Tag Archives: myometrium

Inhibition of Na(+),K(+)-ATPase and activation of myosin ATPase by calix[4]arene C-107 cause stimulation of isolated smooth muscle contractile activity

T. O. Veklich1, R. D. Labyntseva1, O. A. Shkrabak1, O. V. Tsymbalyuk2,
R. V. Rodik3, V. I. Kalchenko3, S. O. Kosterin1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Institute of High Technologies, Taras Shevchenko National University of Kyiv, Ukraine;
3Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: veklich@biochem.kiev.ua; otsymbal@bigmir.net; manli@ioch.kiev.ua

Received: 04 Jule 2019; Accepted: 29 November 2019

The discovery of compounds that might modify myometrial contractility is an important area of researches. In our previous experiments, we found that some representatives of macrocyclic compounds fami­ly – calix[4]arenes – can modify the enzymatic and transport activity of membrane-bound cation-transport ATP hydrolases. The aim of this work was to study and compare the effect of calix[4]arene C-107 on the enzymatic activities of Mg2+-dependent ATPases of the uterine smooth muscle, namely: ouabain-sensitive Na+,K+-ATPase, plasma membrane Ca2+-independent “basal” Mg2+-ATPase, ATPase of the actomyosin complex and myosin subfragment-1, with effect on the contractile activity of the myometrium. It was shown that calix[4]arene C-107 efficiently inhibited myometrium Na+,K+-ATPase (I50 = 54 ± 6 nM) selectively to other ATP-hydrolases of the plasma membrane and simultaneously activated the enzymatic activity of the myosin ATPase of smooth muscles (A50 = 9.6 ± 0.7 μM). Such reciprocal biochemical effects led to the stimulation of the smooth muscle contractile activity that was demonstrated by the tensometric method using different isolated smooth muscles. Calix[4]arene С-107 was shown to stimulate the increase of the tonic component of myometrium contractions induced by oxytocin, as well as contractions of the caecum muscles induced by high-potassium solution or acetylcholine, and to maintain increased tension for a long time. Thus, calix[4]arene C-107 is a prospective compound for enhancing the smooth muscle basal tone and/or contraction in case of hypotonic dysfunctions.

Сalix[4]arene chalcone amides effects on myometrium mitochondria

S. G. Shlykov1, A. M. Kushnarova-Vakal1, A. V. Sylenko1,
L. G. Babich1, О. Yu. Chunikhin1, O. A. Yesypenko2,
V. I. Kalchenko2, S. O. Kosterin1

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

Received: 19 November 2018; Accepted: 14 March 2019

Mitochondria are a key player in a wide range of the most important functions of the cell. Calixarenes are supramolecular compounds that have been widely used in bioorganic chemistry and biochemistry. The aim of this work was to study the effects of calix[4]arenes with two (С-1012, С-1021), three (С-1023, С-1024) and four (С-1011) chalcone amide groups on the myometrial mitochondria membranes polarization, Ca2+ concentration in the matrix of these organelles ([Ca2+]m ) and on the average hydrodynamic diameter of mitochondria. It was shown that permeabilized myometrium cells incubation with calix[4]arenes containing two or more chalcone amide groups, was accompanied by an increased level of myometrial mitochondria membranes polarization. All studied calix[4]arenes increased [Ca2+]m values in the absence and in the presence of exogenous Ca2+. The values of [Ca2+]m in the absence of exogenous Ca2+ were higher at mitochondria incubation in Mg2+-containing, than in Mg2+,ATP-containing medium. Incubation of isolated mitochondria with the studied calix[4]arenes resulted in changes of mitochondria volume: at incubation with С-1012, С-1021, C-1023 the average hydrodynamic diameter was decreased, while with С-1011 it was increased. Thus, we have shown that a short-term (5 min) incubation of mitochondria in the presence of 10 µM calix[4]arenes, which contain from two to four chalcone amide groups, increased the level of mitochondria membranes polarization, ionized Ca concentration in the matrix and had different effects on the mitochondrial volume.

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 relationship between the ionized Ca concentration and mitochondrial functions

L. G. Babich1, S. G. Shlykov1, A. M. Kushnarova-Vakal1, N. I. Kupynyak2, V. V. Manko2, V. P. Fomin3, S. O. Kosterin1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: babich@biochem.kiev.ua;
2Ivan Franko National University of Lviv, Ukraine;
3University of Delaware, Newark, USA

