Ca2+-ATPases (SERCA): energy transduction and heat production in transport ATPases

The sarcoplasmic reticulum Ca2+-ATPase is able to cleave ATP through two different catalytic routes. In one of them, a part of the chemical energy derived from ATP hydrolysis is used to transport Ca2+ across the membrane and part is dissipated as heat. In the second route, the hydrolysis of ATP is completed before Ca2+ transport and all the energy derived from ATP hydrolysis is converted into heat. The second route is activated by the rise of the Ca2+ concentration in the vesicle lumen. In vesicles derived from white skeletal muscle the rate of the uncoupled ATPase is several-fold faster than the rate of the ATPase coupled to Ca2+ transport, and this accounts for both the low Ca2+/ATP ratio usually measured during transport and for the difference of heat produced during the hydrolysis of ATP by intact and leaky vesicles. Different drugs were found to selectively inhibit the uncoupled ATPase activity without modifying the activity coupled to Ca2+ transport. When the vesicles are actively loaded, part of the Ca2+ accumulated leaks to the medium through the ATPase. Heat is either produced or released during the leakage, depending on whether or not the Ca2+ efflux is coupled to the synthesis of ATP from ADP and Pi.     

Uncoupled ATP hydrolysis and thermogenic activity of the sarcoplasmic reticulum Ca2+-ATPase: coupling effects of dimethyl sulfoxide and low temperature

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the energy derived from ATP hydrolysis. During catalysis, part of the energy is used to translocate Ca(2+) across the membrane, and part is dissipated as heat. At 35 degrees C the heat released during the hydrolysis of each ATP molecule varies depending on the formation of a Ca(2+) gradient across the membrane. With leaky vesicles (no gradient) the heat released varies between 9 and 12 kcal/mol of ATP cleaved, and with intact vesicles (gradient), the heat released increases to 20-24 kcal/mol of ATP. After Ca(2+) accumulation, 82% of the Ca(2+)-ATPase activity is not coupled to Ca(2+) transport, and the ratio between Ca(2+) transported and ATP cleaved is 0.3. The addition of 20% dimethyl sulfoxide (v/v) to the medium or decreasing the temperature from 35 to 20 degrees C abolishes the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient. This is accompanied by a simultaneous inhibition of the uncoupled ATPase activity and an increase of the Ca(2+)/ATP ratio from 0.3 to 1.3-1.4. It is concluded that the uncoupled Ca(2+)-ATPase is responsible for both the low Ca(2+)/ATP ratio measured during transport and the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient.     

Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.     

Ca(2+) release and heat production by the endoplasmic reticulum Ca(2+)-ATPase of blood platelets. Effect of the platelet activating factor.

Different sarco/endoplasmic reticulum Ca(2+)-ATPases isoforms are found in blood platelets and in skeletal muscle. The amount of heat produced during ATP hydrolysis by vesicles derived from the endoplasmic reticulum of blood platelets was the same in the absence and presence of a transmembrane Ca(2+) gradient. Addition of platelets activating factor (PAF) to the medium promoted both a Ca(2+) efflux that was arrested by thapsigargin and an increase of the yield of heat produced during ATP hydrolysis. The calorimetric enthalpy of ATP hydrolysis (DeltaH(cal)) measured during Ca(2+) transport varied between -10 and -12 kcal/mol without PAF and between -20 and -24 kcal/mol with 4 microM PAF. Different from platelets, in skeletal muscle vesicles a thapsigargin-sensitive Ca(2+) efflux and a high heat production during ATP hydrolysis were measured without PAF and the DeltaH(cal) varied between -10 and -12 kcal/mol in the absence of Ca(2+) and between -22 up to -32 kcal/mol after formation of a transmembrane Ca(2+) gradient. PAF did not enhance the rate of thapsigargin-sensitive Ca(2+) efflux nor increase the yield of heat produced during ATP hydrolysis. These findings indicate that the platelets of Ca(2+)-ATPase isoforms are only able to convert osmotic energy into heat in the presence of PAF.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37 degrees C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 microM CaCl2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca2+ uptake, a second phase of Ca2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca2+ influx of sarcoplasmic reticulum near steady state of Ca2+ uptake was measured by pulse labeling with 45Ca2+. The Ca2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca2+ uptake in normal medium, Ca2+ influx was balanced by Ca2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca2+ exchange rate at the first plateau of Ca2+ uptake was about half of that in normal medium. When the second phase of Ca2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 micrograms/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca2+ uptake was also observed. These data suggest that the Ca2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca2+ efflux, which subsequently stimulates Ca2+ exchange.     

