The sarcoplasmic calcium pump — A most efficient ion translocating system

In contrast to the sodium-potassium transporting plasma membranes, the sarcoplasmic membranes (SR) are highly specialized structures into which only two major intrinsic proteins, a calcium transporting protein and a calcium binding protein are embedded. The calcium transporting protein is a highly asymmetric molecule. It binds two calcium ions with a very high affinity at its external, and two calcium ions with low affinity at the internal section of the molecule. ATP is bound with high affinity to an external binding site, inducing a conformational change. When the vesicular membranes are exposed to solutions containing Ca⁺⁺, Mg⁺⁺ and ATP, ATP is hydrolyzed and simultaneously calcium ions are translocated from the external medium into the vesicular space. When calcium ions are translocated in the opposite direction, ATP is synthesized. The calcium-ATP ratio for ATP cleavage as well as for ATP synthesis is 2. Thus, the SR membranes can transform reversibly chemical into osmotical energy. Inward and outward movements of calcium ions are relatively slow processes connected with the appearance and disappearance of different phosphorylated intermediates. One phosphorylated intermediate is formed by phosphoryltransfer from ATP when calcium ions are present in the medium. In contrast, when calcium ions are absent from the external medium, two different intermediates can be formed by the incorporation of inorganic phosphate. Only when calcium ions present in the internal space of the vesicles are released, the incorporation of inorganic phosphate gives rise to an intermediate whose phosphoryl group can be transferred to ADP.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Effect of acylphosphates on Ca2+ uptake by sarcoplasmic reticulum vesicles

The data presented in this paper concern a kinetic study of the calcium uptake by sarcoplasmic reticulum vesicles and of the hydrolysis of the substrates which support the process. The results show that substrates which are different from ATP, acetylphosphate, and carbamylphosphate are able to support calcium transport. The technique used to follow the process allows us to detect continuously the changes in the concentration of the calcium present in the external medium. In our experimental conditions the calcium uptake supported by all the high energy substrates tested proceeds for several seconds at a constant rate, presumably corresponding to the “steady state” of the process; furthermore the calcium transport is clearly Ca²⁺ and Mg²⁺ dependent: the lowering of the Ca⁺ concentration in the medium from 10^?4 to 10^?5m causes a remarkable reduction of the V of the calcium transport and an apparent increase of the affinity of the sarcoplasmic reticulum vesicles for the acylphosphates; in the absence of Mg²⁺, none of the substrates is able to support the calcium uptake which increases in the presence of rising amounts of Mg²⁺ in the reaction medium. Furthermore, both the calcium transport and the substrate hydrolysis appear to follow the Michaelis-Menten kinetics in the presence of acylphosphates but not in the presence of ATP. The hydrolytic activity of sarcoplasmic reticulum vesicles on ATP and acylphosphates reveals a clear Mg²⁺ dependence; furthermore, in the absence of free Ca²⁺ and in the presence of 5 mm Mg²⁺, the high energy substrates tested reveal a different susceptibility to the hydrolitic attack by sarcoplasmic reticulum vesicles.     

Calcium pool size modulates the sensitivity of the ryanodine receptor channel and calcium-dependent ATPase of heavy sarcoplasmic reticulum to extravesicular free calcium concentration

