Effects of low-molecular-weight aluminum complexes on brain tissue calcium homeostasis

The in vitro effects of low-molecular-weight aluminum complexes (citrate, lactate, and ATP complex) on the Ca²⁺ uptake and aluminum-induced lipid peroxidation of brain tissue show that the modification of the calcium homeostasis is determined by the nature of the ligand and that there is no correlation between the aluminum-induced lipid peroxidation and the Ca²⁺ uptake. The same characteristics have been shown by a similar study performed with Ehrlich carcinoma cells. The electrophoretic analyses of the aluminum lactate-albumin and aluminum lactate-ATP interactions indicate an aluminum transfer from the lactate to the albumin and ATP ligands. The increased Ca²⁺ uptake when ATP is present in the incubation medium with aluminum citrate and aluminum lactate corroborates the suggested mediator role of ATP in cellular calcium homeostasis modification induced by iron.     

Stabilization of calcium uptake in rat rod outer segments by taurine and ATP

Summary. Calcium ion (Ca²⁺) uptake was measured in rod outer segments (ROS) isolated from rat retina in the presence of varying concentrations of CaCl₂ in the incubation buffer (1.0–2.5 mM). It is known that taurine increases Ca²⁺ uptake in rat ROS in the presence of ATP and at low concentrations of CaCl₂ (Lombardini, 1985a); taurine produces no significant effects when CaCl₂ concentrations are increased to 1.0 and 2.5 mM. With the removal of both taurine and ATP, Ca²⁺ uptake in rat ROS increased significantly in the presence of 2.5 mM CaCl₂. Taurine treatment in the absence of ATP was effective in decreasing Ca²⁺ uptake at the higher levels of CaCl₂ (2.0 and 2.5 mM). Similar effects were observed with ATP treatment. The data suggest that taurine and ATP, alone or in combination, limit the capacity of the rat ROS to take up Ca²⁺ to the extent that a stable uptake level is achieved under conditions of increasing extracellular Ca²⁺, indicating a protective role for both agents against calcium toxicity. Received January 25, 2000/Accepted January 31, 2000     

Cyclosporin a Increases Mitochondrial Calcium Uptake Capacity in Cortical Astrocytes but not Cerebellar Granule Neurons

Isolated brain mitochondria are a heterogeneous mixture from different cell types and these subsets may have differing sensitivities to Ca²⁺-induced membrane permeability transition (MPT) and to inhibition of the MPT by cyclosporin A (CsA). This study tested the hypothesis that mitochondria within primary cultures of astrocytes and neurons exhibit different energy-dependent Ca²⁺ uptake capacities and different degrees to which CsA increases their uptake capacity. Astrocytes and neurons were suspended in a cytosol-like medium containing respiratory substrates, ATP, and Mg²⁺ in the presence of digitonin to selectively permeabilize the plasma membrane. Uptake of added Ca²⁺ by mitochondria within the cells was measured by Calcium Green 5N fluorescent monitoring of the medium [Ca²⁺]. Permeabilized astrocytes had a fourfold higher Ca²⁺ uptake capacity, relative to neurons and a twofold higher content based on relative contents of mitochondria assessed by measurements of mitochondrial DNA and cytochrome oxidase subunit 1 protein. In astrocytes the Ca²⁺ uptake capacity was increased twofold by preincubation with 2–5 μM CsA, while in neurons CsA had no effect. Similar results were obtained using measurements of the effects of added Ca²⁺ on mitochondrial membrane potential. FK506, a drug similar to CsA but without MPT inhibitory activity, had no effect on either cell type. These results are consistent with the presence of a calcium-induced MPT in astrocytes, even in the presence of ATP, and indicate that the MPT in cerebellar granule neurons is resistant to CsA inhibition. Some of the protective effects of CsA in vivo may therefore be mediated by preservation of mitochondrial functional integrity within astrocytes.     

ATP in cellular calcium-overload by trivalent metal ions

Extracellular Ca²⁺-influx induced by trivalent metal ions (Fe³⁺, Al³⁺, Cr³⁺, In³⁺, Ga³⁺, and La³⁺) in Ehrlich carcinoma cells is enhanced by ATP. This action seems to be related to the high coordination capacity of the ATP ligand that inhibits the polymerization of the solvated cations taking place at physiological pH, and consequently permits their biological activity. A general relationship between induced lipid peroxidation and increased calcium uptake was not found. These results emphasize the ATP role in the toxicity of trivalent metals, and its possible involvement, via cellular calcium overload, in a neurodegenerative process, such as Alzheimer's and Parkinson's diseases, in whose etiology the implication of aluminum and iron has been suggested.     

