Effect of caffeine on azide-sensitive Mg2+, ATP-dependent increase of tetracycline fluorescent response in modeling of Ca ions accumulation in mitochondria

The influence of caffeine on the Mg2+, ATP-dependent Ca(2+)-uptake was investigated in the experiments, conducted on mitochondria isolated from myometrium of nonpregnant estrogenized rats. NaN3-sensitive CTC fluorescence increasing (lambda f = = 520 nm) was used as a test for active Ca2+ transport. Kinetics of NaN3-sensitive Mg2+, ATP-dependent component of CTC fluorescence change fits to the pattern of the first-order reaction either in the absence or in the presence of caffeine (20 mM). Caffeine (0-20 mM) inhibited both the stationary level (settled on the 2-3d min. of incubation) and the initial rate V0, and rate constant k of CTC fluorescence change. Magnitude of the apparent inhibition constant I0.5 for caffeine is 10.41 +/- 1.81 mM, inhibition process has weak positive cooperativity--the value of apparent Hill coefficient for caffeine is equal to 1.2 +/- 0.3. Data obtained suggest that caffeine inhibits both stationary Ca2+ capacity of mitochondria and the rate of NaN3-sensitive Mg2+, ATP-dependent Ca(2+)-accumulation in case of myometrium. These data could be useful for further investigation of molecular and membrane mechanisms of caffeine action on the intracellular Ca2+ homeostasis in uterus smooth muscle and its contractive activity.     

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

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.     

Cationic specificity of a Ca2+-accumulating system in smooth muscle cell mitochondria

In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process.     

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

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.     

Utilization of purified myometrium cell plasma membrane Ca2+, Mg2+-ATPase for comparative estimation of the efficiency of energy-dependent Ca2+-transport inhibitors

With the aim of comparative estimation of efficacy of well-known inhibitors of energy-dependent Ca(2+)-transporting systems their effects were investigated on the activity of purified Ca2+, Mg(2+)-ATPase of the myometrium cell plasma membranes. From the approved inhibitors (eosin Y, o-vanadate, thapsigargin, cyclopiazonic acid, ruthenium red, sodium azide) only eosin Y and o-vanadate are potent inhibitors of myometrium sarcolemma Ca(2+)-pump: the values of Ki equal 0.8 and 4.7 microM, respectively. Thapsigargin and cyclopiazonic acid as well as ruthenium red in concentrations inhibiting, respectively, endo(sarco)plasmic reticulum Ca(2+)-pump and energy-dependent Ca(2+)-transport in mitochondria had no effect on the Ca2+, Mg(2+)-ATPase of the uterus smooth muscle cell plasma membrane. Sodium azide (10 mM) blocking completely Ca(2+)-transport in mitochondria inhibited activity of the plasma membrane Ca(2+)-transporting ATPase by 14%.     

Calcium-magnesium interactions in pancreatic acinar cells.

Although the role of calcium (Ca2+) in the signal transduction and pathobiology of the exocrine pancreas is firmly established, the role of magnesium (Mg2+) remains unclear. We have characterized the intracellular distribution of Mg2+ in response to hormone stimulation in isolated mouse pancreatic acinar cells and studied the role of Mg2+ in modulating Ca2+ signaling using microspectrofluorometry and digital imaging of Ca2+- or Mg2+-sensitive fluorescent dyes as well as Mg2+-sensitive intracellular microelectrodes. Our results indicate that an increase in intracellular Mg2+ concentrations reduced the cholecystokinin (CCK) -induced Ca2+ oscillations by inhibiting the capacitive Ca2+ influx. An intracellular Ca2+ mobilization, on the other hand, was paralleled by a decrease in [Mg2+]i, which was reversible upon hormone withdrawal independent of the electrochemical gradients for Mg2+, Ca2+, Na+, and K+, and not caused by Mg2+ efflux from acinar cells. In an attempt to characterize possible Mg2+ stores that would explain the reversible, hormone-induced intracellular Mg2+ movements, we ruled out mitochondria or ATP as potential Mg2+ buffers and found that the CCK-induced [Mg2+]i decrease was initiated at the basolateral part of the acinar cells, where most of the endoplasmic reticulum (ER) is located, and progressed from there toward the apical pole of the acinar cells in an antiparallel fashion to Ca2+ waves. These experiments represent the first characterization of intracellular Mg2+ movements in the exocrine pancreas, provide evidence for possible Mg2+ stores in the ER, and indicate that the spatial and temporal distribution of intracellular Mg concentrations profoundly affects acinar cell Ca2+ signaling.     

