Taurine activation of a bicarbonate-dependent,ATP-supported calcium uptake in frog rod outer segments

Isolated frog rod outer segments (ROS) with a leaky plasma membrane showed a bicarbonate-dependent, ATP-activated⁴⁵Ca accumulation. This calcium uptake requires magnesium and is specific for ATP; Other nucleotides, ITP, GTP, UTP and the non-hydrolysable analogue of ATP --methylene ATP did not substitute for ATP.⁴⁵Ca accumulation was inhibited by mersalyl, ethylmaleimide, ruthenium red, oligomycin and dicyclohexylcarbodiimide and was unaffected by ouabain. Addition of taurine to the incubation medium enhanced⁴⁵Ca uptake in a concentration-dependent manner; increases of more than 100% being produced by 25 mM taurine. The taurine-induced stimulation of⁴⁵Ca uptake was also sensitive to the tested inhibitors. The effect of taurine was only exerted on the bicarbonate-dependent, ATP-activated⁴⁵Ca uptake. Calcium accumulation observed in the absence of ATP or in a tris-buffered medium was unaffected by taurine. Other amino acids, glycine, GABA, -alanine, glutamic acid and the taurine analogue guanidinoethyl-sulfonate did not stimulate⁴⁵Ca uptake. These results suggest that taurine is affecting a Mg-ATPase activity responsible for calcium accumulation in frog ROS.     

Adenosine Triphosphate-Dependent Calcium Uptake by Rat Submaxillary Gland Microsomes

Microsomes from rat submaxillary glands are able to take up calcium from the suspension media. Calcium uptake is greatly increased by the presence of ATP. This effect of ATP is not detected at 0°C. ADP cannot replace ATP to potentiate calcium uptake. ATP-dependent calcium uptake is not observed in the absence of magnesium. ATP-dependent calcium uptake is enhanced by oxalate and, to a lesser degree, by inorganic phosphate. Total calcium per milligram of microsomal protein observed when tests were performed without oxalate closely parallels the amounts for skeletal and cardiac muscles reported by several authors. Calcium uptake in salivary gland microsomes is slower than in muscle microsomes. Speculations are considered about the role of ATP-dependent calcium uptake. It is suggested that a decrease in intracellular free calcium levels returns these cells to the resting state after secretion.     

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     

Active transport of calcium in thymocytes]

Calcium uptake was studied on thymocytes from rabbits with 45Ca loading followed by rapid filtration. Datta shown that the substrates of the mitochondrial electron transport chain did not allowed calcium uptake. Exogenous ATP was required to observe a large influx of calcium in thymocytes. Succinate, magnesium and phosphate ions increased this ATP induced influx of calcium, which was not completely inhibited with atractyloside or ruthenium red.     

Calcium uptake and calcium release by subcellular fractions of smooth muscle. II. Kinetics of calcium uptake by microsomes and mitochondria from pig coronary artery and guinea pig ileum.

Calcium uptake by the microsomal and mitochondrial fractions of pig coronary artery and guinea pig ileum was studied in the presence of ATP, ATP plus oxalate and without ATP and oxalate. Microsomes and mitochondria of both smooth muscles were found to be unable to accumulate appreciable amounts of calcium in the absence of ATP. Oxalate noticeably stimulated the calcium uptake of the mitochondrial fraction from pig coronary artery but had little effect on calcium uptake by the microsomal fraction of this smooth muscle. The calcium uptake of microsomes and mitochondria from guinea pig ileum was not or only slightly enhanced by oxalate. There are typical kinetics regarding the time course and the extent of calcium uptake by microsomes and mitochondria from pig coronary artery and guinea pig ileum. In comparison, considerable qualitative and quantitative differences between both smooth muscles are observed. The high ATP-dependent calcium uptake capacity of the mitochondria from pig coronary artery and guinea pig ileum are a further argument for the hypothesis that these organelles may play an important role in the contraction-relaxation mechanism of smooth muscle.     

