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The processes of regulation of smooth muscle contractile function depend greatly on the efficiency of maintaining Са2+-homeostasis by systems of active ionic transport of the plasma membrane (calcium pump and Na+,Ca2+-exchanger) and intracellular stores (sarcoplasmic reticulum and mitochondria). In this work, the effect of calixarene C-716 (macrocyclic compound capable of blocking functioning of the plasma membrane calcium pump) on the kinetics of relaxing K+-induced contraction of rat myomet­rium smooth muscles, while inhibiting the systems of active transport of calcium ions of intracellular Са2+-stores, were investigated. It was established that the application of 100 M calixarene at the maximum of the phase component of the K+-induced contractile response did not change the kinetic parameters of the relaxation process (the normalized maximal velocity of relaxation phase Vnr and the half-maximal time of relaxation τ1/2). The tonic component of K+-induced contraction remained without statistically significant changes. Blocking of Са2+-accumulating function of mitochondria by the protonophore СССР caused a significant increase in both the parameter Vnr and the tonic component of K+-induced contraction, while the τ1/2 index remained at the control level. Blocking calcium pump of sarcoplasmatic reticulum with thapsigargin (TG) caused a conside­rable increase in the τ1/2 ,Vnr parameters and the tonic component of the K+-induced contraction. Combination of TG and СССР induce further increase in the Vnr parameter and the tonic component of K+-induced contraction. The action of calixarene С-716 (100 M) at the background of the impact of TG and СССР was accompanied by further increase in the tonic contraction, Vnr remained at the increased level, and the τ1/2 index decreased. Possible mechanisms for changing the mechanokinetic parameters of relaxing K+-induced contraction of myometrium is discussed. Analysis of the results of our experimental data and the data of other researchers allow predicting that an increase in the rate of relaxation of myometrium smooth muscles at the action of calixarene C-716 under the conditions of blockage of intracellular Ca2+ depots is associated with a significant activation of NO synthesis by constitutive forms of NO-synthases, whereas an increase in the tonic component of K+-contraction under these conditions is probably due to an elevated concentration of Ca2+ ions in the myocytes.

This work shows the existence of a phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) bound form of the cardiac sarcolemmal Na+/Ca2+ exchanger. That was demonstrated in Western blots and cross-immunoprecipitation by using specific antibodies against the NCX1 exchanger (NCX1) and against PtdIns-4,5-P2. In addition, PtdIns-4,5-P2 bound to the Na+/Ca2+ exchanger and the Na+/Ca2+ exchange fluxes displayed a similar MgATP regulation: (a) both increase by 100–130% when membrane vesicles are incubated (15–20 s at 37 °C) with 1 mm MgATP and 1 µm Ca2+ (b) in the presence of 100 µm Ca2+, MgATP fails to stimulate the exchange fluxes and does not modify the levels of PtdIns-4,5-P2 bound to the exchanger. In addition, in the absence of Ca2+, the net synthesis of total membrane PtdIns-4,5-P2 is greatly reduced compared with that in the presence of 1 µm Ca2+. Furthermore, in the absence of Ca2+ there is no effect of MgATP on the levels of PtdIns-4,5-P2 bound to the exchanger. These results indicate that, in bovine heart, MgATP-stimulation of Na+/Ca2+ exchange is associated with intracellular Ca2+-dependent levels of PtdIns-4,5-P2 bound to the exchanger molecule.

α-Crystallin, an endogenous low-molecular-weight protein with chaperone activity, exerted protective effects on membrane systems of Ca2+ transport into the sarcoplasmic reticulum of skeletal muscles. Protective action of α-crystallin depended on the body state. This effect was not observed in the control and after adaptation to stress, while after stress, especially against the background of adaptation, α-crystallin increased the rate of Ca2+ transport into the sarcoplasmic reticulum and thermal resistance of Ca2+ pump. The mechanisms of α-crystallin activation during stress are discussed.

