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Plasma membrane Ca2+,Mg2+-ATPase is an important element of general myometrium tonus control mechanism, which also makes a contribution to muscle tension relaxation after its contraction. Expiriments were done on the myometrial cell plasma membrane suspension, which was treated with 0.1% digitonin solution. The authors have investigated the inhibitory action of calix[4]arene C-90 (5,11,17,23-tetra(trifluor) methyl(phenylsulphonylimino)-methylamino-25,26,27,28-tetra propoxi-calix[4]arene) on the Ca2+,Mg2+-ATPase activity (the magnitude of 10.5 was 20.2 +/- 0.5 mkM). The inhibitory action of calix[4]arene C-90 on the activity of Ca2+, Mg2+-ATPase is explained as cooperative action of four trifluormethyl(phenylsulfonylimino)methylamino groups that are spatially oriented on the calix[4]-arene base rather than with the action of tetra-phenol macrocycle or separate pharmacophore sulphonilamidin groups. Considering established kinetic pattern of calix[4]arene C-90 inhibitory action on the plasma membrane Ca2+,Mg2+-ATPase activity, stationary kinetical model of basal calcium concentration control in unexcited uterus myocytes was developed. It is assumed that obtained results may be promising for creation of new generation ("supramolecular") pharmacological agent - uterus basal tonus stimulator - on the base of calix[4] arene C-90.

The inhibition of the Na+,K(+)-ATPase activity of the myometrium cell plasma membranes with calixarene C-107 (5,17-diamino(2-pyridyl) methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxycalix[4]arene) was investigated. It has been shown that calixarene C-107 reduced the Na+,K(+)-ATPase activity more efficiently than ouabain did, while it did not practically influence the "basal" Mg(2+)-ATPase activity of the same membrane. The magnitude of the cofficient of inhibition I0.5 was 33 +/- 4 nM, Hill coefficient was 0.38 +/- 0.06. The model calixarene C-150--the calixarene "scaffold" (26,28-dihydroxy-25,27-dipropoxycalix[4]arene), and the model compound M-3 (4-hydroxyaniline(2-pyridine)methylphosphonic acid)--a fragment of the calixarene C-107, had practically no influence on the enzymatic activity of Na+,K(+)-ATPase and Mg(2+)-ATPase. We carried out the computer simulation of interaction of calixarenes C-107 and the mentioned model compound with ligand binding sites of the Na+,K(+)-ATPase of plasma membrane and structure foundation of their intermolecular interaction was found out. The participation of hydrogen, hydrophobic, electrostatic and pi-pi (stacking) interaction between calixarene and enzyme aminoacid residues, some of which are located near the active center of Na+,K(+)-ATPase, was discussed.

The aim of our investigation was to determine structural features of calix[4]arene C-99 which are important for its inhibition properties relative to Na+,K(+)-ATPase of uterus myocite plasma membrane. Therefore we studied the effect of calix[4]arenes C-296, C-297, C-424, C-425, C-426, C-427, which are structurally similar to this inhibitor, on the mentioned enzyme activity. We have shown that calixarenes C-296 and C-297 which have two additional propoxy groups on the lower rim of macrocycle are less effective inhibitors of Na+,K(+)-ATPase relative to calixarene C-99. Calixarenes C-425 and C-427 which have on the upper rim of macrocycle three and four phosponic residues, respectively, also inhibit Na+,K(+)-ATPase activity less effectively as compared to calixarene C-99. Both calixarenes: C-424, which has only two carbonate residues on the upper rim, and C-426, which has on the upper rim ketomethilphosphonate residues instead of hydroxymethilphosphonate residues of calixarene C-99, do not affect Na+,K(+)-ATPase activity. We have made respective conclusions concerning the role of certain chemical groups of calixarene C-99 during its interaction with Na+,K(+)-ATPase.

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.

Calix[4]arene C-97 (code is shown) is the macrocyclic compound which has lipophilic intramolecular higly-structured cavity formed by four aromatic cycles, one of which on the upper rim is modified by methylene bisphosphonic group. It was shown that calix[4]arene C-97 (100 microM) efficiently inhibits ATPase activity of myosin subfragment-1 from pig myometrium, the inhibition coefficient I(0.5) being 83 +/- 7 microM. At the same time, this compound at 100 microM concentration significantly increases the effective hydrodynamic diameter of myosin subfragment-1, that may be indicative of intermolecular complexation between the calix[4]arene and myosin head. Computer simulation methods (docking, molecular dynamics, involving the Grid) have been used to clarify structural basis of the intermolecular interaction of calix[4]arene C-97 with myosin subfragment-1 of the myometrium; participation of hydrophobic, electrostatic and pi-pi (stacking) interactions between calix[4]arene C-97 and amino acid residues of myosin subfragment-1, some of them being located near the active site of the ATPase has been found out.

