Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka 814-0193, Japan
Submitted 4 December 2003 ; accepted in final form 17 March 2004
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ABSTRACT |
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prostanoid receptor agonists; electrophysiology; motile activity; bone resorption
PGE2 exerts its effects through specific receptors that consist of four subtypes designated EP1, EP2, EP3, and EP4 (25), and osteoblasts have been shown to express several of these PGE2 receptor subtypes (32, 33). Recent studies using EP receptor-knockout animals and selective EP receptor agonists demonstrated that EP4 and/or EP2 mediate both anabolic (37, 39) and catabolic (24, 33) actions of PGE2.
Evidence is mounting that PGE2 also has a direct effect on osteoclasts and negatively regulates their bone-resorbing activity (5, 6, 10, 14, 22). Chambers and coworkers (5, 6, 10) demonstrated that PGE2 causes cytoplasmic retraction and reduction of motility (i.e., movement of the peripheral cell membrane lamellipodia) in isolated osteoclasts, thereby inhibiting bone resorption. Kaji et al. (14) reported that PGE2 inhibits bone resorption in the absence of osteoblasts but stimulates bone resorption in the presence of osteoblasts. A study using highly purified rabbit osteoclasts showed that osteoclasts express EP4 and EP2 receptors and that both receptors participate in the inhibitory effect of PGE2 on osteoclast activity (22).
Several types of ion channels have been identified in osteoclasts, and their physiological roles in the activities of osteoclasts have been reported (19). Recently, we demonstrated (15) that membrane depolarization induced by exposing osteoclasts to high-K+ solution decreases intracellular Ca2+ concentration ([Ca2+]i) and causes cytoplasmic retraction and reduction of motility. This result demonstrates the importance of cell membrane potential for controlling [Ca2+]i and morphology in osteoclasts. Because membrane potential is regulated by the activities of several ion channel types expressed in osteoclasts, activation and deactivation of these channels is likely to determine osteoclast morphology. Several agents that modulate bone resorption have also been shown to modulate ion channels in osteoclasts (20, 27, 29). Therefore, to understand the role of PGE2 in osteoclast physiology and pathology, an understanding of its actions on ion channels is desirable. There is no previous report on PGE2-mediated effects on osteoclast ion channels to our knowledge.
In the present study, we applied the whole cell patch-clamp technique to isolated rat osteoclasts and examined the effects of PGE2 on ion channel activity and the role of these channels in PGE2-induced morphological changes. We found that PGE2 acts directly on rat osteoclasts and stimulates outwardly rectifying Cl channels via a cAMP-dependent mechanism through EP2 and EP4 receptors. This pathway contributes to reduction of cell area and loss of osteoclast motility.
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MATERIALS AND METHODS |
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Electrophysiological measurements. The coverslip with adherent cells was placed in a recording chamber (volume 1 ml) mounted on an inverted microscope (TE 300; Nikon, Tokyo, Japan) and continuously superfused (1 ml/min) with standard extracellular solution (HEPES-buffered solution) containing (in mM) 134 NaCl, 6 KCl, 10 glucose, 0.5 MgCl2, 1.25 CaCl2, and 10 HEPES, adjusted to pH 7.3 with Tris. In some preliminary experiments a bicarbonate-buffered solution was used, which was prepared by replacing HEPES in the standard extracellular solution with 14.5 mM NaHCO3. This solution was gassed with 5% CO2-95% O2 and had a pH of 7.3. The patch pipette solution for recording of membrane potential and whole cell currents (shown in Fig. 1A) contained (in mM) 140 KCl, 3 MgCl2, 2 ATP (disodium salt), 0.3 EGTA, and 10 HEPES, adjusted to pH 7.3 with Tris. For experiments designed to eliminate K+ currents, a K+-free solution was prepared by replacing K+ in the extracellular and patch pipette solutions with equimolar amounts of Na+ and Cs+, respectively.