Effector action of ethanol on the accumulation of Ca2+ in intracellular structures of uterine smooth muscle

The work is devoted to the investigation of ethanol direct effect on the transmembrane Ca2+ metabolism in the intracellular structures of myometrium. In the experiments in vitro it has been shown that the Mg2+, ATP-dependent system for Ca2+ accumulation in endoplasmic reticulum is more sensitive then Ca(2+)-accumulating system in mitochondria. It has also been found that the oxytocin insensitive part of Mg2+, ATP-dependent Ca2+ accumulation of the endoplasmic reticulum is less resistant to ethanol inhibition than the oxytocin sensitive one. The data above revealed allow to discuss mechanism of ethanol action on the intracellular Ca2+ homeostasis in myometrium.     

Accumulation of Ca ions in intracellular structures of rat myometrium during subacute, acute, and chronic administration of ethanol

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.     

Passive Ca2+ fluxes across the membrane of sarcoplasmatic reticulum of skeletal muscles. The effect of calcium channel blockers

It was found that the initial rate of passive KC1-stimulated Ca2+ influx into sarcoplasmic reticulum (SR) vesicles follows the saturation kinetics at Ca2+ concentrations of 8-10 mM. The inhibitory effect of Ca2+ channel blockers (La3+, Mn2+, Co2+, Cd2+, Mg2+) on passive Ca2+ influx into SR vesicles is competitive with respect to Ca2+. These blockers also inhibit the initial fast phase of Ca2+ efflux from Ca2+-loaded SR vesicles. Verapamil (0.1-0.5 mM) added to the incubation mixture has no effect on passive Ca2+ fluxes across the SR vesicle membrane or on Ca2+ binding and ATP-dependent Ca2+ accumulation. However, preincubation of SR vesicles with verapamil (18 hours, 4 degrees C) or its introduction into the medium for SR vesicle isolation leads to the inhibition of passive Ca2+ fluxes.     

Adenosine triphosphate - dependent calcium binding and accumulation by guinea pig cardiac sarcolemma.

Sarcolemma isolated from guinea pig heart ventricles possessed ATP-dependent Ca2+ binding and accumulation (+ oxalate) activities which were not inhibited by sodium azide, oligomycin, or ruthenium red. Ca2+ binding and accumulation by sarcolemma were sensitive to pH, the optimum being about pH 6.8. The concentrations of ATP required for half-maximal binding and accumulation were 94.3 and 172 muM, respectively. Mg2+ up to 5 mM significantly enhanced both activities but was inhibitory at higher concentrations (greater than 10 mM). Sarcolemmal Ca2+ binding and accumulation were stimulated 100% by K+, half-maximal enhancement occurring at 5-10 mM K+. Ca2+ binding and accumulation were both saturable processes and the respective apparent Km values for Ca2+ were 16.4 and 14.3 muM. Ca2+ binding by sarcolemma was a rapid process and the bound Ca2+ was released upon depletion of ATP in the medium. It is suggested that the sarcolemmal Ca2+ transport system may well be of significance in regulation of the contraction-relaxation cycle of cardiac muscle.     

The activation of adrenal cortex mitochondrial malic enzyme by Ca2+ and Mg2+.

Adrenal cortex mitochondria prepared by a standard method do not exhibit malic enzyme activity. Addition of physiological concentrations of Ca2+ and Mg2+ enables these mitochondria to reduce added NADP+ by malate to form free NADPH. Half-maximum activation of the mitochondrial malic enzyme requires 0.3 mM Ca2+ and 1 mM Mg2+. Solubilized mitochondrial malic enzymes is independent of Ca2+ and has a K M of 0.2 mM for Mg2+. The Ca2+ effect is dependent on an initial period of active Ca2+ uptake which also causes other changes in respiratory properties similar to those observed with mitochondria from other tissues. After Ca2+ accumulation has taken place, free Ca2+, but not additional accumulation, is still required for malic enzyme activity. The requirement for Mg2+ can be met by Mn2+ (1 mM). This concentration of Mn2+ alone yielded only a slight activation of mitochondrial malic enzyme while higher concentrations of Mn2+ alone gave good activation of the mitochondrial malic enzy.e The NADPH generated by the Ca2+-Mg2+ activated malic enzyme effectively supports the 11beta-hydroxylation of deoxycorticosterone, whereas in the presence of malate, or malate plus Mg2+ but absence of Ca2+, the energy linked transhydrogenase supplies all the required NADPH. The activated malic enzyme appears to be more efficient than transhydrogenase in generating NADPH to support 11beta-hydroxylation. Cyanide and azide have been found to inhibit solubilized mitochondrial malic enzyme.     