The inhibitory action of calixarene C-107 (5,17-diamino(2-pyridyl)methylphosphono- 11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxy-calix[4]arene) on Na+, K(+)-ATPase activity kinetic properties of myometrium perforated plasma membrane was investigated. It has been shown that the calixarene C-107 inhibiting Na+, K(+)-ATPase does not change the kinetic parameters (Km, nH) of reaction velocity dependence on substrate concentration. The constant Ka of enzyme activation by MgCl2 has complex dependence on calixarene C-107 concentration: it increases twice with growth of calixarene concentration up to 50 nM and decreases to the control level with further growth of calixarene concentration. The Hill cooperativity coefficient nH of activation by MgCl2 does not vary in the presence of calixarene C-107. Both ATP and MgCl2 have no influence on Na+, K(+)-ATPase constant of inhibition by calixarene C-107, but an increase of concentration of the mentioned physiological compounds causes the growth of cooperativity coefficient nH of enzymatic reaction inhibition by calixaren C-107.

In the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution we investigated the influence of calixarene C-107 (5,17-diamino(2-pyridyl)methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxyca-lix[4]arene) on the Na+,K(+)-ATPase activity. It was shown that this calixarene increased the affinity of the enzyme for the sodium pump conventional inhibitor - ouabain: the magnitudes of the seeming constant of inhibition I0.5 changed from 26.9 +/- 1.3 mM to 10.9 +/- 0.6 mM. However the ouabain itself did not influence on the affinity of the Na+,K(+)-ATPase for calixarene C-107.

It has been shown that calix[4]arene C-99 inhibited myosin subfragment-1 ATPase of myometrium. This inhibition is noncompetitive as to ATP and Mg2+. At the same time, this compound reduces the seeming enzymatic hydrolysis maximum rate of nucleoside triphosphate with respect to ATP and Mg2+. With the help of computer design the interaction of mentioned calix[4]arene with myosin subfragment-1 of myometrium has been investigated. Several mechanisms involved in the calix[4]arene C-99 inhibition of myosin head ATPase were supposed and participation of hydrogen, hydrophobic and electrostatic interactions in these mechanisms was discussed.

The comparative study of influence of ouabain and calixarene C107, and the structure component of this calixarene--fragment M3, in the conditions of in vitro and chronic action in vivo on Na+, K(+)-ATPase activity was carried out on the fractions of plasmatic membranes (PM) of the rat hepatocytes. A general property in the conditions in vitro is the ability of calixarene C107 and ouabain (both substances were in the concentration of 1 mM) to inhibit PM Na+, K(+)-ATPase of rat hepatocytes. However, in the case of activities of calixarene C107 and ouabain in the conditions in vivo heterogeneous action on Mg2(+)-ATPase and Mg2+, Na+, K(+)-ATPase activities takes place: total activity in the conditions of injection of increased concentrations of ouabain remains without changes, but Mg2(+)-ATPase activity significantly grows; in analogous conditions under the action of calixarene C107 both these activities decrease twice in comparison with control. Both under the in vitro and in vivo conditions, M3 fragment (the structural component of C107) does not change the values of investigated enzymatic activities. The biochemical mechanisms of calixarene C107 action on Na+, K(+)-ATPase activity in PM of rat hepatocytes are discussed.

