Analysis of decay kinetics of the cytosolic calcium transient induced by oxytocin in rat myometrium smooth muscle cells.

The method of kinetic analysis of the relaxation phase of the mechanical response of the smooth muscle previously proposed by Burdyga and Kosterin was applied to study the dynamics of the decay of oxytocin-induced calcium transients in cytosol of the rat myometrium smooth muscle cell detected by a fluorescence signal generated by a calcium-sensitive probe fluo-4 using a laser scanning confocal microscope. The experimental data were well linearized in the coordinates ln [(Fm - F)/F] vs lnt (F and Fm are the current fluorescence intensity of the calcium probe and the fluorescence intensity at the maximum of the calcium transient, respectively, while t is the time). The empirical parameters n and τ were determined by which the maximal normalized relaxation rate Vn was calculated for five different ROIs (regions of interest) in the myocyte cytosol. It proved to be almost the same for all ROIs. The maximal normalized relaxation rate calculated from the fluorescence intensity was always lower than that calculated from the corresponding calcium concentration, i.e. the cytosolic Ca2+ concentration in the relaxation phase decreases faster than the corresponding fluorescence intensity. The value of the maximal normalized relaxation rate calculated both from the fluorescence intensity and from the force of oxytocin-induced contractions of isolated rat uterus longitudinal smooth muscles (according to Tsymbalyuk and Kosterin) was exactly the same. This indicates that in the relaxation phase, the decreasing curves of both the fluorescence intensity and the contraction forces coincide.

Mitochondrial ryanodine-sensitive Ca2+ channels of rat liver.

To examine ryanodine-sensitive Ca2+ channels in mitochondria of rat hepatocytes and their role in energy state of the cells via investigation of the ryanodine effect on mitochondrial membrane potential. Oxygen consumption was measured by polarography using the Clark electrode. The substrates of oxidation such as pyruvate (5mM), α-ketoglutarate (5mM), or succinate (5mM) were used. Oxidative phosphorylation was stimulated by the addition of adenosine diphosphate (200nM). Mitochondrial membrane potential was measured using a voltage-sensitive fluorescent probe tetramethylrhodamine-methyl-ester (0.1µM) and was analyzed by a flow cytometer. To evaluate the intact mitochondria, we used carbonil cyanide m-chlorophenyl hydrazone (CCCP, 10µM). Changes in the ionized calcium concentration in rat liver mitochondria were measured using a fluorescent probe Fluo-4 AM. Effect of ryanodine on oxygen consumption of rat liver mitochondria depends on the oxidation substrate and the incubation time. Oxidation of pyruvate in the presence of ryanodine (0.05µM) decreased the membrane potential of rat liver mitochondria by 38.4%. At higher concentrations, ryanodine (0.1µM or 1µM) led to decrease of membrane potential by 51.7% and 42.8%, respectively. In contrast, oxidation of α-ketoglutarate in the presence of ryanodine (0.05µM) increased mitochondrial membrane potential by 16.8%. However, at higher concentrations, ryanodine (0.1µM or 1µM) triggered a decreasing of membrane potential by 42.5% and 31.0%, respectively. Therefore, ryanodine at various concentrations (0.05µM, 0.1µM, or 1µM) causes differential effects on Ca2+ concentration in the mitochondria matrix under oxidation of pyruvate or α-ketoglutarate. The data suggest the presence of ryanodine receptors in mitochondrial membrane of rat hepatocytes. Their inhibition with higher concentrations of ryanodine leads to decreasing of intra-mitochondrial Ca2+ concentration and affecting the energy state of mictochondria in hepatocytes.

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

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 µÐœ 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.

CALMODULIN ANTAGONISTS EFFECT ON Ca2+ LEVEL IN THE MITOCHONDRIA AND CYTOPLASM OF MYOMETRIUM CELLS.

