Interactions of phosphate groups of ATP and Aspartyl phosphate with the sarcoplasmic reticulum Ca2+-ATPase: an FTIR study

Phosphate binding to the sarcoplasmic reticulum Ca2+-ATPase was studied by time-resolved Fourier transform infrared spectroscopy with ATP and isotopically labeled ATP ([beta-18O2, betagamma-18O]ATP and [gamma-18O3]ATP). Isotopic substitution identified several bands that can be assigned to phosphate groups of bound ATP: bands at 1260, 1207, 1145, 1110, and 1085 cm(-1) are affected by labeling of the beta-phosphate, bands likely near 1154, and 1098-1089 cm(-1) are affected by gamma-phosphate labeling. The findings indicate that the strength of interactions of beta- and gamma- phosphate with the protein are similar to those in aqueous solution. Two bands, at 1175 and 1113 cm(-1), were identified for the phosphate group of the ADP-sensitive phosphoenzyme Ca2E1P. They indicate terminal and bridging P-O bond strengths that are intermediate between those of ADP-insensitive phosphoenzyme E2P and the model compound acetyl phosphate in water. The bridging bond of Ca2E1P is weaker than for acetyl phosphate, which will facilitate phosphate transfer to ADP, but is stronger than for E2P, which will make the Ca2E1P phosphate less susceptible to attack by water.     

Structures of the Ca-ATPase complexes with ATP, AMPPCP and AMPPNP. An FTIR study

We studied binding of ATP and of the ATP analogs adenosine 5′-(β,γ-methylene)triphosphate (AMPCP) and β,γ-imidoadenosine 5′-triphosphate (AMPPNP) to the Ca²⁺-ATPase of the sarcoplasmic reticulum membrane (SERCA1a) with time-resolved infrared spectroscopy. In our experiments, ATP reacted with ATPase which had AMPPCP or AMPPNP bound. These experiments monitored exchange of ATP analog by ATP and phosphorylation to the first phosphoenzyme intermediate Ca₂E1P. These reactions were triggered by the release of ATP from caged ATP. Only small differences in infrared absorption were observed between the ATP complex and the complexes with AMPPCP and AMPPNP indicating that overall the interactions between nucleotide and ATPase are similar and that all complexes adopt a closed conformation. The spectral differences between ATP and AMPPCP complex were more pronounced at high Ca²⁺ concentration (10 mM). They are likely due to a different position of the γ-phosphate which affects the β-sheet in the P domain.     

Functional Approach to the Catalytic Site of the Sarcoplasmic Reticulum Ca2+-ATPase: Binding and Hydrolysis of ATP in the Absence of Ca2+

Isolated sarcoplasmic reticulum vesicles in the presence of Mg(2+) and absence of Ca(2+) retain significant ATP hydrolytic activity that can be attributed to the Ca(2+)-ATPase protein. At neutral pH and the presence of 5 mM Mg(2+), the dependence of the hydrolysis rate on a linear ATP concentration scale can be fitted by a single hyperbolic function. MgATP hydrolysis is inhibited by either free Mg(2+) or free ATP. The rate of ATP hydrolysis is not perturbed by vanadate, whereas the rate of p-nitrophenyl phosphate hydrolysis is not altered by a nonhydrolyzable ATP analog. ATP binding affinity at neutral pH and in a Ca(2+)-free medium is increased by Mg(2+) but decreased by vanadate when Mg(2+) is present. It is suggested that MgATP hydrolysis in the absence of Ca(2+) requires some optimal adjustment of the enzyme cytoplasmic domains. The Ca(2+)-independent activity is operative at basal levels of cytoplasmic Ca(2+) or when the Ca(2+) binding transition is impeded.     

Gangliosides activate the phosphatase activity of the erythrocyte plasma membrane Ca2+-ATPase

