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Am J Physiol Cell Physiol 293: C885-C896, 2007. First published June 6, 2007; doi:10.1152/ajpcell.00602.2006
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Role of endosomal Na+-K+-ATPase and cardiac steroids in the regulation of endocytosis

Tomer Feldmann,1 Vladimir Glukmann,1 Eleonora Medvenev,1 Uri Shpolansky,2 Dana Galili,2 David Lichtstein,2 and Haim Rosen1

1The Kuvin Center for the Study of Infectious and Tropical Diseases, Institute of Microbiology, and 2Department of Physiology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel

Submitted 5 December 2006 ; accepted in final form 5 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Plasma membrane Na+-K+-ATPase, which drives potassium into and sodium out of the cell, has important roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically interact with the pump and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds are present in mammalian tissues, synthesized in the adrenal gland, and considered to be new family of steroid hormones. In this study, the mechanism of Na+-K+-ATPase involvement in the regulation of endocytosis is explored. We show that the effects of various CS on changes in endosomal pH are mediated by the pump and correspond to their effects on endosomal membrane traffic. In addition, it was found that CS-induced changes in endocytosed membrane traffic were dependent on alterations in [Na+] and [H+] in the endosome. Furthermore, we show that various CS differentially regulate endosomal pH and membrane traffic. The results suggest that these differences are due to specific binding characteristics. Based on our observations, we propose that Na+-K+-ATPase is a key player in the regulation of endosomal pH and endocytosed membrane traffic. Furthermore, our results raise the possibility that CS-like hormones regulate differentially intracellular membrane traffic.

bufalin; ouabain; endosomal pH

IONIC ELECTROCHEMICAL GRADIENTS across cellular membranes (e.g., H+ in the mitochondria or Na+, K+, and Ca2+ in the plasma membrane) are major driving forces for numerous cellular functions. These gradients are established and controlled by primary active transporters such as Na+-, K+-, H+-, or Ca2+-activated ATPases. Plasma membrane Na+-K+ATPase hydrolyzes ATP and uses the free energy generated to drive potassium into the cell and sodium out of the cell, against their electrochemical gradients. Consequently, this enzyme has an important role in regulating cell volume, the plasma membrane electrical potential ({Delta}{Psi}), and cytoplasmic pH and Ca2+ levels through Na+/H+ and Na+/Ca2+ exchangers, respectively (for reviews, see Refs. 16 and 25). Na+-K+-ATPase is a heteromeric protein composed of a catalytic {alpha}-subunit that binds Na+, K+, and ATP and beta- and {gamma} (FXYD)-subunits that can modulate substrate affinity (1, 15, 25, 42). It is well established that specific steroids, originally identified in plants and amphibians (e.g., digitalis, cardenolides, and bufadienolides), collectively termed here cardiac steroids (CS), bind to a specific site on the {alpha}-subunit and inhibit ATP hydrolysis and ion transport (4). The pharmacological profile of these steroids as Na+-K+-ATPase inhibitors has been extensively studied and well defined (28, 34). In the past decade, several groups have identified CS-like compounds in animal and human tissues, and their synthesis in and release from the adrenal gland was proven (for reviews, see Refs. 13 and 20). The endogenous CS-like compounds are considered to function as hormones and have been implicated in salt and water homeostasis and the regulation of blood pressure (5, 9, 39).

Recent studies have supported the notion that Na+-K+-ATPase participates in physiological processes distinct from its role in ion homeostasis: research on heart tissue, kidney and lung epithelial cells, and smooth muscle cells have implied that Na+-K+-ATPase interacts with other proteins as an intracellular signal transducer, thereby affecting numerous cellular functions (3, 48). In addition, CS have been shown to induce intracellular slow Ca2+ oscillations associated with the activation of NF-{kappa}B (24). Furthermore, ouabain-induced toxicity in OS cells was directly associated to signal transduction mechanisms and dissociated from the inhibition of ATPase activity by the steroid (44).

Endocytosis is a process in which cells internalize material from the environment and from cell surface receptors. It has been well established that after internalization of extracellular components by clathrin-coated vesicles (CCVs), receptor-ligand complexes, membrane proteins, and lipids are delivered to early endosomes in the peripheral cytoplasm. Early endosomes are multifunctional organelles that regulate the sorting and transport of membrane components between the plasma membrane and various intracellular compartments. These include recycling and late endosomes, lysosomes, and the trans-Golgi network. The prevalent model suggests that combinations of different members of highly conserved protein families [soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE), coat complexes, Rho and Rab] in distinct membranal compartments are the basis for sorting specificity (12, 23, 31).

Recently, we discovered that CS, at physiological concentrations (nM), induce changes in intracellular membrane traffic by inhibiting recycling within the late endocytic pathway (35). The ability of CS to induce these changes in membrane traffic in human cells was markedly reduced following transfection with the rat Na+-K+-ATPase {alpha}1-subunit, which has a low-affinity binding site for CS. Thus, we concluded that the CS-induced changes in membrane traffic are mediated by Na+-K+-ATPase (35). In the present study, we explored the mechanisms underlying the involvement of Na+-K+-ATPase and CS in membrane traffic. We showed that the CS-induced effect is mediated by changing the activity of the enzyme in endosomes and thereby acidifying the endosome. We propose that these events are the molecular basis for the alterations in CS-induced changes in membrane traffic.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials and antibodies. CS and geneticin disulfate salt (G418) were purchased from Sigma-Aldrich. 86Rb was purchased from New England Nuclear. All fluorescence probes were purchased from Molecular Probes. Monoclonal anti-Na+-K+-ATPase {alpha}-subunits, clone M7-PB-E9, were purchased from Sigma-Aldrich (A276), and rabbit polyclonal antibodies against Rab5 (sc-309), Rab7 (sc-6563), and Rab11 (sc-6565) were obtained from Santa Cruz Biotechnology. Polyclonal Cy5- and Cy2-conjugated AffiniPure, peroxidase-conjugated goat anti-mouse IgG F(ab)2 fragment, polyclonal Cy3-conjugated AffiniPure, and goat anti-rabbit IgG F(ab)2 fragment were purchased from Jackson ImmunoResearch Laboratories. ECL kits were from Pierce. Serum, cell culture medium, and antibiotics were provided by Biological Industries.