Effect of caffeine and cyclosporin A on the transmembrane exchange of Ca ions in smooth muscle mitochondria

The effects of cyclosporin A and caffeine on the active and passive transport of Ca2+ in mitochondria isolated from adult rat myometrium were studied by fluorescent technique using Ca2+-sensitive probe tetracycline (TC). It was shown that 5 microM cyclosporin increases Ca2+ accumulation by the mitochondria matrix. But it fails to exhibit such effect when 20 mM caffeine was also present in the incubation medium, while the inhibitory action of caffeine on the accumulation of Ca2+ reveals nevertheless in the absence or presence of cyclosporin A. In case of the preliminary incubation of mitochondria with 10 mM caffeine before the initiation of transport process one could also observe the inhibition of kinetic parameters of the active accumulation of Ca2+ by the mitochondria. It was also shown, that caffeine stimulates passive efflux of Ca2+ from the myometrium mitochondria. Thus we conclude, that the stimulating effect of cyclosporin on Ca2+ accumulation by the myometrium mitochondria is sensitive to caffeine, while caffeine has no direct effect on Ca2+-uniporter, but it evidently disturbs the barrier function of the inner mitochondria membrane in such way, that stimulating effect of cyclosporin A cannot develop.     

Kinetic model of spermine effect on the Mg2+, ATP-dependent transport of Ca ions in the smooth muscle mitochondria

In experiments, carried out with the use of a radioactive label (45Ca2+) on suspension of rat uterus myocytes treated with digitonin solution (0.1 mg/ml), influence of spermine on the Mg2+, ATP-dependent Ca2+ transport in the mitochondria was investigated. Ca2+ accumulation in the mitochondria was tested as such which was blocked by ruthenium red (10 microM) and was not sensitive to thapsigargin (100 nM). It was shown, that dependence of initial speed of Ca ions accumulation in the mitochondria on spermine concentration (0.1-10 mm) is described by a bell-shaped curve. Spermine concentration being increased in the range of 0.1-1 mM the stimulation of Ca2+ accumulation was observed, at the further increase in polyamine concentration up to 10 mM the suppression of this process took place. On the basis of the analysis of the authors' experimental results and the literature data the model of complex spermine action on Ca2+ accumulation in mitochondria was proposed and analyzed. The existence of two spermine binding sites on mitochondrial membrane--S1 and S2 occupation of which is connected to activation and inhibition of Ca(2+)-unipoter, accordingly, was taken into account. The kinetic analysis of the model which has been made in an equilibrium mode, allowed to calculate some important quantitative parameters describing spermine influence on Ca ions accumulation in mitochondria. It is supposed, that the proposed model can be useful in the further research of polyamine influence on transmembrane exchange of Ca ions in mitochondria.     

Properties of the smooth muscle cell endoplasmic reticulum calcium pump

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.     

Characterization of calcium transport by basal plasma membranes from human placental syncytiotrophoblast.

We have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full-term human placenta. These purified membranes were enriched 25-fold in Na+/K(+)-adenosine triphosphate (ATPase), 37-fold in [3H] dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+ uptake was observed, which followed Michaelis-Menten kinetics, with a Km Ca2+ of 0.18 +/- 0.05 microM and Vmax of 0.93 +/- 0.11 nmol/mg/min. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentration-dependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver-Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+/Mg2+ exchange mechanism. This ATP-independent Ca2+ uptake was not sensitive to 10(-6) M nitrendipine nor to 10(-4) M verapamil. An ATP-dependent Ca2+ transport was also detected in these BPM, whose Km Ca2+ was 0.09 +/- 0.02 microM and Vmax 3.4 +/- 0.2 nmoles/mg/3 min. This Ca2+ transport requires Mg2+, the optimal concentration of Mg2+ being approximately 1 mM. Preincubation of the membrane with 10(-6) M calmodulin strongly enhanced the initial ATP-dependent Ca2+ uptake. Finally, no Na+/Ca2+ exchange process could be demonstrated.     

ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+,K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase

In order to characterize low affinity ATP-binding sites of renal (Na+,K+) ATPase and sarcoplasmic reticulum (Ca2+)ATPase, the effects of ATP on the splitting of the K+-sensitive phosphoenzymes were compared. ATP inactivated the dephosphorylation in the case of (Na+,K+)ATPase at relatively high concentrations, while activating it in the case of (Ca2+)ATPase. When various nucleotides were tested in place of ATP, inactivators of (Na+,K+)ATPase were found to be activators in (Ca2+)ATPase, with a few exceptions. In the absence of Mg2+, the half-maximum concentration of ATP for the inhibition or for the activation was about 0.35 mM or 0.25 mM, respectively. These values are comparable to the previously reported Km or the dissociation constant of the low affinity ATP site estimated from the steady-state kinetics of the stimulation of ATP hydrolysis or from binding measurements. By increasing the concentration of Mg2+, but not Na+, the effect of ATP on the phosphoenzyme of (Na+,K+)ATPase was reduced. On the other hand, Mg2+ did not modify the effect of ATP on the phosphoenzyme of (Ca2+)ATPase. During (Na+,K+)ATPase turnover, the low affinity ATP site appeared to be exposed in the phosphorylated form of the enzyme, but the magnesium-complexed ATP interacted poorly with the reactive K+-sensitive phosphoenzyme, which has a tightly bound magnesium, probably because of interaction between the divalent cations. In the presence of physiological levels of Mg2+ and K+, ATP appeared to bind to the (Na+,K+)ATPase only after the dephosphorylation, while it binds to the (Ca2+)-ATPase before the dephosphorylation to activate the turnover.     

Mechanism of eosin Y action of activity of solubilized Ca2+, Mg2+- ATPase from smooth muscle sarcolemma

The eosin Y inhibitory effect on the activity of smooth muscle plasma membrane Ca(2+)-transporting ATPase was studied: effect of this inhibitor on the maximal initial rate of ATP-hydrolase reaction, catalyzed by Ca2+, Mg(2+)-ATPase, on the affinity of enzyme for the reaction reagents (Ca2+, Mg2+, ATP). Dependence of eosin Y inhibitory effect on some physicochemical factors of incubation medium was studied too. It was determined that eosin Y inhibited reversibly and with high specificity purified Ca2+, Mg(2+)-ATPase solubilized from myometrial cell plasma membrane (Ki--0.8 microM), decreased the turnover rate of this enzyme determined both by Mg2+, ATP and Ca2+. This inhibitor had no effect on the enzyme affinity for Ca2+, increased affinity for Mg2+ and decreased affinity for ATP. It was determined that inhibition of Ca2+, Mg(2+)-ATPase by eosin Y depended on pH and dielectric permeability of the incubation medium: increasing of pH from 6.5 to 8.0 reduced the apparent Ki, decreasing of dielectric permeability from 74.07 to 71.19 increased the apparent Ki.     

Ca2+-transporting properties of myometrial plasma cell membrane Ca2+, Mg2+-ATPase reconstituted in liposomes

Purified myometrium cells plasma membrane Ca2+, Mg(2+)-ATPase was reconstitute in liposomes in functionally active state by the method of cholate dialysis: it showed ATP-hydrolase activity increased by 0.8 microM A23187 average 4 times and it showed Mg2+, ATP-dependent Ca(2+)-transporting activity. Reconstituted system transported Ca2+ at an initial rate of 114.4 +/- 16.3 nmol.min-1.mg-1 with the stoichiometry Ca2+: ATP = 1: (3.2-3.7). Calmodulin increased by 30% the initial rate of Ca(2+)-accumulation by the proteoliposomes with reconstituted Ca2+, Mg(2+)-ATPase; 0.1 mM orthovanadate decreased by 80% Ca(2+)-accumulation by this system. Ca2+, Mg(2+)-ATPase reconstituted in liposomes is just Ca(2+)-transporting ATPase of the plasma membrane. Obtained enzyme preparate can be utilised for study of the properties of this important energy-dependent Ca(2+)-transporting system of smooth muscle cell.     

Effect of ethanol on the activity of the energy-dependent Ca2+-transporting system of myometrial cells

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.     

ATP-dependent Ca2+-uptake by the plasma membrane fraction of the myometrium

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.