The previous studies showed that gangliosides modulated the ATPase activity of the PMCA from porcine brain synaptosomes [Yongfang Zhao, Xiaoxuan Fan, Fuyu Yang, Xujia Zhang, Arch. Biochem. Biophys. 427 (2004) 204-212]. The effects of gangliosides on the hydrolysis of p-nitrophenyl phosphate (pNPP) catalyzed by the erythrocyte plasma membrane Ca(2+)-ATPase, which was characterized as E(2) conformer of the enzyme, were studied. The results showed that pNPPase activity was stimulated up to seven-fold, depending upon the different gangliosides used with GD1b>GM1>GM2>GM3 approximately Asialo-GM1. Under the same conditions, the ATPase activity was also activated, suggesting that gangliosides should modify both E(1) and E(2) conformer of the enzyme. The Ca(2+), which drove the enzyme to E(1) conformation, inhibited the pNPPase activity, but with the similar half-maximal inhibitory concentrations (IC(50)) in the presence and the absence of gangliosides. Moreover, the pNPPase activity was also inhibited by the raise in ATP concentrations. Gangliosides caused a large increase in V(max), but had no effect on the apparent affinity (K(m)) of the enzyme for pNPP. The kinetic analysis indicated that gangliosides could modulate the erythrocyte PMCA through stabilizing E(2) conformer.     

Lack of nitric oxide synthase depresses ion transporting enzyme function in cardiac muscle

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.     

Gangliosides modulate the activity of the plasma membrane Ca(2+)-ATPase from porcine brain synaptosomes

We systematically examined the effects of gangliosides on the plasma membrane Ca(2+)-ATPase (PMCA) from porcine brain synaptosomes. Our results showed that GD1b (two sialic acid residues) stimulated the activity, GM1 (one sialic acid residue) slightly reduced the activity, while asialo-GM1 (no sialic acid residue) markedly inhibited it, suggesting that sialic acid residues of gangliosides are important in the modulation of the PMCA. We also examined the oligosaccharide effects by using GM1, GM2, and GM3 whose only difference was in the length of their oligosaccharide chain. GM1, GM2, and GM3 reduced the enzyme activities, whereas GM2 and GM3 were potent inhibitors. Gangliosides affect both affinity for Ca(2+) and the Vmax of enzyme. It was observed that GD1b and GM2 increased the affinity of the enzyme for Ca(2+). GD1b, GM2 affected the Vmax with an increase of GD1b, but decreases of GM2. The study of the affinity for ATP and the Vmax of enzyme in the presence of gangliosides showed that GD1b and GM2 had little effect on the ATP binding to the enzyme, but the Vmax was apparently changed. Moreover, the effects of gangliosides are additive to that of calmodulin, suggesting that the modulation of PMCA by gangliosides should be through a different mechanism. The conformational changes induced by gangliosides were probed by fluorescence quenching. We found that fluorescent quenchers (I(-) and Cs(+)) with opposite charges had different accessibility to the IAEDANS binding to the PMCA in the presence of gangliosides. An apparent red shift (25nm) with increased maximum of fluorescence spectrum was also observed in the presence of GD1b.     

Ca2+ oscillations stimulate an ATP increase during fertilization of mouse eggs

Mammalian eggs and embryos rely upon mitochondrial ATP production to survive and proceed through preimplantation development. Ca(2+) oscillations at fertilization have been shown to cause a reduction of mitochondrial NAD+ and flavoproteins, suggesting they might also cause changes in cytosolic ATP levels. Here, we have monitored intracellular Ca(2+) and ATP levels in fertilizing mouse eggs by imaging the fluorescence of a Ca(2+) dye and luminescence of firefly luciferase. At fertilization an initial increase in ATP levels occurs with the first Ca(2+) transient, with a second increase occurring about 1 h later. The increase in cytosolic ATP was estimated to be from a prefertilization concentration of 1.9 mM to a peak value of 3 mM. ATP levels returned to prefertilization values as the Ca(2+) oscillations terminated. An increase in ATP also occurred with other stimuli that increase Ca(2+) and it was blocked when Ca(2+) oscillations were inhibited by BAPTA injection. Additionally, an ATP increase was not seen when eggs were activated by cycloheximide, which does not cause a Ca(2+) increase. These data suggest that mammalian fertilization is associated with a sudden but transient increase in cytosolic ATP and that Ca(2+) oscillations are both necessary and sufficient to cause this increase in ATP levels.     

