Effect of inhibitors of energy-dependent Ca2+-transporting systems on ATP-hydrolase activity of smooth muscle actomyosin

Comparative investigation of of smooth muscle actomyosine ATP-ase sensitivity to some inhibitors of energy-dependent Ca(2+)-transporting systems has been carried out. It is proved that the ATP-ase of actomyosine is nonselectively inhibited by thapsigargin (imaginary inhibition constant Ki is equal 29.4 +/- 5.2 nM), cyclopiazonic acid (Ki = 626 +/- 118 nM), eosin Y (Ki = 70 +/- 14 nM) and p-chlormercurybenzoate (Ki = 380 +/- 151 nM). The data obtained could be used for the further development of the ideas about regularities of Ca(2+)-dependent control of the smooth muscles contraction-relaxation.     

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 calmodulin on myometrial cell membrane Ca2+,Mg2+-ATPase activity

Myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase purified by an affinity chromatography on calmodulin-sepharose 4B is calmodulin-dependent enzyme. Concentration of calmodulin required for half-maximal activation of enzyme was about 26 nM. By unlike to the enzymes originated from other tissues sensitivity to the calmodulin of the myometrial sarcolemma Ca(2+)-transporting ATPase was lower: calmodulin increased Vmax of ATPase about 1.25-fold, the apparent constant of the activation of enzyme by Ca2+ failed to alter independently on the phospholipid presenting at the enzyme isolation.     

Effect of fluoroaluminate on the ATP-hydrolysing and Ca2+-transporting activity of the purified Ca2+, Mg2+-ATPase of myometrium cell plasma membranes

Fluoroaluminate, known modulator of G-proteins, inhibits ATP-hydrolase activity of purified solubilized Ca2+, Mg(2+)-ATPase from myometrium cell plasma membranes and Ca(2+)-transporting activity of this enzyme reconstituted into azolectin liposomes: 10 mM NaF plus 10 microM AlCl3 inhibited the primary activity by 95% and--by 81%. Inhibition of purified both solubilized and reconstituted Ca2+, Mg(2+)-ATPases by fluoroaluminate evidences for the possibility of direct interaction AlF4- with this enzyme without involvement of G-protein. The sensitivity to fluoroaluminate of sarcolemmal Ca2+, Mg(2+)-ATPase from myometrium is similar to that of Ca2+, Mg(2+)-ATPase from stomach smooth muscle.     

Reduction of Ca2+-transporting systems in memory T cells

Antigen-specific B and T lymphocytes make up the material grounds of immune memory, their main functional distinction from the so-called "naive" cells is due to the rapid and enhanced response to the antigen-pathogen. An essential distinction between the memory and naive T cells is different sensitivity of these two subpopulations of T lymphocytes to Ca2+-ionophores. Comparative analysis of Ca2+ responses of the immune memory T lymphocytes and naive T cells of mouse CBA/J line to the addition of Ca2+-mobilizing agents concanavalin A, thapsigargin, and ionomycin was carried out. These compounds in concentrations increasing [Ca2+]i in naive cells had no effect on [Ca2+]i in memory cells. Thus, the Ca2+ entrance into memory cells was not activated by exhaustion of intracellular resources. Estimation of intracellular resources of Ca2+, mobilized by ionomycin and thapsigargin in Ca2+ free medium has shown the absence in memory T cells of the intracellular Ca2+ pool, which may be one of factors of their resistance to ionophores. Reduction of the system of Ca2+ influx into memory T cells was shown using the SH-reagent thimerosal. Memory T cells appear to be resistant to "Ca2+ -paradox." Their incubation with 0.5 mM EDTA in the presence or absence of Ca2+ -mobilizing compounds followed by addition of 2 mM CaCl2 did not result in induction of Ca2+ influx into these cells.     