We have examined calcium cycling and associated ATP consumption by isolated heavy sarcoplasmic reticulum (HSR) vesicles incubated in conditions believed to exist in resting muscle. Our goals were to estimate the magnitude of calcium cycling under those conditions and identify the main mechanisms involved in its regulation. The integrity of the HSR vesicles was documented by the retention of [¹⁴C]-sucrose and electron microscopy. HSR actively exchanged Ca²⁺ with the medium through a partially open ryanodine-binding channel (RyR), as evidenced by the rapid attainment of a steady-state gradient between HSR and medium, which was promptly increased by the closure of the channel with ruthenium red (RR) or collapsed by its opening with caffeine. The ATP dependency was evidenced by the sustained ATP consumption after the steady state was attained and by the abrogation of the gradient following inhibition of the pump with thapsigargin (Tg) or the omission of ATP. When HSR vesicles were incubated in a comparatively large pool of calcium (≈1 μmol/mg HSR protein), ATP consumption was 1–1.5 μmol × [min × mg protein]⁻¹ at 0.1 μM free Ca²⁺. Under such conditions, the main regulator of the sarcoplasmic Ca²⁺-dependent ATPase (SERCA) was extravesicular-free Ca²⁺ concentration, with a four- to fivefold increase between 0.1 and 2 μM Ca²⁺, whereas RyR channel activity and the replenishment of the HSR vesicles had only a modest effect on ATP consumption. When calcium pool size was reduced to 0.1 μmol/mg HSR protein, a steady state was established at a lower level of HSR calcium. In spite of a slightly lower free extravesicular Ca²⁺ at equilibrium (≈0.07 μM following an initial concentration of 0.1 μM), both ATP consumption and the open probability of the RyR channel were increased by a factor of three to five. Compared to the large calcium pool, the sensitivity of both RyR channel and SERCA to extravesicular free Ca²⁺ concentration as well as to caffeine and RR was markedly enhanced. Conclusions: (1) In conditions present in resting muscle, HSR calcium is in dynamic equilibrium with the medium through a partially open RyR channel, which requires continuous ATP hydrolysis. (2) The availability of calcium is a major determinant of the sensitivity of both RyR channel and SERCA to free extravesicular Ca²⁺ and possibly other stimuli. (3) These observations are consistent with the concept that calcium cycling in resting muscle may account for a significant fraction of muscle energy demands and further suggest that restricting calcium availability may enhance the energetic demands of this process. J. Cell. Physiol. 175:283–294, 1998. © 1998 Wiley-Liss, Inc.     

The effect of ATP on calcium efflux in dialyzed barnacle muscle fibres

Calcium efflux has been studied in barnacle muscle fibres under internal dialysis conditions. Prolonged dialysis of these fibres, with a medium free of ATP and containing 2 mM cyanide and 1 mM iodoacetate, causes the ATP in the perfusion effluent to fall to less than 20 μM. The mean calcium efflux from fibres dialyzed with EGTA buffered solution containing 0.3 μM ionized Ca and no ATP is 0.6 pmol · cm⁻² · s⁻¹. A two-fold stimulation of the calcium efflux is observed when ATP is added to fibres previously dialyzed with an ATP-free medium. Withdrawal of Na⁺ and Ca²⁺ from the external medium causes a marked drop in the Ca²⁺ efflux in the presence of internal ATP.     

The effect of calcium ionophore A23187 on the ATP level of human erythrocytes

Incubation of red cells at 37° with the ionophore A23187 results in a loss of ATP that is dependent on the concentrations of A23187 and Ca²⁺ in the medium. ATP hydrolysis is greatest at micromolar concentrations of Ca²⁺ and decreases as Ca²⁺ in the medium is raised to millimolar levels. The ATP depletion is due to stimulation of calcium ATPase by A23187-mediated Ca²⁺ influx into the cell. The biphasic nature of Ca²⁺-stimulated ATP depletion in whole cells reflects the activity of Ca²⁺-ATPase in membrane preparations at varying Ca²⁺ concentrations. The ionophore can be removed by washing the cells with plasma or bovine serum albumin-containing medium and the ATP levels restored to normal by reincubating with 5 mM adenosine for 1 hr.     

A permeability change of myelin membrane vesicles towards cations is induced by MgATP but not by phosphorylation of myelin basic protein

The existence of an endogenous protein kinase activity and protein phosphatase activity in myelin membrane from mammalian brain has now been well established. We found that under all conditions tested the myelin basic protein is almost the only substrate of the endogenous protein kinase in myelin of bovine brain. The protein kinase activity is stimulated by Ca²⁺ in the micromolar range. Optimal activity is reached at a free Ca²⁺ concentration of about 2 μM. Myelin membrane vesicles were prepared and then shown to be sealed by a light-scattering technique. After preloading with ⁴⁵Ca²⁺, ⁸⁶Rb⁺, or ²²Na⁺, the self-diffusion (passive outflux) of these ions from myelin membrane vesicles was measured. Ionophores induced a rapid, concentration-dependent outflux of 80–90% of the cations, indicating that only a small fraction of the trapped ions was membrane bound. There was no difference in the diffusion rates of the three cations whether phosphorylated (about 1 mol phosphate per myelin basic protein) or non-phosphorylated vesicles were tested. In contrast, a small but significant decrease in permeability for Rb⁺ and Na⁺ was measured, when the vesicles were pretreated with ATP and Mg²⁺.     

