1Division of Pathology, Department of Pediatric Laboratory Medicine, Research Institute, Hospital for Sick Children and University of Toronto, Toronto M5G 1X8; and 2Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1
Submitted 8 April 2003 ; accepted in final form 20 June 2003
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ABSTRACT |
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airway chemoreceptor; nicotinic acetylcholine 3
2,
4
2, and
7 receptors; whole cell patch clamp; in situ hybridization; immunohistochemistry
Pulmonary NEB are presumed airway oxygen sensors that may be involved in the autonomic regulation of breathing, especially during the neonatal period (16). Classic NEB cells are composed of innervated clusters of amine (serotonin, 5-HT) and neuropeptide-containing cells that respond to hypoxia by release of 5-HT (21). Studies on NEB cells in both culture and lung slices have shown that these cells express a membrane-delimited O2 sensor and that K+ channel activity plays an important role in hypoxia chemotransduction (20, 44, 47). We report here immunohistochemical, molecular, and electrophysiological evidence that NEB cells express a variety of nAChR composed of various subunits, including 3,
4,
7, and
2, raising the possibility of a heterogeneous population of ion channels. These findings underscore the importance of cholinergic mechanisms in NEB cell function under normal and pathological conditions.
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MATERIALS AND METHODS |
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In Situ Hybridization for nAChR 2-Subunit
To cross-identify the cells expressing the mRNA signal and to identify cells expressing the nAChR 2-subunit, we used immunohistochemistry followed by nonisotopic in situ hybridization (NISH). Tissues were fixed in 10% buffered formalin and embedded in paraffin. First, anti-serotonin (5-HT) monoclonal antibody (Sera-Lab, Crawley Down, Sussex, United Kingdom) was used to localize NEB cells. The 5-HT antibody was diluted 1:100, and the reaction was detected with avidin-biotin complex. Subsequently, in situ hybridization was performed on the same sections using digoxigenin-labeled antisense or sense (used as control) RNA probes for the nAChR
2-subunit as described previously (45). The appropriate cRNA probes were generated from a cDNA for rat nAChR
2-subunit (a gift from Dr. J. Boulter, Univ. of California Los Angeles). Briefly, sections of hamster fetal lung were treated with protease VIII to unmask the mRNA signal. Detection of the signal was achieved with DIG Nucleic Acid Detection Kit (Roche Molecular Biochemicals, Boehringer Mannheim, Mannheim, Germany), giving a dark purple color.
Immunofluorescence for nAChR 2-,
7-, and
4-Subunits
For immunohistochemical localization of 7 or
2 nAChR subunits in NEB cells, and to differentiate NEB cells from surrounding epithelial cells, we used double-label immunofluorescence on lung tissue fixed in 4% paraformaldehyde and embedded in paraffin, as previously reported (22). The sections were washed in PBS before being exposed to blocking solution containing 10% normal goat and rabbit serum albumin in PBS for 30 min at room temperature, followed by overnight incubation at 4°C in a cocktail of the primary antisera. The primary antibodies used included 1) anti-AChR
7 (1:200 dilution), a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 367-502 of human origin (Santa Cruz Biotechnology, Santa Cruz, CA); 2) anti-AChR
2 (C-20; 1:200 dilution), a goat polyclonal antibody raised against a peptide mapping at the COOH terminus of the
2-subunit of nAChR of human origin (Santa Cruz Biotechnology; 3) rat anti-serotonin (5-HT) antibody IgG (1:100 dilution; Medicorp, Montreal, PQ, Canada) as a marker of NEB cells; 4) anti-AChR
4 (1:50 dilution), a rabbit polyclonal antibody raised against a recombinant protein corresponding to a amino acids 342-474 of human origin (Santa Cruz Biotechnology); and 5) anti-calcitonin gene-related peptide (CGRP), a rabbit polyclonal antibody (1:200 dilution), against synthetic rat CGRP (Chemicon, Temecula, CA). The secondary antibodies consisted of FITC-conjugated rabbit anti-mouse IgG (1:300 dilution; Dako, Glostrup, Denmark), FITC-conjugated rabbit anti-goat IgG (1:400 dilution; Incstar, Stillwater, MA), and Texas red-conjugated goat anti-rabbit IgG (1:400 dilution; Jackson Immunoresearch Laboratories, West Grove, PA), respectively. Secondary antibodies were diluted in PBS containing 0.7% BSA. Samples were covered with Vectashield Mounting Medium (Vector Laboratories, Burlington, ON, Canada) before being viewed under an Olympus BX60 microscope (Carsen Group). RSimage software (Roper Scientific, Tucson, AZ) was used for image acquisition, and Adobe PhotoShop 6.0 software was used to process the images. As a positive control for nAChR
2- and
7-subunits, we used paraffin-embedded sections of rat and hamster brain tissues fixed in 4% paraformaldehyde according to the immunostaining protocol recommended by the manufacturer. For localization of the
4-subunit of nAChR, we used paraffin-embedded sections of tissue fixed in methanol, as recommended by the manufacturer. Lung tissue fixed in methanol was found to be suitable for immunolocalization of CGRP but not for serotonin (unpublished observations). As a positive control, a section of rat brain fixed in methanol was used per the manufacturer's instructions. As negative controls, the primary antisera were omitted.