Opposing interactions of ionophores (valinomycin and monensin) on calcium ion uptake in rat retinal preparations

Valinomycin is a potent inhibitor of taurine-stimulated ATP-dependent calcium ion uptake in rat retinal membrane preparations but had no effect on ATP-dependent calcium ion uptake in the absence of taurine and no effect on ATP-independent calcium ion uptake. The presence of potassium ions in the buffer systems were required for valinomycin to be inhibitory. On the contrary, monensin stimulated calcium ion uptake in the ATP-dependent system but had no effect on ATP-independent calcium ion uptake. The crude retinal homogenate was also fractionated into various subcellular components. The fraction which contained photoreceptor cell synaptosomes (P₁) had a higher, specific activity for taurinestimulated ATP-dependent calcium ion uptake than the crude homogenate or either the fractions which contained synaptosomes derived from the plexiform layer (P₂) or rod outer segments (ROS). No differences in calcium ion uptake were observed in the various subcellular fractions compared to the homogenate when assayed for ATP-dependent calcium ion uptake. Valinomucin inhibited both ATP-dependent and taurine-stimulated ATP-dependent calcium ion uptake in the P₁, P₂, and ROS fractions while monensin stimulated the ATP-dependent calcium ion uptake in the subcellular fractions.     

Hormone sensitive calcium uptake by liver microsomes

The effects of glucagon and insulin on hepatic microsomal calcium uptake were investigated. Microsomes isolated from perfused rat liver accumulated calcium in the presence of ATP and oxalate. Addition of glucagon to the perfusate significantly increased calcium uptake by microsomes subsequently isolated. In contrast, addition of insulin to the perfusate resulted in a decreased microsomal calcium uptake and inhibition of the glucagon effect. Because the effects of glucagon and insulin on hepatic microsomal calcium uptake are opposite, as are the metabolic effects of these hormones, it is likely that the observed differences are of physiological importance.     

Phosphorylation from inorganic phosphate and ATP synthesis of sarcoplasmic membranes

The incorporation of inorganic phosphate in the fragmented sarcoplasmic membranes induced by the removal of calcium ions bound to high affinity binding sites at the cytoplasmic surface of the membranes gives rise to the formation of two species of phosphoenzyme. The properties of the phosphoproteins formed depend on the absence or the presence of a gradient of calcium ions across the membranes. The phosphoenzymes differ by the affinity of the protein for phosphate, the enthalpy of formation, the kinetics of phosphate incorporation, and by the sensitivity to ionophores and ADP. In the absence of a calcium gradient less than 0.5 nmol phosphoenzyme per mg protein are formed in media containing less than 5 mM phosphate at pH7 and 10 degrees C. Under the same conditions approximately 2 nmol of phosphoenzyme per mg protein are formed with an initial rate of 0.5 nmol mg-1-s-1 when a calcium gradient exists. When the gradient is abolished by the addition of the ionophore X537A, the level of phosphoprotein drops to the same value as observed in the absence of a gradient. On addition of ADP at concentrations increasing from 0.3 to 10 muM continuous ATP formation is activated to its maximum rate, and simultaneously, the level of phosphoprotein declines. These concentrations of ADP scarcely affect phosphoprotein formed in the absence of a gradient, the phosphoryl residue of which is displaced when the concentration of ADP exceeds 10 micrometer without the formation of an equivalent amount of ATP. Minimum mechanisms for the formation of gradient-independent and gradient-dependent phosphoprotein are discussed.     

Studies on the Germination of Pine Pollen (Pinus mugo) in vitro. II. Effects of Different Ions

Studies on the initial germination of pollen of Pinus mugo showed no significant influence of ions on O₂ uptake and uptake of ³²P-labelled phosphate. At the onset of tube growth O₂ uptake decreased in the absence of calcium. In inorganic media tube growth and ³²P uptake were reduced in the absence of calcium or boric acid. In the absence of calcium a requirement for magnesium was observed. When the medium was deprived of polyvalent ions with EDTA, growth and ³²P uptake ceased. The presence of calcium in the medium was found to be essential for the maintenance of the structural and functional integrity of the cell membranne. — The ion requirement was more pronounced when tube growth was stimulated with sucrose. Calcium, magnesium, boric acid, and nitrate (as nitrogen source) were essential constitutents of the medium. The stimulation due to calcium required either magnesium or boric acid. — A density effect was observed which can be related to diffusible substances from the pollen into the medium. This was not observed when calcium and magnesium were present in the medium. The phenomenon is explained as an enrichment of the medium with diffusible substances from non-germinated dead pollen. — Germination and the tube growth were found to be greatly dependent on a short period of equilibration of pollen at room temperature before sowing.     