It is known that Ca(2+)-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 Ca in mitochondria and cytoplasm of uterine smooth muscle cells using spectrofluorometry and confocal microscopy. It was shown that myometrium mitochondria, in the presence of ATP and MgCl2 in the incubation medium, accumulate Ca ions in the matrix. Incubation of mitochondria in the presence of CCCP inhibited cation accumulation, but did not cease it. Calmodulin antagonist such as trifluoperazine (100 µm) considerably increased the level of ionized Ca in the mitochondrial matrix. Preliminary incubation of mitochondria with 100 µM Ca2+, 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 µM was accompanied by depolarization of mitochondrial membrane and an increase in the concentration of ionized Ca 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 Ca concentration in both the mitochondrial matrix and the cell cytoplasm.

[Calix[4]arenes C-136 and C-137 hyperpolarize myometrium mitochondria membranes].

Calixarenes--supramolecular compounds interacting with bioactive molecules and ions that causes the changes in biochemical and biophysical processes. The aim of this work was to study the effects of calix[4]arenes C-136, C-137 and C-138 on the level of polarization of rat myometrium mitochondria membrane. Structure of synthesized calix[4]arene molecules was confirmed by the methods of 1H NMR and infra-red spectroscopy. Calix[4]arenes C-136 and C-137 possess two chalcone amide moieties at the lower rim, while the calix[4]arene C-138--only one. In case of calix[4]arenes C-136 and C-137 take place, accordingly, absence or presence of phenolic hydroxyl groups at the lower rim on the calix[4]arene skeleton. It was shown that calix[4]arenes C-136, C-137 and C-138 form micelles in a water medium and in the dimethylformamide (DMF). The irradiation of micelles with argon laser on flow cytometer results in appearance of autofluorescence. In the water medium calix[4]arene micelles interact with positively charged potential-sensitive fluorescent probe TMRM, that can testify to the presence of negative charge in these structures. However calix[4]arene micelles in DMF solution do not interact with TMRM. Mitochondrial membrane potential was measured using fluorescent dyes MTG and TMRM with confocal microscopy and fluorescent dye TMRM with flow cytometry. Experiments were conducted on myometrium cells in culture and on suspension of digitonin-permeabilized uterus myocytes. It was shown that a fluorescent signal was stable during time of experiment. Calix[4]arenes C-136 and C-137 (10 µM) hyperpolarize mitochondria membranes. A maximal effect was 173%. At the same time calix[4]arene C-138 did not influence on mitochondria membrane potential. Connection comes into question between structural organization of investigated calix[4]arene molecules and their influence on polarization of mitochondria membrane.

[Modulators of transmembrane calcium exchange in myometrium mitochondria change their hydrodynamic diameter].

The influence of modulators of calcium exchange in mitochondria--oligomycin, Mg2+ and ruthenium red (RuR)--on the myometrium mitochondria size and granularity was studied in the work. The study of the mitochondria size was carried out using the photon correlation spectroscopy. It was shown that the average hydrodynamic diameter was 655 +/- 14 nm (n = 5; control). The addition of oligomycin (1 microg/ml)--the inhibitor of ATP-synthase F0-component, increases the mitochondria average hydrodynamic diameter to 913 +/- 75 nm (n = 5), that is by 39% more than the control. In the presence of RuR (10 microM) (Ca2(+)-uniporter inhibitor) and Mg2+ (7 mM) the mitochondria average hydrodynamic diameter increases to 788 +/- 28 and 788 +/- 38 nm (n = 5) respectively, that is by 17% more than the control. Using flow cytometry it was shown, that oligomycin (1 microg/ml) causes the increase of side scattering of the mitochondria. Addition of RuR (10 microM) and Mg2+ (7 mM) does not lead to significant changes in side scattering of the mitochondria. So it was shown that oligomycin significantly increases mitochondria granularity, but Mg2+ and RuR have no influence on this parameter

[Effect of Mg ions and spermine on ATP-dependent Ca2+ transport in myometrial intracellular structures. I. Comparative study of Ca2+ accumulation in mitochondria and sarcoplasmic reticulum]. / Vliianie ionov Mg i spermina na ATP-zavisimyi transport Ca2+ vo vnutrikletochnykh strukturakh miometriia. 1. Sravnitel'noe izuchenie akkumuliatsii Ca2+ v mitokhondriiakh i sarkoplazmaticheskom retikulume.