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Osmolarity of all solutions was measured with a freezing-point depression osmometer (Osmometer Automatic; Knauer, Berlin, Germany) and adjusted to 290300 mosM with mannitol. Hypotonic solution (210 mosM) was made by reducing NaCl concentration in the extracellular K+-free solution to 100 mM. Low-NaCl (100 mM) isotonic solution (290 mosM) was prepared by addition of 80 mM mannitol to the hypotonic solution for control experiments. In some experiments, 100 mM NaCl in the hypotonic solution was replaced entirely with the same concentration of NaI, NaBr, or sodium gluconate.
Membrane potential and currents were recorded with the whole cell configuration patch-clamp technique (12) with an amplifier (Axopatch 200A; Axon Instruments, Foster City, CA). Currents were filtered at 1 kHz and digitized at a sampling frequency of 25 kHz. Data acquisition and analysis were performed with pCLAMP 8.0 software (Axon Instruments). All electrophysiological experiments were performed at 2627°C.
Patch pipettes were prepared with a pipette puller (P-97; Sutter Instrument, Novato, CA), and their tips were heat polished with a microforge (MF83; Narishige Scientific Instrument Laboratory). Values of electrode resistance of the patch pipette filled with the patch pipette solution were in the range of 2.55 M. Series resistance (6080%) was compensated to minimize the clamp speed. An Ag-AgCl reference electrode was connected to the extracellular solution through a 3 M KCl-agar salt bridge to minimize changes in liquid junction potentials. Membrane potential was then corrected for liquid junction potentials as previously described (17). The zero-current potential before formation of the gigaseal was set to 0 mV.
Morphological analysis.
The pure osteoclast preparation was placed in a plastic petri dish filled with -MEM containing 5 mM HEPES, adjusted to pH 7.3 with Tris. In some preliminary experiments, we used
-MEM containing 14.5 mM NaHCO3, equilibrated with 5% CO2-95% O2 to maintain a pH of 7.3, as the bath solution. In these experiments, FBS was omitted from
-MEM (serum-free culture medium) to avoid any influence by this ingredient. The petri dish was then placed in a temperature-controlled incubation chamber (Tokai Hit, Japan) mounted on an inverted phase-contrast microscope (TMS; Nikon) and incubated at 37°C. The microscope was connected through a charge-coupled device (CCD) camera (CS8310; Tokyo Electronic Industry, Tokyo, Japan) to a time-lapse video recorder (KV 7168; Toshiba, Tokyo, Japan). Osteoclasts were preincubated with serum-free culture medium for
1 h before the recordings were started. PGE2 or other test agents were added directly to the serum-free culture medium. Images of osteoclasts were digitized at intervals of 2 min and stored in a Macintosh G4 computer (Apple). Image analysis software (NIH Image, version 1.62) was used for measurement of osteoclast planar area. The planar area A(t) of an osteoclast at time t (in minutes) was quantitated by tracing the outlines of individual cells and normalized to 100% at the beginning of the recording. Osteoclasts continuously move their membrane periphery by protracting and retracting lamellipodia. This phenomenon has been described in terms of motility (1), and we assessed this motility on the basis of the method originally described by Alam et al. (1). An outline of each cell obtained at time t was overlaid on the subsequent outline at time t + 2. The nonoverlapping area (
A) was measured with NIH Image and expressed relative to A(t). The change in
A/A(t) was used as an index of cell motility.
Drugs.
PGE2 receptor agonists, ONO-DI-004 (EP1 agonist), ONO-AE1-259 (EP2 agonist), ONO-AE-248 (EP3 agonist), and ONO-AE1-329 (EP4 agonist), were kindly supplied by Ono Pharmaceutical (Osaka, Japan). PGE2, forskolin, guanosine 5'-O-(2-thiodiphosphate) (GDPS) trilithium salt, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), phorbol 12-myristate 13-acetate (PMA), Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS), and tamoxifen were purchased from Sigma (St. Louis, MO). N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide dihydrochloride (H-89), ionomycin, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) were obtained from Calbiochem (La Jolla, CA). The above drugs were dissolved in dimethyl sulfoxide (DMSO; Sigma) and later diluted with extracellular solutions or the patch pipette solutions to reach final concentration, which resulted in a final DMSO concentration of <0.05%. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; Sigma) was dissolved directly in the extracellular solution. All other chemicals were obtained from Sigma.
