Tag Archives: smooth muscle

The heterogeneity of the Na(+),K(+)-ATPase ouabain sensitivity in microsomal membranes of rat colon smooth muscles

A. A. Kaplia

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

The dose dependence of the Na+,K+-АТРase ouabain inhibition in the rat colon smooth muscle permeabilized microsomes has been analyzed according to the model of two independent binding sites of inhibitor to determine the activity of separate molecular forms of the enzyme that differ by affinity for cardiac glycosides. The two-phase inhibition curve with moderate content of the high-affinity activity component was revealed. The apparent inhibition constant of the low-affinity component corresponds to the value for the rat kidney microsomal Na+,K+-АТРase (α1-isoform). The specific role of the α2- and α1- Na+,K+-АТРase catalytic subunit isoforms in colonic smooth muscle electromechanical coupling is considered.

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.

The biosynthesis of nitric oxide from L-arginine. Nitric oxide formation features and its functional role in mitochondria

G. V. Danylovych, T. V. Bohach, Yu. V. Danylovych

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

Modern data on biochemical patterns of nitric oxide biosynthesis in mammal cells from L-arginine in normoxic conditions is described.  The attention of the authors is given to the results of the recent years on the structure and regulation features isoforms of NO-synthase. The emphasis is put on the latest conception of the compartmentalization of certain isoforms of these enzymes in cells and on the possibility of the directed transport of nitric oxide in the vascular wall. The central place in the review is devoted to issues on the endogenous formation of NO in mitochondria and its potential physiological significance. Our own results on the identification of NO in mitochondria of the uterine smooth muscle, biochemical characteristics of this process and NO possible role in Са2+ transport regulation by organelles are presented and discussed.

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.

Nitric oxide as the regulator of intracellular homeostasis in the uterus myocytes

Yu. V. Danylovych

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

The published data on the mechanisms and regulation of active and passive Ca2+ transport in the myometrium have been analyzed. Particular attention is paid to the cGMP-dependent and independent pathways of action of nitric oxide or its derivatives on intracellular Ca2+ homeostasis of uterine smooth muscle and its contractile activi­ty. Information on the effect of nitric oxide on Ca2+-transport systems of other types of smooth muscles is provided in a comparative aspect. Based on own experimental results and literature data a scheme of NO action in the myometrium is suggested in which nitric oxide or its derivatives cause­ Ca2+-dependent polarization of the sarcolemma. In accordance with our results, this effect may be based on the increase of sarcolemma Ca2+ permeability under the influence of NO or its derivatives and the stimulation of at least the initial passive transport of the cation in the myocytes mediated by dihydropyridine-sensitive channels. Additional factors that contribute to the polarization of the membrane are the increase of protons transport from the muscle cells and stimulation of Na+, K+-ATPase. Acting on the sarcoplasmic reticulum, nitrosactive compounds activate the inclusion of calcium in this compartment and inhibit Ca2+-induced release of the cation. The latter effects are able to provide compensation for NO-induced Ca2+ increase in myocytes and supress the electro-mechanical coupling at Ca2+ release from the reticulum. NO-derivates also inhibit a key link in the smooth muscle contractile act – the formation of the Ca2+-calmodulin complex.

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.

Laureates of the Palladin Prize of the National Academy of Sciences of Ukraine (1991–1992)

V. M. Danilova, R. P. Vynogradova

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

The article presents biographical information and analysis of scientific activity of laureates of the Palladin Prize of NAS of Ukraine G. V. Donchenko (1991) and S. O. Kosterin (1992) who were awarded for the monographs “Biochemistry of ubiquinone” and “Calcium transport in smooth muscles”, respectively. These monographs summarized the results of the original researches and the data of the world scientific literature.

Ca(2+) accumulation study in isolated smooth muscle mitochondria using Fluo-4 AM

O. V. Kolomiets, Yu. V. Danylovych, G. V. Danylovych, S. O. Kosterin

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

The opportunity of Ca2+-sensitive fluorescent dye Fluo-4 AM and spectrofluorimetry method application for the study of energy-dependent Ca2+ accumulation in mitochondria from uterus smooth muscle is proved. It has been found that the presen­ce of mitochondrial preparation increases time-dependent fluorescent response considerably and this effect depends on Ca2+ concentration in the medium. Thus, in these conditions, deesterification active probe is formed which is sensitive to Ca2+. It is shown that the accumulation of calcium ions in mitochondria in the presence of Mg-ATP and succinate depends on exogenous Ca2+ concentration and is characterized by substrate saturating­. The apparent activation constant of Ca2+ accumulation is 53.9 ± 6.9 mM, which corresponds to the physiological concentration of the cation in the cell next to mitochondria. Transit addition of Ca2+-ionophore A23187 to the incubation medium caused a rapid release of ionized cation from mitochondria. When proton gradient on the inner mitochondrial membrane is dissipated by protonophore CCCP, in the case of suppressing the generation of the gradient by oligomycin and in the presence of ruthenium red that inhibits Ca2+ mitochondrial accumulation systems, Ca2+ entry is significantly reduced. The results indicate the prospects of using Fluo-4 AM to study the properties of the Ca2+ accumulation system in isolated mitochondria of the myometrium.

Calmodulin antagonists effect on Ca(2+) level in the mitochondria and cytoplasm of myometrium cells

S. G. Shlykov, L. G. Babich, M. E. Yevtushenko, S. O. Karakhim, S. O. Kosterin

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

It is known that Са2+-dependent regulation of this cation exchange in mitochondria is carried out with participation of calmodulin. We had shown in a previous work using two experimental models: isolated mitochondria and intact myometrium cells, that calmodulin antagonists reduce the level of mitochondrial membrane polarization. The aim of this work was to investigate the influence of calmodulin antagonists on the level of ionized Са in mitochondria and cytoplasm of uterine smooth muscle cells using spectrofluorometry and confocal microscopy. It was shown that myometrium mitochondria, in the presence of АТР and MgCl2 in the incubation medium, accumulate Са ions in the matrix. Incubation of mitochondria in the presence of СССР inhibited cation accumulation, but did not cease it. Calmodulin antagonist such as trifluoperazine (100 µМ) considerably increased the level of ionized Са in the mitochondrial matrix. Preliminary incubation of mitochondria with 100 µМ Са2+, before adding trifluoperazine to the incubation medium, partly prevented influence of the latter on the cation level in the matrix. Incubation of myometrium cells (primary culture) with another calmodulin antagonist calmidazolium (10 µМ) was accompanied by depolarization of mitochondrial membrane and an increase in the concentration of ionized Са in cytoplasm. Thus, using two models, namely, isolated mitochondria and intact myometrium cells, it has been shown that calmodulin antagonists cause depolarization of mitochondrial membranes and an increase of the ionized Са concentration in both the mitochondrial matrix and the cell cytoplasm.