It was found that calixarene C-107 (5,17-diamino(2-pyridyl)methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxycalix[4]arene) could effectively reduce Na+,K(+)-ATPase activity of the myometrium cell plasmatic membranes (the value of the apparent constant of inhibition I0.5 was 33 +/- 4 nM) while it practically did not influence the "basal" Mg2(+)-ATPase activity of the same membrane. In comparative experiments, we have shown that the model calixarene C-150--the calixarene "scaffold" (26,28-dihydroxy-25,27-dipropoxycalix[4]arene), and the model compound M-3 (4-hydroxyaniline(2-pyridine)methylphosphonic acid)--a fragment of the calixarene C-107, had practically no influence on the enzymatic activities of Na+,K(+)-ATPase and Mg(2+)-ATPase over a wide range of concentrations. Hence, the influence of calixarene C-107 on Na+,K(+)-ATPase activity was caused by the joint action of two aminophosphonic substituents on the upper rim of the calixarene bowl. The isomer of calixarene C-107--calixarene C-160 (5,11-diamino(2-pyridyl)methylphosphono-17,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxycalix[4]arene) also did not influence the Na+,K(+)-ATPase and Mg(2+)-ATPase activities of plasmatic membrane of myometrium cells. We carried out molecular modeling of calixarenes C-107 and C-160 and showed differences in interatomic distance between aminophosphonic substituents of mentioned calixarenes. We came to the conclusion that spatial structure of calixarene C-107, namely localization of two aminophosphonic substituents in 5,17 position of the upper rim of this calixarene, is crucial for inhibition of Na+,K(+)-ATPase activity. Using laser correlation spectroscopy it was found that the 100 microM solution of calixarene C-107 and 2.5% DMSO had microparticles with size range from 100 nm to 10 microm. Plasma membrane vesicles had average hydrodynamic diameter 401 +/- 17 nm, but after interaction of these vesicles with calixarene C-107 we have registered the creation of some particles with sizes greater than 10 microm. Therefore membrane vesicles agglutinated to each other and/ or to calixarene microparticles.

We studied the effect of calix[4]arenes C-97, C-99 and C-107 (codes are shown) functionalized by: one fragment of methylene-bisphosphonic, two fragments of hydroxy-phosphonic and two fragments of amino(methyl)phosphonic acids, respectively, on the enzymatic activity of actomyosin ATPase and ATPase of subfragment-1 (head) of myosin from smooth muscle of the uterus. It has been shown that calixarene C-107 at a concentration of 100 microM activated enzymatic activity of actomyosin ATPase by 230 +/- 12% (the value of the apparent constant of activation A0.5 = 9.6 +/- 0.7 microM). At the same time, 100 microM calixarenes C-97 and C-99 inhibited the activity by 70 +/- 8% and 50 +/- 9%, respectively (the value of the apparent constants of inhibition being I0.5 = 84.0 +/- 2.0 and 98.8 +/- 1.3 microM). In the experiments carried out with the myosin subfragment-1 ATPase it was shown that 100 microM calixarene C-107 increased ATP hydrolysis more than twice (A0.5 = 25 +/- 4 microM) and 100 microM calixarene C-99 inhibited activity by 77 +/- 4% (I0.5 = 43 +/- 8 microM). Photon correlation spectroscopy has shown an increase of average hydrodynamic diameters (D(av)) of subfragment-1 in the presence of calixarene C-107. This correlates with an increase of calixarene concentration. In addition, in the presence of calixarene C-107 one could observe a time-dependent increase of D(av) in the smooth muscle myosin head. The data presented demonstrates that the calixarenes which we have studied, can influence uterus smooth muscle at the level of the contractile proteins, namely the ATPase of the catalytic domain of the myosin head.

The inhibition of alkaline phosphatases by calix[4]arenes functionalysed at the macrocyclic upper rim by one or two methylenebisphosphonic acid fragments has been investigated. It is established, that calix[4]arene bismethylenebisphosphonic acid displayed stronger inhibition of alkaline phosphatase from bovine intestine mucosa than calix[4]arene methylenebisphosphonic acid. At the same time, the both inhibitors showed almost similar levels of inhibitory activities in respect of bovine kidney alkaline phosphatase or E. coli alkaline phosphatase. The tested compounds were docked computationally to the active site of the E. coli alkaline phosphatase. On the basis of results obtained the possible binding modes of inhibitors were analysed.

Calixarenes, owing to the ability to form supramolecular complexes with biologically important molecules and ions, can influence a course of biochemical processes and, accordingly, be considered as perspective molecular platforms for creation of physiologically active compounds. The work purpose is to study calixarene C-91 influence on systems of active Ca ions transport which are localized in subcellular membrane structures (mitochondria, sarcoplasmic reticulum, plasma membrane) of myometrial cells. It has been shown, that calixarene C-91 addition to incubation medium led to an increase in Ca2+ accumulation level in mitochondria. The maximal stimulating effect was 173% and it was observed at 100 microM concentration. It is suggested, that calixarene C-91 can enter mitochondria with the subsequent precipitation of Ca ions in a matrix therefore calcium capacity increases, and as a consequence, higher Ca2+ accumulation in these structures is observed. In a wide range of concentration (1-100 microM) calixarene C-91 did not influence a level of Ca2+ accumulation in sarcoplasmic reticulum of myometrial cells. Titration of solubilized Ca2+, Mg2+-ATPase by calixarene C-91 (0,1-100 microM) did not cause changes in its activity. Thus, calixarene C-91 increases Ca2+ accumulation level in mitochondria, but practically does not influence calcium pumps activity of a plasma membrane and sarcoplasmic reticulum of myometrial cells.