Oxytocin-induced Ca2+ responses in human myometrial cells

Complex spatiotemporal changes in intracellular Ca2+ were monitored in an immortalized human myometrial cell line (PHM1-41) and first-passage human myometrial cells after oxytocin stimulation (1. 0-1000 nM). Laser cytometry revealed intracellular Ca2+ oscillations in both culture systems starting at 1.0 nM, which were followed by repetitive Ca2+ transients by 10-15 min that lasted for at least 90 min. The amplitude of the initial Ca2+ spike was dose dependent, while the frequency of Ca2+ oscillations identified by Fast Fourier Transform (FFT) tended to increase with dose. Removal of oxytocin resulted in termination of oscillations. Analysis of the sources of the Ca2+ involved in oscillations indicated that the major contribution to oscillation frequencies of 相似文献   

Effect of progesterone on intracellular Ca2+ homeostasis in human myometrial smooth muscle cells

Although it iswell known that progesterone alters uterine contractility and plays animportant role in maintenance of pregnancy, the biochemical mechanismsby which progesterone alters uterine contractility in human gestationare less clear. In this investigation we sought to identifyprogesterone-induced adaptations in human myometrial smooth musclecells that may alter Ca²⁺signaling in response to contractile agents. Cells were treated withvehicle or the progesterone analog medroxyprogesterone acetate (MPA)for 5 days, and intracellular freeCa²⁺ concentration([Ca²⁺]_i)was quantified after treatment with oxytocin (OX) or endothelin (ET)-1.OX- and ET-1-induced increases in[Ca²⁺]_iwere significantly attenuated in cells pretreated with MPA in adose-dependent manner. Progesterone receptor antagonists prevented theattenuated Ca²⁺ transients inducedby MPA. ET_A andET_B receptor subtypes were expressed in myometrial cells, and treatment with MPA resulted insignificant downregulation of ET_Aand ET_B receptor binding. MPA didnot alter ionomycin-stimulated increases in[Ca²⁺]_iand had no effect on inositol trisphosphate-dependent or -independent release of Ca²⁺ from internalCa²⁺ stores. We conclude thatadaptations of Ca²⁺ homeostasis inmyometrial cells during pregnancy may include progesterone-inducedmodification of receptor-mediated increases in[Ca²⁺]_i.     

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 ATP-hydrolase activity of myometrial actomyosin

In order of estimating some regularities of ethanol effective action on the uterus smooth muscles contractile proteins the effects of spiritus introduced into incubation medium on myometrium actomyosine ATP-hydrolase activity and superprecipitation was studied. ATP-hydrolase activity was displayed as more sensitive to ethanol action; its dependence on ethyl spiritus concentration had three-phase character expressed in two inhibiting and one activating sites. While defining the kinetic parameters of ATP hydrolysis reaction catalysed by uterus myometrium the correlation between inhibition the ATP-hydrolase activity of myometrium contractile complex under introducing into incubation medium 2% ethanol and decreasing the affinity of actomyosine to Mg2+ was made; the highest activating effect of ethanol on ATP-hydrolase activity of actomyosine complex in the presence of 8% ethanol correlated with increasing the affinity of actomyosine to Mg2+ and ATP.     

GTP-sensitivity of the energy-dependent Ca2+ storage pool in permeabilized pancreatic acinar cells

Isolated rabbit pancreatic acinar cells, permeabilized by saponin treatment and incubated in the presence of 0.1 microM free Ca2+, accumulated 3.3 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Part of this energy-dependent pool could be released by GTP in a polyethylene glycol-dependent manner. The kinetics of GTP-induced release of Ca2+ showed a biphasic pattern with an initial rapid phase followed by a sustained slower phase. In contrast, IP3-induced release of Ca2+ was completed within 30 s following addition of IP3. No reuptake of Ca2+ was observed following GTP- or IP3-induced release of Ca2+. The GTP effect was independent of IP3 and not inhibited by Ca2+, indicating that the IP3-operated Ca2+ channel is not involved in GTP-induced release of Ca2+. The size of the IP3-releasable pool was not affected by GTP, indicating that GTP, when added to permeabilized acinar cells, does not promote the coupling between IP3-insensitive and IP3-sensitive Ca2+ accumulating organelles. Thus, in permeabilized acinar cells, GTP and IP3 act on different Ca2+ sequestering pools. Interestingly, however, comparison of the size of the GTP-releasable pool with that of the IP3-releasable pool for the cell preparations used in the present study, revealed an inversed relationship, indicating that at the time of permeabilization the GTP-releasable pool can be coupled to a greater or lesser extent to the IP3-releasable pool. This suggests that, in the intact cell, a GTP-dependent mechanism may exist that controls the size of the IP3-releasable pool by coupling IP3-insensitive to IP3-sensitive organelles. Moreover, this suggests that the extent of coupling is preserved during permeabilization.     