Statistical analysis. Data are expressed as means ± SE for n observations. Statistical differences were analyzed with Student's t-test, and a value of P < 0.05 was considered to be significant.
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RESULTS |
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The Cl dependence of the current stimulated by 1 µM PGE2 was determined by varying [Cl]o. As shown in Fig. 2Ba, reversal potentials of the PGE2-stimulated outwardly rectifying current shifted with [Cl]o. Mean reversal potentials were 1.1 ± 1.2 (n = 5), 16.3 ± 1.8 (n = 3) and 33.8 ± 2.8 (n = 3) mV for 143.5, 73.5, and 33.3 mM [Cl]o, respectively (Fig. 2Bb), and these values were close to the predicted reversal potentials for these [Cl]o. A least-squares fit for the reversal potentials against [Cl]o on a semilogarithmic scale had a slope of 44 mV per 10-fold change in [Cl]o, suggesting that the stimulated current by PGE2 is mainly carried by Cl.
Anion permeability of Cl channels stimulated by PGE2.
Relative anion permeability of the Cl channels stimulated by PGE2 was determined (Fig. 3). Values for the relative membrane permeability of each anion against Cl (PX/PCl) were estimated from the reversal potential changes by the following equation derived from the Goldman-Hodgkin-Katz equation:
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Sensitivity of PGE2-stimulated Cl current to Cl channel blockers. To examine the effects of the Cl channel blockers DIDS, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and NPPB on the PGE2-stimulated Cl current (Fig. 4, A and B), these blockers were applied to the bath after the current was maximally stimulated by PGE2. DIDS (30 µM) reversibly inhibited the PGE2-induced Cl current in a voltage-dependent manner, being more effective on the outward current than on the inward current (Fig. 4A; 44 ± 5% inhibition at 70 mV vs. 20 ± 6% inhibition at 70 mV; n = 5). The following application of 100 µM DIDS caused further suppression of the PGE2-stimulated current. Another stilbene derivative, SITS, also inhibited the PGE2-induced Cl current in a voltage-dependent manner (data not shown). NPPB (30 µM), an arylaminobenzoate, also inhibited the PGE2-induced Cl current and affected outward and inward currents equally (Fig. 4B; 60 ± 4% inhibition at 70 mV vs. 55 ± 6% inhibition at 70 mV; n = 5). Figure 4C shows the effect of tamoxifen, an inhibitor of the swelling-activated Cl channels (26), on the PGE2-stimulated Cl current. Application of tamoxifen (10 µM) caused only weak inhibition of the PGE2-stimulated Cl current. The following application of a higher concentration of tamoxifen (100 µM) further inhibited the PGE2-stimulated Cl current in a voltage-independent manner (Fig. 4C; 89 ± 8% inhibition at 70 mV vs. 82 ± 6% inhibition at 70 mV; n = 5).
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Effect of PGE2 on osteoclast membrane potential. Because activation of Cl channels in osteoclasts causes membrane depolarization (17), we examined the effect of PGE2 on the membrane potential of osteoclasts bathed in standard extracellular solution. Seven of ten cells examined possessed a resting membrane potential between 65 and 73 mV (68 ± 2 mV; n = 7). Three cells showed a resting potential of 5 to 38 mV. In cells possessing membrane potential of around 70 mV, application of PGE2 (1 µM) produced a long-lasting depolarization to 0 ± 3 mV (n = 4) with a latency of 12 min. Simultaneous application of DIDS (100 µM) suppressed the PGE2-induced membrane depolarization (n = 4).
