[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.

Effects of eosin Y on the catalytic and functional activities of Mg2+,ATP-dependent calcium pump of smooth muscle cell plasma membrane.

Experiments with highly purified preparations of Ca2+,Mg2+-ATPase solubilized from sarcolemma and a fraction of inside-out sarcolemmal vesicles were performed to study the kinetics of inhibitory effects of eosin Y (0-100 microM) on the catalytic and transport activity of Mg2+,ATP-dependent calcium pump of myometrial cell plasma membrane. For both the Ca2+,Mg2+-dependent ATP hydrolysis and the Mg2+, ATP-dependent accumulation of Ca2+ the apparent inhibitory constant Ki was 0.8 microM. However, eosin Y used at concentrations of up to 100 microM had absolutely no effect on Na+-Ca2+ exchange in the plasma membrane fraction. This inhibitor decreased the turnover rate of purified Ca2+,Mg2+-ATPase determined both by Mg2+ and ATP. However, the affinity of purified Ca2+,Mg2+-ATPase for Mg2+ increased, and its affinity for ATP decreased in the presence of eosin Y. In the case of Mg2+,ATP-dependent Ca2+ transport, eosin Y decreased the turnover rate of the calcium pump whose affinity for Mg2+ and ATP displayed virtually no dependence on the presence of the inhibitor in the incubation medium. The possibility of the use of eosin Y in model experiments for identification of Mg2+, ATP-dependent and Na+-dependent calcium fluxes from smooth muscle cells into the intercellular milieu is discussed.

[Effect of organic solvents on catalytic and functional activity of transport Ca2+,Mg2+-ATPase from smooth muscle cell membrane]. / Vliianie organicheskikh rastvoritelei na kataliticheskuiu i funktsional'nuiu aktivnost' transportnoi Ca2+-Mg2+-ATFazy plazmaticheskoi membrany gladkomyshechnykh kletok.

It was shown that organic solvents (dioxane, acetone, ethanol, dimethylsulfoxide) at concentrations of < 10% suppress the activity of transport Ca2+, Mg(2+)-ATPase solubilized from plasmatic membranes of smooth muscle cells and Mg(2+)-ATP-dependent accumulation of Ca2+ ions in inverted membrane vesicles. It was found that one of the reasons for the inhibition of enzymatic and transport activity of Ca2+, Mg(2+)-ATPase by the action of these solvents is an increase in the attractive force between oppositely charged active center of the enzyme and the product (products) of the ATP-hydrolase reaction, which is induced by a decrease in the dielectric permeability of incubation medium.

[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.

[Kinetic uniformity of the ATP hydrolysis reaction catalyzed by Mg2+-ATPase in smooth muscle cell membrane]. / Kineticheskie zakonomernosti reaktsii gidroliza ATP, kataliziruemoi Mg2+-ATPazoi plazmaticheskoi membrany gladkomyshechnykh kletok.

The kinetic properties of Mg(2+)-ATPase (EC 3.6.1.3) from myometrium cell plasma membranes have been studied. Under conditions of enzyme saturation with ATP (0.5-1.0 mM) or Mg2+ (1.0-5.0 mM) the initial maximal rates of the Mg(2+)-dependent enzymatic ATP hydrolysis, V0 ATP and V0 Mg, are 27.4 +/- 3.3 and 25.2 +/- 4.1 mumol Pi/hour/mg of protein, respectively. The apparent Michaelis constant, Km, for ATP and of the apparent activation constant, K alpha, for Mg2+ are equal to 28.1 +/- 2.6 and 107.0 +/- 26.0 microM, respectively. The bivalent metal ions used at 1.0 mM suppress the Mg(2+)-dependent hydrolysis of ATP whose efficiency decreases in the following order: Cu2+ > Zn2+ = Ni2+ > Mn2+ > Ca2+ > Co2+. Alkalinization of the incubation medium from pH 6.0 to pH 8.0 stimulates the Mg(2+)-dependent hydrolysis of ATP. It has been found that Mg(2+)-ATPase has the properties of an H(+)-sensitive enzymatic sensor which is characterized by a linear dependence between the initial maximal rate of the reaction, V0, and the pH value. The feasible role of plasma membrane Mg(2+)-ATPase in some reactions responsible for the control of proton and Ca2+ homeostasis in myometrium cells has been investigated.

[Possible mechanism of the regulation of smooth muscle relaxation by calcium ions]. / Vozmozhnyi mekhanizm reguliatsii rasslableniia gladkoi myshtsy ionami kal'tsiia.