Cell culture. NT2 cells (human neuronal precursor cells) were obtained from Stratagene Cloning Systems. Cells were grown in T25 tissue culture flasks in DMEM supplemented with 2 mM glutamine, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 5% CO2 at 37°C, as previously described (30, 35).

Fluorescence microscopy and internalization assays. Confocal and conventional microscopy were performed as previously described (35). Images were acquired with a cooled SensiCAM charge-coupled device camera and analyzed using IP Plus 4.1 version (Signal Analytics) software. Unlabeled cells were used to determine autofluorescence. The image background was corrected as follows: two or three regions were selected from cell-free areas in each field, and the average intensity of these regions was considered the background value for that field. This value was then subtracted from each pixel in the field. An integrated fluorescence power reading for each image was recorded after background correction. Images were saved in TIFF format and transferred to Adobe Photoshop version 5.5 software for printing. Internalization assays were performed using FM1-43 and transferring as previously described (35). The fluorogenic styryl dye FM1-43 reversibly stains membranes in relation to their activity and is an ideal probe for the study of endocytosis and exocytosis. To visualize endocytic membrane traffic, cells cultured on glass coverslips were incubated in the presence of FM1-43 (5 µg/ml) or transferrin Alexa fluor (50 µg/ml) for 5 and 10 min, respectively. Unbound probe was removed, and cells were washed twice and reincubated for 4 h at 37°C in the presence or absence of the tested CS. Cells were then either fixed as described below and subjected to indirect immunofluorescence analysis or examined by standard live cell imaging (35).

Endosomal pH measurements. The basis of intracellular pH measurements is the pH-dependent fluorescence intensity of certain fluorochromes. We used a mixture of fluorescein- and Alexa fluor 633-labeled transferrin probes. The pH sensitivity of fluorescein (at 490-nm absorbance and 518-nm emission) together with the pH insensitivity of Alexa fluor 633 (at 632-nm absorbance and 647-nm emission) provided a precise and reliable means for pH measurements. These measurements served as a calibration curve for the measurements of changes in endosomal pH. All the experiments involving in vivo pH measurements included a synchronization step of 10 min at 12°C, during which time the cells bound the pH sensors. Measurements of the changes in endosomal pH were performed at 20–22°C, a temperature that delays the rate of the process.

Immunodetection of Na+-K+-ATPase {alpha}-subunit and Rab proteins. Cells were grown on glass coverslips for 24 h and then incubated under various experimental conditions. Incubation was terminated by fixing cells with PBS containing 1.5% glutaraldehyde and 13% sucrose for 15 min at room temperature. After being rinsed three times with PBS, cells were incubated in PBS containing 0.5% sodium borohydride for 5 min at room temperature and washed three times with PBS, once with PBS containing 0.015% saponin, and once with PBS containing 0.015% saponin and 1% ovalbumin. Samples were then incubated with primary antibody (1:50) at 4°C for 12–36 h, followed by an incubation with secondary antibody (1:300) at room temperature for 2 h. After being rinsed with PBS, plates were examined under fluorescence microscopy.

RT-PCR analyses of Na+-K+-ATPase isoforms. Total RNA was extracted from NT2 cells and used for RT-PCR according to a protocol similar to that of Krichevsky et al. (23). Gene-specific primers used for Na+-K+-ATPase isoforms were as follows: 5'-TGTACCTGGGTGTGGTGCTA-3' and 5'-GTTATCCACCTTGCAGCCAT-3' for {alpha}1, 5'-CCGCCTGATCTTTGACAACT-3' and 5'-CCTGTCCATAGCTGTCCTCC-3' for {alpha}2, 5'-AAGCTCATCATTGTGGAGGG-3' and 5'-ATTGCTGGTCAGGGTGTAGG-3' for {alpha}3, 5'-GGGAGGCTTAGGTTTGAAGC-3' and 5'-AAGATTCAGCCCAGAGGGAT-3' for beta1, 5'-AACTCCGCATCAACAAAACC-3' and 5'-CTTCAGGCTGGGTTGAGAAG-3' for beta2, and 5'-CCCTGCATACGAAGTTGGAT-3' and 5'-CACCATGACGAAGAACGAGA-3' for beta3. One microgram of total RNA was used for reverse transcription in a 10-µl reaction mix. The amplification profile involved denaturation at 93°C for 1 min, primer annealing at 60°C for 1 min, and extension at 72°C for 1 min. This cycle was repeated 30 times. PCR products were then separated on an agarose gel (22).

Measurement of K+ uptake using 86Rb. 86Rb uptake was performed as previously described (27). Experiments were performed under standard growth conditions in complete growth medium. NT2 cells were plated at 104 cells/ml in 24-well plates (1 ml/well). After 48–72 h, the medium was replaced with 0.5 ml of fresh medium, and 2 µCi of 86Rb were added in the presence or absence of 100 µM ouabain. In some experiments, cells were preincubated with different concentrations of ouabain or bufalin for 5 h before the addition of 86Rb. Incubation was carried out for 10 min and terminated by aspirating the incubation medium, followed by four rapid washes with 1 ml of an ice-cold 100 mM MgCl2 solution. Cells were lysed by the addition of 100 µl ethanol. Following its evaporation (30 min), 500 µl of water were added to each well, the contents were transferred to vials, and the radioactivity was determined in a {gamma}-counter. 86Rb uptake was normalized to the protein concentration measured in adjacent wells using a Bio-Rad protein assay kit. A quadruplicate set of samples was run simultaneously to determine ouabain- or bufalin-sensitive 86Rb+ uptake. This was calculated by subtracting the uptake in the presence of the steroid (ouabain- or bufalin-insensitive uptake) from the total 86Rb+ uptake.