In experiments, which were carried out with the use of a radioactive label (45Ca2+) on the suspension of rat uterus myocytes treated by digitonin solution (0.1 mg/ml), influence of Mg ions and spermine on Mg2+, ATP-dependent Ca2+ transport in mitochondria and sarcoplasmic reticulum was investigated. Ca2+ accumulation in mitochondria (1324 +/- 174 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to thapsigargin (100 nM) and was blocked by ruthenium red (10 microM). Oxalate-stimulated Ca2+ accumulation in sarcoplasmic reticulum (136 +/- 17 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to ruthenium red and was blocked by thapsigargin. It has been shown, that initial speed and level of energy-dependent Ca2+ accumulation in mitochondria considerably exceeded the values of these parameters for sarcoplasmic reticulum Ca2+-accumulation system. Ca2+ accumulation kinetic in mitochondria was characterized by a steady-state phase (for 5-10 min. of incubation) while accumulation kinetic of this cation in sarcoplasmic reticulum corresponded to zero order reaction. Increase of Mg2+ concentration up to 5 mM led to activation of Ca2+-accumulation systems in mitochondria and sarcoplasmic reticulum (values of activation constants K(Mg) for Mg2+ were 2.8 and 0.6 mM, accordingly). Concentration dependence of spermine action on Ca2+ accumulation in mitochondria was described by a dome-shaped curve with a maximum at 1 mM spermine. In case of sarcoplasmic reticulum Ca2+ pump only the inhibition phase was tested at spermine concentration above 1 mM. However values of inhibition constants for both transporting systems were practically identical--5.2 +/- 0.6 and 5.7 +/- 0.7 mM, accordingly. Hence, Mg ions carry out the important role in regulation of energy-dependent Ca2+ transporting systems both in uterus smooth muscle mitochondria and sarcoplasmic reticulum. Spermine acts first of all on mitochondrial calcium uniporter.

[Influence of Mg ions and spermine on ATP-dependent Ca2+ transport in myometrial intracellular structures. II. Comparative study of spermine, Mg ions and cyclosporin A effects on Ca2+ transport in mitochondria]. / Vliianie ionov Mg i spermina na ATR-zavisimyi transport Ca2+ vo vnutrikletochnykh strukturakh miometriia. II. Sravnitel'noe izuchenie deistviia spermina, ionov Mg i tsiklosporina A na transport Ca2+ v mitokhondriiakh.

In experiments carried out with the use of the radioactive label (45Ca2+) on suspension of the rat uterus myocytes processed by digitonin solution (0.1 mg/ml), influence of spermine and cyclosporin A on Mg2+, ATP-dependent Ca2+ transport in mitochondria at different Mg2+ concentration were investigated. Ca2+ accumulation in mitochondria was tested as such which was not sensitive to thapsigargin (100 nM) and was blocked by ruthenium red (10 microM). It has been shown, that spermine (1 mM) stimulates Mg2+, ATP-dependent Ca2+ accumulation in mitochondria irrespective of Mg2+ concentration (3 or 7 mM) in the incubation medium. At the same time cyclosporin A (5 microM) effects on Ca2+ accumulation in mitochondria depend on Mg2+ concentration in the incubation medium: at 3 mM Mg2+ the stimulating effect was observed, and at 7 mM Mg2+ - the inhibitory one. In conditions which led to the increase of nonspecific mitochondrial permeability and, accordingly, to dissipation of electrochemical potential (it was reached by 5 min. preincubation of myocytes suspension in the medium that contained 10 microM Ca2+, 2 mM phosphate and 3 or 7 mM Mg2+, but not ATP) significant inhibition of Mg2+, ATP-dependent Ca2+ accumulation in mitochondria was observed. The inhibition to the greater degree was observed when medium ATP and Mg2+ were absent simultaneously in the preincubation. Thus the quality of spermine effects on Ca2+ accumulation was kept: stimulation in the presence both of 3 mM and 7 mM Mg2+. Ca2+ accumulation did not reach the control level when 3 mM Mg2+ and 1 mM spermine was present and ATP absent in the preincubation medium. However, in the presence of 7 mM Mg2+ and 1 mM spermine practically full restoration (up to a control level) of Ca2+ accumulation was observed. At the same time with other things being equal such restoration was not observed at simultaneous absence of ATP and Mg2+ in the preincubation medium. The quality of cyclosporin A effects on Ca2+ accumulation in mitochondria was also kept: stimulation - in the presence of 3 mM Mg2+, inhibition - in the presence of 7 mM Mg2+ in the preincubation medium. And, at last, in the presence of cyclosporin A irrespective of the fact which preincubation medium was used, Ca2+ accumulation level practically did not depend on Mg2+ concentration.

