CALIX[4]ARENES AS MODULATORS OF ENERGY-DEPENDENT CA2+_ACCUMULATION AND FUNCTIONING OF THE ELECTRON TRANSPORT CHAIN IN SMOOTH MUSCLE MITOCHONDRIA.

The influence of supramolecular macrocyclic compounds calix[4]arenes (C-97, C-99, C-107) at a concentration of 100 nM in the process of energy-dependent Ca²âº-transport in isolated mitochondria of smooth muscle, as well as autofluorescence mitochondrial coenzyme NADH, FAD and hydrodynamic diameter of these organelles was investigated. Using Ca²âº-sensitive fluorescent dye Fluo-4 AM it was shown that the selected calix[4]arenes can suppress energy-dependent accumulation of Ca²âº by mitochondria. Accumulation of Ca²âº (80 jiM in the medium) accompanied by the growth of the fluorescent probe response from a conventional unit to a value of 1,57±0,04 (n=5). Calix[4]arenes C-97, C-99, C-107 falls fluorescent signal below the 0,88±0,08, 0,92±0,08 and 0,78±0,04 respectively. Thus, the selected calix[4]arenas lead to release of previously accumulated Ca2+ from mitochondria. Under the influence of C-97 and C-99 fluorescent signal from NADH reduced to -0,11±0,02 and -0,12±0,02, respectively, in relation to the reference value - -0,05±0,01 (n=5). Analysis of fluorescence response NADH and FAD in a suspension of isolated mitochondria suggests that the effects of test compounds on the functional activity of the electron transport chain is associated with the initial stimulation of its 1-th complex and subsequent inhibition of Ca²âº-dependent NAD- containing Krebs cycle dehydrogenases. Along with this, the use of photon correlation spectroscopy to assess changes in the volume of mitochondria (their hydrodynamic diameter) under the action of selected calix[4]arenes has shown that interference with the electron transport chain leads to changes in the osmotic balance between the matrix of the mitochondria and the external environment. The result is the growth of isolated organelles volume. In particular, the hydrodynamic diameter of mitochondria increased by 22±6 % and 34±8 % (n=5) in presence of C-97 or C-99. The conclusion was done about the advisability of further studies of the calyx[4]arenes effect on smooth muscle Ca²âº-homeostase and mitochondrial bioenergetics in order to find effective modifiers of their func- tional activity.

Calix[4]arene C-90 and its analogs activate ATPase of the myometrium myosin subfragment-1.

Numerous female reproductive abnormalities are consequences of disorders in uterus smooth muscle (myometrium) contractile function. In this work, we described activators of ATPase, which could be used for development of effective treatments for correcting this dysfunction. Myosin ATPase localized in the catalytic domain of myosin subfragment-1 transforms a chemical energy deposited in macroergic bonds of ATP into mechanical movement. It was shown that сalix[4]arene C-90 and its structural analogs functionalized at the upper rim of macrocycle with four or at least two N-phenylsulfonуltrifluoroacetamidine groups, are able to activate ATP hydrolysis catalyzed by myometrium myosin subfragment-1. It was shown with the method of computer modeling that N-phenylsulfonуltrifluoroacetamidine groups of calix[4]arene C-90 interact with responsible for binding, coordination and the hydrolysis of ATP amino acid residues of myosin subfragment-1. The results can be used for further research aimed at using calix[4]arene C-90 and its analogs as pharmacological compounds that can effectively normalize myometrium contractile hypofunction.

FURIN INHIBITORS BASED ON THE DERIVATIVES OF CALIX[4]ARENE CX3im.

The aim of this work was to study antifurin activity of some derivatives of calix[4]arenes modified on the upper rim of the macrocycle by positively charged or uncharged groups. It was found that calixarene CX3im derivatives containing positively charged N-methylimidazolium cycles were indeed able to inhibit furin (K(i) = 58.2 µM). The magnitude of the effects depended also on the hydrophobicity of the substituents located on the lower rim of the macrocycle. The findings indicated the possibility of creating furin inhibitors of new generation based on the calix[4]arene platform.