Kinetic characterization and substrate requirement for the Ca2+ uptake system in platelet membrane

An ATP-dependent mechanism for Ca²⁺ uptake in human platelet membrane fractions has been identified and characterized. Ca²⁺ uptake into a membrane fraction is shown to be stimulated at low concentrations of ATP and Ca²⁺ and to require magnesium ions. Initial rate kinetics, using Eadie-Scatchard analysis, indicated a single class of calcium uptake sites in the presence of ATP, with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for free [Ca²⁺] of 0.145 μM. Ca²⁺ uptake in the presence of several ATP concentrations demonstrates that ATP binds to at least two sites, representing high and low affinities of 3.21 and 80.1 μM, respectively. The neuroleptic drug fluphenazine inhibited ATP-stimulated calcium uptake ($\text{IC}{}_{50}\text{= 55 μ}\text{M}$), suggesting this ATP-dependent Ca²⁺ uptake system may provide a useful ion-transport model with which to study neuroleptic therapy in humans.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles II. Ca2+ efflux in the energized state of the calcium pump

Ca²⁺ efflux from sarcoplasmic reticulum vesicles was studied by measurements of net Ca²⁺ uptake, ⁴⁵Ca²⁺ flux and hydrolysis of energy-rich phosphate. The maximal Ca²⁺ uptake capacity (150–200 nmol/mg protein at pH 6.7, 10 mM MgCl₂ and μ=0.26) was independent of the nature and concentration of the energy-donating substrate (ATP or carbamyl phosphate) and of temperature (15–35°C), suggesting coupling between influx and efflux of Ca²⁺. In the presence of high concentrations of ATP, this efflux of Ca²⁺ was much higher than the passive Ca²⁺ permeation, measured after ATP or Ca²⁺ depletion of the reaction medium. Ca²⁺ efflux was imperceptible at vesicle filling levels below 35–40 nmol Ca²⁺/mg protein, and uncorrelated to the inhibition of the Ca²⁺-ATPase by high intravesicular Ca²⁺ concentrations. Analysis of the data indicated that Ca²⁺ efflux under our conditions probably is associated with one of the Ca²⁺-ATPase partial reactions occurring after dephosphorylation, rather than with a reversal of the Ca²⁺ translocation step in the phosphorylated state of the enzyme. Furthermore, passive Ca²⁺ permeation may be concurrently reduced during the enzymatically active state. It is proposed that both Ca²⁺ efflux and passive Ca²⁺ permeation (Ca²⁺ outflow) proceed via the same channels which are closed (occluded) during part of the Ca²⁺-ATPase reaction cycle.     

Tri-Calciphor (16,16-dimethyl-15-dehydroprostagalndin B1 trimer)-mediated mitochondrial Ca2+ movements: modulation by phosphate

The trimeric derivative of 16,16-dimethyl-15-dehydroprostaglandin B₁ (termed tri-Calciphor), which protects tissues against ischemic damage, induced Ca²⁺ efflux and swelling in mitochondria in the absence of phosphate, Mg²⁺ and ATP. When glutamate/malate rather than succinate was the substrate, higher tri-Calciphor concentrations were required for the ionophoretic activity. Ca²⁺ efflux and mitochondrial swelling induced by tri-Calciphor were completely inhibited by ATP, phopsphate and Mg²⁺ added together, and partially inhibited with phosphate plus either ATP or Mg²⁺. Between 0 and 7 μM added Ca²⁺ and in the presence of phosphate, ATP and Mg²⁺, tri-Calciphor stimulated the uptake of Ca²⁺ by mitochondria and increased the efficiency of buffering of extramitochondrial Ca²⁺. Thus depending on the assay conditions, two different effects involving Ca²⁺ movements and mitochondria are observed with tri-Calciphor.     