[Effect of ethanol on intracellular Ca2+ metabolism]. / Effect of ethanol on intracellular Ca2+ metabolism.

The aim of this review is to summarize current thinking on ethanol effects on the Ca2+ homeostasis in the excitable tissue cells. It has been shown that acute exposure to ethanol decreases cytoplasmic Ca2+ concentration due to Ca2+ channels inhibition and Ca2+ pumps activation. Whereas chronic exposure to ethanol increases the intracellular Ca2+ concentration in cells due to activation of passive Ca2+ transport systems and inhibition of energy-dependent Ca2+ transport systems. The emphasis is place on a possible role of pharmacologic agents that preserve Ca2+ homeostasis in protecting against ethanol-induced diseases.

[Effect of ethanol on accumulation of Ca ions in intracellular structures of rat myometrium under conditions of first creating ethanol-dependent models]. / Effektronoe vliianie étanola na akkululiatsiiu ionov Ca vo vnutrikletochnykh strukturakh miometriia krys v usloviiakh predvaritel'nogo sozdaniia étanolzavisimykh modelei.

In the experiments which have been conducted on digitonin-treated myometrium cell suspensions of nonpregnant rats the direct influence of ethanol in concentration 0-10% on the Ca2+ accumulation in mitochondria and endoplasmic reticulum have been studied. Studies have been conducted on the different models, such as, control (model I), subacute (model II), acute (model III) and chronic ethanol consumption (model IV). It has been shown for all models that the dependence of Ca2+ accumulation by mitochondria on the concentration of ethanol in incubation medium is bell-shaped. Acute and chronic ethanol consumption resulted in statistically reliable decrease in the amount of accumulated cations. Nevertheless the I50 values were the same for the models I-III and were 8-9%, although in the case of model IV this one was only 4.0 +/- 0.6%. The increase of ethanol concentration in the incubation medium caused of Ca2+ accumulation decreasing in the endoplasmic reticulum for all studied models, the values of I50 also decreased for models II-IV (2.8 +/- 0.2; 2.5 +/- 0.2 and 2.3 +/- 0.3% respectively) relative to the control (3.8 +/- 0.2%). At the level of model I oxytocin-inhibited component of the endoplasmic reticulum Ca2+ uptake was more stable to the ethanol effects than oxytocin-independent one. Although the sensitivity of the first one to the ethanol effects at the level of models II-IV rose, that parameter for the oxytocin-independent component was not changed. The mechanisms of ethanol effects on Ca2+ accumulation in the myometrium intracellular structures have been discussed.

Adenylate cyclase of myometrium and its regulation by cAMP-dependent protein kinase.

The purpose of this study was to examine activity of adenylate cyclase (AC) and its cAMP-dependent phosphorylation by cAMP-dependent protein kinase (PKA) in the membrane of rabbit myometrium. Isoproterenol (IP) significantly increased AC activity of nonpregnant rabbits myometrium plasma membranes. However, during pregnancy AC of myometrium plasma membranes did not respond to IP. Phosphorylation of the myometrium membrane by PKA was followed by significantly decreased response of AC to IP.

[Effect of ethanol on the activity of the energy-dependent Ca2+-transporting system of myometrial cells]. / Vliianie étanola na aktivnost' énergozavisimykh Ca2+-transportiruiushchikh sistem kletok miometriia.

In order of estimating some regularities of ethanol direct (effectory) effect to transmembrane calcium metabolism in the myometrium the action of this substance on the energy-dependent Ca(2+)-transporting systems of the uterine myocytes subcellular structures has been studied. The systems of Mg2+, ATP-dependent Ca2+ transport regarding their sensitivity to ethanol inhibitory effect were displayed as satisfying the following sequences: endoplasmic reticulum calcium pump > plasma membrane solubilized Ca2+, Mg2+, ATP-ase > mitochondrial Ca(2+)-accumulating system = plasma membrane calcium pump. Alongside with the latter, the oxytocin-insensitive component of Mg2+, ATP-dependent Ca2+ accumulation in the endoplasmic reticulum was defined to be less resistant to inhibitory effect of ethanol if compared with the oxytocin-sensitive one. On the base of the data received some mechanisms of ethanol effectory action on the intracellular calcium homeostasis in the myometrium cells are under the discussion.