Receptor subtypes and intracellular signal transduction pathways involved in PGE2-induced stimulation of Cl current.
To determine the PGE2 receptor subtypes (EPs) involved in the PGE2 stimulation of Cl current, the stimulatory effects of EP1, EP2, EP3, and EP4 receptor agonists on Cl current were examined. Figure 5A shows dose-response relationships between amplitude of outwardly rectifying current recorded at 80 mV and concentration of various EP agonists. The EP2 agonist ONO-AE1-259 stimulated the outwardly rectifying current in a concentration-dependent manner (Fig. 5A). The stimulatory effect was comparable to that of PGE2. The EP4 agonist ONO-AE1-329 also stimulated this current (Fig. 5A), but the maximal effect was much smaller than that of PGE2 or the EP2 agonist. Reversal potentials of the ONO-AE1-259 (1 µM)- and ONO-AE1-329 (1 µM)-induced currents were shifted to positive potentials by lowering [Cl]o, and least-squares fits against [Cl]o on a semilogarithmic scale had slopes of 47 (n = 3) and 45 (n = 3) mV per 10-fold change in [Cl]o, respectively. The estimated PX/PCl values of the ONO-AE1-259 (1 µM)-induced current were 1.50 ± 0.08 (n = 4) for I, 1.05 ± 0.04 (n = 3) for Br, and 0.25 ± 0.09 (n = 3) for gluconate. Those of the ONO-AE1-329 (1 µM)-induced current were 1.46 ± 0.08 (n = 3) for I, 1.01 ± 0.02 (n = 3) for Br, and 0.28 ± 0.11 (n = 3) for gluconate. Thus currents induced by ONO-AE1-259 and ONO-AE1-329 displayed the same anion permeability sequence of I > Br Cl. DIDS (30 µM) and NPPB (30 µM) reversibly suppressed the ONO-AE1-259 (1 µM)-induced current in voltage-dependent (50 ± 5% inhibition at 70 mV vs. 16 ± 3% inhibition at 70 mV; n = 3) and -independent (55 ± 6% inhibition at 70 mV vs. 51 ± 4% inhibition at 70 mV; n = 3) manners, respectively. The ONO-AE1-329 (1 µM)-induced current was also inhibited by DIDS (50 µM) in a voltage-dependent manner (60 ± 5% inhibition at 70 mV vs. 28 ± 5% inhibition at 70 mV; n = 3). Thus electrophysiological and pharmacological properties of currents induced by ONO-AE1-259 and ONO-AE1-329 were very similar to those of currents induced by PGE2. On the other hand, no significant stimulatory effects on Cl current were observed with EP1 and EP3 agonists ONO-DI-004 and ONO-AE-248 (Fig. 5A).
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Effects of PGE2 and EP agonists on cell planar area and motility of isolated rat osteoclasts. To assess the action of PGE2 on osteoclast morphology, we examined PGE2 effects on cell planar area and motility with pure osteoclast preparations (see MATERIALS AND METHODS). The morphological changes were observed in HEPES-buffered medium to match the experimental conditions of our electrophysiological experiments. Figure 6A represents time-lapse video images of bulgy serrated osteoclasts recorded before application, during application, and after removal of 1 µM PGE2. Although osteoclasts showed intense motility, ceaselessly protruding and retracting lamellipodia before application of PGE2, a sustained decrease or increase in cell planar area did not occur. DMSO (0.05%) did not change averaged values of osteoclast planar area or motility. Application of 1 µM PGE2 produced a progressive decrease in planar area (19 ± 5% inhibition at 15 min; n = 5), reduced osteoclast motility (21 ± 3% inhibition at 15 min; n = 5) (Fig. 6B), and resulted in a spiny appearance of the cell periphery (Fig. 6A, center). Effects of PGE2 on cell planar area and motility reached a maximum within 10 min after application of PGE2 and were sustained during the application (Fig. 6B). Removal of PGE2 reversed both effects (Fig. 6A, right). The effects of PGE2 on cell planar area and motility were also examined in bicarbonate-buffered medium, and results qualitatively similar to those in HEPES-buffered medium were obtained: PGE2 (1 µM) reduced osteoclast planar area (14 ± 5% inhibition at 15 min; n = 4) and inhibited motility (21 ± 3% inhibition at 15 min; n = 4). These results suggest that PGE2-induced morphological changes are relevant to osteoclast physiology. Because the preparation contained only osteoclasts (pure osteoclast preparation), these results also confirm that PGE2 acts directly on osteoclasts.