This study evaluated whether annexins I, II and VI possess Ca2+ transport activity in phospholipid membranes by the burst method, and the activity of each was compared with that of annexin V. Briefly, in the presence of 400 microM Ca2+, each annexin at 50 nM was added to large unilamellar vesicles (LUV) which were then burst in fura-2 solution with 0.2% Triton X-100, followed by examination of Ca2+ signals. Annexins I, II, V and VI were all shown to express, each to a different degree, Ca2+ activity toward phosphatidylserine/phosphatidyl- ethanolamine-LUV. Ca2+ signal intensity increased dependent on annexin concentration, and the Ca2+ transport activity of annexin V and VI was higher than that of annexin I and II. However, none of annexin I, II, V and VI expressed Ca2+ transport activity in LUV produced using phosphatidylcholine. Ca(2+)-incorporated LUV with no annexin showed signals whose intensity was proportional to Ca2+ concentration. The Ca2+ transport activity of the annexins could be effectively measured by the burst method. Ca2+ signal intensity would thus appear to be unique for each of the annexins and to be determined by the particular function and specificity of each of those considered in this study.

Frog sympathetic ganglion neurons exhibit a novel Ca2+ uptake mechanism, release-activated calcium transport or RACT, which is manifest in both cytosolic and store [Ca2+] signals as greatly accelerated Ca2+ uptake after Ca2+ release from internal stores. RACT is activated by Ca2+ release but not by Ca2+ entry and serves to selectively refill Ca2+ stores after release. RACT lowers cytosolic [Ca2+] with a rate constant about 1.6 times that of the SERCA pump with empty ER. RACT is thapsigargin-insensitive, was eliminated by ryanodine, but was not affected by blocking mitochondrial or plasma membrane Ca2+ transport. A Ca2+ flux model with RACT in the ER membrane reproduced the cytosolic and store [Ca2+] responses to all stimuli.

Effects of prednisolone, estradiol, and testosterone on the transport of Ca2+ and the respiration induced by it in the heart and liver mitochondria of rats were studied. Prednisolone and testosterone were found to reduce the Ca-accumulating capacity of the mitochondria, the rates of ion entry and exit, and the rate of Ca2+-induced respiration. Estradiol, while inhibiting Ca2+ transport across mitochondrial membrane, did not influence the respiration in the phase of Ca2+ absorption, but accelerated it in the phase of ion exit. These data suggest that due to their lipophilic properties, the steroids become incorporated in the mitochondrial membrane, thereby changing its viscosity and permeability and limiting the mobility of transmitter proteins.

In this work, we compared the effect of K+ on vesicles derived from the longitudinal (LSR) and terminal cisternae (HSR) of rabbit white muscle. In HSR, K+ was found to inhibit both the Ca2+ accumulation and the heat released during ATP hydrolysis by the Ca2+-ATPase (SERCA1). This was not observed in LSR. Valinomycin abolished the HSR Ca2+-uptake inhibition promoted by physiological K+ concentrations, but it did not modify the thermogenic activity of the Ca2+ pump. The results with HSR are difficult to interpret, assuming that a single K+ is binding to either the ryanodine channel or to the Ca2+-ATPase. It is suggested that an increase of K+ in the assay medium alters the interactions among the various proteins found in HSR, thus modifying the properties of both the ryanodine channel and SERCA1.

Ca2+ permeability mediated by voltage-dependent anion-selective channel protein 1 (VDAC1) can be tested by reconstitution of purified VDAC1 into liposomes. Here, we describe a setup for this membranal system, which has been used to study the transport activity of various transporters, including VDAC1, and allows detection of the passage of molecules across the lipid bilayer. Despite the disadvantage of needing radiolabeled molecules, this system is highly desirable when the transport properties of noncharged molecules and/or active transporters are studied.