[Comparative study of the effect of aliphatic alcohols on energy-dependent accumulation of Ca2+ in intracellular membranes of smooth muscle cells]. / Sravnitel'noe issledovanie vliianiia alifaticheskikh spirtov na énergozavisimuiu akkumuliatsiiu Ca2+ vo vnutrikletochnykh membrannykh strukturakh gladkoi myshtsy.

The effects of ethanol and other aliphatic alcohols on energy-dependent Ca2+ transport in endoplasmic reticulum and mitochondria were studied in digitonin-treated myometrium cells. The Ca2+ uptake in mitochondria increased (on 15-20%) with increasing methanol, ethanol and propanol concentrations in medium, whereas further rise of concentration inhibited this process. Treatments of myometrial cells with short-chain alcohols caused an inhibition of calcium uptake in endoplasmic reticulum. Butanol inhibited both calcium uptake in mitochondria and endoplasmic reticulum. Ca2+ accumulation in intracellular pools is inhibited by aliphatic alcohols in the following order of potency: butanol > propanol > ethanol > methanol. It is concluded that modifying effect of aliphatic alcohols on energy dependent calcium accumulation in intracellular membrane structures is defined as on origin of Ca(2+)-transporting system and (or) properties of these membrane structures so on properties of alcohols.

[Accumulation of Ca ions in intracellular structures of rat myometrium during subacute, acute, and chronic administration of ethanol]. / Akkumuliatsiia ionov Ca vo vnutrikletochnykh strukturakh mimetriia krys pri podostrom, ostrom i khronichskom vvedenii étanola.

The effects of subacute, acute and chronic ethanol exposure on the activity of Ca(2+)-accumulating systems of mitochondria and endoplasmic reticulum in myometrial cells of nonpregnant estrogen-treated rats were studied. It has been shown that the activity of Ca(2+)-accumulating system of mitochondria was higher than the activity of Ca(2+)-accumulating system of endoplasmic reticulum in myometrial cells from control, acute and subacute treated with ethanol rats. Under ethanol chronical assumption both Ca(2+)-accumulation in mitochondria and Ca(2+)-transporting activity of endoplasmic reticulum are inhibited. In the latter ease Mg2+, ATP-dependent Ca(2+)-pump lost its sensitivity to oxytocin.

[Properties of the smooth muscle cell endoplasmic reticulum calcium pump]. / Svoistva kal'tsievogo nasosa éndoplazmaticheskogo retikuluma gladkomyshechnky kletok.

In experiments with 45Ca2+ conducted on digitonin-treated (0.1 mg/ml) myometrium cells suspension, the properties of ruthenium red-insensitive, oxalate- or phosphate-stimulated and thapsigargin- or cyclopiasonic acid-suppressed Mg2+, ATP-dependent calcium pump of myometrium sarcoplasmic reticulum was studied. The Ca2+ accumulation increased linearly in time up to 10 min, the average initial rate was 80-130 pmol Ca2+/10(6) cells per min. In the presence of 10 mM oxalate the values of the activation constant KMg for Mg2+ and K(m) for ATP were 0.6 and 1.0 mM, respectively. The relative efficiency of the different cations in insuring of the ATP-dependent Ca2+ accumulation was Mg2+ > Mn2+ = Co2+ >> Ni2+; the Ca2+ accumulation was not observed in the presence of 3 mM Zn2+ or Cu2+. We observed the suppression of calcium pump activity by different inhibitors such as thapsigargin, cyclopiazonic acid, p-chloromercuribenzoic acid, eosin Y ad Na3 VO4: the values of K0.5 were 2.0 nM, 0.3 microM, 0.6 microM, 0.8 microM and 45 microM respectively. The conclusion was made that suspension of myometrial cells treated with digitonin represent a suitable experimental model for studying the properties of myometrium sarcoplasmic reticulum calcium pump.