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Signal transduction pathways involved in PGE2-induced morphological changes. Application of forskolin (10 µM) reduced osteoclast planar area and motility (Fig. 7A). DBcAMP (1 mM) also reduced cell planar area and motility. Pretreatment with Rp-cAMPS (50 µM) for 30 min inhibited the PGE2-induced reduction of osteoclast planar area and motility (Fig. 7B). Figure 7C summarizes the effects of PKA inhibitors on 1 µM PGE2-induced reduction of cell planar area and motility. Pretreatment of osteoclasts with H-89 (1 µM) also reduced the PGE2 effects on cell planar area and motility.
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DISCUSSION |
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PGE2 exerts its effects through specific membrane receptors coupled to GTP-binding proteins (25). Four subtypes of PGE2 receptors with different intracellular signaling pathways have been identified. The EP1 subtype induces [Ca2+]i mobilization, both EP2 and EP4 subtypes stimulate adenylate cyclase, and the EP3 subtype mainly inhibits adenylate cyclase (25). Among specific agonists for each of the four subtypes of PGE2 receptors, ONO-AE1-259, an EP2 agonist, was most effective in stimulating the Cl current and reducing cell planar area and motility. Our results are most consistent with the presence of EP2 receptor on osteoclasts: 1) GDPS, Rp-cAMPS, and H-89 inhibit the PGE2-induced current, and 2) forskolin and DBcAMP mimic the PGE2-induced current.
ONO-AE1-329, an EP4 agonist, also had a stimulatory effect on Cl current and caused a reduction of cell planar area, but both effects were much weaker than those of ONO-AE1-259. It thus appears that PGE2-induced changes in osteoclast electrophysiology and morphology are mediated predominantly by the EP2 subtype and to a lesser degree by the EP4 subtype. In contrast to our findings, Mano et al. (22) reported that direct action of PGE2 on rabbit osteoclasts was mediated by the EP4 subtype and that EP4 mRNA was abundantly expressed in rabbit osteoclasts, whereas the other types of EP mRNAs, including EP2 mRNA, were detected only in minor amounts. Although this discrepancy may be attributed to differences in animal species, further experiments at the molecular or protein level are required to elucidate the different mechanisms of PGE2 action in rats and rabbits.
Properties of PGE2-stimulated Cl current in osteoclasts.
The PGE2-stimulated Cl current examined in this study was characterized by outward rectification, rapid activation, time-dependent inactivation at strong depolarizing potentials (Fig. 2), an anion permeability sequence of I > Br Cl > gluconate (Fig. 3), and block by DIDS, NPPB, and tamoxifen (Fig. 4, AC). The Cl current stimulated by forskolin also showed characteristics similar to those of the PGE2-stimulated Cl current. The electrophysiological and pharmacological properties of the PGE2-stimulated Cl current examined in our study also resemble those of the current induced by hyposmotic stimulation (swelling-activated Cl currents) demonstrated previously in a variety of cells (23, 26, 36, 38) including mouse osteoclasts (31). In addition, any further stimulatory effect of PGE2 on the current was abolished after currents were induced by hyposmotic solution (Fig. 4D), indicating that PGE2-induced current and hyposmotically activated current share a common pathway. The presence of such a common pathway for cAMP- and swelling-activated Cl currents has also been suggested in rat carotid body cells (4).