Ca2+/H+-exchange mechanisms in myometrial plasmalemma cells

Transport of 45Ca2+ into vesicules of hog myometrium plasmolemma under dissipation conditions of opposite-directed transmembrane gradient of protons (delta pH) was investigated. When studying some time regularities of the process, H+ dissipation was determined to have little effect on the initial velocity Vo (18 and 25 nmol Ca2+/1 mg of protein per 1 min at delta pH = 0 and delta pH = 1.5, respectively) and the time of semiaccumulation of cation (1.1 and 2.1 min). Estimation of Ca2+ accumulation concentrational dependence in the vesicules in Vo (30 s) revealed that Ca2+ input into vesicules was limited by binding the cation with carboxyl residues of Ca2+ channel external part. This effect is a consequence of the absence of Ca(2+)-transport systems in the vesicules on the background of quick filling of the intervesicular space by the cation as well as discrimination of Ca2+ sorption process by the vesicules inner surface under operating in the Vo regime. The value K0.5 = 0.5 microM for Ca2+ obtained conforms to physiological meaning of the imagined Kd, Ca2+ binding with four glutamate residues of Ca2+ channel external part. Dissipation of the artificial delta pH = 1.5 on the vesicular membrane leads to increasing the affinity for Ca2+ (to 0.1 microM at constant value of Vmax (40 nmol Ca2+/1 mg of protein per 1 min). We have also demonstrated irreversibility of the process tested and substrate specificity. The results obtained permit to suppose that delta pH dissipation provides for some conformational changes of the channel structure resulting in increasing Ca2+ affinity for the transporting system as well as increases the membrane permeability for the cation. The latter means the interrelation of two most important signal molecules such as Ca2+ and H+ in the cell is capable to occur on the level on Ca2+ separate channels.     

Reversibility of energy-dependent Ca2+ accumulation in mitochondria

Ca2+ accumulation in energized rat liver mitochondria has been studied after the blockage of mitochondrial permeability transition pore (MPTP) by cyclosporin A. It is shown that Ca2+ transport is coupled to the countertransport of protons: from the matrix of mitochondria in the medium in the course of Ca2+ accumulation, and, on the contrary, from the medium to mitochondrial matrix after membrane depolarization. In standard incubation medium containing K+, Cl-, oxidation substrate (glutamate) and inorganic phosphate (H2PO4(-)) the observed stoichiometry of the exchange is 1Ca2+ : 1H+. In accordance with this exchange ratio, proton, as well as cation, transport follows the same first-order kinetics, which is characterized in both cases by very close values of reaction half-times and rate constants. It is shown that reversion of Ca2+ -uniporter, sensitive to ruthenium red, is necessary for Ca2+ - efflux from the matrix ofdeenergized mitochondria when MPTP is blocked by cyclosporin A. It is also shown that Ca2+ -uniporter reversion takes place only after membrane depolarization and permeabilization by protonophore CCCP. Calcium release from mitochondria in the presence of CCCP is accompanied by proton flow into the matrix. Both calcium and proton fluxes are sensitive to Ca2+ uniporter blocker, ruthenium red, which gives the evidence of the identity of Ca2+ -efflux and influx pathways. The data obtained lead to the conclusion that calcium-proton exchange is necessary for Ca2+ -uniporter reversion and the reversibility of energy-dependent Ca2+ -uptake in mitochondria.     

Gene knockout studies of Ca2+-transporting ATPases.