pH-dependent Ca2+ and Na+ transport via Na-Ca+K exchange was measured in isolated and purified bovine retinal rod outer segments (ROS). Ca2+ transport was measured (1) as 45Ca uptake via reverse Na-Ca+K exchange or via Ca-Ca exchange and (2) as a Ca(2+)-induced rise in cytosolic free Ca2+ concentration via reverse Na-Ca+K exchange measured with the fluorescent Ca(2+)-indicating dye fluo-3. Na+ transport was inferred from a Na(+)-induced lowering of cytosolic free [Ca2+] measured with fluo-3. Both Na+ and Ca2+ influx into ROS mediated by the Na-Ca+K exchanger showed a steep (40-100-fold increase in influx rate when the pH was increased from 6.0 to 8.8) pH dependency when measured at low occupancy of the external transport site with Na+ or Ca2+, respectively. High occupancy of the transport site with either Na+ or Mg2+ caused a dramatic decrease in the pH dependence of either Ca2+ or Na+ influx. These observations corroborate the proposed presence of a common Na+ and Ca2+ transport site of the ROS Na-Ca+K exchanger.

BACKGROUND Myocardial stunning has been associated with a greater than twofold increase in intracellular free [Mg2+] from 0.6 to 1.5 mM. The effect of this increase in free [Mg2+] on the function of the sarcoplasmic reticulum (SR) Ca2+ pump was assessed in SR isolated from Langendorff perfused, isovolumic rabbit hearts after 15 minutes of global ischemia. METHODS AND RESULTS Our results indicate that myocardial stunning results in a shift in the Ca2+ sensitivity of oxalate-supported, Ca2+ transport over the entire range of free [Ca2+] associated with the cardiac cycle. Using 0.6 mM free Mg2+ as control, maximal rates of Ca2+ transport occurred at 1 microM free Ca2+ (control, 519 +/- 32; stunned, 337 +/- 37 nmol Ca2+.min-1.mg-1). At 0.56 microM free Ca2+, SR Ca2+ transport was reduced from a control of 351 +/- 49 to 263 +/- 12 nmol Ca2+.min-1.mg-1 at 0.6 mM free [Mg2+]. Moreover, an increase in the free [Mg2+] from 0.6 to 1.5 mM results in a greater shift in the Ca2+ activation curve with no change in the level of maximal activation. Ca2+ transport at 0.56 microM free Ca2+ was shifted in the stunned SR from 263 +/- 12 to 138 +/- 29 nmol Ca2+.min-1.mg-1 at 0.6 and 1.5 mM free Mg2+, respectively. CONCLUSIONS These results indicate that an increase in free [Mg2+] after stunning in combination with the inherent defect in the SR Ca2+ ATPase may reduce the ability of the cell to regulate Ca2+ to a greater extent than previously observed. This impairment in Ca2+ regulatory function may contribute directly to the increase in diastolic tone and indirectly to the reduced systolic function characteristic of the stunned myocardium.

Membrane vesicles capable of energized Ca2+ pumping have been reconstituted from cardiac sarcoplasmic reticulum (SR). Cardiac SR was solubilized with Triton X-100 in a detergent to protein weight ratio of 0.8, and membranous vesicles were reconstituted by removal of detergent with Bio-Beads SM-2 (a neutral porous styrene-divinylbenzene copolymer). The reconstituted vesicles exhibited ATP-dependent oxalate-facilitated Ca2+ accumulation with rates and efficiency comparable to the best reconstituted skeletal muscle preparation (Ca2+-loading rate = 1.65 +/- 0.31 mumol mg-1 min-1, Ca2+-activated ATPase activity = 2.39 +/- 0.25 mumol mg-1 min-1, efficiency (Ca2+/ATP) = 0.69 +/- 0.09). Phospholamban in the reconstituted vesicles was phosphorylated with added catalytic subunit of cAMP-dependent protein kinase to almost the same extent as that in original vesicles. However, phosphorylation of phospholamban had no effect on the Ca2+ accumulation of the reconstituted vesicles. This is to be contrasted with a decrease in the half-maximal concentration of Ca2+ for Ca2+ accumulation (KCa) in the original vesicles from 1.35 +/- 0.08 microM to 0.75 +/- 0.12 microM by cAMP-dependent phosphorylation of phospholamban. On the other hand KCa for the reconstituted vesicles was about 0.5 microM and remained unchanged by phosphorylation, indicating that the Ca2+ pump in the reconstituted vesicles is already fully activated. These results suggest that in normal cardiac SR, phospholamban in the dephosphorylated state acts as a suppressor of the Ca2+ pump and that phosphorylation of phospholamban serves to reverse the suppression.