It is well known that cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels are activated by a cAMP-dependent pathway (13). However, the electrophysiological properties of CFTR Cl channels, showing a linear I-V relationship in symmetrical Cl concentration, insensitivity to extracellular DIDS, and an anion permeability sequence of Cl > I (2, 7, 13), are unlike those of the PGE2-stimulated current demonstrated in our study. Therefore, the channel responsible for the PGE2-stimulated current in rat osteoclasts differs from the CFTR Cl channel. A recent report by Diewald et al. (8) shows that CLC-7 Cl channels expressed in Xenopus oocytes display outwardly rectifying properties similar to the Cl current described in the present study. However, the electrophysiological and pharmacological properties of CLC-7 Cl channels have not yet been established. Thus it is presently uncertain whether or not the Cl current stimulated by PGE2 is carried by CLC-7 Cl channels.
Possible contribution of Cl channels to PGE2-induced changes in osteoclast morphology. Osteoclasts alternate between resorptive and motile states (16), both of which are necessary for effective bone resorption. Cl channels have been documented to play important roles in bone resorption during the resorptive state of osteoclasts. During bone resorption, osteoclasts transport H+ into the resorption lacuna through vacuolar H+ pumps located in the ruffled border membrane (35). Because this H+ pump is electrogenic (9), countermovement of appropriate ions is required to maintain the activity of the H+ transport at a constant level and to prevent the accumulation of electric charges (3). Thus Cl channels act as a charge transport pathway for electrical neutralization against the charge accumulation associated with the H+ transport. For example, the Cl channel blocker DIDS inhibits bone resorption (11, 18). Also, disruption of the CLC-7 Cl channel in mice leads to severe osteopetrosis that is due to failure of osteoclasts to secrete H+ (21). These studies demonstrate that the CLC-7 Cl channel provides a Cl-conductive pathway that acts in conjunction with the H+ pump for electroneutral HCl secretion into the resorption lacuna by resorptive-state osteoclasts. In addition, Cl efflux through Cl channels would also serve to maintain the inward Cl gradient that generates the driving force for the Cl/HCO3 exchange mechanism.
In contrast, little is known about the roles of Cl channels in nonresorbing/motile-state osteoclasts. Recently, we (15) demonstrated that bulgy, serrated osteoclasts maintain higher basal [Ca2+]i compared with flat, round osteoclasts and that exposure of these cells to a high-K+ solution, which causes cell membrane depolarization, reduces [Ca2+]i, cell planar area, and motility. Both inhibition of inwardly rectifying K+ channels and activation of outwardly rectifying Cl channels may induce membrane depolarization in osteoclasts (17, 27). Our results demonstrate that the PGE2-induced depolarization in osteoclasts is due to activation of outwardly rectifying Cl channels.
In many cell types including osteoclasts, it has been suggested that Cl channels also contribute to cell volume regulation and that their activation leads to the loss of cytoplasmic osmolytes and water, resulting in a cell volume decrease (28, 31). The physiological consequences of this effect for osteoclast function and bone resorption remain to be investigated.
Electrophysiological and morphological analyses of PGE2 actions were performed with HEPES-buffered (or in some experiments bicarbonate buffered) solutions. We found a good correlation between electrophysiological and morphological changes induced by PGE2 regarding time course, reversibility, and cAMP dependence. These results suggest a correspondence between electrophysiological and morphological effects of PGE2. Qualitatively similar results were also observed in our experiments using bicarbonate-buffered solutions, indicating that the electrophysiological and morphological changes induced by PGE2 are of physiological significance.
It is likely that activation of the Cl channel by PGE2 will lower [Ca2+]i levels by depolarizing the membrane potential of bulgy, serrated osteoclasts and thereby reduce osteoclast motility as observed in this study. Because there is a good correlation between osteoclast motility and bone-resorbing activity (40), suppression of osteoclast motility by PGE2 via EP2 receptor activation is likely to reduce bone resorption.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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