[3H]ouabain and [3H]digoxin equilibrium binding to the guinea pig synaptosomal fraction. A crude synaptosomal membrane preparation was prepared as previously described (19). A 200-µl volume of diluted synaptosomes (~85 µg protein) was incubated at 37°C with 300 µl of binding solution, resulting in a final concentration of 30 mM Tris·HCl buffer (pH 7.4), 0.2 mM EDTA, 80 mM NaCl, 4 mM MgSO4, 2 mM ATP (Tris salt, vanadium free), and 0.91 nM [3H]ouabain (20.5 Ci/mmol) or 0.57 nM [3H]digoxin (25 Ci/mmol) in the presence of various concentration of nonradioactive steroid. In some experiments, 1 mM KCl was added to the incubation media. After 60 min of incubation (at which saturation binding was obtained), reactions were terminated by vacuum filtration on Whatman GF/B filters (Whatman, Maidstone, UK). Filters were washed twice with 3 ml Tris buffer, dried, and counted in a liquid scintillation counter. In experiments in which the effect of pH on binding was determined, low-pH buffers (5.5, 6.0, and 6.5) were prepared with MES sodium salt; higher pH buffers (7.0 and 7.4) were made with Tris·HCl. To ensure a change in pH in intrasynaptosomal compartments, the H+ ionophore CCCP was added to all reaction mixes. Specific binding was calculated by subtracting the binding observed in the presence of 100 µM unlabeled steroid from that observed in its absence, which represented 98% of the total binding. The equilibrium dissociation constant (Kd) and maximum binding sites (Bmax) were determined as the best fit from a single-site model using LIGAND software (36).

Dissociation kinetic experiments. Synaptosomes were incubated in binding solution as described above. Following 60 min, at which equilibrium binding was achieved, a dilution medium containing (final concentration) 10 µM unlabeled steroid, 80 mM KCl, 85 mM manitol, 3 µM EDTA, and 10 mM KH2POH (pH 7.4) was added. Mixtures were incubated at 37°C for different periods of time, and reactions were terminated by filtration on Whatman GF/B filters as described above. The dissociation rate constant (k–1) was calculated from the slope of ln Bt/Beq versus time plots, where Beq is the specific steroid bound at equilibrium and Bt is the specific steroid bound at different time points after the addition of dilution media.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Inhibition of plasma membrane Na+-K+-ATPase activity does not elicit changes in membrane traffic. The classical effect of CS is their binding to Na+-K+-ATPase, consequently inhibiting its activity. Thus, the most likely hypothesis for the mechanism of the CS-induced changes in endocytosed membrane recycling is the inhibition of plasma membrane Na+-K+-ATP activity. This may result in the dissipation of ionic gradients, an increase in intracellular Ca2+, alterations in plasma membrane {Delta}{Psi}, etc., events that may be involved in intracellular membrane traffic. If so, it is reasonable that inhibition of plasma membrane Na+-K+-ATPase activity per se will cause changes in endocytosed membrane traffic.

The expression and activity of Na+-K+-ATPase in NT2 cells were determined. As shown in the inset of Fig. 1, mRNA for the major three isoforms of the {alpha}-subunit and the three isoforms of the beta-subunit of Na+-K+-ATPase are expressed in NT2 cells. This finding supports a recent report (46) on the distribution of the isoforms in the human brain. Na+-K+-ATPase activity (expressed as ouabain-sensitive 86Rb uptake) in NT2 cells incubated in DMEM-F-12 is shown in Fig. 1A. Both ouabain and bufalin inhibited enzyme activity in a dose-dependent manner, with inhibition apparent only at concentrations higher than 10 nM. The IC50 of bufalin was 30-fold lower than that of ouabain (0.1 and 3 µM, respectively).


Figure 1
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  		Fig. 1. Inhibition of plasma membrane Na+-K+-ATPase activity does not induce changes in endocytosed membrane traffic. Effects of increasing concentrations of ouabain and bufalin (Buf) on Na+,K+-ATPase activity are shown. Enzyme activity was measured as ouabain-sensitive 86Rb uptake (means ± SD, n = 4) as described in MATERIALS AND METHODS. The rate was measured after a 10-min incubation. Inset: representative RT-PCR of different Na+-K+-ATPase isoforms. Total RNA extraction and the sets of primers used were as described in MATERIALS AND METHODS. Product sizes were as follows: 257, 403, 257, 214, 184, and 318 kb for the {alpha}1-, {alpha}2-, {alpha}3-, beta1-, beta2-, and beta3-isoforms, respectively. B: effects of a reduction in extracellular K+ and effects of bufalin on Na+-K+-ATPase activity in NT2 cells. Cells were incubated for 5 h in 5 or 1 mM extracellular [K+] in the presence or absence of bufalin. 86Rb was then added, and its uptake was determined after 10 min of incubation as described in MATERIALS AND METHODS. Data are means ± SD of 4 experiments. C: effects of a reduction in extracellular K+ and effects of bufalin on FM1-43 accumulation in NT2 cells. Cells were incubated as in B, and FM1-43 accumulation was determined as described in MATERIALS AND METHODS. Data represent means ± SD of 3 experiments. Significantly different from control (Cont) at 5 mM KCl: *P < 0.001 and **P < 0.05.

 
To test the involvement of plasma membrane Na+-K+-ATPase activity in CS-induced changes in endocytosed membrane traffic, the two parameters were examined in the same experiments under similar conditions. Na+-K+-ATPase activity was measured by 86Rb uptake (27), and intracellular membrane traffic was determined by following the translocation and accumulation of the styryl dye FM1-43 in the cells (35). These two parameters were manipulated by lowering extracellular K+ concentration ([K+]o) and by adding bufalin. Indeed, a reduction in [K+]o in the growth medium from 5 to 1 mM lowered Na+-K+-ATPase activity by >50% after 5 h (Fig. 1B). Similar inhibition was obtained by the addition of 1 nM bufalin to the 5 or 1 mM [K+]o medium (Fig. 1B). The addition of 1 nM bufalin to the growth medium caused a 300% increase in the intracellular accumulation of FM1-43 after 5 h of incubation. On the other hand, a reduction in [K+]o from 5 to 1 mM had no effect on FM1-43 accumulation (Fig. 1C). Thus, inhibition of plasma membrane Na+-K+-ATPase activity per se does not cause changes in endocytosed membrane traffic.