Influences of calcium deficiency and cerium on the conversion efficiency of light energy of spinach

Chloroplast absorbs light energy and transforms it into electron energy, and then converts it into active chemical energy and stable chemical energy. In the present paper, we investigated the effects of Ce(3+), which has the most significant catalytic effects and similar characteristics with Ca(2+), on light energy conversion of spinach chloroplasts under Ca(2+)-deficient stress. The results illuminated that the Hill reaction activity, electron flow both photosystems and photophosphorylation rate of spinach chloroplasts reduced significantly under Ca(2+)-deficient condition, and activities of Mg(2+)-ATPase and Ca(2+)-ATPase on the thylakoid membrane were severely inhibited. Meanwhile, the activity of Rubisco, which is the key enzyme of photosynthetic carbon assimilation, was also prohibited. However, Ce(3+) decreased the inhibition of calcium deprivation the electron transport rate, the oxygen evolution rate, the cyclic and noncyclic photophosphorylation, the activities of Mg(2+)-ATPase, Ca(2+)-ATPase and Rubisco of spinach chloroplasts. All above implied that Ca(2+)-depletion could disturb light energy conversion of chloroplasts strongly, which could be reversed by Ce(3+).     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Plasma membrane calcium pump regulation by metabolic stress

The plasma membrane Ca(2+)-ATPase (PMCA) is an ATP-driven pump that is critical for the maintenance of low resting [Ca(2+)](i) in all eukaryotic cells. Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism, has the capacity to cause ATP depletion and thus inhibit PMCA activity. This has potentially fatal consequences, particularly for non-excitable cells in which the PMCA is the major Ca(2+) efflux pathway. This is because inhibition of the PMCA inevitably leads to cytosolic Ca(2+) overload and the consequent cell death. However, the relationship between metabolic stress, ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted. There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion. In particular, there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function. Moreover, membrane phospholipids, mitochondrial membrane potential, caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA. The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca(2+) overload and cytotoxicity.     

Use of helper enzymes for ADP removal in infrared spectroscopic experiments: application to Ca2+-ATPase

Adenylate kinase (AdK) and apyrase were employed as helper enzymes to remove ADP in infrared spectroscopic experiments that study the sarcoplasmic reticulum Ca(2+)-ATPase. The infrared absorbance changes of their enzymatic reactions were characterized and used to monitor enzyme activity. AdK transforms ADP to ATP and AMP, whereas apyrase consumes ATP and ADP to generate AMP and inorganic phosphate. The benefits of using them as helper enzymes are severalfold: i), both remove ADP generated after ATP hydrolysis by ATPase, which enables repeat of ATP-release experiments several times with the same sample without interference by ADP; ii), AdK helps maintain the presence of ATP for a longer time by regenerating 50% of the initial ATP; iii), apyrase generates free P(i), which can help stabilize the ADP-insensitive phosphoenzyme (E2P); and iv), apyrase can be used to monitor ADP dissociation from transient enzyme intermediates with relatively high affinity to ADP, as shown here for ADP dissociation from the ADP-sensitive phosphoenzyme intermediate (Ca(2)E1P). The respective infrared spectra indicate that ADP dissociation relaxes the closed conformation immediately after phosphorylation partially back toward the open conformation of Ca(2)E1 but does not trigger the transition to E2P. The helper enzyme approach can be extended to study other nucleotide-dependent proteins.     

Mapping the BKCa channel's "Ca2+ bowl": side-chains essential for Ca2+ sensing

There is controversy over whether Ca(2+) binds to the BK(Ca) channel's intracellular domain or its integral-membrane domain and over whether or not mutations that reduce the channel's Ca(2+) sensitivity act at the point of Ca(2+) coordination. One region in the intracellular domain that has been implicated in Ca(2+) sensing is the "Ca(2+) bowl". This region contains many acidic residues, and large Ca(2+)-bowl mutations eliminate Ca(2+) sensing through what appears to be one type of high-affinity Ca(2+)-binding site. Here, through site-directed mutagenesis we have mapped the residues in the Ca(2+) bowl that are most important for Ca(2+) sensing. We find acidic residues, D898 and D900, to be essential, and we find them essential as well for Ca(2+) binding to a fusion protein that contains a portion of the BK(Ca) channel's intracellular domain. Thus, much of our data supports the conclusion that Ca(2+) binds to the BK(Ca) channel's intracellular domain, and they define the Ca(2+) bowl's essential Ca(2+)-sensing motif. Overall, however, we have found that the relationship between mutations that disrupt Ca(2+) sensing and those that disrupt Ca(2+) binding is not as strong as we had expected, a result that raises the possibility that, when examined by gel-overlay, the Ca(2+) bowl may be in a nonnative conformation.     