Functional evidence of a transmembrane channel within the Ca2+ transport ATPase of sarcoplasmic reticulum.

Ca2+ efflux can be studied conveniently following dilution of sarcoplasmic reticulum (SR) vesicles preloaded with 45Ca2+ by active transport. The rates of efflux are highly dependent on ATPase substrates and cofactors (Pi, Mg2+, Ca2+ and ADP) in the efflux medium. On the other hand, phenothiazines stimulate efflux through a passive permeability channel with no coupled catalytic events. Efflux activation by manipulation of catalytically active ATPase ligands, as well as by the catalytically inactive phenothiazines, can be prevented by thapsigargin, which is a highly specific inhibitor of the Ca(2+)-ATPase. This demonstrates that the passive channel activated by phenothiazines is an integral part of the ATPase, and can operate either uncoupled or coupled to catalytic events.     

Quercetin interaction with the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin.     

Magnesium permeability of sarcoplasmic reticulum. Mg2+ is not countertransported during ATP-dependent Ca2+ uptake by sarcoplasmic reticulum

Magnesium transport across sarcoplasmic reticulum (SR) vesicles was investigated in reaction mixtures of various composition using antipyrylazo III or arsenazo I to monitor extravesicular free Mg2+. The half-time of passive Mg2+ efflux from Mg2+-loaded SR was 100 s in 100 mM KCl, 150 S in 100 mM K gluconate, and 370 S in either 100 mM Tris methanesulfonate or 200 mM sucrose solutions. The concentration and time course of Mg2+ released into the medium was also measured during ATP-dependent Ca2+ uptake by SR. In reaction mixtures containing up to 3 mM Mg2+, small changes in free magnesium of 10 microM or less were accurately detected without interference from changes in free Ca2+ of up to 100 microM. Three experimental protocols were used to determine whether the increase of free [Mg2+] in the medium after an addition of ATP was due to Mg2+ dissociated from ATP following ATP hydrolysis or to Mg2+ translocation from inside to outside of the vesicles. 1) In the presence of ATP-regenerating systems which maintained constant ATP to ADP ratios and normal rates of active Ca2+ uptake, the increase of Mg2+ in the medium was negligible. 2) Mg2+ released during ATP-dependent Ca2+ uptake by SR was similar to that observed during ATP hydrolysis catalyzed by apyrase, in the absence of SR. 3) In SR lysed with Triton X-100 such that Ca2+ transport was uncoupled from ATPase activity, the rate and amount of Mg2+ release was greater than that observed during ATP-dependent Ca2+ uptake by intact vesicles. Taken together, the results indicate that passive fluxes of Mg2+ across SR membranes are 10 times faster than those of Ca2+ and that Mg2+ is not counter-transported during active Ca2+ accumulation by SR even in reaction mixtures containing minimal concentrations of membrane permeable ions that could be rapidly exchanged or cotransported with Ca2+ (e.g. K+ or Cl-).     