Lung Slice Preparation
For electrophysiological studies, the lungs were cut into small pieces and embedded in 2% agarose (FMC Bioproducts, Rockland, ME). Sectioning was performed with tissue immersed in ice-cold Krebs solution that had the following composition (in mM): 140 NaCl, 3 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, and 5 glucose, pH 7.3, adjusted with NaOH (7). Transverse lung slices (200-300 µm) were cut with a Vibratome (Ted Pella, Redding, CA).
Electrophysiological Techniques and Solutions
For electrophysiological recordings, the lung slices were transferred to a recording chamber mounted on the stage of a Nikon microscope (Optiphot-2UD; Nikon, Tokyo, Japan). The perfusing Krebs solution had the following composition (in mM): 130 NaCl, 3 KCl, 2.5 CaCl2, 1 MgCl2, 10 NaHCO3, 5 HEPES, and 10 glucose, pH 7.35-7.4. To identify NEB cells in fresh lung tissue, the slices were incubated with vital dye, neutral red (0.02 mg/ml) for 15 min at 37°C, as previously described (20). To isolate inward currents, an internal pipette solution with the following composition was used (in mM): 130 CsCl, 1 CaCl2, 2 MgCl2, 10 EGTA, 10 HEPES, and 4 MgATP, pH adjusted to 7.2 with CsOH. The chamber, which had a volume of 1 ml, was perfused continuously with Krebs solution at a rate of 6-7 ml/min. All recordings were made from submerged lung slices at a temperature of 30 ± 2°C.
Drugs were applied to the perfusate, and their delivery to the cells was controlled by separate valves. The following drugs used in this study were obtained from Sigma (Oakville, ON, Canada): nicotine, ACh, atropine, choline, mecamylamine, 1,1-dimethyl-4-phenylpiperazinium (DMPP), and hexamethonium. The drugs obtained from RBI (Sigma-Aldrich Canada, Oakville, ON, Canada) included methyllycaconitine (MLA) and dihydro--erythroidine (DH
E). Stock solutions (1-10 mM) of all the drugs were prepared on the day of the experiment in twice-distilled water and diluted with Krebs solution to their final concentration before use.
An Axopatch 200B amplifier (Axon Instruments, Foster, CA) was used to record for whole cell currents (voltage clamp) or membrane potential (current clamp). Whole cell patch recordings were performed as described by Hamill et al. (25). The data were filtered at 5 KHz. The level of the fluid over the slices was kept low to minimize stray capacitance. Voltage and current clamp protocols, data acquisition, and analysis were performed using pClamp6 software and DigiData 1200B interface (Axon Instruments). All data values are given as means ± SE.
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RESULTS |
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We combined immunostaining for 5-HT using the immunoperoxidase method with NISH to localize the 2-subunit of nAChR in NEB cells. Typically, 5-HT immunoreactive NEB formed small cell clusters localized within the airway mucosa (Fig. 1A, a). After NISH was applied using antisense probe for the
2-subunit of nAChR on the same section, a strong mRNA signal (dark purple) was localized in 5-HT immunoreactive NEB cells (Fig. 1A, b). Although the adjacent airway epithelial cells also expressed a weak signal for mRNA for nAChR
2-subunit (light purple signal), there was no 5-HT immunoreactivity in non-NEB cells (Fig. 1A, a).
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For localization of the 7-subunit of nAChR in NEB cells, we used a dual-labeling immunohistochemistry method with antibodies against the
7-subunit (Texas red labeled) combined with anti-5-HT antibody (FITC labeled) as a marker of NEB cells. In agreement with a previous report (40), immunostaining for the
7-subunit was localized diffusely in airway epithelial cells (Fig. 1B, a). When the same section was examined for FITC-labeled 5-HT, positive immunoreactivity was restricted to NEB cell clusters (Fig. 1B, b). In merged images (yellow),
7 and 5-HT immunoreactivities were colocalized in NEB cells but not in adjacent epithelium (Fig. 1B, c). We also used a double-immunolabeling method to colocalize the
7- and
2-subunits of nAChR in the same lung tissue sections. Although there was diffuse positive labeling of airway epithelium with individual antibodies against either the
7- or
2-subunit (Fig. 1C, a and b), in merged images, strong colocalization of the two subunits was seen mostly in NEB cells, whereas adjacent epithelium was only weakly positive (Fig. 1C, c). Antibody against the
4-subunit of nAChR gave a positive reaction in NEB cells in lung tissue fixed in methanol but was negative in tissue fixed in 4% paraformaldehyde. Immunostaining for the
4-subunit was strongly positive in NEB cells, whereas adjacent epithelium appeared negative (Fig. 1D, a). We used CGRP as a marker for NEB cells in methanol-fixed tissue since immunostaining for serotonin was negative. Expression of CGRP in NEB cells is shown in Fig. 1D, b, and coexpression of the
4-subunit with CGRP appears as a yellow signal in NEB cell cytoplasm on a merged image (Fig. 1D, c).