The cardenolide ouabain does not induce changes in endocytosed membrane traffic. We (35) have previously shown that Na+-K+-ATPase is involved in CS-induced changes in endocytosed membrane recycling. To support this conclusion, we screened different steroids for their ability to induce these changes. A positive correlation between the capability of steroids to induce changes in endocytosed membrane traffic and to inhibit Na+-K+-ATPase activity would favor Na+-K+-ATPase involvement. The relative potency of steroids to induce the accumulation of FM1-43 is shown in Table 1. As expected, corticosterone (100 nM), which lacks the lactone ring of CS, had no effect. On the other hand, proscillaridin A, digoxin, and digoxigenin induced changes in membrane traffic (Table 1) with a high correlation to their established ability to bind to and inhibit Na+-K+-ATPase activity (28, 34). Astonishingly, the most established CS inhibitor of Na+-K+-ATPase activity, ouabain, even at 100 nM, had no effect on endocytosed membrane traffic. This ineffectiveness was confirmed in 20 separate experiments using 3 different batches of the steroid. Clearly, these results raise the question as to the involvement of Na+-K+-ATPase in the mechanisms underlining CS-induced changes in endocytosed membrane traffic.


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  		Table 1. Relative potency of cardiac steroids in inducing FM1-43 accumulation in NT2 cells

 
Ouabain antagonizes the changes in endocytosed membrane traffic induced by other CS. The paradoxical observation that ouabain did not affect endocytosed membrane traffic, despite the involvement of Na+-K+-ATPase in this phenomenon (see above), can be reconciled if ouabain interferes in the effect induced by other CS. Thus, the effect of ouabain on CS-induced accumulation of FM1-43 was tested (Fig. 2). In agreement with our previous observations, digoxin (100 nM) induced a vast increase in FM1-43 accumulation in NT2 cells, indicative of inhibition of endocytosed membrane recycling (Fig. 2, A and D). Ouabain (100 nM) did not induce any change in FM1-43 accumulation (Fig. 2B). However, the simultaneous addition of ouabain and digoxin (100 nM each) abolished the digoxin effect (Fig. 2, C and D). Similar results were obtained with bufalin (Fig. 2E). The inhibitory effect of ouabain on digoxin- and bufalin-induced FM1-43 accumulation was dose dependent (Fig. 2, D and E). The competition between different CS shows that the effect of digoxin and bufalin on endocytosed membrane traffic is mediated via their direct interaction with the classical CS binding site on Na+-K+-ATPase.


Figure 2
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  		Fig. 2. Inhibition of digoxin-induced changes in membrane traffic by ouabain. NT2 cells were grown on glass coverslips for 24 h. The medium was then replaced with medium containing digoxin, ouabain, or a mixture of the two steroids. Following 20 min of incubation, the medium was removed, and cells were incubated for 5 min in PBS containing FM1-43. The reagent was then removed, and cells were washed twice with PBS and incubated for an additional 4 h in the presence of the steroids. Images were obtained using a fluorescence microscope. A–C: merged images of the phase-contrast and fluorescence signals. Quantification of the ouabain effect on digoxin-and bufalin-induced changes in FM1-43 accumulation are shown in D and E, respectively.

 
CS and monensin induce identical and synergistic changes in endocytosed membrane traffic. The experiments described above and our previously published data (35) support the notion that CS-induced changes in endocytosed membrane traffic are mediated by Na+-K+-ATPase but not by the inhibition of this enzyme activity at the plasma membrane. There are two possible mechanisms for CS-induced changes of membrane traffic: one involves CS inhibition of Na+-K+-ATPase activity in intracellular organelles and the other, in which CS-Na+-K+-ATPase interactions elicit second messenger pathways, is independent of Na+-K+-ATPase activity. This second possibility has been proposed in other systems (24, 39, 48). It is reasonable to assume that inhibition of Na+-K+-ATPase activity in intracellular compartments leads to changes in intracellular Na+ distribution. To test the involvement of intracellular Na+ in CS-induced changes in endocytosed membrane traffic, we used the monovalent ionophore monensin. This ionophore induces the transport of Na+ and H+ across the plasma membrane (18, 32). It has been previously shown that monensin blocks transferrin recycling by causing the internalized ligand to accumulate in large vesicles in the perinuclear regions of the cells, downstream to the early endosome (41). Since monensin and CS affect intracellular Na+ metabolism and induce transferrin accumulation in the perinuclear region, we hypothesized that the two compounds act in the same subcellular compartment. To test this hypothesis, we determined the colocalization of transferrin and various Rab proteins in monensin-induced vesicles. As shown in Fig. 3A, monensin, like bufalin, induced transferrin accumulation in NT2 cells. This effect was observed with 50 nM monensin but not with 5 nM monensin. In control cells, ~20% of the transferrin was colocalized with Rab11 and ~50% with Rab7, whereas in monensin-treated cells >90% of the transferrin was colocalized with both Rab11 and Rab7 (Fig. 3B). These results are virtually identical to those obtained for the bufalin-induced vesicles (see above), indicating that the two compounds inhibit membrane recycling in the same late endocytic compartment, characterized by the presence of Rab7 and Rab11. We next characterized the relationship between CS- and monensin-induced alterations in endocytosis. As shown in Fig. 3C, 5 nM bufalin induced a 10-fold increase in transferrin accumulation compared with that in control cells, whereas 5 nM monensin had no effect. Interestingly, the simultaneous addition of 5 nM bufalin and 5 nM monensin caused a 57-fold increase in transferrin accumulation, indicating the synergistic effect of the two compounds. The Na+-H+ transport properties of monensin, the identical subcellular compartment affected by monensin and CS, and the synergistic effect of the two compounds support the notion that intracellular Na+ and H+ metabolism is involved in the mechanisms governing the CS-induced changes in endocytosed membrane traffic.


Figure 3
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  		Fig. 3. Monensin (Mon) and bufalin synergistically induce transferrin (Tf) accumulation in the same subcellular compartment. The dose response of monensin-induced accumulation of transferrin in perinuclear vesicles is shown in A. Colocalization of transferrin and Rab proteins was determined as described in MATERIALS AND METHODS. The effects of monensin on the colocalization of transferrin and Rab11 and Rab7 are shown in B. The effects of bufalin (5 nM) and/or monensin (5 nM) on the accumulation of transferrin in perinuclear vesicles are shown in C.

 

Figure 5
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  		Fig. 5. Inhibition of bufalin-induced changes in membrane traffic by bafilomycin A1 (Bafi). The bufalin-induced accumulation of FM1-43 or transferrin-Alexa fluor 594 conjugate in NT2 cells was performed as described in Fig. 2. A–D: merged images of the phase-contrast and FM1-43 fluorescence signals. Quantification of the bafilomycin A1 effects on bufalin-induced accumulation of FM1-43 or transferrin-Alexa fluor 594 are shown in E and F, respectively.