A structural model for the catalytic cycle of Ca(2+)-ATPase

Ca(2+)-ATPase is responsible for active transport of calcium ions across the sarcoplasmic reticulum membrane. This coupling involves an ordered sequence of reversible reactions occurring alternately at the ATP site within the cytoplasmic domains, or at the calcium transport sites within the transmembrane domain. These two sites are separated by a large distance and conformational changes have long been postulated to play an important role in their coordination. To characterize the nature of these conformational changes, we have built atomic models for two reaction intermediates and postulated the mechanisms governing the large structural changes. One model is based on fitting the X-ray crystallographic structure of Ca(2+)-ATPase in the E1 state to a new 6 A structure by cryoelectron microscopy in the E2 state. This fit indicates that calcium binding induces enormous movements of all three cytoplasmic domains as well as significant changes in several transmembrane helices. We found that fluorescein isothiocyanate displaced a decavanadate molecule normally located at the intersection of the three cytoplasmic domains, but did not affect their juxtaposition; this result indicates that our model likely reflects a native E2 conformation and not an artifact of decavanadate binding. To explain the dramatic structural effect of calcium binding, we propose that M4 and M5 transmembrane helices are responsive to calcium binding and directly induce rotation of the phosphorylation domain. Furthermore, we hypothesize that both the nucleotide-binding and beta-sheet domains are highly mobile and driven by Brownian motion to elicit phosphoenzyme formation and calcium transport, respectively. If so, the reaction cycle of Ca(2+)-ATPase would have elements of a Brownian ratchet, where the chemical reactions of ATP hydrolysis are used to direct the random thermal oscillations of an innately flexible molecule.     

Kinetic control of multiple forms of Ca(2+) spikes by inositol trisphosphate in pancreatic acinar cells.

The mechanisms of agonist-induced Ca(2+) spikes have been investigated using a caged inositol 1,4,5-trisphosphate (IP(3)) and a low-affinity Ca(2+) indicator, BTC, in pancreatic acinar cells. Rapid photolysis of caged IP(3) was able to reproduce acetylcholine (ACh)-induced three forms of Ca(2+) spikes: local Ca(2+) spikes and submicromolar (<1 microM) and micromolar (1-15 microM) global Ca(2+) spikes (Ca(2+) waves). These observations indicate that subcellular gradients of IP(3) sensitivity underlie all forms of ACh-induced Ca(2+) spikes, and that the amplitude and extent of Ca(2+) spikes are determined by the concentration of IP(3). IP(3)-induced local Ca(2+) spikes exhibited similar time courses to those generated by ACh, supporting a role for Ca(2+)-induced Ca(2+) release in local Ca(2+) spikes. In contrast, IP(3)- induced global Ca(2+) spikes were consistently faster than those evoked with ACh at all concentrations of IP(3) and ACh, suggesting that production of IP(3) via phospholipase C was slow and limited the spread of the Ca(2+) spikes. Indeed, gradual photolysis of caged IP(3) reproduced ACh-induced slow Ca(2+) spikes. Thus, local and global Ca(2+) spikes involve distinct mechanisms, and the kinetics of global Ca(2+) spikes depends on that of IP(3) production particularly in those cells such as acinar cells where heterogeneity in IP(3) sensitivity plays critical role.     

Characterization of the palytoxin effect on Ca2+-ATPase from sarcoplasmic reticulum (SERCA)

The effect of palytoxin was studied in a microsomal fraction enriched in longitudinal tubules of the sarcoplasmic reticulum membrane. Half-maximal effect of palytoxin on Ca²⁺-ATPase activity yielded an apparent inhibition constant of approx. 0.4 μM. The inhibition process exhibited the following characteristics: (i) the degree of inhibition was dependent on membrane protein concentration; (ii) no protection was observed when the ATP concentration was raised; (iii) dependence on Ca²⁺ concentration with a decreased maximum catalytic rate; (iv) it occurred in the absence of Ca²⁺ ionophoric activity. Likewise, the inhibition mechanism was linked to: (i) rapid enzyme phosphorylation from ATP in the presence of Ca²⁺ but lower steady-state levels of phosphoenzyme; (ii) more drastic effect on phosphoenzyme levels when the toxin was added to the enzyme in the absence of Ca²⁺; (iii) decreased phosphoenzyme levels at saturating Ca²⁺ concentrations; (iv) no effect on kinetics of phosphoenzyme decomposition. The palytoxin effect is related with lock of the enzyme in the Ca²⁺-free conformation so that progression of the catalytic cycle is impeded.     