Schwann Cells Exhibit P2Y Purinergic Receptors that Regulate Intracellular Calcium and Are Up-Regulated by Cyclic AMP Analogues

Abstract: Schwann cells establish close contact with axons during development, and this is maintained throughout life. Signaling by neurotransmitters may play an important role in Schwann cell-axon interaction. Schwann cells were examined for the presence of neuroligand receptors that are linked to increases in levels of cytoplasmic calcium. Schwann cell cultures were prepared from neonatal rat sciatic nerve and, after 0.25, 1, 4, 7, and 14 days in vitro (DIV), loaded with the calcium indicator dye fura 2-AM. The influence of neuroligands on the cytosolic free calcium concentration ([Ca²⁺]_i) was then examined at each time point using a video-based imaging system. Approximately 80–95% of all freshly isolated Schwann cells responded to 10 µM ATP with a three-fold rise in [Ca²⁺]_i. Bradykinin, glutamate, and histamine had no or only partial and inconsistent responses. The ATP-induced calcium response disappeared within 4 DIV. Culturing cells in the presence of cyclic AMP (cAMP) analogues (which induce proliferation and differentiation in vitro) restored the ability of Schwann cells to respond to ATP with increased [Ca²⁺]_i. In the presence of cAMP analogues the extent of recovery of ATP responsiveness was dependent on serum concentration. Fifty to ninety percent of cells regained calcium responsiveness to ATP when grown in medium containing cAMP analogues and 1% serum. These cells also exhibited immunoreactivity to P₀ antibody, characteristic of the myelinating lineage. In contrast, only 15–30% of the Schwann cells regained calcium responsiveness when grown in medium containing cAMP analogues and 10% serum. Under these conditions all Schwann cells exhibited immunoreactivity to antibodies against nerve growth factor receptor, characteristic of the nonmyelinating lineage, although some also contained galactocerebroside immunoreactivity. The correlation between the recovery of the ATP response and the recovery of stage-specific markers suggests that Schwann cell ATP receptor expression may be a developmental process, preferentially associated with Schwann cells moving toward the myelinating lineage.     

Purinergic agonist ATP is a comitogen in thyroid FRTL-5 cells

Several growth factors may stimulate proliferation of thyroid cells. This effect has, in part, been dependent on calcium entry. In the present study using FRTL-5 cells, we show that in addition to its effect on calcium fluxes, ATP acts as a comitogen in these cells. In medium containing 5% serum, but no TSH, ATP stimulated the incorporation of ³H-thymidine in a dose- and time-dependent manner in the cells. At least a 24-h incubation with ATP was necessary to observe the enhanced (30–50%) incorporation of ³H-thymidine and an increased (30%) cell number. The effect of ATP was dependent on insulin in the incubation medium. Furthermore, ATP enhanced the TSH-mediated incorporation of ³H-thymidine. The effect of ATP was apparently mediated via a G-protein dependent mechanism, as no stimulation of thymidine incorporation was observed in cells treated with pertussis toxin. The effect of ATP was not dependent on the activation of protein kinase C (PKC), as ATP was effective in cells with downregulated PKC. ATP rapidly phosphorylated mitogen activated protein (MAP) kinase in FRTL-5 cells. In addition, ATP stimulated the expression of a 62 kDa c-fos dependent protein in a dose- and time-dependent manner. Our results thus suggest that extracellular ATP, in the presence of insulin, may be a cofactor in the regulation of thyroid cell proliferation, probably by phosphorylating MAP kinase and stimulating the expression of c-fos. © 1996 Wiley-Liss, Inc.     