Calcium modulates the ATP and ADP hydrolysis in human placental mitochondria

This study evaluated the effect of Ca2+ on the extramitochondrial hydrolysis of ATP and ADP by the extramitochondrial ATPase in isolated mitochondria and submitochondrial particles (SMPs) from human term placenta. The effect of different oxidizable substrates on the hydrolysis of ATP and ADP in the presence of sucrose or K+ was evaluated. Ca2+ increased phosphate release from ATP and ADP, but this stimulation showed different behavior depending on the oxidizable substrate present in the incubation media. Ca2+ stimulated the hydrolysis of ATP and ADP in the presence of sucrose. However, Ca2+ did not stimulate the hydrolysis of ADP in the medium containing K+. Ca2+ showed inhibition depending on the respiratory substrate. This study suggests that the energetic state of mitochondria controls the extramitochondrial ATPase activity, which is modulated by Ca2+ and respiratory substrates.     

Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+

When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions.     

Interactions of physiological ligands with the Ca pump and Na/Ca exchange in squid axons

We have studied the interaction of physiological ligands other than Nai and Cai with the Ca pump and Na/Ca exchange in internally dialyzed squid axons. The results show the following. (a) Internal Mg2+ is an inhibitor of the Nao-dependent Ca efflux. At physiological Mg2+i (4 mM), the inhibition amounts to approximately 50%. The inhibition is partial and noncompetitive with Cai, and is not affected by Nai or ATP. The ATP-dependent uncoupled efflux is unaffected by Mgi up to 20 mM. Both components of the Ca efflux require Mg2+i for their activation by ATP. (b) At constant membrane potential, Ki is an important cofactor for the uncoupled Ca efflux. (c) Orthophosphate (Pi) activates the Nao-dependent Ca efflux without affecting the uncoupled component. Activation by Pi occurs only in the presence of Mg-ATP or hydrolyzable ATP analogues. Pi under physiological conditions has no effect on the uncoupled component; nevertheless, at alkaline pH, it inhibits the Ca pump, probably by product inhibition. (d) ADP is a potent inhibitor of the uncoupled Ca efflux. The Nao-dependent component is inhibited by ADP only at much higher ADP concentrations. These results indicate that (a) depending on the concentration of Ca2+i, Na+i Mg2+i, and Pi, the Na/Ca carrier can operate under a low- or high-rate regime; (b) the interactions of Mg2+i, Pi, Na+i, and ATP with the carrier are not interdependent; (c) the effect of Pi on the carrier-mediated Ca efflux resembles the stimulation of the Nao-dependent Ca efflux by internal vanadate; (d) the ligand effects on the uncoupled Ca efflux are of the type seen in the Ca pump in red cells and the sarcoplasmic reticulum.     

ATP-Mg/Pi carrier activity in rat liver mitochondria.

The ATP-Mg/Pi carrier in liver mitochondria is activated by micromolar Ca2+ and mediates net adenine nucleotide transport into and out of the mitochondrial matrix. The purpose of this study was to characterize certain features of ATP-Mg/Pi carrier activity that are essential for understanding how the mitochondrial adenine nucleotide content is regulated. The relative importance of ATP and ADP as transport substrates was investigated using specific trap assays to measure their separate rates of carrier-mediated efflux with Pi as the external counterion. Under energized conditions ATP efflux accounted for 88% of total ATP+ADP efflux. With oligomycin present to lower the matrix ATP/ADP ratio, ATP efflux was eliminated and ADP efflux was relatively unaffected. Mg2+ was stoichiometrically required for ATP influx and is probably transported simultaneously with ATP. Ca2+ and Mn2+ could substitute for the stoichiometric Mg2+ requirement. ADP influx and Pi-induced adenine nucleotide efflux were unaffected by external Mg2+. Experiments with Pi analogues suggested that Pi is transported as the divalent anion, HPO4(2-). The results show that ATP-Mg and divalent Pi are the major transport substrates; the most probable transport mechanism for the ATP-Mg/Pi carrier is an electroneutral exchange. The results are consistent with the hypothesis that the direction and magnitude of net adenine nucleotide movements are determined mainly by the (ATP-Mg)2- and HPO4(2-) concentration gradients across the inner mitochondrial membrane.     