Further Characterization of the Red Beet Plasma Membrane Ca-ATPase Using GTP as an Alternative Substrate

The GTP-driven component of Ca²⁺ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca²⁺-translocating ATPase and assess its utility as a probe for this transport system. Uptake of ⁴⁵Ca²⁺ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum, sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca²⁺ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca²⁺-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of ⁴⁵Ca²⁺-uptake by exogenous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven ⁴⁵Ca²⁺ uptake represents the capacity of the plasma membrane Ca²⁺-translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ-³²P]-GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca²⁺-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca²⁺-ATPases present in animal cells.     

Alkalinization within sarcoplasmic reticulum during the uptake of calcium ions.

The pH indicator, bromothymol blue, was incorporated into sarcoplasmic reticulum vesicles which bind more than 90% of the total added dye. The sequestered dye does not respond to changes in external pH upon addition of acid to the medium, since the decrease of absorbance at 616 nm is very slow. The absorbance of sequestered dye at 616 nm increases suddenly after triggering the transport of Ca²⁺ by ATP at a rate much higher than that of Ca²⁺ uptake, and declines when Ca²⁺ has been accumulated. When the uptake of Ca²⁺ is followed in the presence of oxalate, the absorbance of the indicator declines after the first phase of Ca²⁺ uptake. The results suggest that a transient alkalinization occurs rapidly inside the vesicles and reflects the formation of a transmembrane proton gradient responsible for sustaining the Ca²⁺ transport.     

Calcium uptake into acini from rat pancreas: Evidence for intracellular ATP-dependent calcium sequestration

Summary Intracellular ATP-dependent Ca²⁺ sequestration mechanisms were studied in isolated dispersed rat pancreatic acini following treatment with saponin or digitonin to disrupt their plasma membranes. In the presence of⁴⁵Ca²⁺ concentrations <10^–6 mol/liter, addition of 5 mmol/liter ATP caused a rapid increase in⁴⁵Ca²⁺ uptake exceeding the control by fivefold. ADP mimicked the ATP effect by 50 to 60%, whereas other nucleotides such as AMP-PNP, AMP-PCP, CTP, UTP, ITP, GTP, cAMP and cGMP did not. Maximal ATP-promoted Ca²⁺ uptake was obtained at 10^–5 mol/liter Ca²⁺ uptake by mitochondrial inhibitors was dependent on the Ca²⁺ concentration, indicating the presence of different Ca²⁺ storage systems. Whereas the apparent half-saturation constant found for mitochondrial Ca²⁺ uptake was 4.5×10^–7 mol/liter, in the presence of antimycin and oligomycin (nonmitochondrial uptake) it was 1.4×10^–8 mol/liter. In the absence of Mg²⁺ both ATP- and ADP-promoted Ca²⁺ uptake was nearly abolished. The Ca²⁺ ionophore and mersalyl blocked Ca²⁺ uptake. Electron microscopy showed electrondense precipitates in the rough endoplasmic reticulum of saponintreated cells in the presence of Ca²⁺, oxalate and ATP, which were absent in intact cells and in saponin-cells without ATP or pretreated with A23187. The data suggest the presence of mitochondrial and nonmitochondrial ATP-dependent Ca²⁺ storage systems in pancreatic acini. The latter is likely to be located in the rough endoplasmic reticulum.     

Uptake of calcium by microvillous membranes of intestinal epithelial cells

Uptake or “binding” of Ca²⁺ ions by microvillous membranes occurs in the absence or presence of ATP. In its absence uptake is independent of temperature (3–37°C), reduced by the presence of other cations and is dependent upon pH and the number and type of “binding” sites of which there are at least two classes. Under certain conditions uptake of Ca²⁺ is increased by ATP. The degree of stimulation is dependent upon both the concentration of Ca²⁺ and ATP, the time and temperature of incubation and is partly dependent upon the presence of Mg²⁺ ions.     

Identification and Characterization of the Ca-ATPase which Drives Active Transport of Ca at the Plasma Membrane of Radish Seedlings

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca²⁺ uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MI De Michelis [1987] Plant Physiol 83: 994-1000). A Ca²⁺-dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca²⁺-dependent ATPase is stimulated by the Ca²⁺ ionophore A₂₃₁₈₇ and is localized at the plasma membrane. Ca²⁺-dependent ATPase activity and ATP-dependent Ca²⁺ uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca²⁺ (half-maximal rate at about 70 nanomolar), and MgATP (K_m 15-20 micromolar). Ca²⁺ uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca²⁺ uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca²⁺-ATPase described in this paper is the enzyme which drives active transport of Ca²⁺ at the plasma membrane of higher plants.     