Calcium Efflux from Internally Dialyzed Squid Giant Axons

Calcium efflux has been studied in squid giant axons under conditions in which the internal composition was controlled by means of a dialysis perfusion technique. The mean calcium efflux from axons dialyzed with 0.3 µM calcium and 5 mM ATP was 0.26 pmol/cm²·s at 22°C. The curve relating the Ca efflux with the internal Ca concentration had a slope of about one for [Ca]_i lower than 0.3µM and a slope smaller than one for higher concentrations. Under the above conditions replacement of [Na]_o and [Ca]_o by Tris and Mg causes an 80% fall in the calcium efflux. When the axons were dialyzed with a medium free of ATP and containing 2 mM cyanide plus 5µg/ml oligomycin, analysis of the perfusion effluent gave values of 1–4 µM ATP. Under this low ATP condition, replacement of external sodium and calcium causes the same drop in the calcium efflux. The same effect was observed at higher [Ca]_i, (80 µM). These results suggest that the Na-Ca exchange component of the calcium efflux is apparently not dependent on the amounts of ATP in the axoplasm. Axons previously depleted of ATP show a significant transient drop in the calcium efflux when ATP is added to the dialysis medium. This effect probably represents the sequestering of calcium by the mitochondrial system. The consumption of calcium by the mitochondria of the axoplasm in dialyzed axons was determined to be of the order of 6.0 x 10^-7 mol Ca⁺⁺/mg of protein with an initial rate of 2.6 x 10^-8 mol Ca⁺⁺/min·mg of protein. Axons dialyzed with 2 mM cyanide after 8–10-min delays show a rise in the calcium efflux in the presence of "normal" amounts of exogenous ATP. This effect seems to indicate that cyanide, per se, can release calcium ions from internal sources.     

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.     

Studies on ATP hydrolysis in medium for histochemical demonstration of ATPase activity

Summary Nonenzymatic ATP hydrolysis in medium of Wachstein and Meisel for histochemical demonstration of ATPase activity was investigated. In this medium considerable amounts of phosphorus are released without the participation of the enzyme. ATP hydrolysis in Wachstein-Meisel's medium increase with the concentration of Pb⁺⁺ and decrease at its small concentrations. The degree of ATP hydrolysis appeared to increase with increase both temperature and pH. At high concentration of ATP (5.76 mM) the degree of ATP hydrolysis in Wachstein-Meisel's medium is lower than at 1.44 mM ATP. 10.0 mM Ca⁺⁺ or 3.6 mM Fe⁺⁺ speed up ATP hydrolysis after 30- and 60-minute incubation. In the presence of 3.6 mM Co⁺⁺ or 2.6 mM Cu⁺⁺ ATP hydrolysis in Wachstein-Meisel's medium increased throughout the whole period examined. On the contrary, 3.6 mM Fe⁺⁺⁺ decreases ATP hydrolysis in this medium.10.0 mM F^– raises the degree of ATP hydrolysis which is, however, lowered in the presence of 2.5 mM pCMB or 3.6 mM KCN. 2.0 mM cysteine highly inhibits the process of nonenzymatic ATP hydrolysis in Wachstein-Meisel's medium.These data show that the histochemical reaction for ATPase activity in Wachstein-Meisel's medium does not originate exclusively from the hydrolysis of ATP in the presence of Pb⁺⁺, but take rise, above all, as a result of an enzymatic reaction.     

Thiophosphorylation and phosphorylation of saponin-permeabilized cultured chromaffin cells

There is increasing evidence that phosphorylation of cellular proteins plays a role in the control of events surrounding secretion in neurons and chromaffin cells. In previous studies, we have used thiophosphorylation of cell proteins as a means of fixing cellular phosphorylation reactions in the phosphorylated state. Thiophosphorylation of permeabilized chromaffin cells with adenosine-5′-O-(3-thiotriphosphate) results in irreversible inhibition of secretion. Thiophosphate is incorporated primarily by two cellular proteins of 58 and 47 kDa. Calcium enhanced thiophosphorylation of the 47 kDa protein but not the 54 kDa protein. This pattern of thiophosphorylation differed markedly from that for phosphorylation under similar treatment conditions. The phosphoprotein composition of the cells depended upon the medium calcium and ATP concentration. In the absence of exogenous ATP, fewer phosphoproteins were seen in calcium stimulated cells than in unstimulated cells. Proteins labelled with ³²P or ³⁵S migrated to the same position on polyacrylamide gels containing sodium dodecyl sulfate. In the presence of exogenous ATP, ³²P incorporation was similar for both control and calcium-stimulated cells and was found primarily in a 64 kDa protein. Incorporation of [³²P]phosphate by calcium-stimulated cells was reduced to the same extent by pretreatment of the cells with either adenosine-5′-O-(3-thiotriphosphate) or ATP.The different electrophoretic banding patterns for thiophosphorylation and phosphorylation are likely due to the irreversibility of the thiophosphorylation reaction and reversibility of the phosphorylation reaction. The inability to turn over thiophosphate groups, in association with changes in secretion, may permit identification of those phosphoproteins that are putatively involved in secretion.     