 
CS-induced changes in endosome acidification. A hypothesis linking Na+-K+-ATPase activity in the early endosome to ATP-dependent regulation of endosome acidification was proposed more than 17 years ago: an in vitro study (11) on the transport properties and ionic permeability of early and late endosomes led to the suggestion that Na+-K+-ATPase activity is a key player in the determination of intraendosomal pH. This hypothesis was supported by experiments in living cells, demonstrating the effect of ouabain on the regulation of endosomal pH (7). Based on these observations, we raised the possibility that the mechanism for CS-induced changes in endocytosed membrane traffic is mediated by the activity of endosomal Na+-K+-ATPase. This leads to the prediction that CS, such as bufalin and digoxin, will affect the kinetics of changes in endosomal pH in correlation with their ability to change endocytosed membrane traffic. Furthermore, ouabain, which does not affect endocytosed membrane traffic, should show a different pattern in influencing endosomal pH. To test these possibilities, CS-induced changes in endosomal pH were monitored using a fluorescence transferrin-pH sensor (Fig. 4). To follow changes in the kinetics of endosomal pH, we used the experimental protocol described previously (40). In these experiments, cells were incubated with the fluorescent transferrin-pH sensor for 3 min at 12°C. The unbound sensor was then extensively washed, and the pH was monitored for 15 min at 20–22°C. Under these conditions, most of the transferrin was translocated from the plasma membrane via the CCV to the early endosome and then rapidly recycled to the plasma membrane (40). Thus, pH monitoring, using the transferrin-sensor, depicts changes in pH along this pathway. In control cells (Fig. 4A), the pH dropped by ~1.4 units within 7 min. This indicated that, within this time period, most of the transferrin was translocated into early endosomes. This was followed by alkalization to the original pH within an additional 7 min, pointing to the recycling of transferrin to the plasma membrane. These observations are in agreement with the results of other investigators (7, 11). The addition of bufalin (10 nM) or ouabain (100 nM) to the cells induced increased acidification of the endosomal pH by 1.6 units, reaching a minimum pH of 5.5 at 7 min (Fig. 4A). After this time, ouabain-treated cells showed a gradual alkalization of the endosome, with the pH reverting to the value at time 0, as in control cells. In bufalin-treated cells, however, the endosomal pH remained acidic at ~5.5 (Fig. 4A). NT2 cells transfected with rat Na+-K+-ATPase {alpha}-subunit cDNA did not show CS-induced changes in endocytosed membrane traffic (35). Accordingly, the addition of 10 nM bufalin to these cells did not elicit the sustained endosomal acidification (Fig. 4B), as opposed to control cells. These results show that the acidification event is affected by the interaction of bufalin and ouabain with Na+-K+-ATPase, while the subsequent alkalization is inhibited by bufalin but not by ouabain.


Figure 4
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  		Fig. 4. Cardiac steroids affect endosomal pH. The pH was determined by the ratio between the fluorescence intensity of a mixture of transferin-Alexa fluor 633 conjugate and transferrin-FITC conjugate as described in MATERIALS AND METHODS. Experiments were conducted using confocal microscopy, and data were normalized according to the mean fluorescence intensity of 10 fields of FITC at pH 7. Left: kinetics of endosomal pH. NT2 cells were grown on glass coverslips for 24 h. The medium was then replaced with fresh medium containing ouabain or bufalin or without cardiac steroid and incubated for 25 min at 37°C. Subsequently, cells were incubated with a mixture of transferin-Alexa fluor 633 conjugate and transferrin-FITC conjugate for 4 min at 12°C. Cells were then washed twice with cold PBS and transferred to PBS at room temperature (20–22oC) for the determination of pH changes using confocal microscopy. Right: bufalin-induced changes in endosomal pH are dependent on Na+-K+-ATPase. Cells were transfected with pCI-neo or with rat Na+-K+-ATPase {alpha}1-subunit cDNA and pCI-neo (pCI-Ouabr) as previously described (35). The effect of bufalin on the reversion of endosomal pH to neutrality (expressed by the difference between the pH at time 0 and at 15 min) in transfected cells is shown. wt, wild type.

 
Endosome acidification is required but not sufficient for CS-induced changes in membrane traffic. H+-ATPase activity is a major component in endosomal acidification (45). Bafilomycin A1, a specific inhibitor of this enzyme, has been shown to inhibit acidification of various intracellular vesicles (2). If bafilomycin A1 affects CS-induced changes in endocytosed membrane traffic, this would indicate that alterations in endosomal pH are involved in the mechanisms underlying this phenomenon. As shown in Fig. 5, 10 nM bufalin induced a marked increase in FM1-43 accumulation, whereas 10 nM bafilomycin A1, at a concentration that has been shown to be highly specific for H+-ATPase inhibition (2), had no effect. However, the simultaneous addition of bufalin and bafilomycin A1 (both at 10 nM) significantly inhibited the bufalin-induced effect on endocytosed membrane traffic, as demonstrated by the accumulation of FM1-43 (Fig. 5D). Similar results were obtained when changes in endocytosed membrane traffic were monitored using transferrin as a probe (data not shown). The inhibitory effect of bufilomycin A1 on bufalin-induced changes in FM1-43 and transferrin accumulation was dose dependent, as shown in Fig. 5, E and F. These experiments demonstrate that CS-induced changes in membrane traffic require H+-ATPase activity to acidify the endosome.