The biochemical functions of intracellular and plasma membrane Ca2+-transporting ATPases in the control of cytosolic and organellar Ca2+ levels are well established, but the physiological roles of specific isoforms are less well understood. There appear to be three different types of Ca2+ pumps in mammalian tissues: the sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), which sequester Ca2+ within the endoplasmic or sarcoplasmic reticulum, the plasma membrane Ca2+-ATPases (PMCAs), which extrude Ca2+ from the cell, and the putative secretory pathway Ca2+-ATPase (SPCA), the function of which is poorly understood. This review describes the results of recent analyses of mouse models with null mutations in the genes encoding SERCA and PMCA isoforms and genetic studies of SERCA and SPCA dysfunction in both humans and model organisms. These studies are yielding important insights regarding the physiological functions of individual Ca2+-transporting ATPases in vivo.     

Stimulation of intracellular Ca2+ oscillations by diacylglycerol in human myometrial cells

Stimulation of G-protein coupled membrane receptors linked to phospholipase C results in production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from the endoplasmic reticulum, which triggers increased Ca2+ influx across the plasma membrane, so-called capacitative calcium entry. DAG can also activate plasma membrane calcium-permeable channels but the mechanism is still not fully understood. In the pregnant human myometrial cell line PHM1 and in primary myometrial cells, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, induced variable oscillatory patterns of intracellular free Ca2+. Similar behavior was seen with Sr2+ entry. The Ca2+ oscillations were not blocked by a broad spectrum of protein kinase C inhibitors, including chelerytrine, bisindolylmaleimide I and calphostin C, and were enhanced and prolonged by RHC-80267, an inhibitor of diacylglycerol lipase. The OAG-induced oscillatory response was not dependent on Ca2+ release from the endoplasmic reticulum but required extracellular Ca2+. Our results indicate that diacylglycerol directly activates cation channels in PHM1 and primary myometrial cells and promotes intracellular Ca2+ oscillations by actions independent of intracellular Ca2+ -ATPase activity and protein kinase C involvement.     

The effect of total ischemia on the Ca2+-transporting activity of the sarcoplasmic reticulum of the rat myocardium

Different caffeine and calcium concentrations have been studied for their influence on Ca2(+)-pumping function of sarcoplasmic reticulum in a homogenate from control and ischemic rat myocardium. Ca2(+)-transporting system of sarcoplasmic reticulum terminal cisternae membranes from the ischemic myocardium was found to be more sensitive to Ca2+ and caffeine action, inhibiting Ca2+ uptake velocity, as compared to control. This may be one of causes leading to the contractibility disorder under myocardium ischemia.     

The binding of Ca2+ to Ca2+-transporting microsomes derived from bovine uterine sarcoplasmic reticulum

An obligatory early step in the transport of calcium across the internal membranes of smooth muscle cells is the binding of calcium to the Ca,Mg-ATPase. The characterization of calcium binding to sarcoplasmic reticulum from smooth muscle has not been reported. Calcium binding to a bovine myometrium preparation was investigated using Scatchard analysis and a computer program utilizing weighted least squares curve fitting and an exact mathematical model of binding. This permitted objective measurement of goodness of fit and showed that best fit was obtained using a two site model. Magnesium did not change the affinity for calcium of the two sites; but reduced the number of low affinity sites to half.     

Contribution of the Mg2+-, ATP- AND Na+-dependent Ca2+-transport systems to the regulation of Ca2+ concentration in myometrial cells

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.     

Regulation by Ca2+-calmodulin-dependent phosphorylation of passive transport of Ca2+ in the myometrial sarcolemma

Closed vesiculate preparations of pig myometrium sarcolemma (predominantly with inside-out orientation) are characterized by passive permeability for Ca2+. The kinetics of Ca2+ release from the vesicles is exponential. Using the grapho-analytical subtraction method, the kinetic parameters of this reaction were determined. Myometrium sarcolemma contains endogenous Ca2+-calmodulin-dependent protein kinase and phosphoprotein phosphatase which is inhibited by sodium o-vanadate. The Ca2+-calmodulin-dependent phosphorylation stimulates passive Ca2+ release from sarcolemmal vesicles. In the course of phosphorylation the capacity of the pool providing for rapid Ca2+ release increases by 61%, the initial rate of Ca2+ release showing a 28% increase. Trifluoroperazine, an inhibitor of Ca2+-calmodulin-dependent processes, eliminates the activating effect of phosphorylation on the rate of Ca2+ release from sarcolemmal vesicles.