Role of Mg2+ in the Ca2+-Ca2+ exchange mediated by the membrane-bound (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum vesicles were preloaded with either 45Ca2+ or unlabeled Ca2+. The unidirectional Ca2+ efflux and influx, together with Ca2+-dependent ATP hydrolysis and phosphorylation of the membrane-bound (Ca2+, Mg2+)-ATPase, were determined in the presence of ATP and ADP. The Ca2+ efflux depended on ATP (or ADP or both). It also required the external Ca2+. The Ca2+ concentration dependence of the efflux was similar to the Ca2+ concentration dependences of Ca2+ influx, Ca2+-dependent ATP hydrolysis, and phosphoenzyme formation. The rate of the efflux was approximately in proportion to the concentration of the phosphoenzyme up to 10 microM Ca2+. These results and other findings indicate that the Ca2+ efflux represents the Ca2+-Ca2+ exchange (between the external medium and the internal medium) mediated by the phosphoenzyme. In the range of 0.6-5.2 microM Mg2+, no appreciable Ca2+-Ca2+ exchange was detected although phosphoenzyme formation occurred to a large extent. Elevation of Mg2+ in the range 5.2 microM-4.8 mM caused a remarkable activation of the exchange, whereas the amount of the phosphoenzyme only approximately doubled. The kinetic analysis shows that this activation results largely from the Mg2+-induced acceleration of an exchange between the bound Ca2+ of the phosphoenzyme and the free Ca2+ in the internal medium. It is concluded that Mg2+ is essential for the exposure of the bound Ca2+ of the phosphoenzyme to the internal medium.     

Fast efflux of Ca2+ mediated by the sarcoplasmic reticulum Ca2(+)-ATPase.

The Ca2(+)-ATPase found in the light fraction of sarcoplasmic reticulum vesicles can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. This reaction was inhibited by the phenothiazines trifluoperazine, chlorpromazine, imipramine, and fluphenazine and by the beta-adrenergic blocking agents propranolol and alprenolol. The inhibition was reversed by raising either the Pi or the Mg2+ concentration in the medium and was not affected by the presence of K+. Phosphorylation of the Ca2(+)-ATPase by Pi was also inhibited by ruthenium red and spermidine. These compounds compete with Mg2+, but, unlike the phenothiazines, they did not compete with Pi at the catalytic site, and the inhibition was abolished when K+ was included in the assay medium. The efflux of Ca2+ from loaded vesicles was greatly increased by the phenothiazines and by propranolol and alprenolol. In the presence of 200 microM trifluoperazine, the rate of Ca2+ efflux was higher than 3 mumol of Ca2+/mg of protein/10 s. The activation of efflux by these drugs was antagonized by Pi, Mg2+, K+, Ca2+, ADP, dimethyl sulfoxide, ruthenium red, and spermidine. The increase of Ca2+ efflux caused by trifluoperazine was not correlated with binding of the drug to the membrane lipids. It is concluded that the Ca2+ pump can be uncoupled by different drugs, thereby greatly increasing the efflux of Ca2+ through the ATPase. Displacement of these drugs by the natural ligands of the ATPase blocks the efflux through the uncoupled pathway and limits it to a much smaller rate. Thus, the Ca2(+)-ATPase can operate either as a pump (coupled) or as a Ca2+ channel (uncoupled).     

Coupling of ATP synthesis to reversal of rat liver microsomal Ca2+-ATPase

The reversal of the rat liver microsomal Ca2+-ATPase transport cycle was studied. Microsomes were loaded with 45Ca2+ (approximately 30 nmol/mg of protein) in an ATP-dependent process, and the time dependency of the microsomal 45Ca2+ efflux was determined with various ADP and inorganic phosphate (Pi) concentrations. Pseudo-first-order rate constants (K'e) for 45Ca2+ efflux were determined. Although there was considerable 45Ca2+ efflux in the absence of added ADP or Pi, the addition of ADP or Pi alone had minimal effects upon the K'e; in contrast, a 2.5-fold increase in the K'e was observed in the presence of both ADP and Pi. The apparent Km values for ADP and Pi were 4 microM and 0.22 mM, respectively. Stimulation of 45Ca2+ efflux by ADP and Pi was associated with ATP synthesis. The calcium ionophore A23187 prevented ATP synthesis, which indicates that the Ca2+ gradient facilitates the coupling of ATP synthesis to Ca2+ efflux.     