The paper is devoted to comparative analysis of the influence of a new class of macrocyclic compounds - calixarens on enzymatic activity of two ATP-hydrolase systems localized in the plasmatic membrane of contractile (myocytes of the uterus) and mobile (spermatozoids) cells--Na+, K+ -ATPase and basal Mg2+ -ATPase. The experiments performed on plasmatic membrane suspensions of myometrium and spermatozoids treated with detergent the authors studied the influence of calixarens C-97, C-99, C-107 (identified by the codes), functionalized with fragments of alpha-hydroxyphosphonic, alpha-aminophosphonic and methylenbisphosphonic acids accordingly, on enzymatic activity. The results have shown that C-97 and C-107 calixarenphosphonic acids in 100 microM concentration (97-99%) inhibit Na+, K+ -ATPase activity in both cases almost completely. C-99 (100 microM) calixaren appeared to be less effective with regard of its influence on the enzymatic systems under study: in the case of plasmatic membranes of myometrium suspension the activity of Na+, K+ -ATPase was decreased by 84-88%, and in the case of spermatozoids suspension--just by 15-20% of the control. All the studied calixarens (for both objects) in the maximal concentration (100 microM) practically did not influence the activity of basal Mg2+ -ATP-ase. The calixarens inhibited the enzymatic activity of Na+, K+ -ATPase more effectively than ouabain: in the first case the value of apparent inhibition constant I(0,5) was 25-100 nM, and in the second case--20-100 microM. The inhibition influence of calixarens on Na+, K+ -ATPase activity is characterized by the phenomenon of negative cooperativity (Hill's coefficient nH <1); the influence of ouabain in the case of plasmatic membranes of myometrium suspension is also characterized by negative cooperativity (nH < 1), and in case of spermatozoids suspension--by positive cooperativity (nH >1). The above results show that the studied calixarens are effective inhibitors of Na+, K+ -ATPase plasmatic membrane of contractive and mobile cells (C-97, C-99, C-107 calixarens in case of myocytes of uterus, and C-97, C-107 calixarens in case of spermatozoids).

Investigation the influence of calyx[4]arenes C-90, C-91, C-97 and C-99 (codes are indicated) on the enzymatic activity of four functionally different Mg2+ -dependent ATPases from smooth muscle of the uterus: actomyosin ATPase, transporting Ca2+, Mg2+ -ATPase, ouabain-sensible Na+, K+ -ATPase and basal Mg2+ -ATPase. It was shown that calixarenes C-90 and C-91 in concentration 100 microM act multidirectionally on the functionally different Mg2+ -dependent ATP-hydrolase enzymatic systems. These compounds activate effectively the actomyosin ATPase (Ka = 52 +/- 11 microM [Ukrainian character: see text] 8 +/- 2 microM, accordingly), at the same time calixarene C-90 inhibited effectively activity of transporting Ca2+, Mg2+ -ATPase of plasmatic membranes (I(0,5) = 34.6 +/- 6.4 microM), but influence on membrane-bound Na+, K+ -ATPase and basal Mg2+ -ATPase. Calixarene C-91 reduce effectively basal Mg2+ -ATPase activity, insignificantly activating Na+, K+ -ATPase but has no influence on transporting Ca2+, Mg2+ -ATPase activity of plasmatic membranes. Calixarenes C-97 and C-99 (100 microM), which have similar structure, have monodirectional influence on activity of three functionally different Mg2+-dependent ATPases of the myometrium: actomyosin ATPase and two ATPases, that related to the ATP-hydrolases of P-type--Ca2+, Mg2+ -ATPase and Na+, K+ -ATPase of plasmatic membranes. Basal Mg2+ -ATPase is resistant to the action of these two connections. Results of comparative experiments that were obtained by catalytic titration of calixarenes C-97 and C-99 by actomyosin ATPase (I(0,5) = 88 +/- 9 and 86 +/- 8 microM accordingly) and Na+, K+ -ATPase from plasmatic membranes (I(0,5) = 33 +/- 4 and 98 +/- 8 nM accordingly) indicate to the considerably more sensitiveness of Na+, K+ -ATP-ase to these calixarenes than ATPase of contractile proteins. Thus, it is shown that calixarenes have influence on activity of a number of important enzymes, involved in functioning of the smooth muscle of the uterus and related to energy-supplies of the process of the muscle contracting and support of intracellular ionic homeostasis. The obtained results can be useful in further researches, directed at the use of calixarenes as pharmaceutical substance, able to normalize the contractile function of the uterus at some pregnancy pathologies in women's.