Calix[4]arene C-90 selectively inhibits Ca2+,Mg2+-ATPase of myometrium cell plasma membrane.

The supramolecular compound calix[4]arene C-90 (5,11,17,23-tetra(trifluoro)methyl(phenylsulfonylimino)-methylamino-25,26,27,28-tetrapropoxycalix[4]arene) is shown to efficiently inhibit the ATP hydrolase activity of Ca2+,Mg2+-ATPase in the myometrium cell plasma membrane fraction and also in a preparation of the purified enzyme solubilized from this subcellular fraction. The inhibition coefficient I0.5 values were 20.2 ± 0.5 and 58.5 ± 6.4 µM for the membrane fraction and the solubilized enzyme, respectively. The inhibitory effect of calix[4]arene C-90 was selective comparatively to other ATPases localized in the plasma membrane: calix[4]arene C-90 did not influence the activities of Na+,K+-ATPase and "basal" Mg2+-ATPase. The inhibitory effect of calix[4]arene C-90 on the Ca2+,Mg2+-ATPase activity was associated with the cooperative action of four trifluoromethylphenylsulfonylimine (sulfonylamidine) groups oriented similarly on the upper rim of the calix[4]arene macrocycle (the calix[4]arene "bowl"). The experimental findings seem to be of importance for studies, using calix[4]arene C-90, of membrane mechanisms of regulation of calcium homeostasis in smooth muscle cells and also for investigation of the participation of the plasma membrane Ca2+-pump in control of electro- and pharmacomechanical coupling in myocytes.

[Kinetics of inhibitory effect of calix[4]arene C-90 on activity of transporting plasma membrane Ca2+, Mg2+-ATPase of smooth muscle cells].

In experiments on the suspension of myometrium cell plasma membrane, processed by 0.1% digitonin, the inhibitory action of calix[4]arene C-90 (5,11,17,23-tetra(threeftor)methyl(phenilsulphonilimino)-methylamino-25,26,27,28-tetrapropoxy-calix[4]arene) on the activity of Ca2+,Mg2+-ATPase was investigated. The authors also examined the influence of calix[4]arene in different concentration on affinity of enzyme (Ca2,Mg2+-ATPase) for the ATP and ions of Mg and Ca, and its influence on cooperative effect and maximum velocity of ATP hydrolysis. It is shown that calix[4]arene does not influence the affinity of Ca2+,Mg2+-ATPase for the ATP, which means that these two compounds have different binding centers. Also calix[4]arene has no influence on affinity and cooperative effect of Ca ions, if it is used in concentration lower than 50 µM. Calix[4]arene slightly increases coefficient of Ca2+,Mg2+-ATPase activation by magnesium chloride. In all three cases, where ATP, Mg and Ca ions are used to test the impact of calix[4]arene, maximum velocity of ATP hydrolysis significantly decreases. All these results clarify that calix[4]arene implements its inhibitory action through mechanism of uncompetitive inhibition of Ca2+,Mg2+-ATPase activity.

[Kinetic properties of calixarene C-90 action on the myometrial plasma membrane Ca2+,Mg2+-Atpase activity and on the Ca2+ concentration in unexcited cells of the myometrium].

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 calix[4]arene C-107 is highly effective supramolecular inhibitor of the Na+,K(+)-ATPase of plasma membranes].

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.

[Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane 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.

[Application of calixarenes for DNA transfection in cells].

First results of non-ionic and poly-cationic calixarenes utilization for gene transfection are presented and analyzed in this survey. State of the art in the field of scientific searching for new non-viral vectors are shown in the general form. The prospects of supramolecular concept in design agents for transfection are demonstrated. Some relationships between calixarene architecture and calixarene ability to promote gene transfection are revealed, namely: formation of supramolecular self-assembled aggregates at water media facilitates hierarchical formation of complexes with DNA molecules. Latter particles will effectively transfect genes if they are nano-sized and positively-charged.

[The calixarene C-107 increases the affinity of the Na+,K(+)-ATPase in plasmatic membrane of smooth muscle cells to the ouabain].