CS causes the retention of transferrin in the early endosome. The CS-induced alterations in acidification described above may result from changes in pH within a given subcellular compartment or changes in the sorting of the pH sensor to different compartments. Bufalin, for example, may cause increased acidification and recycled membrane retention within the early endosome or change the sorting of the transferrin sensor to a more acidic compartment, such as the late endosome or lysosome. To explore this issue, we assessed the colocalization of the pH sensor (fluorescent transferrin) with specific Rab proteins. We used Rab5, Rab7, and Rab11 as markers for early, late, and recycling endosomes, respectively. The colocalization experiments were performed using the same experimental protocol as described for the pH measurements, and samples taken at 1, 7, and 15 min after the temperature was raised to 20–22°C were tested. Representative examples of such experiment depicting the colocalization of Rab5 with transferrin at the 15-min time point with and without bufalin are shown in Fig. 6, A–F. Quantifications of these experiments for Rab5, Rab7, and Rab11 are shown in Fig. 5, G–I, respectively. As expected, in control cells, colocalization of transferrin with Rab5 was low (~15%) after 1 min, maximal (~50%) at 7 min, and declined to ~5% after 15 min In contrast, colocalization with Rab7 was very low (<5%) at 1 and 7 min and markedly increased to ~50% at 15 min Colocalization of transferrin with Rab11 was ~10% at 1 min, ~20% at 5 min, and <5% at 15 min. These results are in accord with the established pathway of transferrin sorting in the cell. Indeed, 7 min after the temperature was raised, most of the transferrin was translocated into the early endosome (colocalized with Rab5) and at 15 min the remaining transferrin was transferred to the late endosome (colocalized with Rab7). The addition of bufalin (20 nM) to the incubation medium resulted in a significant increase in the colocalization of transferrin with Rab5 after 7 min (>60%), which continued to increase up to 70% after 15 min (Fig. 6G), whereas the colocalization pattern with Rab7 did not change. Bufalin treatment increased the colocalization of transferrin with Rab11 after 7 and 15 min by 20–40%, respectively (Fig. 6I). Thus, the most remarkable effect of bufalin is the retention of transferrin within the early endosome, as manifested by its colocalization with Rab5 at the 15-min time period.


Figure 6
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  		Fig. 6. Bufalin induced the retention of transferrin at the early endosome. NT2 cells were grown, treated with bufalin, and incubated with transferrin-Alexa fluor 633 conjugate as described in Figs. 2 and 4. Cells were taken for immunocytochemical analyses at 1, 7 and 15 min following the transfer to 20–22°C. A–F: experiments using confocal microscopy of the colocalization of transferrin and Rab5 15 min after transfer to 20°C. Quantifications of the colocalizations of Rab5, Rab7, or Rab11 with transferrin are shown in G–I, respectively.

 
Na+-K+-ATPase is localized in the early endosome. The Na+-K+-ATPase subcellular localization was determined by immunocytochemistry (Fig. 7). The colocalization of Na+-K+-ATPase {alpha}-subunits with Rab5 was determined using specific monoclonal antibodies. Clearly, as shown in Fig. 7, A–C, Na+-K+-ATPase {alpha}-subunits colocalized (yellow color) with Rab5. Biochemical fractionation of intracellular vesicles using a floating sucrose gradient and Western blot analysis resulted in comparable results showing the presence of Na+-K+-ATPase in both early and late endosomes (data not shown and Refs 8 and 43).


Figure 7
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  		Fig. 7. Na+-K+-ATPase is present in endosomal compartments. Colocalization of Na+-K+-ATPase and Rab5a was tested by immunocytochemistry and confocal microscopy. The yellow signals in the merged images represent the colocalization between Na+-K+-ATPase and Rab proteins.

 
Differences in binding characteristics favor the binding of digoxin but not ouabain in the endosomal compartment. As described above, bufalin and digoxin, but not ouabain, elicited the accumulation of transferrin in the endosomal compartment (Fig. 2 and Table 1). In addition, bufalin but not ouabain prevented the pH from reverting to a neutral value in the early endosome (Fig. 4). A possible explanation for these differences is that the process of internalization and sorting of Na+-K+-ATPase from the plasma membrane to the late endosome favors ouabain but not digoxin dissociation. To explore this issue, we determined the binding characteristics of ouabain and digoxin (for which [3H]ligands are available) under several relevant experimental conditions. Since a relatively large amount of membrane is required for these experiments, they were performed on guinea pig brain synaptosomes. This membrane preparation consists of pinched-off isolated nerve terminals prepared from cortical brain neurons, which contain essentially mitochondria, enzymes, and vesicles of neurotransmitters. Retaining most of the physiological behavior of normal nerve terminals, such as the maintenance of ATP levels and the release of neurotransmitters, these synaptosomes are widely used for enzyme activity and receptor binding research (47). The equilibrium binding experiments of [3H]ouabain and [3H]digoxin to the synaptosomal fraction are shown in Fig. 8A. The results best fit a one-site model with parameters of Kd = 35.9 ± 5.4 and 81.09 ± 20.27 nM and Bmax = 54.6 ± 5.46 and 111.1 ± 22.2 pmol/mg protein for ouabain and digoxin, respectively. Although [3H]digoxin binding revealed significantly more (P < 0.03) binding sites, the binding affinities of the two steroids were essentially the same. Dissociation experiments (Fig. 8B) showed that the ouabain k–1 was more than threefold greater than that of digoxin (0.0053 ± 0.0007 vs. 0.0015 ± 0.0011 min–1, P < 0.019).


Figure 8
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  		Fig. 8. Characterization of [3H]ouabain and [3H]digoxin binding to guinea pig brain synaptosomal fractions. The preparation of synaptosomes and binding methods are described in MATERIALS AND METHODS. A: [3H]steroid binding under equilibrium in the presence of increasing concentrations of unlabeled ligand. Data are means ± SE of 5 experiments, each performed in triplicate. B: dissociation of [3H]steroid from synaptosomal fractions. Synaptosomes were equilibrated at 37°C with 2 nM [3H]steroid under optimal binding conditions, and samples were taken for filtration at various times after the addition of 10 µM unlabeled steroid, as described in MATERIALS AND METHODS. Data are presented as ln B/Beq versus time, where Beq is the specific steroid bound at equilibrium and B is the specific steroid bound at several time points. Dissociation rate contant (k–1) values were calculated as the slopes of the plots. Data are means ± SE of 4 experiments, each performed in triplicate. C: effect of [K+] on [3H]steroid binding to guinea pig brain synaptosomal fractions. Samples were incubated at 37°C with 2 nM [3H]steroid for 60 min in the presence or absence of 1 mM KCl. Data are presented as %binding in the absence of [K+]. Data represent means ± SE of 3 experiments, each performed in triplicate. The addition of 1 mM [K+] to the binding buffer significantly reduced [3H]ouabain (**P < 0.00001) and [3H]digoxin (*P < 0.001) binding. D: effect of pH on [3H]steroid binding to guinea pig brain synaptosomal fractions. Equilibrium binding at different pH values was estimated as described in MATERIALS AND METHODS. Data are means ± SE of 4 experiments, each performed in triplicate. [3H]ouabain binding was significantly lower than [3H]digoxin binding at pH 5.5 (*P < 0.0065).