In the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution the authors investigated influence of the calix[4]arenes C-97 and C-107 (codes are shown) on ouabain effect on the Na+,K+-ATPase activity. It was shown that calixarenes in concentration 100 tiM inhibited by 97-98% the enzymatic Na+,K+-ATPase activity, while they did not practically influence on the basal Mg2+-ATPase activity, and suppressed much more effective than ouabain the sodium pump enzymatic activity: in the case of the action of the calixarenes the value of the apparent constant of inhibition I0.5 was < 0.1 microM while for ouabain it was 15-25 microM. The negative cooperative effect was typical of the inhibitory action of calixarenes, as well as ouabain: the value of Hills factor nH = 0.3-0.5 <1. The modelling compound M-3 (0.1 microM 4 microM)--a fragment of the calixarene C-107--did not practically influence the enzymatic activities as Na+,K+-ATPase and basal Mg2+-ATPase. Hence the influence of calixarene C-107 on the Na+, K+-ATPase activity is caused by cooperative action of two fragments M-3 and effect of calixarene bowl, rather than by simple action of the fragment M-3. Calixarenes C-97 and C-107, used in concentration corresponding to values of I0.5 (40 and 60 nM, accordingly), essentially stimulated inhibiting action of ouabain on the specific Na+, K+-ATPase activity in the memrane fraction. Under coaction of ouabain with calixarene C-97 or C-107 there was no additive effect of the action of these inhibitors on the Na+,K+-ATPase activity. Calixarene C-97 brought in the incubation medium in concentration of 10 nM not only led to inhibition of the Na+,K+-ATPase activity relative to control, but also simultaneously increased the affinity of the enzyme for the cardiac glycoside: the magnitudes of the apparent constant of inhibition I0.5 were 21.0 +/- 5.2 microM and 5.3 +/- 0.7 microM. It is concluded, that highly effective inhibitors of the Na+,K+-ATPase activity--calixarenes C-97 and C-107 can enhance the effect of the sodium pump conventional inhibitor--ouabain, increasing the affinity of the enzyme for the cardiac glycoside (on the example of calixarene C-97).

Effect of calix[4]arenes C-97, C-99, C-107, functionalized by fragments of alpha-hydroxy-phosphonic, alpha-aminophosphonic- and methylene-bisphosphonic acid on enzymatic activity of oubaine-sensitive Na+, K+-ATPase and oubaine-resistant basal Mg2+- ATPase (specific activity - 10.6 +/- 0.9 and 18.1 +/- 1.2 micromol Pi/h per 1 mg of protein, respectively; n = 7) was studied in experiments made on the suspension of myometrium cell plasma membranes treated by 0.1% solution of digitonin. It was found that calixarene-phosphonic acids in concentration of 100 microM inhibited enzymatic activity of Na+, K+-ATPase by 86-98% and did not practically affect activity of Mg2+-ATPase. These calixarenes were more efficient than oubaine in suppressing enzymatic activity of the sodium pump: in case of the effect of calixerenes the value of the appearence constant of inhibition I0.5 was < 0.1 microM. Calixarene-methylene-bisphosphonic acid (calixarene C-97; I0.5 =33 +/- 4 microM (n = 6) takes the most efficient inhibitory effect on Na+,K+-ATPase activity among the studied calixarenes. A phenomenon of negative cooperation: the Hill coefficient value etaH =0.1-0.5<1 is characteristic of both the inhibiting effect of calixarenes and oubaine. Reguliarities of calixarenes C-97 effect on enzymatic activity of Na+,K+-ATPase were studied. As it appeared its inhibiting effect cannot be caused by trivial factors - potentially possible binding of Mg ions by it and (or) this substance effect on Mg2+ interaction with ATP4- in the incubation medium. Calixerene C-97 does not also decrease the enzyme affinity for Mg ions or ATP. However this calixerenes decreases the affinity of Na+,K+-ATPase for Na ions (the value of activation constant K(Na+)) from 50 +/- 4 (control) to 76 +/- 6 microM in the control and under the effect of calixerene, respectively). A conclusion is made that calixerene C-97 is highly-efficient (with respect to oubaine) and selective (with respect to lack of its effect on basal Mg2+-ATPase) inhibitor of Na+,K+-ATPase of plasma membrane. In the practical aspect it may be used in concentration of 1-10 microM in biochemical membranology when testing and studying kinetic and catalytic properties of the sodium pump in case of such experimental model, as the plasma membrane fraction.