Effects of the nature and orientation of a side chain in cyclic octapeptides on Ca2+ transport were examined by using cyclo[L-Lys(Z)-Sar-L-Leu-Sar]2 (C8-L), cyclo[L-Lys(Z)-Sar]4 (C8KS), and their diastereomer cyclic octapeptides, cyclo[L-Lys(Z)-Sar-D-Leu-Sar]2 (C8-D) and cyclo[L-Lys(Z)-Sar-D-Lys(Z)-Sar]2 (C8Kk). All these cyclic octapeptides were found to take a single conformation in CDCl3, and the conformation was C2-symmetric for C8-L and C8-D, and C4-symmetric for C8KS and C8Kk. They formed a complex with Ca2+. Upon complexation, C8KS accompanied isomerization of peptide bonds, but C8-D retained the arrangement of peptide bonds. The amount of Ca2+ extracted from an aqueous solution to a chloroform solution by all L cyclic octapeptide C8-L or C8KS was about twice that of Na+, but 6-8-fold smaller than that by C8-D or C8Kk including D units. These cyclic octapeptides were capable of transporting Ca2+ through a lipid membrane above the phase transition temperature, and the transport rate decreased in the order of C8Kk-C8KS greater than C8-D greater than C8-L.

Microsomal fractions isolated from coleoptiles of dark or far-red light grown corn show ATP-dependent Ca 2+ accumulation. The microsomal transport from dark but not from far-red light grown tissue could be stimulated by calmodulin. Ca 2+ accumulation into mitochondria from coleoptiles of far-red light grown corn in also inhibited as compared to the dark controls. Light irradiation of isolated microsomes and mitochondria had no effect on either Ca 2+ uptake nor efflux.

The pattern of Ca2+ accumulation by tumor mitochondria (MC) was investigated under various experimental conditions. In the absence of penetrating anions tumor MC were shown to take up Ca2+ in only one fifth the amount taken up by liver MC. In the presence of acetate this difference was greater still. Inorganic phosphate (Pin) abolished the observed defects of Ca2+ transport and increased the Ca2+ capacity of the tumor MC considerably. By contrast with liver MC, Pin also had a stabilizing effect on membrane permeability of the tumor MC; this may be the cause of the increasee Ca2+ capacity of these MC.

Attempts were made to elucidate the relation between lens opacification resulting from inhibition of Ca2+ transport and high calcium accumulation in the lens. Mouse lenses were incubated with naphthalenesulfonamide compounds (W-5, W-7, W-12 and W-13) which act as calmodulin antagonists. When lens Ca2+-ATPase activity was inhibited by those calmodulin antagonists, an accumulation of calcium in the lens was observed. This phenomenon depended on the chemical structure of calmodulin antagonist. In lenses treated with W-7, more than 97% of excess calcium was bound. Insoluble protein increased from 8.5 to 17% of the total protein and the degree of lens opacification increased to 85.1%. These results suggested that dysfunction of the Ca-pump system leads to calcium accumulation, thus in turn inducing protein denaturation which in turn is followed by lens opacification.

The effect of aging on Ca2+ -transport in synaptosomal preparations from rat brains was assessed by measuring the accuinulation of radioactive 45Ca within these particles. Four groups of rats at 6, 12 24 and 30 months of age were used for this study. Synaptosomal particles were isolated from the cerebral cortes of each animal and the radioactive 45Ca inside the particles were ineasured after incubating the particles with media containing an energy source and 45Ca C12. Results indicated that the transport of 45Ca was lower in the younger rats than the older groups. A 20% increase uw consistent with the old rats (30 mo) as compared with the young ones (6 mo). The increase in Ca2+ -transport across synaptic plasma membranes may be related to the transmitter release and behavioral activity after senescence.