 
Since internalization of Na+-K+-ATPase exposes the pump to high [K+] in cells, the effect of an increase in [K+] on total binding was examined. The addition of [K+] to the incubation medium (1 mM final concentration) reduced the binding of the two steroids (Fig. 8C). However, whereas [3H]digoxin binding decreased by 17.6%, [3H]ouabain binding declined to 54.3% of the control binding in the absence of [K+]. As described above, intraendosomal acidification to pH 5.5 is a process involved in membrane traffic. Thus, in the course of its traffic, the Na+-K+-ATPase-CS complex is exposed to an acidic pH, which may differentially affect ouabain or digoxin binding. To investigate this possibility, [3H]digoxin and [3H]ouabain binding to synaptosomes were determined at different pH values (Fig. 8D). Whereas [3H]digoxin binding did not change significantly, [3H]ouabain binding was reduced as the acidity increased, reaching 41% of binding at pH 5.5 compared with binding at pH.7. These results are in accordance with a previous elegant pioneering study (49) by Yoda and coworkers, who demonstrated an increase in the [3H]ouabain dissociation rate with increased acidity. Cumulatively, these findings suggest that digoxin binding may last longer when plasma membrane Na+-K+-ATPase is internalized and sorted to early endosomes compared with ouabain. These differences may account for the observed disparity between these steroids with respect to transferrin accumulation and pH changes.


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It is well established that the regulation of endocytosed membrane traffic is governed by ligand-receptor interactions at the plasma membrane and the activity of different members of protein families of SNARE, coat complexes, Rho and Rab, in distinct membranal compartments (12, 23). Recently, we discovered that the interaction of CS with Na+-K+-ATPase is also involved in the regulation of endocytosed membrane traffic (35). The present study was aimed at exploring the mechanisms underlying this phenomenon.

Na+-K+-ATPase and endocytosed membrane traffic. The inhibition of plasma membrane Na+-K+-ATPase per se is not the mechanism responsible for CS-induced changes in endocytosed membrane traffic. This can be concluded from the experiments demonstrating that the inhibition of plasma membrane Na+-K+-ATPase activity, by a reduction in extracellular K+, did not induce changes in membrane traffic (Fig. 1). This conclusion is further supported by the surprising observation that ouabain, the classical Na+-K+-ATPase inhibitor, did not elicit changes in membrane traffic even at 500 nM (Table 1 and Fig. 2 and data not shown). Furthermore, the observation that ouabain antagonized the effects induced by other CS (Fig. 2) serves as direct evidence supporting our previous conclusion, based on transfection experiments (35), that the interaction of CS with Na+-K+-ATPase regulates endocytosed membrane traffic. In addition, the experiments using the ionophore monensin (Fig. 3) demonstrated that changes in intracellular Na+ are part of the mechanisms regulating endocytosed membrane traffic. Taken together, these findings led us to focus on Na+-K+-ATPase in intracellular compartments. Our results demonstrate unequivocally that Na+-K+-ATPase is present and active in endosomes. The presence in early and late endosomes was demonstrated by colocalization experiments with Rab proteins using confocal microscopy (Fig. 7). These results are in complete agreement with other studies (8, 43) demonstrating the presence of Na+-K+-ATPase in endosomes of epithelial cells using biochemical means. Endosomal Na+-K+-ATPase activity was manifested by in situ analysis demonstrating CS-induced changes in endosomal acidification (Fig. 6).

Involvement of endosomal Na+-K+-ATPase activity in the regulation of endosomal acidification. It is well established that endocytosis is associated with highly specific regulated changes in intravesicular pH. The mechanisms governing the regulation of pH within the endocytic compartments (early endosome: pH 6.2–6.5 vs. late endosome: pH 5.5) are not clear. A possible role for Na+-K+-ATPase activity in regulating early endosome acidification was proposed 17 years ago by Mellman and his colleagues (11) on the basis of in vitro experiments on transport properties and the ionic permeability of early and late endosomes. This suggestion was supported by a report (7) showing that in A549 cells, ouabain induced a drop in endosomal pH. On the contrary, studies (38,0) with K562, Sc9, and HD3 cell lines led to the conclusion that Na+-K+-ATPase does not regulate acidification of endocytic vesicles. Data from the present study provide strong evidence for the involvement of Na+-K+-ATPase in endosomal pH regulation, demonstrating that 1) CS (ouabain and bufalin) enhance the acidification of the early endosome (Fig. 4); 2) bufalin, but not ouabain, alters endocytosed membrane traffic and induces, in addition to enhanced acidification, a delay in the subsequent alkalization of early endosomes (Fig. 4); and 3) bufalin does not induce endosomal acidification in NT2 cells transfected with rat Na+-K+-ATPase {alpha}-subunit cDNA (Fig. 4).

Differential effects of CS. All CS bind to and inhibit the activity of Na+-K+-ATPase. Despite these common features, differences in the actions of various CS have been known for many years. For example, a long-term infusion of ouabain produced hypertension in rats, whereas digoxin did not elicit this effect (21). In another study, it was shown that the resting mean arterial pressure was significantly increased by long-term subcutaneous ouabain plus high-salt (8%) intake and that this effect could be prevented when digoxin was given concomitantly (14); human nongastric H+-K+-ATPase was inhibited by bufalin, digoxin, and digitoxin but virtually resistant to digoxigenin and ouabagenin (26); digoxin and digitoxin but not ouabain were substrates for the P-glycoprotein transporter (MDR1) (29); and ouabain and bufalin affected differentially the intracellular signaling protein 14-3-3 in the rat lens (22). The molecular basis for the differences between various CS is not known. In the present study, we found that the regulation of endosomal acidification and endocytosed membrane traffic are differentially affected by CS. Whereas bufalin, proscillaridin A, digoxin, and digoxigenin induced the accumulation of endocytosed membrane components, ouabain had no effect (Table 1). Furthermore, ouabain antagonized these CS-induced changes (Fig. 2). Thus, it is possible to draw the conclusion that changes in cellular functions induced by other CS but not by ouabain are likely to be associated with CS-induced alterations in membrane traffic, and vice versa.