Transport mechanism for calcium and phosphate in ram spermatozoa

Calcium uptake into ejaculated ram spermatozoa is highly enhanced by the addition of extracellular phosphate. Under identical conditions, extracellular calcium stimulates the uptake of phosphate by the cells. Both calcium and phosphate uptake are comparably inhibited by the sulfhydryl reagent mersalyl. The I50 was found to be 6.36 and 10.14 nmol mersalyl per mg protein for phosphate and calcium uptake, respectively. Calcium uptake is inhibited by mersalyl whether phosphate is present or not. Extracellular fructose causes a 5-fold increase in calcium uptake. When fructose and phosphate are present in the cell's medium, there is an additive effect, which indicates that two independent systems are involved in calcium transport into the cell. Ruthenium red, which blocks Ca2+ transport into the mitochondria, causes 70% and 95% inhibition of calcium uptake in the absence or in the presence of fructose, respectively. Ruthenium red does not affect phosphate uptake unless calcium was present in the incubation medium. The stimulatory effect of fructose upon calcium uptake can be mimicked by L-lactate and can be inhibited by the glycolytic inhibitor 2-deoxyglucose. Fructose and L-lactate stimulate mitochondrial respiration in a comparable way. Oligomycin, which inhibits mitochondrial ATP synthesis, does not inhibit Ca2+ uptake. This indicates that ATP is not involved in the mechanism by which mitochondrial respiration stimulates Ca2+ uptake. The calcium channel blocker, verapamil, inhibits Ca2+ uptake in the presence or absence of extracellular phosphate. The phosphate-dependent calcium transport mechanism is more sensitive to verapamil than is the phosphate-independent transporter. In summary, the data indicate that the plasma membrane of mammalian spermatozoa contains a calcium/phosphate symporter, a phosphate-independent calcium carrier and a calcium-independent phosphate carrier.     

Regulation of calcium transport in bovine spermatozoa

Calcium uptake into bovine epididymal spermatozoa is enhanced by introducing phosphate in the suspending medium (Babcock et al. (1975) J. Biol. Chem. 250, 6488-6495). This effect of phosphate is found even at a low extracellular Ca2+ concentrations (i.e., 5 microM) suggesting that phosphate is involved in calcium transport via the plasma membrane. Bicarbonate (2 mM) cannot substitute for phosphate, and a relatively high bicarbonate concentration (20 mM) causes partial inhibition of calcium uptake in absence of Pi. In the presence of 1-2 mM phosphate, 20 mM bicarbonate enhances Ca2+ uptake. The data indicate that the plasma membrane of bovine spermatozoa contains two carriers for Ca2+ transport: a phosphate-independent Ca2+ carrier that is stimulated by bicarbonate and a phosphate-dependent Ca2+ carrier that is inhibited by bicarbonate. Higher phosphate concentrations (i.e., 10 mM) inhibit Ca2+ uptake into intact cells (compared to 1.0 mM phosphate) and this inhibition can be relieved partially by 20 mM bicarbonate. This effect of bicarbonate is inhibited by mersalyl. Calcium uptake into the cells is enhanced by adding exogenous substrates to the medium. There is no correlation between ATP levels in the cells and Ca2+ transport into the cell. ATP levels are high even without added exogenous substrate and this ATP level is almost completely reduced by oligomycin, suggesting that ATP can be synthesized in the mitochondria in the absence of exogenous substrate. Calcium transport into the sperm mitochondria (washed filipin-treated cells) is absolutely dependent upon the presence of phosphate and mitochondrial substrate. Bicarbonate cannot support Ca2+ transport into sperm mitochondria. There is good correlation between Ca2+ uptake into intact epididymal sperm and into sperm mitochondria with the various substrates used. This indicates that the rate of calcium transport into the cells is determined by the rate of mitochondrial Ca2+ uptake and respiration with the various substrates.     

A study of calcium pump activity of lysosomes from rat renal cortex

Fractions rich in either primary or secondary lysosomes were prepared from rat renal cortex by differential centrifugation and evaluated for their capacity for net calcium uptake. No uptake was observed in the absence of ATP. A vigorous uptake did take place in the presence of ATP but it was largely prevented by azide and other inhibitors of mitochondrial calcium uptake, suggesting that it was attributable to contamination by mitochondria. Evidence was obtained for an inhibitory influence of the secondary lysosomal fraction on mitochondrial calcium uptake.     

A study of calcium pump activity of lysosomes from rat renal cortex.

Fractions rich in either primary or secondary lysosomes were prepared from rat renal cortex by differential centrifugation and evaluated for their capacity for net calcium uptake. No uptake was observed in the absence of ATP. A vigorous uptake did take place in the presence of ATP but it was largely prevented by azide and other inhibitors of mitochondrial calcium uptake, suggesting that it was attributable to contamination by mitochondria. Evidence was obtained for an inhibitory influence of the secondary lysosomal fraction on mitochondrial calcium uptake.     