Kinetics of Ca2+ energy-dependent transport in sarcolemma and mitochondrion fractions of myometrium was studied. On the basis of the results obtained the mechanism of calcium control of smooth muscle relaxation was analysed. In terms of this mechanism kinetic curves of myometrium relaxation were calculated. It follows from their pattern that the mitochondria play the role of the main intercellular depo of Ca2+, while the calcium pump of the sarcolemma carries out fine regulation of this process making its contribution to relaxation at its later stage.

[The role of sarcolemma and mitochondria in calcium-dependent control of myometrium relaxation]. / Rol' sarkolemmy i mitokhondrii v obespechenii kal'tsievogo kontrolia rasslableniia miometriia.

Using atomic absorption spectroscopy, it was shown that the amount of firmly bound Ca2+ in cattle mitochondria and myometrium sarcolemma is 160 +/- 10 and 30 +/- 10 mumol/kg of wet tissue, respectively. The Ca2+ 1 accumulating capacity of mitochondria (350 nmol per mg of protein) markedly exceeds that of sarcolemmal vesicles (30 nmol per mg of protein). Using a Ca2+-EGTA buffer, it was found that the affinity of ionized Ca for the mitochondrial transport system (Km = 5.69 microM) is higher than that for the Na+-Ca2+ system of sarcolemma exchange (Km = 30 microM), but is markedly lower than that for the Mg2+, ATP-dependent Ca2+ efflux (Km = 0.35 microM). A kinetic analysis demonstrated that the sarcolemmal Ca2+ pump is incapable of causing complete relaxation of the smooth muscle within the physiologically significant time, whereas the Ca2+ transport system of mitochondria evokes this process within 21 s. However, the contribution of the Ca2+ pump to the regulation of the Ca2+ content in myocytes is paralleled with the accumulation of Ca2+ in mitochondria and is realized at low concentrations of this cation in the myoplasm, i.e., at late steps of relaxation. A mechanism of Ca2+ control over myometrium relaxation is proposed. The system of non-electrogenic Na+-Ca2+ exchange maintains Ca2+ concentration in the myoplasm as high as 10(-5) M. Mitochondria which accumulate the bulk of Ca2+ rapidly decrease its concentration in the cytoplasm down to 10(-6)-10(-7) M; at these values, the activity of the sarcolemmal Ca2+ pump with a high affinity for the transfer substrate is manifested. In this way, the Ca2+ pump accomplishes fine regulation of Ca2+ concentration in the myocytes.

[Equilibrium characteristics of calcium binding on the inner surface of membrane vesicles]. / Ravnovesnye kharakteristiki sviazyvaniia kal'tsiia na vnutrennei poverkhnosti membrannykh vezikul.

A method is proposed for determining equilibrium parameters of calcium binding on the inner surface of membrane vesicules. Based on the studies of concentration relationship of calcium content in vesicules of myometrium sarcolemma (at the equilibrium state) there were determined the values of maximal capacity of Ca2+-binding (12.79 nmole/mg of protein) and the dissociation constants of Ca2+-binding centre complex (62.3 mkM) on the inner surface of the vesicules, and the value of intravesicular volume as well (25.7 mkl/mg of protein).

[Effect of divalent metal ions--stimulants of uterine contractile activity--on the Mg2+, ATP-dependent transport of Ca2+ in the myometrial sarcolemma fraction]. / Vliianie dvukhvalentnykh ionov metallov--stimuliatorov sokratitel'noiaktivnosti matki na Mg2+, ATF-zavisimyi transport Ca2+ vo fraktsii sarkolemmy miometriia.

Mn2+, Co2+, Zn2+ and Cu2+ were shown to inhibit the Mg2+-ATP-dependent accumulation of 45Ca2+ in the fraction of cytoplasmic membranes. Mechanism of inhibitory effect of the Me2+ is considered. It is assumed that the cations, exhibiting higher affinity to ATP as compared with Mg2+, contributed to dissociation of the actual substrate of the enzymatic reaction Mg2+-ATP as well as to formation of the chelate complexes of the Me2+-ATP type; these complexes participated in the transport of Ca2+ less effectively as compared with the true substrate Mg2+-ATP. Among the essential factors responsible for an increase in the contractile activity of uterus in response to divalent ions of metals, accumulated into cells, elevation of Ca2+ concentration appears to occur in myocytes due to inhibition of the Mg2+-ATP-dependent output of Ca2+ from myoplasm into interstitial space.

[Contribution of the Mg2+-, ATP- AND Na+-dependent Ca2+-transport systems to the regulation of Ca2+ concentration in myometrial cells]. / Vklad sistem Mg2+, ATP- i Na+-zavisimogo transporta Ca2+ v reguliatsiiu ego kontsentratsii v kletkakh miometriia.