Mitochondria isolated from some 14 different higher plants and fungi were examined for their capacity to carry out respiration-dependent accumulation of Ca2+. Additions of Ca2+ give little or no stimulation of state 4 respiration of plant mitochondria, although the added Ca2+ was largely accumulated. Accumulation of Ca2+ required phosphate and, in most cases, was stimulated by Mg2+ and ADP or ATP. Ca2+ uptake was abolished by respiratory inhibitors and uncoupling agents. The ratio of Ca2+ ions taken up per pair of electrons per energy-conserving site was normal at about 2.0 for mitochondria from sweet potato and white potato; mitochondria from other plants showed somewhat lower ratios. Accumulated Ca2+ was only very slowly released from previously loaded plant mitochondria. Respiration-inhibited sweet potato mitochondria show both high-affinity and low-affinity Ca2+ binding sites sensitive to uncouplers, La3+, and ruthenium red and thus resemble animal mitochondria. Most other plant mitochondria lack high affinity sites. In general, mitochondria from sweet potato and white potato tubers resemble those from animal tissues, but mitochondria from carrots, beets, turnips, onions, cabbage, artichokes, cauliflower, avocados, mung bean and corn seedlings, and mushrooms show rather low affinity and activity in accumulation of Ca2+, probably due to lack of a specific Ca2+ carrier.

The comparative analysis of responses of memory and naive T lymphocytes to Ca 2+ -mobilizing agents, namely Con A, thimerosal, thapsigargin and ionomycin, was carried out. The effect of these agents on both types of T cells differed qualitatively and quantitatively. The lack of intracellular Ca 2+ stores in memory T cells was shown. Ca 2+ -mobilizing agents did not induce influx of Ca 2+ in memory T cells from outside and this was the reason for their stability to Ca 2+ ionophores. It was also shown that memory T cells were resistant to the 'Ca 2+ paradox'. z 1999 Federation of European Biochemical Societies.

(1) Vanadate (pentavalent vanadium) inhibits with high affinity ( K 0.5 = 3 μ M ) the ATP-dependent Ca2+ efflux in reconstituted ghosts from human red cells. (2) To inhibit Ca2+ efflux vanadate has to have access to the inner surface of the cell membrane. (3) The inhibitory effect of vanadate is potentiated by intracellular Mg2+ and by intracellular K+. (4) Ca2+ in the external medium antagonizes the inhibitory effect of vanadate.

Plots relating the initial rate of mitochondrial Ca2+ transport to the Ca2+ concentration (kinetic plots) have a hyperbolic shape in a Ca2+ concentration range of 2.5-100 muM as measured in sucrose or KCl media. In the presence of Mg2+ or a polyamine spermine, which both are competitive inhibitors of Ca2+ binding to low affinity sites at the membrane surface, the shape of the plots becomes sigmoidal. At higher concentrations of these agents linear kinetic plots are obtained as measured in a sucrose medium. In a KCl medium the sigmoidality of the kinetic plots is enhanced by an increase in the Mg2+ or spermine concentration. It is suggested that Mg2+ and spermine affect the kinetics of Ca2+ transport by interfering with Ca2+ binding to low affinity sites of the membrane surface and that the binding of Ca2+ to these sites is the first step of the mitochondrial Ca2+ transport.

Ca2+ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na+-loaded membrane vesicles were suspended in KHCO3/KOH buffer (pH 10) containing Ca2+, rapid uptake of Ca2+ was observed. The apparent Km value for Ca2+ measured at pH 10 was about 7 microM, and the Km value shifted to 24 microM when measured at pH 7.4. The efflux of Ca2+ was studied with Ca2+-loaded vesicles. Ca2+ was released when Ca2+-loaded vesicles were suspended in medium containing 0.4 M Na+. Ca2+ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K+-valinomycin in the presence of Na+. These results indicate the presence of Ca2+/Na+ and H+/Na+ antiporters in the alkalophilic Bacillus A-007

Publisher Summary This chapter highlights the characteristics of Ca2+ transport in yeast. Calcium transport has been characterized in yeast by several groups. The study of binding of cations to yeast cells is important because it provides information about the relevance of this phenomenon in transport. Experiments were carried out to determine the effects of several inhibitors or cations that seem to eliminate the possibility of a relationship between binding and transport. Some cations affect similarly the binding of Ca2+ but have different effects on transport; a different effect was found also in the inhibition of Ca2+ transport by cations of different kinds that, depending on their valency only, have similar effects on the binding to the cells; uranyl, for instance, was found before to have a marked effect on binding, with a rather small effect on the transport of Ca2+. It was found also that, as the temperature was increased, the rate of transport increased, but the increase was sharper at temperatures that could be shown to produce a change in the fluorescence polarization of diphenylhexatriene that seems to be an adequate indicator of the fluidity of the membrane.