Further Characteristics of the ATP-Stimulated Uptake of Calcium into Chromaffin Granules

Abstract: The ATP-stimulated uptake of ⁴⁵Ca²⁺ [and [³H](-)-noradrenaline ([³H]NA)] into chromaffin granules and that into mitochondria are driven by a protonic gradient ΔμH⁺, composed of the components ΔpH (concentration gradient of protons) and ΔΨ(electrical potential difference). The granular ATPase pumps protons into the matrix (ΔpH inside acid, ΔΨ positive), but the mitochondrial ATPase ejects protons from the matrix (ΔpH alkaline, ΔΨ negative inside). To show different driving forces of uptake, the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ (and [³H]NA) into chromaffin granules was compared with the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into mitochondria (adrenomedullary or rat liver). In the presence of nitrate, the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into chromaffin granules is higher than in the presence of acetate, because the lyotropic anion nitrate stimulates the granular ATPase and increases ΔpH (acid inside). Compared with nitrate, the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into mitochondria is higher in the presence of the proton-carrying anion acetate, which, after permeation, provides protons for ejection by the ATPase. In the absence of ATP, a valinomycin-mediated potassium influx (ΔΨ inside positive) stimulates the granular uptake of [³H]NA, which has an electrogenic component, but not the granular uptake of ⁴⁵Ca²⁺, which is electroneutral. The electrogenic uptake of ⁴⁵Ca²⁺ into mitochondria is stimulated by a valinomycin-mediated potassium efflux (ΔΨ negative inside). The ATP-stimulated uptake of ⁴⁵Ca²⁺ into chromaffin granules is sensitive to ruthenium red, suggesting a carrier-mediated mechanism of uptake, and it is sensitive to atractyloside, indicating the simultaneous uptake of ATP. After collapse of ΔpH by ammonia, the ATP-stimulated uptake of ⁴⁵Ca²⁺ into chromaffin granules is abolished, but not that into mitochondria. In the presence of ammonia, the rate of the ATP-stimulated uptake of [³H]NA is very low, and an ATP-independent uptake of ⁴⁵Ca²⁺ into chromaffin granules is observed which is similar to the ATP-independent Ca²⁺/Na⁺ exchange at the granular membrane.     

Calcium uptake by placental plasma membrane vesicles

The Placental plasma membrane vesicles are capable of accumulating up to 190 mM Ca²⁺. This is 24-fold higher than the external Ca²⁺ concentration.This process is dependent on ATP hydrolysis by the placental Ca²⁺-ATPase.The $\text{P}{}_{\mathrm{i}}\text{Ca}$ ratio is dependent on the external Ca²⁺ concentration, and reaches the value of 2 at 10 mM Ca²⁺.Phosphate (5 mM) can double Ca²⁺ uptake when measured in the presence of 5 mM Ca²⁺.Mg²⁺; increased Ca²⁺ uptake only at low Ca²⁺ concentrations, and had no significant effect at 5 mM Ca²⁺.     

Regulation of Ca2+ homeostasis by glucose metabolism in rat brain

In a previous communication we reported that glucose deprivation from KHRB medium resulted in a marked stimulation of Ca²⁺ uptake by brain tissue, suggesting a relationship between glucose and Ca²⁺ homeostasis in brain tissue [17]. Experiments were carried out to investigate the significance of glucose in Ca²⁺ transport in brain cells. The replacement of glucose with either D-methylglucoside or 2-deoxyglucose, non-metabolizable analogues of glucose, resulted in stimulation of Ca²⁺ uptake just as by glucose deprivation. These data show that glucose metabolism rather than glucose transfer was necessary to stimulate Ca²⁺ uptake in brain tissue. Inhibition of glucose metabolism with either NaF, NaCN, or iodoacetate resulted in stimulation of Ca²⁺ uptake similar to that produced by glucose deprivation. These results lend further support for the concept that glucose metabolism is essential for Ca²⁺ homeostasis in brain. Anoxia promotes glucose metabolism through glycolytic pathway to keep up with the demand for ATP by cellular processes (the Pasteur effect). Incubation of brain slices under nitrogen gas did not alter Ca²⁺ uptake by brain tissue, as did glucose deprivation and the inhibitors of glucose metabolism. We conclude that glucose metabolism resulting in the synthesis of ATP is essential for Ca²⁺ homeostasis in brain. Verapamil and nifedipine which block voltage-gated Ca²⁺ channels, did not alter Ca²⁺ uptake stimulated by glucose deprivation, indicating that glucose deprivation-enhanced Ca²⁺ uptake was not mediated by Ca²⁺ channels. Tetrodotoxin which specifically blocks Na⁺ channels, abolished Ca²⁺ uptake enhanced by glucose deprivation, but had no effect on Ca²⁺ uptake in presence of glucose (controls). These results suggest that stimulation of Ca²⁺ uptake by glucose deprivation may be related to Na⁺ transfer via Na-Ca exchange in brain.     