Functional coupling of Ca(2+) channels to ryanodine receptors at presynaptic terminals. Amplification of exocytosis and plasticity

Ca(2+)-induced Ca(2+) release (CICR) enhances a variety of cellular Ca(2+) signaling and functions. How CICR affects impulse-evoked transmitter release is unknown. At frog motor nerve terminals, repetitive Ca(2+) entries slowly prime and subsequently activate the mechanism of CICR via ryanodine receptors and asynchronous exocytosis of transmitters. Further Ca(2+) entry inactivates the CICR mechanism and the absence of Ca(2+) entry for >1 min results in its slow depriming. We now report here that the activation of this unique CICR markedly enhances impulse-evoked exocytosis of transmitter. The conditioning nerve stimulation (10-20 Hz, 2-10 min) that primes the CICR mechanism produced the marked enhancement of the amplitude and quantal content of end-plate potentials (EPPs) that decayed double exponentially with time constants of 1.85 and 10 min. The enhancement was blocked by inhibitors of ryanodine receptors and was accompanied by a slight prolongation of the peak times of EPP and the end-plate currents estimated from deconvolution of EPP. The conditioning nerve stimulation also enhanced single impulse- and tetanus-induced rises in intracellular Ca(2+) in the terminals with little change in time course. There was no change in the rate of growth of the amplitudes of EPPs in a short train after the conditioning stimulation. On the other hand, the augmentation and potentiation of EPP were enhanced, and then decreased in parallel with changes in intraterminal Ca(2+) during repetition of tetani. The results suggest that ryanodine receptors exist close to voltage-gated Ca(2+) channels in the presynaptic terminals and amplify the impulse-evoked exocytosis and its plasticity via CICR after Ca(2+)-dependent priming.     

Capsazepine elevates intracellular Ca2+ in human osteosarcoma cells, questioning its selectivity as a vanilloid receptor antagonist

Capsazepine is thought to be a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on different cell types is unclear. In human MG63 osteosarcoma cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Capsazepine caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. Capsazepine-induced [Ca(2+)](i) rise was partly reduced by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was composed of extracellular Ca(2+) influx and intracellular Ca(2+). In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of capsazepine on [Ca(2+)](i) was inhibited by 75%. Conversely, pretreatment with capsazepine to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not capsazepine-induced, [Ca(2+)](i) rise. Overnight treatment with 1-100 microM capsazepine inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, capsazepine increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing intracellular Ca(2+) release from the endoplasmic reticulum via a phospholiase C-independent manner. Capsazepine may be mildly cytotoxic.     

Ca2+ regulation of ion transport in the na+/ca2+ exchanger

The binding of Ca(2+) to two adjacent Ca(2+)-binding domains, CBD1 and CBD2, regulates ion transport in the Na(+)/Ca(2+) exchanger. As sensors for intracellular Ca(2+), the CBDs form electrostatic switches that induce the conformational changes required to initiate and sustain Na(+)/Ca(2+) exchange. Depending on the presence of a few key residues in the Ca(2+)-binding sites, zero to four Ca(2+) ions can bind with affinities between 0.1 to 20 μm. Importantly, variability in CBD2 as a consequence of alternative splicing modulates not only the number and affinities of the Ca(2+)-binding sites in CBD2 but also the Ca(2+) affinities in CBD1.     

Regulation of Ca2+ mobilization by prolactin in mammary gland cells: possible role of secretory pathway Ca2+-ATPase type 2

Regulatory role of prolactin (PRL) on Ca2+ mobilization in human mammary gland cell line MCF-7 was examined. Direct addition of PRL did not affect cytoplasmic Ca2+ concentration ([Ca2+]i); however, treatment with PRL for 24h significantly decreased the peak level and duration time of [Ca2+]i elevation evoked by ATP or thapsigargin (TG). Intracellular Ca2+ release by IP3 or TG in permeablized cells was not decreased after PRL-treatment, indicating that the Ca2+ release was not impaired by PRL treatment. Extracellular Ca2+ entry evoked by ATP or TG was likely to be intact, because entry of extracellular Ba2+ was not affected by PRL treatment. Among Ca2+-ATPases expressed in MCF-7 cells, we found significant increase of secretory pathway Ca2+-ATPase type 2 (SPCA2) mRNA in PRL-treated cells by RT-PCR experiments including quantitative RT-PCR. Knockdown of SPCA2 by siRNA in PRL-treated cells showed similar Ca2+ mobilization to that in PRL-untreated cells. The present results suggest that PRL facilitates Ca2+ transport into Golgi apparatus and may contribute the supply of Ca2+ to milk.