Studies of mitochondrial calcium movements using chlorotetracycline

1. The association of calcium with isolated rat liver mitochondrial membranes under various metabolic conditions was monitored using the fluorescent chelate probe, chlorotetracycline. Chlorotetracycline fluorescence increased markedly during energized calcium uptake in the absence of a permeant anion. Uncoupler and a respiratory chain inhibitor caused a rapid decrease in chlorotetracycline fluorescence when added either before or after calcium. During calcium uptake experiments concentrations of calcium exceeding 100 μM caused a transient fluorescence increase followed by an extensive decrease in fluorescence.

2. Changes in the chlorotetracycline-associated fluorescence of the mitochondrial suspensions were correlated with the uptake of exogenous ⁴⁵Ca. A positive correlation was observed between fluorescence and energized ⁴⁵Ca uptake in the absence of permeant anions. Addition of the permeant anion, phosphate, caused an extensive decrease in chlorotetracycline fluorescence but an enhanced uptake of exogenous ⁴⁵Ca.

3. The interaction of endogenous mitochondrial calcium with the fluorescent chelate probe was studied under a number of experimental conditions using mitochondria labeled during preparation with ⁴⁵Ca. Endogenous ⁴⁵Ca was lost rapidly from the mitochondria upon treatment with uncoupler, antimycin A, and A23187. Potassium phosphate and EGTA had no effect on the endogenous calcium as measured by either the ⁴⁵Ca content of the mitochondria or the fluorescence of the probe.

4. Mitochondria treated with antimycin A lost most of their endogenous ⁴⁵Ca within 3 min; subsequent energization of the mitochondria resulted in a partial uptake of the released ⁴⁵Ca but caused nearly a complete return of the chlorotetracycline fluorescence to the original level. Addition of phosphate did not change the fluorescence level but resulted in an almost complete accumulation of the ⁴⁵Ca previously released.

5. Following this energized uptake of ⁴⁵Ca, EGTA, p-trifluoromethoxyphenyl hydrazone of carbonyl cyanide, A23187 and calcium chloride all caused a nearly complete loss of the ⁴⁵Ca from the mitochondria and, with the exception of calcium chloride, caused an extensive decrease in the fluorescence level. Hence, the apparent location and/or properties of the endogenous calcium in this rat liver mitochondrial system were altered significantly by manipulation of the energetic state of the mitochondrial membrane.     

Study of the mitochondrial phosphate carrier in the course of calcium phosphate accumulation: A requirement for Mg2+ and ADP of its sensitivity to thiol reagents

1. The accumulation of calcium phosphate driven by succinate oxidation is ADP-dependent. In its absence the accumulation stops after a short incubation time and the oxygen uptake is permanently stimulated. This uncoupled oxygen uptake is insensitive to the inhibitors of phosphate transport, like mersalyl and N-ethylmaleimide. When ADP plus Mg²⁺ are added to the medium, or when ADP is added in the initial presence of magnesium, the inhibitory action of the thiol reagents on oxygen uptake is re-established. ADP alone or Mg²⁺ alone are without any effect.2. Phosphate/phosphate exchange has been studied, in the absence of ADP, when calcium phosphate accumulation had stopped and oxygen uptake is uncoupled. Under these conditions the exchange process becomes insensitive to thiol reagents. Sensitivity is recovered solely in the presence of ADP plus Mg²⁺.3. When mitochondrial swelling is studied according to the method of Chappell, it also appears that the phosphate carrier loses it sensitivity to mersalyl in the absence of ADP, which confirms the data obtained with phosphate/phosphate exchange experiments. When ADP plus Mg²⁺ are added (or present), together with mersalyl, the action of the thiol inhibitor is recovered. ADP and magnesium are inactive separately. EGTA plus Mg²⁺ (but not EGTA plus ADP) may substitute for ADP plus Mg²⁺ in this process.4. A possible interaction between the magnesium binding site and the phosphate carrier is considered and discussed.     

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

ATP-dependent uptake of nitrate in Nostoc muscorum and inhibition by ammonium ions

A high rate of nitrate uptake was observed in Nostoc muscorum when cells were grown on elemental nitrogen as compared to that when they were grown on nitrate or ammonium. The uptake of nitrate was light dependent. However, supplementation with ATP (50 μM) stimulated nitrate uptake both in light and darkness. ADP, under similar conditions had no effect. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2-n-heptyl-4-hydroxyquinoline, (HOQNO) and KCN inhibitied nitrate uptake in light which could be partially reversed by adition of ATP. Inhibitiion by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler of photophosphorylation, was complete and could not be restored by the addition of ATP. N,N′-dicyclohexylcarbodiimide (DCCD), a specific inhibitor of ATPase, blocked nitrate uptake in the presence or absence of externally added ATP. Although no nitrate uptake was observed under anaerobic conditions in dark, addition of ATP resulted in uptake of nitrate, which was similar in magnitude to that observed under aerobic condition in the light, and was inhibited by DCCD. Ammonium ions inhibited the uptake of nitrate in the absence of ATP but in its presence there was simultaneous uptake of nitrate and ammonium ions. However, uptake of ammonium ions alone was not affected by presence or absence of ATP in the external medium. It was concluded that nitrate ion uptake was energy dependent and may be linked with a proton gradient which can be formed either by photophosphorylation or ATP hydrolysis.     