In order to characterize Ca2+-transport in red cells stored in ACD Ca2+-loading and Mg2+-depletion by the ionophore A23187, CaATPase activity determination in intact cells and an accurate Ca2+-influx technique were adapted to preserved blood. Active Ca2+-efflux (pump) was measured in rejuvenated cells loaded by Ca2+ with A23187. The rate of Ca2+-pump declined only slightly during 3 weeks of storage (from 80 ± 15 to 66 ± 17 μmoles Ca2+/l. cells/min) and a marginal trend of decrease in the Ca:ATP ratio was observed (from 1.96 ± 0.15 to 1.88±0.11). Passive Ca2+-influx (leak) was studied in regenerated cells in which the Ca2+-pump was blocked with 0.2 mm lanthanum. Ca2+-influx showed a slow increase during the first 3 weeks of storage (from 0.4±0.16 to 1.25±0.4 μmoles Ca2+/l. cells/min), later it increased rapidly. Passive Ca2+ leak and exchange transport were studied in unregenerated, phosphate ester-depleted cells. In this case passive Ca2+-influx increased 2–3-fold as early as 1–2 d after storage in ACD. This initial increase was followed by a continuous slow enhancement that reached a flux of 3.5±0.7 μmoles Ca2+/l. cells/min after 3 weeks of storage. The passive Ca2+-permeability increase that occurred during storage could be readily compensated by the Ca2+-pump without causing metabolic imbalance. The Ca2+-transport, of unregenerated stored cells, however, showed impairment under certain conditions (A23187+EDTA and lanthanum treatments, ghost preparation). The Ca2+-induced shape changes were reversible and ran parallel with the cell Ca2+ level during Ca2+-pumping up to 5 weeks of storage. This finding indicates a direct relationship between cell Ca2+ and shape.

The regulation of Ca2+ concentration in different cells involves two Ca2+ pumps. The presence of such mechanisms in human platelets is still controverted. We then investigated this question by using plasma and intracellular membranes obtained after simultaneous isolation by centrifugation ca 40% sucrose from a mixed 100,000 g membrane fraction.The Ca2+ uptake by the different membrane vesicles has been studied. Both membrane fractions took up Ca2+ and the Ca2+ transport systems exhibited a high affinity towards Ca 2+.However, the two associated Ca2+ transport systems showed a different time course and exhibited different oxalate sensitivity. The plasma membranes are not permeable to potassium oxalate, while the Ca2+ uptake was stimulated by potassium oxalate in intracellular membranes.Two Ca2+ activated ATPase activities are associated with the isolated membrane fractions and appeared different for the following parameters : 1) a different time course of the two enzyme activities; 2)a similar apparent affinity towards Ca2+ (10−7 M), though inhibition of the Ca2+ ATPase activity was only observed in intracellular membranes at 10−6 M Ca2+ ; 3)a different pH dependence with a maximum at pH 7 for the enzyme of intracellular membranes and pH 8 for the enzyme of plasma membranes; 4)a 10 fold difference in the ATP requirement of the enzymes, thus the maximal response was obtained with 20 uM for the intracellular membrane enzyme and with 200 uM for the plasma membrane enzyme ; 5) a different affinity for various nucleotides as energy donors with a higher specificity of the plasma membrane enzyme towards ATP ; 6) a different vanadate inhibition-dose reponse which did not exceed 60% for the plasma enzyme while it reached 100% for the intracellular enzyme.Taken together, these studies agree with the possible role of both a plasma membrane and a dense tubular system Ca2+ -ATPases in the regulation of Ca2+ concentration in human platelets.