Studies with sarcolemma from cattle myometrium containing inside-out cytoplasmic vesicles, using Ca2+-EGTA buffer, showed that the affinity of ionized Ca2+ for the Mg2+- or ATP-dependent transport is higher than that for the Na+-Ca2+ exchange system (Kd = 3,2 X 10(-6) and (4.3-5.3) X 10(-5) M), respectively. The Km values for MgATP are 2.15 mM. Oxytocin added to the homogenization medium containing rabbit and cattle myometrium cells, i.e. during the formation of closed sarcolemmal fragments, resulted in inhibition of Mg2+, ATP-dependent accumulation of 45Ca2+ by plasma membranes. However, an addition of oxytocin to the incubation medium did not affect the kinetics of active accumulation of Ca2+. It was assumed that the system of non-electrogenic Na+-Ca2+ exchange in the myometrium possessing a low affinity for Ca2+ provides for the maintenance of ionized Ca2+ concentration in the myocytes at 10(-5) M. Therefore, this system cannot induce relaxation of mechanical tension of the uterus. Further decrease of Ca2+ in the myoplasm from 10(-5) to 10(-7) M and, correspondingly, the relaxation of myometrium is provided for by the Mg2+, ATP-dependent efflux of Ca2+ from the myocytes having a high affinity for this cation. The decrease of the activity of ATP-dependent Ca2+-pump by oxytocin is the cause of Ca2+ elevation in the myoplasm and, consequently, of myometrium contraction.

[Effect of sodium gradient on calcium uptake by plasma membranes of the myometrium]. / Vliianie gradienta natriia na pogloshchenie kal'tsiia vo fraktsii plazmaticheskikh membran miometriia.

The effects of sodium gradient on Ca2+ uptake by plasma membranes of the myometrium were studied. It was found that the changes in the composition of the incubation mixture reduce 45Ca2+ uptake by Na+-loaded vesicles in the following order: sucrose greater than Li+ greater than K+ greater than Na+. The ratio of 45Ca2+ uptake by Na+-loaded vesicles in a KCl-containing medium, i. e. Nai/Ke, to the 45Ca2+ uptake by Na+-loaded vesicles in a NaCl-containing medium (Nai/Nae) or by K+-loaded vesicles in a KCl-containing medium (Ki/Ke) is 1.9 and 1.3, respectively. NaCl (100 mM) in the presence of 1 mM EGTA stimulates the release of 45Ca2+ accumulated in the course of the ATP-dependent process much stronger than does KCl taken at the same concentration. The plots for the dependence of the rate of 45Ca2+ influx into the vesicles on [Nai+] and [Cae2+] are characterized by a plateau. LaCl3 (0.1 mM) added to the Na+-loaded vesicles after their 5 min incubation with 45Ca2+ and KCl decreases the calcium uptake down to 70%; when the Na+-loaded vesicles are incubated in a medium wit NaCl and 45Ca2+ this uptake is decreased down to 50%. Studies on the ionophore A-23187 and Na-DS demonstrated that no 45Ca2+ influx into the vesicles stimulated by sodium gradient occurs against the electrochemical potential gradient. The role of sodium gradient in Ca2+ transport in myometrium cells is discussed.

[ATP-dependent Ca2+-uptake by the plasma membrane fraction of the myometrium]. / ATP-zavisimoe nakoplenie Ca2+ fraktsiei plazmaticheskikh membran miometriia.

The specific activities of Mg2+, Ca2+-ATPase in the plasma membrane fraction of rabbit and cattle myometrium are 8.30 +/- 0.80 and 2.36 +/- 0.48 mkmoles of Pi per mg of protein, respectively. This fraction possesses a higher (in comparison with other subcellular fractions) capacity for ATP-dependent uptake of 45Ca2+ (9.37 +/- 1.66 and 6.86 +/- 0.96 nmoles of 45Ca2+ per mg of protein in 15 min for rabbit and cattle myometrium, respectively); the ratio of ATP-dependent uptake of Ca2+ to adsorbed Ca2+ is also high. Phosphate increases Ca2+ uptake in the presence of ATP and Mg2+. The ionophore A-23187 added to the incubation mixture without ATP and Mg2+ sharply increases Ca2+ binding. An addition of the ionophore at the 15th min of the ATP-dependent Ca2+ uptake causes a complete and rapid release of the accumulated Ca2+. The release of Ca2+ can be also caused by an addition of Na-DS or EGTA to the incubation mixture. This suggests that Ca2+ is accumulated through the plasma membrane inside the closed structures. It was assumed that myometrial sarcolemma plays an essential role in regulation of intracellular Ca2+ concentration in the uterus at rest and that the active Ca2+ efflux from the cells is controlled by the Mg2+, Ca2+-ATPase system.