Evidence for Mg2, ATP-dependent accumulation of Ca2+ by the intracellular non-mitochondrial calcium store in uterine smooth muscle cells.

Using carbachol contracture as the experimental model for testing the properties of the intracellular calcium store in intact tissue and 45Ca2+ accumulation in the chemically skinned by digitonin smooth muscle cells isolated from oestrogen-dominated rat uterus the evidence for the presence of Mg2+, ATP-dependent Ca2+ pump in the non-mitochondrial store has been found which is supposed to play a key role in the process of refilling' of the store on the cytoplasmic level. The experiments performed on intact muscle showed that the functional activity of the carbachol-releasable Ca2+ store is critically dependent on Ca2+ entry. It is found that Ca2+ entry via voltage operated Ca2+ channels or on the Na(+)-Ca2+ exchange was needed to refill the store in this tissue. However, when Ca2+ extrusion systems located in the plasma membrane were inhibited by La3+, the store retained its ability to discharge and reaccumulate Ca2+ released on the regular basis suggesting the presence of the energy-dependent Ca2+ accumulating system in the store. The process of the store refilling was totally inhibited by cyclopiazonic acid. Chemically skinned uterine smooth muscle cells demonstrated the presence of Mg2+, ATP-dependent accumulation of Ca2+ in the non-mitochondrial (ruthenium red insensitive) intracellular store(s) potentiated by Ca(2+)-precipitating anions (potassium oxalate and phosphate), in a time- and concentration dependent way which was inhibited by Ca(2+)-ionophore A 23187 (5 microM) and cyclopiazonic acid with Ki = 0.4 microM. It is suggested that in the uterine smooth muscle of the oestrogen-dominated rats, nonmitochondrial receptor-operated intracellular calcium store is represented by endoplasmic reticulum.

Suspension of smooth muscle cells treated with digitonin as a model for studying the myometrial endoplasmic reticulum calcium pump.

The effect of the nonionic detergent digitonin on ruthenium red-insensitive, oxalate-stimulated, and thapsigargin-suppressed accumulation of Ca2+ by suspension of myometrial cells from ATP- and Mg(2+)-containing incubation medium was studied using passive loading with 45Ca2+. The dependence of Ca2+ accumulation by the cells on the concentration of digitonin is bell-shaped, the maximal Ca2+ uptake being observed at 0.05-0.1 mg/ml digitonin. Myocytes chemically skinned with digitonin represent a suitable experimental model for studying kinetic properties of the uterine myocyte endoplasmic reticulum Mg2+, ATP-dependent Ca2+ pump and assessing sensitivity of this Ca(2+)-transporting system to various effectors including the uterotonic octapeptide oxytocin.

[Effect of n-palmitoylethanolamine on energy-dependent transport of Ca2+ in intracellular structures of myometrium and their phospholipid composition]. / Vliianie n-pal'mitoilétanolamina na énergozavisimyi transport Ca2+ vo vnutrikletochnykh strukturakh miometriia i ikh fosfolipidnyi sostav.

N-palmitoylethanolamine (NPE) was studied for its effect on the systems of energy-dependent transport of Ca2+ in the intracell structures (mitochondria, endoplasmic reticulum) of permeabilized cells of smooth muscles of the rat uterus as well as on the lipid composition of myocytes. NPE in concentration of 10(-5) M partially (by 30-50%) inhibited energy-dependent accumulation of Ca2+ in endoplasmic reticulum and mitochondria of myometrium cells permeabilized by means of treatment of myocytes suspension by digitonin (0.1 mg/ml). NPE modifies the lipid composition of permeabilized myocytes when determining the increase of the amount of inorganic phosphorus of total phospholipids by 57.3% at the expense of considerable accumulation of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. It is supposed that the effect of NPE on the systems of energy-dependent transport of Ca2+ is achieved through the modification of phospholipid composition of a cell, while modulating effect of NPE on the active transmembrane transfer of Ca2+ in the intracellular structure can be an important link in the general mechanism of the effect of this compound on Ca2+ metabolism in myometrium and on Ca(2+)-depended control of the contracting function of the uterus.

