Tag Archives: uterus

Influence of heavy metal ions on the ATPase activity of actomyosin complex and myosin subfragment-1 from smooth muscle of the uterus

R. D. Labyntseva, O. M. Bobrovska, O. Ju. Chunikhin, S. O. Kosterin

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: labyntseva@biochem.kiev.ua

The effect of divalent cations – Со2+, Сu2+, Mn2+ and Ni2+ (5 mМ) on the activity of actomyosin complex ATPase and ATPase of subfragment-1 (S1,head) of myosin from smooth muscle of the uterus was studied. It has been shown that Co2+, Mn2+ and Ni2+ inhibited, while Cu2+ activates the enzyme activity of both actomyosin and myosin S1.  Mg and Mn ions had practically no effect on the emission intensity of eosin Y associated with actomyosin, while one could observe the most marked suppression of emission of related fluorescent probe in the presence of Cu  cations and less pronounced suppression in the presence of Co2+.  In the presence of Mn, Co and Ni cations the average hydrodynamic diameter (HD) of actomyosin complex and  of subfragment-1 of the smooth muscle of the uterus is virtually identical to the HD in the presence of Mg2+. In the presence of Cu  cations there is a considerable (ten-fold) increase in the size of the protein particles that may be a result of their aggregation.  The results obtained evidence for the significant changes in the structure and function of the actomyosin complex of the myometrium in the presence of  heavy metals and allow us to assume that the target of the effect of these metals on the contractile proteins is a subfragment-1 of myosin, where the active site of ATPase and actin-binding sites are localized.

Cаlіx[4]аrene С-956 is effective inhibitor of Н(+)-Сa(2+)-exchanger in smooth muscle mitochondria

G. V. Danylovych1, О. V. Kоlomiets1, Yu. V. Danylovych1, R. V. Rodik2, V. I. Kаlchenko2, S. О. Kоsterin1

1Palladin Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua;
2Institute of Organic Chemistry, National Academy of Science of Ukraine, Kyiv

It was shown that calix[4]arene C-956 exhibited a pronounced concentration-dependent (10-100 μM) inhibitory effect on the H+-Ca2+-exchanger of the inner mitochondrial membrane of rat uterine myocytes (Ki 35.1 ± 7.9 μM). The inhibitory effect of calix[4]arene C-956 was accompanied by a decrease in the initial rate (V0) and an increase in the magnitude of the characteristic time (τ1/2) of the ΔрН-induced Са2+ release. At the same time, it did not affect the potential-dependent accumulation of Ca2+ in mitochondria. Thus, the action of calix[4]arene C-956 might be directed on increasing the concentration of Ca ions in the mitochondrial matrix. The calculation of basic kinetic parameters of the Ca2+ transport from isolated organelles (in the case of its non-zero stationary level), based on changes in fluorescence of Ca2+-sensitive dye Fluo-4 AM in mitochondria was performed. The proposed approach can be used for the kinetic analysis of the exponential decrease of the fluorescence response of any probes under the same experimental conditions.

Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na(+), K(+)-ATPase

G. V. Danylovych1, Yu. V. Danylovych1, O. V. Kolomiets1,
S. O. Kosterin1, R. V. Rodik2, S. O. Cherenok2, V. I. Kalchenko2,
A. Ju. Сhunikhin1, V. F. Gorchev1, S. A. Karakhim1

1Palladine Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv
2Institute of Organic Chemistry, National Academy of Science of Ukraine, Kyiv
e-mail: danylovych@biochem.kiev.ua;  vik@bpci.kiev.ua

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.

Structural and functional bases of the intermolecular interaction of calix[4]arene C-97 with myosin subfragment-1 of myometrium

R. D. Labyntseva1, A. A. Bevza1, О. V. Bevza1,
S. O. Cherenok2, 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

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 µM) efficiently inhibits ATPase activity of myosin subfragment-1 from pig myometrium, the inhibition coefficient І0.5 being 83 ± 7 µM. At the same time, this compound at 100 µM 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 π-π (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.

Protective effect of tiacalix[4]arene-tetrasulphonate on heavy metal inhibition of myometrium myosin subfragment-1 ATP-hydrolase activity

R. D. Labyntsevа1, O. V. Bevza1, A. A. Bevza1, A. M. Lulko1,
S. Kharchenko2, V. I. Kalchenko2, S. O. Kosterin1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
е-mail: labyntseva@biochem.kiev.ua;  kinet@biochem.kiev.ua;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
е-mail: vik@ioch.kiev.ua

Heavy metals have a negative effect on the contractility of uterine smooth muscles (myometrium), these effects can lead to various pathologies of a women reproductive system. To overcome these effects the methods for correcting the myometrium contractile activity are  to be developed. Catalyzed by myosin ATPase ATP hydrolysis is the most important reaction in the molecular mechanism of myo­metrium contraction. We have found an inhibitory effect of 0.03-0.3 mM Ni2+, Pb2+ and Cd2+ on enzymatic hydrolysis of ATP by myosin subfragment-1 obtained from swine uterine smooth muscles. We have demonstrated that 100 µM thiacalix[4]arene-tetrasulphonate (C-798) recovered to the control level of ATPase activity of myosin subfragment-1 in the presence of heavy metal cations. One of the most probable mechanisms of C-798 corrective activity is based on its ability to chelate heavy metals, thus cations Pb, Cd and Ni can be removed from the incubation medium. Computer simulation has demonstrated that the protective effect of C-798 may also be the result of weakening the interaction of heavy metal ions with amino acid residues of the myosin molecule near the active site of ATP hydrolase. The obtained results can be used for further research aimed at assessing the prospects of thiacalix[4]arene-tetrasulfonate as pharmacological compounds.