The aim of the study was to show the relationships between ionized Ca concentration ([Ca2+]m) in the mitochondria matrix and functional activity of this organelle. [Ca2+]m was determined using the fluorescent probe Fluo-4, AM. Total level of Ca2+ accumulation in mitochondria was monitored using 45Ca2+ as radioactive tracer. It was shown that incubation of myometrium mitochondria with 3 mM Mg2+ resulted in the low level of [Ca2+]m. Subsequent addition of 100 µM Ca2+ resulted in 8 times increase of [Ca2+]m but in low level of total calcium accumulation. Normalized fluorescence of Ca2+-sensitive probe Fluo-4 in response to the Ca2+ addition was higher than 2.5. At the same time, [Ca2+]m was considerably higher in the medium containing­ 3 mМ АТР and 3 mМ Mg2+. Subsequent addition of 100 µM Ca2+ to the incubation medium resulted in only 2.4 times increase of [Ca2+]m but considerably higher level of total calcium accumulation was observed. Normali­zed fluorescence of Fluo-4 in response to the Ca2+ addition was lower than 1.3. In liver mitochondria higher rate of oxygen consumption was detected in the presence of an oxidative substrate succinate than of pyruvate or α-ketoglutarate. At the presence of an oxidative substrate succinate normalized fluorescence of Fluo-4 in liver mitochondria in response to the Ca2+ addition was lower than 1.3. It was concluded that low level of [Ca2+]m was correlated with low functional activity of this organelle and, vise versa, high level of [Ca2+]m was correlated with high functional activity. It was suggested that normalized fluorescence changes in response to the Са2+ addition could be used as a test of the mitochondrial functional activity: lower normalized fluorescence values − higher functional activity.

Ca(2+)-dependent regulation of the Ca(2+) concentration in the myometrium mitochondria. II. Ca(2+) effects on mitochondria membranes polarization and [Ca(2+)](m)

L. G. Babich, S. G. ShlykoV, A. M. Kushnarova, S. O. Kosterin

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

It is known that Ca2+ accumulation in the mitochondria undergoes complex regulation by Ca2+ itself. But the mechanisms of such regulation are still discussed. In this paper we have shown that Ca ions directly or indirectly regulate the level of myometrium mitochondria  membranes polarization.  The additions of 100 µM Ca2+ were accompanied by depolarization of the mitochondria membranes. The following experiments were designed to study the impact of Ca2+ on the myometrium mitochondria [Ca2+]m. Isolated myometrium mitochondria were preincubated without or with 10 μM Са2+ followed by 100 μM Са2+ addition. Experiments were conducted in three mediums: without ATP and Mg2+ (0-medium), in the presence of 3 mM Mg2+ (Mg-medium) and 3 mM Mg2+ + 3 mM ATP (Mg,ATP-medium). It was shown that the effects of 10 μM Са2+ addition were different in different mediums, namely in 0- and Mg-medium the [Ca2+]m values increased, whereas in Mg,ATP-medium statistically reliable changes were not registered. Preincubation of mitochondria with 10 μM Са2+ did not affect the [Ca2+]m value after the addition of 100 μM Са2+. The [Ca2+]m values after 100 μM Са2+ addition were the same in 0- and Mg,ATP-mediums and somewhat lower in Mg-medium. Preliminary incubation of mitochondria with 10 μM Са2+ in 0- and Mg-mediums reduced changes of Fluo 4 normalized fluorescence values that were induced by 100 μM Са2+ additions, but in Mg,ATP-medium such differences were not recorded. It is concluded that Са2+ exchange in myometrium mitochondria is regulated by the concentration of  Ca ions as in the external medium, so in the matrix of mitochondria. The medium composition had a significant impact on the [Са2+]m values in the absence of exogenous cation. It is suggested that light increase of [Са2+]m before the addition of 100 μM Са2+ may have a positive effect on the functional activity of the mitochondria.

Ca(2+)-dependent regulation of the Ca(2+) concentration in the myometrium mitochondria. I. Trifluoperazine effects on mitochondria membranes polarization and [Ca(2+)](m)

L. G. Babich, S. G. Shlykov, A. M. Kushnarova, S. O. Kosterin

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

Са2+-dependent regulation of Ca2+ exchange in mitochondria is carried out with participation of calmodulin. We have shown previously that calmodulin antagonists reduced the level of mitochondrial membrane polarization and induced increase of the ionized Са concentration in both the mitochondrial matrix and cell cytoplasm. The concentration-dependent  influence of trifluoperazine on the level of polarization of mitochondrial membranes has been shown in this work. The coordinates of the Hill graphs were used to calculate the constant K0.5 and the Hill coefficient. K0.5 was 24.4 ± 5 μM (n = 10). The Hill coefficient was 2.0 ± 0.2, indicating the presence of two centers of the trifluoperazine binding. We have also studied [Ca2+]m changes, when incubating mitochondria in mediums of different composition: without ATP and ions of Mg (0-medium), in the presence of 3 mM Mg (Mg-medium) and 3 mM Mg + 3 mM ATP (Mg,ATP-medium). It was shown that the composition of the incubation medium affected the [Ca2+]m values in the absence of exogenous Ca2+ and did not affect them in the presence of the latter. Preincubation of mitochondria in mediums of different composition with 25 μM trifluoperazine did not affect the [Ca2+]m values both before and after the addition of  100 µМ Са2+ to the  incubation medium. It was concluded, that trifluoperazine depolarized myometrial mitochondria membranes in concentration-dependent manner. However, mitochondria preincubation with 25 μM trifluope­razine accompanied by 50% decrease in membrane polarization did not affect the [Ca2+]m values.

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.

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.