Ca-dependent inhibitory effects of Na and K on Ca transport in sarcoplasmic reticulum vesicles

Effects of Na⁺ and K⁺ on Ca²⁺ transport by sarcoplasmic reticulum vesicles were studied in a medium containing high Mg²⁺ and ATP (2mM) and low Ca²⁺ (0.44μM) concentrations. Under these conditions, Na⁺ and K⁺ inhibit Ca²⁺ uptake. ATPase activity and membrane phosphorylation by ATP. Since the concentrations of ATP and Ca²⁺ used are consistent with relaxation in vivo, the results suggest that under physiological resting conditions the Ca²⁺ pump of the sarcoplasmic reticulum operates below its maximal capacity.     

Ca2+-dependent inhibitory effects of Na+ and K− on Ca2+ transport in sarcoplasmic reticulum vesicles

Effects of Na⁺ and K⁺ on Ca²⁺ transport by sarcoplasmic reticulum vesicles were studied in a medium containing high Mg²⁺ and ATP (2mM) and low Ca²⁺ (0.44μM) concentrations. Under these conditions, Na⁺ and K⁺ inhibit Ca²⁺ uptake. ATPase activity and membrane phosphorylation by ATP. Since the concentrations of ATP and Ca²⁺ used are consistent with relaxation in vivo, the results suggest that under physiological resting conditions the Ca²⁺ pump of the sarcoplasmic reticulum operates below its maximal capacity.     

H+/Ca2+ exchange in rabbit renal cortical endosomes

Summary We have examined the effect of second messengers on ATP-driven H⁺ transport in an H⁺ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1mm) had no effect on H⁺ transport. Acridine orange fluorescence in the presence of 0.5mm Ca²⁺ (+1mm EGTA) was 19±6% of control. Inhibition of ATP-driven H⁺ transport by Ca²⁺ was concentration dependent; 0.25 and 0.5mm Ca²⁺ (+1mm EGTA) inhibited acridine orange fluorescence by 50 and 80%, respectively. Ca²⁺ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca²⁺ did not affect ATP hydrolysis. ATP-dependent Br^– uptake was virtually unchanged in the presence of 0.5mm Ca²⁺ (+1mm EGTA). These vesicles were also shown to transport Ca²⁺ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca²⁺ uptake. These results are consistent with the co-existence of an H⁺ ATPase and an H⁺/Ca²⁺ exchanger on these endosomes, the latter transport system using the H⁺ gradient to energize Ca²⁺ uptake. Attempts to demonstrate an H⁺/Ca²⁺ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca²⁺ uptake could be demonstrated. A Ca²⁺-dependent increase in H⁺ permeability and an ATP-dependent Ca²⁺ uptake might have important implications for the regulation of vacuolar H⁺ ATPase activity as well as the homeostasis of cytosolic Ca²⁺ concentration.     

Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum

The Ca²⁺ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca²⁺ and another represented by Ca²⁺ selectively bound to the membranes. Both of these Ca²⁺ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca²⁺ fraction. The existence of the membrane-bound Ca²⁺ induced by ATP is clearly shown in experiments in which the Ca²⁺ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca²⁺ ionophore, which makes the membrane permeable to Ca²⁺. Thus, in the presence of X-537A all Ca²⁺ accumulated due to ATP is bound to the membranes. This membrane-bound Ca²⁺ represents about 30 nmol/mg protein in the range of external $\text{p}\text{Ca}$ values of 7 to 3.5. The magnitude of this Ca²⁺ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca²⁺ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca²⁺ as a distinct fraction from that which exists intravesicularly.