Uptake of aspartate aminotransferase into mitochondria in vitro depends on the transmembrane pH gradient.

1. The effects of various inhibitors of electron transport and of oxidative phosphorylation and the effects of ionophores on the uptake of native aspartate aminotransferase into mitochondria were investigated. 2. Both antimycin and cyanide completely inhibited the uptake of the enzyme. On the other hand, uptake was stimulated to ATP and by oligomycin; however, the stimulation by ATP is inhibited by oligomycin. 3. The effects of ionophores of the valinomycin type in media containing K+ ions depended on the conditions used. Valinomycin alone stimulated the uptake of the enzyme, but in the presence of phosphate ions uptake was abolished. Nonactin was without effect at a low K+ concentration, but was stimulatory at 100 mM-KCl. Gramicidin also stimulated the uptake process. 4. Nigericin completely abolished uptake of aspartate aminotransferase into mitochondria. 5. The uptake of te enzyme was decreased by 18% in the absence of inhibitors or ionophores when the external pH was increased from 6.9 to 7.6. 6. These results indicate that ATP is not directly involved in the uptake of aspartate aminotransferase into mitochondria, neither is there a requirement for a cation gradient. Rather the uptake depends on the maintenance of a pH gradient across the mitochondrial inner membrane.     

Regulatory role of phosphate and other anions in transport of ADP and ATP by Rickettsia prowazekii.

ADP and ATP were transported in Rickettsia prowazekii by an obligate exchange system without prior hydrolysis. The uptake of ATP and ADP by the obligate exchange system in R. prowazekii was dependent upon the anionic composition of the medium. The rate of transport of ATP was about three times greater than that of ADP in the absence of anions, and the rates of transport of both were about doubled by a variety of anions. However, phosphate anions were able to stimulate greatly the uptake of ADP so that in the presence of these anions, the uptake of ATP and that of ADP were about equal. Millimolar concentrations of anions were required to elicit the stimulation of ADP and ATP transport. The ADP-dependent efflux of ADP and ATP was also greatly stimulated by phosphate anions. The stimulation of ADP and ATP transport required that the anions be present in the external medium, as preincubation of the rickettsiae with phosphate anions was neither necessary nor sufficient. The competitive inhibition of ATP uptake by ADP required phosphate anions, indicating that phosphate anions increased the affinity of ADP for the transport system. The role of phosphate in the regulation of ATP and ADP exchange and its significance are discussed.     

Involvement of intracellular calcium in the phosphate efflux from mammalian nonmyelinated nerve fibers

Summary Phosphate efflux was measured as the fractional rate of loss of radioactivity from desheathed rabbit vagus nerves after loading with radiophosphate. The effects of strategies designed to increase intracellular calcium were investigated. At the same time, the exchangeable calcium content was measured using⁴⁵Ca. Application of calcium ionophore A23187 increased phosphate efflux in the presence of external calcium in parallel with an increase in calcium content. In the absence of external calcium, there was only a late, small increase in phosphate efflux. For nerves already treated with the calcium ionophore, the phosphate efflux was sensitive to small changes in external calcium, in the range 0.2 to 2mm calcium, whereas similar increases in calcium in absence of ionophore gave much smaller increases in phosphate efflux. Removal of external sodium (choline substitution) produced an initial increase in phosphate efflux followed by a fall. The initial increase in phosphate efflux was much larger in the presence of calcium, than in its absence. The difference was again paralleled by an increase in calcium content of the preparation, thought to be due to inhibition of Na/Ca exchange by removal of external sodium. Measurements of ATP content and ATP, ADP, phosphate and creatine phosphate ratios did not indicate significant metabolic changes when the calcium content was increased. Stimulation of phosphate efflux by an increase in intracellular calcium may be due to stimulation of phospholipid metabolism. Alternatively, it is suggested that stimulation of phosphate efflux is associated with the stimulation of calcium efflux, possibly by cotransport of calcium and phosphate.