Biochemical aspects of the visual process. XXXVI. Calcium accumulation in cattle rod outer segments: Evidence for a calcium-sodium exchange carrier in the rod sac membrane

Accumulation of calcium has been studied in bovine rod outer segments (rods), isolated by sucrose density gradient centrifugation. Calcium-depleted rods are obtained by having ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA) present during isolation.Rods thus isolated have a leaky plasma membrane, as shown by the effects of ionophore A23187 and by their light-induced phosphorylation behaviour. The accumulation of ⁴⁵Ca, determined by incubation followed by a single fast washing-filtration procedure, thus represents translocation across the rod sac membrane.Accumulation in non-depleted rods is independent of the external calcium level and of ATP, suggesting exchange of ⁴⁰Ca by ⁴⁵Ca. In depleted rods in the presence of ATP there is net uptake, sigmoidally increasing with the external calcium concentration to the level attained in non-depleted rods. This net uptake is abolished by omission of ATP, its replacement by β,γ-methylene ATP and lowering the temperature to 0° C, suggesting involvement of enzymatic hydrolysis of ATP.Replacement of KCl by NaCl in the medium causes marked inhibition of ⁴⁵Ca uptake, both net uptake and exchange. Oligomycin, ruthenium red, lanthanum and ouabain do not inhibit accumulation.Efflux of ⁴⁵Ca from pre-loaded rods is slow in a KCl medium ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～30}\text{min}$ at 25° C), but is greatly accelerated by addition of NaCl or Ca²⁺ ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{10}\text{s}$ at 25°C).It is concluded that the rod sac membrane contains a carrier system, which is sensitive towards Ca²⁺ and Na⁺ and which requires ATP for net uptake of Ca²⁺ but not for exchange transport of Ca²⁺ with Ca²⁺ or Na⁺.     

ATP Released by Electrical Stimuli Elicits Calcium Transients and Gene Expression in Skeletal Muscle

ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca²⁺ concentration, with an EC₅₀ value of 7.8 ± 3.1 μm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 μm suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y₂ receptor and pannexin-1. As reported previously for electrical stimulation, 500 μm ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca²⁺ homeostasis and muscle physiology.     

Transport of calcium by cells and flagella ofChlamydomonas

Uptake of calcium was studied in the phototactic green algaChlamydomonas reinhardtii with a [⁴⁵Ca]Cl₂ filtration assay system. Uptake by whole cells is biphasic, with an initial rapid phase that probably represents binding to the cell surface, followed by a slow phase of diffusion into the cells. Experiments with inhibitors, ionophores, and drugs all support the conclusion thatChlamydomonas cells have an energy-dependent, outward-oriented calcium pump. In addition, isolated flagella appear to have a similar system that may transport calcium into the medium in the presence of ATP or GTP.     

Kinetics of ion movement in the squid giant axon

The loss of Na²², K⁴², and Cl³⁶ from single giant axons of the squid, Loligo pealii, following exposure to an artificial sea water containing these radioisotopes, occurs in two stages, an initial rapid one followed by an exponential decline. The time constants of the latter stage for the 3 ion species are, respectively, 290, 200, and 175 minutes. The outflux of sodium is depressed while that of potassium is accelerated in the absence of oxygen; the emergence of potassium is slowed by cocaine, while that of sodium is unaffected. One cm. ends of the axons take up about twice as much radiosodium as the central segment; this difference in activity is largely preserved during exposure to inactive solution. Such marked differences are not observed with radiopotassium. From the experimental data estimates are given of the influxes and outfluxes of the individual ions. The kinetics of outflux suggests a cortical layer of measureable thickness which contains the ions in different proportions from those in the medium and which governs the rate of emergence of these ions from the axon as though it contained very few but large (relative to ion dimensions) pores.     

