the same, as for control in the absence of calix [4]arene C-90.That is, V max for the Ca 2+ ,Mg 2+ -ATPase activities in PM and SR in the presence of calix [4]arene C-90 decreased (Fig. 4, a, b).It was found that in the presence of calix The obtained data indicate that calix [4]arene C-90 inhibited the PM and SR pumps through complete noncompetitive mechanism.
Considering that PM and SR pumps play an essential role in control of the Ca ion concentration in the smooth muscle cytoplasm, it is important to find out whether calix [4]arene C-90 would affect intracellular Ca 2+ concentration in the SMC.Therefore our further experiments were performed using confocal microscopy.It was found that in the presence of C-90 (20 µM), a sharp increase in the fluorescent response of Ca 2+ -sensitive probe fluo-4 AM in cell occurred (Fig. 5), then a decrease in the probe quantum yield and restoring of the fluorescence intensity to the initial level were observed.In the control, where calix [4]arene "cup" C-150 (20 µM) was used (according to our previous result [18] calix [4]arene "cup" C-150 did not exhibit pronounced effect on the PM Ca 2+ ,Mg 2+ -ATPase activity), such an increase was not observed (the results have not been presented ).Fluorescence of Hoechst which was localized mainly in the SMC nucleus as well as background fluorescence also did not chang in the presence of calix [4]arene C-150.These results indicate that calix [4]arene C-90 (20 µM) causes a sharp increase in the intracellular Ca 2+ concentration, which is associated with a decrease in activity of Ca 2+ ,Mg 2+ -ATPase in PM and SR.However, during the next 100 s, the Ca 2+ concentration declined to initial level that may be a result of the involvement of myocytes compensatory Ca 2+ -transporting systems, which have a low affinity for Ca 2+ and react to its high intracellular concentration (the mitochondrial Ca 2+ uniporter, the PM Na + -Ca 2+ -exchanger).
Influence of calix [4]arenes on myocytes ion transport systems can determine their impact on the cell shape.Changes in the SMC shape owing to changes in water and osmotic balance can be detected by dynamic light scattering, which records alterations in the SMC effective hydrodynamic diameter.
According to the literature, factors that increase the contractile response of the SM also alter evidently the SMC effective hydrodynamic diameter.As shown in [31], introduction of Ca 2+ (3 mM), treatment with A-23187 (10 µM), tetraethylammonium (1 mM) and 4-aminopyridine (1 mM) decrease the effective hydrodynamic diameter that correlates with the state of SM contraction.According to our results, the introduction of uterotonic drug oxytocin (100 nM) led to a decrease in effective hydrodynamic diameter of the SMC by 23.3 ± 3.2% (M ± m, n = 6) relative to control (Fig. 6, blue bar).The average value of effective hydrodynamic diameter of myocytes (control) was 15183 ± 574 nm (n = 5).Hydrodynamic diameter was measured during 1 min 5 times and then the average value was calculated.Adding DMSO did not lead to significant changes in the studied parameter, namely hydrodynamic diameter altered by 3.5 ± 1.6% compared to control (Fig. 6, green bar).Whereas calix [4]arene C-90 (50 µM) led, similar to the effect of oxytocin (100 nM), to reduction of the hydrodynamic diameter by 27.8 ± 3.8% and 23.3 ± 2.6%, respectively, relative to control (Fig. 6 red and blue bars).
Changes in the SMC hydrodynamic diameter were confirmed by tensometric study of SM [32].In the myocytes suspension cells have a rounded shape, therefore changes in the hydrodynamic diame ter cannot be interpreted as a shortening/lengthening, but can be explained by the rearrangement of the cytoskeleton elements [33,35] that occurs during contraction, which in turn leads to a change in the SMC morphology.Furthermore, changes in the ion active transport may lead to a modification of the water-osmotic balance between cell and incubation medium that may also affect cell volume.
Thus, calix [4]arene C-90 reduces the SMC effective hydrodynamic diameter identically to the effect of uterotonic drug oxytocin.Such change in the hydrodynamic diameter could be interpreted as a combination of events that accompany the SMC contraction/relaxation processes, namely changes in osmotic-water balance, rearrangement of the cytoskele ton elements.Since it has been previously shown that a decrease in the SMC hydrodynamic diameter in the presence of contractile agents correlates with the state of SM contraction, these results suggest a promising use of calix [4]arene C-90 as a regulator of the contractile activity of the uterus SM.
Thus, the obtained experimental data allow us to conclude that calix [4]arene C-90 inhibits Ca 2+ ,Mg 2+ -ATPase activity in PM more significant than the same enzyme in SR.This compound inhibits the PM and SR pumps through complete noncompetitive mechanism.Calix [4]arene C-90 causes the increase in the intracellular Ca 2+ concentration and reduction of the SMC effective hydrodynamic diameter (similar to the effect of uterotonic drug oxytocin).
, the Declaration of Principles on Tolerance (UNESCO, 1995), Universal Declaration on Bioethics and Human Rights (UNESCO , 2005), the Convention for the Protection of Human Rights and Dignity of the Human Being with Regard to the Application of Biology and Medi-cine (Oviedo, Spain, 1997) and the Law of Ukraine On the Protection of Animals against Cruel Treatment approved by Verkhovna Rada of Ukraine in 2002.

Fig. 4 .
Fig. 4. Influence of calix[4]arene C-90 on the dependence of Ca 2+ ,Mg 2+ -ATPase activity in PM (a) and SR (b) of myometrium cells on the Mg ion concentration (n = 5) [4]-arene C-90 (50 µM), a slight increase in the value of activation coefficient k Mg to 1.05 ± 0.07 mM and 0.31 ± 0.02 mM for Ca 2+ ,Mg 2+ -ATPases in PM and SR respectively (M ± m; n = 5) occurred.Although the values of Hill coefficient of both enzymes (1.08 ± 0.06 and 1.00 ± 0.04 in the presence of C-90 for Ca 2+ ,Mg 2+ -ATPases in PM and SR, respectively (M ± m; n = 5)) remained almost unchanged.Thus, the affinity of both ATPases to Mg ions does not depend on the presence of calix[4]arene C-90 in the incubation medium, indicating an absence of competition between Mg ion and C-90.

Fig. 5 .
Fig. 5.The probe fluorescence intensity in the uterus myocytes has been recorded by confocal microscopy: DNA-sensitive Hoechst (1) and Ca 2+ -sensitive fluo-4 AM (2).The C-90 solution (final concentration 20 µM) was introduced at 160 s.The result of a typical experiment is presented

Fig. 6 .
Fig. 6.Alteration in the SMc hydrodynamic diameter caused by the influence of various effectors (n = 6).100% is the control value of the SMC hydrodynamic diameter in the absence of effectors