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.

[Effect of calixarene C-107 on kinetic parameters of Na+, K(+)-ATPase in the plasma membrane of the uterus myocytes].

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.

[Spatial structure of the calixarene-aminophosphonic acids is important for their inhibition of the Na+,K(+)-ATPase activity in plasma membrane of smooth muscle cells].

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.

[Comparative study of in vitro and in vivo effect of ouabain and calixarene C107 on Na+,K(+)-ATPase activity in plasma membranes of rat hepatocytes].

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.

[Effect of calix[4]arenes on the activity of actomyosin ATPase and actomyosin subfragment-1 ATPase from the myometrium].

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.

[Calixarene-dependent hydrolysis of ATP. II. The catalytic properties of reaction stimulated by calixarene C-107].

In this paper we have investigated nonenzimatic hydrolysis of ATP stimulated by calixarene C-107. It has been shown the dependences of the kinetic characteristics from reagent concentration: the maximal value released Pi did not depend on ATP concentration and linearly increased with the growth of calixarene concentration. Besides the growth concentration of ATP or calixarene increased the maximum instantaneous velocity of the reaction and decreased characteristic time. It was identified that univalent cation of Na+, K+, Li+, choline+ and bivalent cation of Ca2+ or Mg2+ did not influence the reaction of ATP hydrolysis, in the presence of other bivalent cation the inhibition of the reaction occurred in line with the sequence: Cu2+ > Ba2+ > Pb2+ > Sr2+ > Ni2+ = Zn2+ > Mn2+ > > Co2+. The alkalization in the range of pH 6.0-8.0 stimulated the ATP hydrolysis. The magnitude of activation energy of the reaction was 50.7 +/- 8.9 kilojoules per mole. The specificity for nucleoside tri- and di-phosphates was not observed. Obtained data can be useful for designing the synthetic ATP-hydrolyzing catalysts and also for subsequent investigation of kinetics, energetics and mechanism of both enzymatic and nonenzymatic ATP hydrolysis reaction.

[Calixarene-dependent hydrolysis of ATP. I. Kinetics and complexation of the calixarene C-107 with nucleoside triphosphate].

It was shown that calix[4]arene bis-aminomethylphosphonic acid C-107 can hydrolyze ATP. The kinetic curve of the ATP hydrolysis induced by calixarene C-107 was nonhyperbolic and had a tendency to plateau (in the course of time) observing from 45-60 minutes of the incubation period when the reaction practically came to the end. The empirical kinetic characteristics of this reaction were calculated. The velocity of calixarene-dependent hydrolysis of ATP exceeds the velocity of spontaneous hydrolysis of ATP at least 14-15 times. The "Host-Guest" complexation of the calixarene C-107 with adenosinetriphosphate in acetonitrile/water (47/53 v/v) solution was investigated by the reversed-phase high performance liquid chromatography. The dissociation constants of the 1:1 "Host-Guest" complexes ofATP-Guest with the calixarene-Host when using two columns Zorbax CN and LiChrosorb RP 18 within 197-231 microM were determined from the capacity factor of the Guest and concentration of the calixarene-Host in the mobile phase. The electrostatic, ion-dipole, dipole-dipole C-H-pi and other weak interactions in the "Host-Guest" complexes were discussed. Obtained data can be a basis for designing the synthetical ATP-hydrolyzing catalysts and also for subsequent investigation of both enzymatic and nonenzymatic ATP hydrolysis reaction,processes of ATP-dependent Ca2+ -transporting in subcellular membrane structures.

[Comparative study of calixarene effect on Na+, K+ -ATPase activity in plasma membrane of contractile and mobile 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).

[Comparative study of calixarene effect on Mg2+ -dependent ATP-hydrolase enzymatic systems from smooth muscle cells of the uterus].

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.

[Calixarene C-91 stimulates Ca2+ accumulation in the myometrium mitochondria].

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.

[Effect of calixarene-phosphonic acid on Na+, K+-ATPase activity in plasma membranes of the smooth-muscle cells].

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.