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.     

Signal Transduction Pathways Coupled to a P2U Receptor in Neuroblastoma × Glioma (NG108-15) Cells

Abstract: Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P₂ purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P₂U receptor in a cultured neuroblastoma × glioma hybrid cell line (NG108-15 cells). The addition of ≥1 μM ATP to NG108-15 cells caused a transient increase in [Ca²⁺]_i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations ≥500 μM also elicited a sustained increase in [Ca²⁺]_i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca²⁺]_i elicited by ATP occurred concomitantly with the hydrolysis off [³²P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca²⁺]_i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP^4-) in the medium and not with the concentration of magnesium-ATP (MgATP^2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca²⁺]_i. ADP, ITP, TTP, GTP, ATP-γS, 2-methylthio ATP, β,γ-imidoATP or 3′-O-(4-benzoyl)benzoylATP, but not CTP, AMP, β,γ-methylene ATP, or adenosine, also caused an increase in [Ca²⁺]_i. In cells labeled with [³²P]P_i or [¹⁴C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [¹⁴C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P₂ nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A₂ and D.     

Interaction between thapsigargin and ATP4− in the regulation of the intracellular calcium in rat submandibular glands

Rat submandibular glands were digested with crude collagenase, and the intracellular calcium concentration of the cellular suspension was measured using fura-2. In the absence of extracellular magnesium and calcium ([Ca²⁺]_o), ATP had no effect; the response to ATP peaked at 1–2.5 mM [Ca²⁺]_o and was inhibited at 5 mM. One millimolar (mM) extracellular ATP did not increase the leak of LDH or fura-2; 10 m?M Coomassie brilliant blue G specifically inhibited the effect of ATP on [Ca²⁺]_in. Depleting intracellular calcium pools with thapsigargin did not affect the response to ATP. Using a Ca²⁺-free/Ca²⁺ reintroduction protocol, it was shown that ATP and thapsigargin increase the uptake of extracellular calcium. The effect of the two agonists was synergistic. Removal of extracellular sodium inhibited the effect of carbachol on [Ca²⁺]_in and the calcium uptake but potentiated the response to ATP. These results suggest that, after binding to purinergic receptors, extracellular ATP^4- increases [Ca²⁺]_in. ATP^4- does not mobilize thapsigargin-sensitive intracellular calcium pools (among which is the IP₃-sensitive calcium pool) but stimulates the uptake of extracellular calcium by a mechanism inhibited by extracellular sodium, probably by opening a nonselective cation channel. © 1994 Wiley-Liss, Inc.     

Calcium and ATP regulation of ion transport in larval frog skin

Ion transport measured as short circuit current (Isc) across the skin of larval frogs is activated by amiloride, acetylcholine, and ATP. In many epithelia, ATP stimulation of Isc involves an increase in intracellular calcium. To define the role of changes in intracellular calcium in ATP stimulation of Isc in larval frog skin, epithelial cells were loaded with calcium by adding 5 μM ionomycin to a 2 mM calcium apical Ringer's solution. Calcium loading had no observable effect on baseline Isc or on stimulation by ATP. Minimizing changes in intracellular calcium by loading the cell with the calcium chelator BAPTA also had no measurable effect on ATP stimulation of Isc. When the apical side was bathed with Ca²⁺-free Ringer's solution, ionomycin increased Isc up to 15 μA. This increase was partially blocked by 2 mM Ca²⁺, 2 mM Mg²⁺, and 10 μM W-7. Other experiments showed that baseline-stimulated and ATP-stimulated Isc were always larger in 2 mM Mg²⁺ Ringer's compared to 2 mM Ca²⁺. In dissociated cells bathed in 2 mM Ca²⁺ Ringer's, ATP had no effect on intracellular calcium as measured by Fluo-LR fluorescence changes. In conclusion, ATP apparently stimulates Isc without concomitant changes in intracellular calcium. This is consistent with a directly ligand-gated receptor at the apical membrane with P2X-like characteristics. Accepted: 21 April 1999