[Effect of inhibitors of energy-dependent Ca2+-transporting systems on calcium pumps of a smooth muscle cell]. / Vliianie ingibitorov énergozavisimykh Ca2+-transportiruiushchikh sistem na kal'tsievye nasosy gladkomyshechnoi kletki.

Comparative investigation of sensitivity of calcium pumps of sarcolemma, endoplasmic reticulum and mitochondria of the smooth muscle to some inhibitors of energy-dependent Ca(2+)-transporting systems has been carried out in experiments made using the isotope method (45Ca2+) on the fraction of plasmatic membranes and suspension of chemically scanned cells of myometrium as well as on the preparation of highly purified Ca2+, Mg(2+)-ATPase which was solubilized from plasmatic membrane of uterus myocytes. It is proved that the calcium pump of mitochondria is selectively inhibited by ruthenium red (imaginary inhibition constant K, is equal to 0.6 microM); it is nonselectively inhibited by o-vanadate (Ki = 0.6 mM), p-chloromercuribenzoate (Ki = 3.2 microM) and cosine (Ki = 2.1 microM), but is not sensitive to the effect of thapsigargin and cyclopiasonic acid. Mg2+, ATP-dependent calcium pump and transport Ca2+, Mg(2+)-ATPase of plasmatic membrane are nonspecifically inhibited by o-vanadate (the value Ki equals 45.0 and 5.0 microM, respectively) and by cosine (Ki = 0.8 microM in the both cases); this calcium pump is also nonspecifically inhibited by p-chlormercury benzoate (Ki = 3.2 microM), but it is not sensitive to the effect of ruthenium red. Mg2+, ATP-dependent calcium pump of endoplasmic reticulum is selectively inhibited by tapsigargin (Ki = 2.0 nM) and cyclopiasonium acid (Ki = 0.3 microM), but, like calcium pumps of mitochondria and plasma membrane, it is nonspecifically inhibited by o-vanadate (Ki = 45.0 microM); by p-chlormercurybenzoate (Ki = 0.6 microM) and eosin (Ki = 0.8 microM), but it is not sensitive to the effect of ruthenium red. Data that have been obtained can be of use for the further development of the ideas about biochemical regularities of Ca(2+)-dependent control of contraction-relax of the smooth muscles, identifications of energy-dependent calcium pumps of smooth cells and finding out of structure-functional organization of these pumps.

[Treatment of intact myometrial cells with oxytocin inhibits Mg2+, ATP-dependent accumulation of Ca2+ in endoplasmic reticulum]. / Obrabotka oksitotsinom intaktnykh kletok miometriia ingibiruet Mg2+,ATP-zavisimuiu akkumuliatsiiu Ca2+ v éndoplazmaticheskom retikulume.

Uterotonic peptide hormone oxytocin has been studied for its effect on ATP-dependent accumulation of Ca2+ nonsensitive to the effect of ruthenium red (10 microM) that is potentiated by Ca2+-precipitating anion - oxalate. That was studied in experiments made on the suspension of myometrium cells from estrogenized rats processed by digitonin (0.1 mg/ml) with the aim permeabilization of plasmatic membrane. The preliminary processing of intact myocytes by oxytocin solution (final concentration 100 mM) to permeabilization of plasmatic membrane causes partial inhibition (stimulated by oxalate by 25-30% (0-10 mM) of Mg2+, ATP-dependent accumulation of Ca2+ in nonmitochondrion intracellular calcium depot of perforates smooth-muscled cells. The value of the component of power-dependent accumulation of Ca2+, that is inhibited by oxytocin, increased with both the incubation time and oxalate concentration. Thapsigargin and cyclopiasonium acid, selective inhibitors of calcium pump of endo(sarco)plasmatic reticulum, when used in saturation concentration (50 mM and 10 mM, respectively), completely inhibit the component of Mg2+, ATP-dependent accumulation of Ca2+ in nonmitochondrial intracellular calcium depot that is stimulated with oxalate (< 10 mM) and inhibited with oxytocin (100 mM). It is concluded that oxytocin partially inhibits Mg2+, ATP-dependent calcium pump of myometrium cell endoplasmic reticulum.