Glucose 6-phosphate and hexokinase can be used as an ATP-regenerating system by the Ca(2+)-ATPase of sarcoplasmic reticulum.

In the presence of hexokinase, vesicles derived from the sarcoplasmic reticulum of skeletal muscle are able to accumulate Ca2+ in a medium containing ADP and glucose 6-phosphate. No significant Ca2+ uptake is observed if one of these components is omitted from the assay medium. Due to its high affinity for ATP, the Ca(2+)-ATPase can use the very low concentrations of ATP formed from glucose 6-phosphate and ADP to form a Ca2+ gradient. This finding indicates that glucose 6-phosphate and hexokinase can be used as an ATP-regenerating system. The Ca2+ uptake supported by glucose 6-phosphate and ADP is inhibited by glucose and D-xylose. Half-maximal inhibition is observed in the presence of 0.4 mM glucose and 100 mM D-xylose. The transport ratio (Ca2+ transported:substrate utilized) is the same for glucose 6-phosphate and ATP. The Ca2+ gradient formed when glucose 6-phosphate and ADP are the substrates can be used to synthesize ATP from ADP and Pi. The concentration of ATP formed after reversal of the Ca2+ pump is much higher than that expected from direct equilibration of the reaction between glucose 6-phosphate and ADP.     

Brown adipose tissue Ca2+-ATPase: uncoupled ATP hydrolysis and thermogenic activity

In this report a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) was identified in rats brown adipose tissue. Electrophoretic analysis of brown fat microssomal protein yields a 110-kDa band that is reactive to SERCA 1 antibody but is not reactive to SERCA 2 antibodies. Nevertheless, the kinetics properties of the brown fat SERCA differ from the skeletal muscle SERCA 1 inasmuch they manifest a different Ca2+ affinity and a much higher degree of ATPase/Ca2+ uncoupling. A SERCA enzyme is not found in white fat. Fatty acids promoted Ca2+ leakage from brown fat vesicles. The heat released during ATP hydrolysis was -24.7 kcal/mol when a Ca2+ gradient was formed across the vesicles membrane and -14.4 kcal/mol in the absence of a gradient. The data reported suggest that in addition to storing Ca2+ inside the endoplasmic reticulum, the Ca2+-ATPase may represent a source of heat production contributing to the thermogenic function of brown adipose tissue.     

The enhancement of Ca2+ efflux from sarcoplasmic reticulum vesicles by urea.

Urea, in nondenaturing concentrations, inhibited Ca2+ uptake by sarcoplasmic reticulum vesicles with no concomitant effect on ATP hydrolysis. This inhibition was antagonized by 5 mM oxalate and 20 mM orthophosphate. At concentrations of 0.2 to 1.0 M, urea induced an increase in the Ca2+ efflux from preloaded vesicles diluted in a medium at pH 7.0 containing 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid, 0.1 mM orthophosphate, and 0.1 mM MgCl2. The urea-induced efflux was arrested by ligands of the (Ca(2+)-Mg2+) ATPase, namely, K+, Mg2+, Ca2+, and ADP, and by ruthenium red and the polyamines spermine, spermidine, and putrescine. In the case of polyamines a dissociation between the effect on the efflux and the net Ca2+ uptake was observed, as only the efflux could be blocked by the drugs. Glycine betaine, trimethylamine-N-oxide, and sucrose antagonized the effects of urea on both the net Ca2+ uptake and the rate of Ca2+ efflux.