Tag Archives: smooth muscle cells
Calix[4]arene С-956 selectively inhibits plasma membrane Са(2+),Mg(2+)-АТРase in myometrial cells
Т. O. Veklich1, O. A. Skrabak1, Yu. V. Nikonishyna1, R. V. Rodik2, V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: veklich@biochem.kiev.ua; manli@ioch.kiev.ua
Using enzymatic assays and kinetic analysis, we demonstrated that 100 µM calix[4]arene C-956 (5,11,17,23-tetra(trifluoro)methyl-(phenylsulfonylimino) methylamino-25,27-dioctyloxy-26,28-dipropoxycalix[4]arene) had the most significant inhibitory effect on the plasma membrane Са2+,Mg2+-АТРase activity compared to effects of other calix[4]arenes, and had no effect on specific activities of other membrane ATPases. Using confocal microscopy and fluorescent probe fluo-4, we observed an increase of the intracellular level of Ca2+ after application of calix[4]arene C-956 to immobilized myocytes. Analysis of the effect of calix[4]arene C-956 on the hydrodynamic diameter of myocytes demonstrated that application of calix[4]arene C-956 solution decreased this parameter by 45.5 ± 9.4% compared to control value similarly to the action of uterotonic drug oxytocin.
The effect of calixarene C-107 on kinetic parameters of Nа(+),K(+)-АТРase of uterus myocyte plasma membrane
T. O. Veklich1, O. A. Shkrabak1, R. V. Rodik2,
V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv;
2Institute of Organic Chemistry, National Academy
of Sciences of Ukraine, Kyiv;
e-mail: kinet@biochem.kiev.ua; vik@bpci.kiev.ua
The inhibitory action of calixarene C-107 (5,17-diamino(2-pyridyl)methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxycalix[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+-АТРase 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.
The calixarene C-107 increases the affinity of the Na(+),K(+)-АТРase activity in plasmatic membrane of smooth muscle cells to the ouabain
T. O. Veklich1, A. A. Shkrabak1, R. V. Rodik2,
V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kinet@biochem.kiev.ua;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: vik@ioch.kiev.ua
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 сalixarene С-107 (5,17-diamino(2-pyridyl)methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxycalix[4]arene) on the Nа+,K+-АТРase 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 Nа+,K+-АТРase for сalixarene С-107.
Comparative investigation of the effect of calix[4]arene C-99 and its analogs on Nа(+),K(+)-ATPase activity of uterus myocite plasma membrane
T. O. Veklich1, A. A. Shkrabak1, S. O. Cherenok2,
V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kinet@biochem.kiev.ua; vik@ioch.kiev.ua
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 Nа+,K+-ATPase of uterus myocite plasma membrane. Therefore we studied the effect of calix[4]-arenes С-296, С-297, С-424, С-425, С-426, С-427, which are structurally similar to this inhibitor, on the mentioned enzyme activity. We have shown that calixarenes С-296 and С-297 which have two additional propoxy groups on the lower rim of macrocycle are less effective inhibitors of Nа+,K+-ATPase relative to calixarene C-99. Calixarenes С-425 and С-427 which have on the upper rim of macrocycle three and four phosponic residues, respectively, also inhibit Nа+,K+-ATPase activity less effectively as compared to calixarene C-99. Both calixarenes: С-424, which has only two carbonate residues on the upper rim, and С-426, which has on the upper rim ketomethilphosphonate residues instead of hydroxymethilphosphonate residues of calixarene C-99, do not affect Nа+,K+-ATPase activity. We have made respective conclusions concerning the role of certain chemical groups of calixarene C-99 during its interaction with Nа+,K+-ATPase.
The inhibitory influence of calix[4]arene of C-90 on the activity of Ca(2+),Mg(2+)-ATPases in plasma membrane and sarcoplasmic reticulum in myometrium сells
T. O. Veklich
Palladin Institute of Biochemistry,National Academy of Sciences of Ukraine, Kyiv;
e-mail: veklich@biochem.kiev.ua
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.
Kinetic regularities of calixarene C-90 action on the myometrial plasma membrane Ca(2+),Mg(2+)-ATPase activity and on the Ca(2+) concentration in unexcited сells of the myometrium
T. O. Veklich1, A. A. Shkrabak1, Yu. Yu. Mazur1, R. V. Rodik2,
V. I. Boyko2, V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kinet@biochem.kiev.ua; vik@ioch.kiev.ua
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 І0.5 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 tetraphenol 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 сalix[4]arene C-107 is highly effective supramolecular inhibitor of the Na+,K+-АТРase of plasmatic membrane
O. V. Bevza1, T. O. Veklich1, O. A. Shkrabak1, R. V. Rodik2, V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kinet@biochem.kiev.ua;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: vik@bpci.kiev.ua
The inhibition of the Na+,K+-АТРase activity of the myometrium cell plasma membranes with calixarene С-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 С-107 reduced the Na+,K+-АТРase activity more efficiently than ouabain did, while it did not practically influence the “basal” Mg2+-АТРase activity of the same membrane. The magnitude of the cofficient of inhibition I0.5 was 33 ± 4 nМ, 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 М-3 (4-hydroxyaniline(2-pyridine)methylphosphonic acid) – a fragment of the calixarene С-107, had practically no influence on the enzymatic activity of Na+,K+-АТРase and Mg2+-АТРаse. 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+-АТРase of plasma membrane and structure foundation of their intermolecular interaction was found out. The participation of hydrogen, hydrophobic, electrostatic and π-π (stacking) interaction between calixarene and enzyme aminoacid residues, some of which are located near the active center of Na+,K+-АТРase, was discussed.
Kinetics of inhibitory effect of calix[4]arene С-90 on activity of transporting plasma membrane Cа(2+),Mg(2+)-ATPase of smooth muscle cells
T. O. Veklich1, A. A. Shkrabak1, Yu. Yu. Mazur1,
R. V. Rodik2, V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kinet@biochem.kiev.ua; vik@ioch.kiev.ua
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.