Differences between ouabain and the other CS studied here were also manifested in their effects on the kinetics of endosomal acidification. Whereas ouabain only enhanced the acidification of early endosomes, bufalin (Fig. 4) and digoxin (data not shown) also delayed the following alkalization. These differential effects can be explained by the different binding characteristics of the steroids: using synaptosomal preparations, we showed that the k–1 of ouabain was threefold higher than that of digoxin. Furthermore, ouabain binding was reduced more significantly than that of digoxin by the addition of [K+] or by lowering the pH (Fig. 8). Thus, it is reasonable to suggest that in the course of internalization and exposure to high intracellular [K+] and low pH, ouabain will be displaced from the binding site, whereas digoxin will retain its binding and Na+-K+-ATPase inhibition capacity. It follows that inhibition of Na+-K+-ATPase activity in the early endosome will result in enhanced acidification, which, in turn, will induce fast ouabain dissociation, removal of Na+-K+-ATPase inhibition, alkalization, and fast endosomal recycling. In the case of digoxin or bufalin, however, the enhanced acidification does not cause significantly increased dissociation of the steroid, and the sustained Na+-K+-ATPase inhibition retains the acidic pH in the early endosome. The implication of this scenario is that the response of different cells to different CS depends on the particular CS and the normal endosomal pH of the specific cell. It was shown by Murphy et al. (38) that cell lines may be grouped into two classes based on observed differences in the pH of early endosomes. Members of the first class (like A549 cells) typically have an early endosomal pH of 6.2, which is sensitive to ouabain, whereas members of the second class (like K562, Sc9, and HD3 cells) have an early endosomal pH of 5.4, which is insensitive to ouabain (38). Based on this ouabain sensitivity, it was concluded that Na+-K+-ATPase is involved in pH regulation of the first but not second class of cells. We suggest, however, that Na+-K+-ATPase is involved in the endosomal pH regulation of all cells and that the lack of ouabain effect in the second class is attributable to the basal acidic pH of the early endosome. Evidently, the different effects of various CS on endosomal pH regulation are cell type-specific phenomena.

As mentioned in the Introduction, CS-like compounds, including ouabain and bufalin derivatives, are present in many mammalian tissues, are synthesized by and secreted from the adrenal gland, and are considered a new family of steroid hormones (9, 13, 20). Thus, the effects of CS described here may represent physiological regulations of intracellular membrane traffic. In this respect, the findings that bufalin enhanced endosomal acidification, delayed pH recovery, and inhibited the recycling of endosomal vesicles, whereas ouabain did not, raise the possibility that the specific CS present in particular tissue will determine the physiological outcome. We are currently studying this tempting hypothesis.

Proposed role for endosomal Na+-K+-ATPase. In accordance with the mechanism proposed by Mellaman and colleagues, we suggest that under normal conditions, Na+-K+-ATPase activity is a major regulator of intraendosomal pH (Fig. 9). The initial pH gradient is established by the activity of H+-ATPase. Since H+-ATPase is electrogenic, its activity and thus intraendosomal acidification will be limited by {Delta}{Psi} (interior positive) across the endosomal membrane. This {Delta}{Psi} may ensue partially from both Na+-K+-ATPase (moving 3 Na ions into and 2 K ions out of the endosome in each cycle) and H+-ATPase activities (37). Thus, inhibition of Na+-K+ATPase activity by CS will decrease {Delta}{Psi} across the endosomal membrane and allow increased acidification. This acidification is maintained as long as Na+-K+-ATPase is inhibited by CS and, therefore, is transient for ouabain, as explained above. The sustained acidification in the early endosome induced by bufalin and digoxin was associated in this study with inhibition of the late endosomal recycling pathway. This correlation does not clarify whether the sustained acidification is a part of the mechanisms involved in CS-induced changes in endocytosed membrane traffic. Our observation that the inhibition of endosomal acidification by the highly specific H+-ATPase inhibitor bafilomycin A1 abolished CS-induced changes in membrane traffic (Fig. 5) support the involvement of acidification in the changes in endocytosed membrane traffic. This conclusion does not exclude, however, a role for CS-Na+-K+-ATPase interactions by other mechanisms involving protein-protein interactions or signal transduction. Cumulatively, our study proves that endosomal Na+-K+-ATPase activity participates in the regulation of endosomal pH and endocytosed membrane traffic.


Figure 9
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  		Fig. 9. Schematic representation of the involvement of endosomal Na+-K+-ATPase in the regulation of endosomal pH. Based on our results and relevant literature (11, 23, 37, 38, 40), we propose the following: under control conditions (left), endosomal pH is established primarily by the activity of H+-ATPase. Since H+-ATPase is electrogenic, its activity is limited by the electrical potential ({Delta}{Psi}; interior positive) across the endosomal membrane. This {Delta}{Psi} ensues partially from both Na+-K+-ATPase and H+-ATPase activities. Inhibition of Na+-K+-ATPase activity by cardiac steroids (right) decreases {Delta}{Psi} across the endosomal membrane and allows increased acidification.

 
Reversible retention in the endosomal system is a mechanism by which cells modulate the protein levels on the cell surface. Several specialized cell types exploit this mechanism to move enzymes and transporters to the cell surface (6). Examples include the effect of insulin on surface levels of glucose transporter 4 in muscle and fat cells (33), translocation of aquaporin-2 in collecting duct cells of the kidney (17), and H+-K+-ATPase in gastric parietal cells (10), which are important for water and acid secretion, respectively. In view of our findings of the involvement of Na+-K+-ATPase and CS in endocytosed membrane traffic, it is tempting to propose that they participate in the regulation of the many processes in which the distribution of proteins between the plasma membrane and intracellular compartment is essential.


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This research was supported by Israel Science Foundation Grant 269/04.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Rosen or D. Lichtstein, The Kuvin Center for the Study of Infectious and Tropical Diseases, Institute of Microbiology, The Hebrew Univ.-Hadassah Medical School, Jerusalem 91120, Israel (e-mail: hrose{at}md2.huji.ac.il or david{at}md2.huji.ac.il)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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A. Y. Bagrov, J. I. Shapiro, and O. V. Fedorova
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