ACh-Induced Inward Current and Depolarization of Membrane Potential in NEB Cells
The resting membrane potential of neonatal hamster NEB cells ranged from -40 to -60 mV (means = -50.4 ± 2.9 mV; n = 50), and the majority of experiments was performed on those with resting potentials more negative than -45 mV. Application of 100 µM ACh to a NEB cell voltage clamped at -60 mV elicited a transient inward current (Fig. 2A). Evoked current ranged from 100 to 450 pA, and the mean peak ACh-induced inward current (IACh) was 319.6 ± 90.8 pA (n = 10) at a holding potential of -60 mV. Desensitization of the nAChR is represented by the decay phase of IACh during prolonged application of the agonist (desensitization was determined by measuring from the peak amplitude of the currents evoked by ACh to after a desensitization of ACh); the decay was fitted with an exponential function, and the mean time constant (
) was 32 ± 4.3 s (n = 7) (36). Under current clamp, mean ACh-induced (100 µM) depolarization was 10.3 ± 1.2 mV (n = 6; Fig. 2B).
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Effects of Nicotine on Inward Currents and Membrane Potential
Nicotine activates two classes of currents in NEB cells. Rapid flow tube application of nicotine or other nicotinic agonists to voltage-clamped NEB cells evoked inward currents at negative potentials in 80% of cells studied (n = 135). The responses were usually one of two types (Fig. 3, A and B). In 88% of NEB cells (61 of 70 cells studied), nicotine (50 µM) induced a transient nicotine-induced inward current (Inic) at the holding membrane potential of -60 mV, followed by a rapid desensitization of the response (Fig. 3A). The mean (± SE) peak amplitude of Inic (50 µM) was 370.2 ± 31 pA (n = 25; range 200-600 pA), and
was 33.2 ± 6s(n = 15). In 11% of NEB cells (9 of 70 cells studied), a distinctly different response to nicotine (25 and 50 µM) was observed (Fig. 3). These currents had two components showing fast and slow desensitization, respectively. The mean peak inward current amplitude of NEB cells with two components was 412.2 ± 61 pA (n = 6), and
was 42.2 ± 4 s (n = 6). The peak inward current amplitude evoked by bath application of 50 µM nicotine vs. holding potential is shown in Fig. 3D, based on cells with one desensitizing component. The nicotine-induced currents reversed near 0 mV and showed prominent inward rectification characteristic of neuronal AChR in other cell types (37, 46, 50, 51). Representative examples of the effect of 50 µM nicotine on membrane potential in NEB cells under current clamp conditions is shown in Fig. 3C. Nicotine consistently depolarized NEB cells, from a initial value of -45 ± 3.1 mV to -6.3 ± 2 mV (n = 6).
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Nicotine dose-response relationship. The concentration dependence of inward currents evoked by bath application of nicotine is shown in Fig. 4A. At a holding potential of -60 mV, rapidly superfused nicotine (0.1-100 µM) induced concentration-dependent currents that displayed desensitization in the continued presence of the agonist. The peak currents ranged from 2.8 to 500 pA for cells with a single desensitizing component. To obtain the dose-response relationship, the mean peak current at each nicotine concentration was normalized to that elicited by 50 µM nicotine. The EC50 for receptor activation by nicotine was 3.8 ± 0.1 µM(n = 4), and the Hill coefficient was 1.1 ± 0.05 (n = 4) (Fig. 4B).
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Pharmacological Characterization of nAChR
Inward currents evoked by nAChR agonist DMPP. For NEB cells, the inward rectifying current-voltage relationship obtained for nicotine responses was similar to that reported for other cells (7, 27, 46, 50). We also tested the effects of another selective nAChR agonist DMPP as shown in Fig. 5. Application of 50 µM nicotine evoked an inward current (Fig. 5, A and C), similar to that of 50 µM DMPP applied to the same cell (Fig. 5B). The mean current induced by 50 µM DMPP was -350 ± 17 pA at a holding potential of -60 mV, and was 35.4 ± 4 s (n = 8, Fig. 5B).
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Blockade of nicotine-induced currents by nAChR antagonists. The current response induced by 50 µM nicotine was reversibly suppressed or abolished by 50 µM mecamylamine, an 3
2 and
4
2 nAChR antagonist (n = 12, Fig. 6, A and B), and by 50 µM DH
E, an
4
2 antagonist (n = 7, Fig. 6D) (4). In these experiments, the antagonists were applied to the cell for 5-10 min in Krebs solution, followed by exposure to the antagonist plus 50 µM nicotine. The blockade was reversible after a 10- to 20-min washout of the drug (Fig. 6, C and E). After nAChR expression in Xenopus oocytes, 50 µM mecamylamine completely blocked the
3
2 nAChR, whereas 5 µM mecamylamine completely blocked those containing
3
4-subunits (11). In NEB cells, the mean current induced by 50 µM nicotine was 444.5 ± 120.4 pA (n = 6) under control conditions and 78.2 ± 30.4 pA (n = 6) after 5 µM mecamylamine, corresponding to a reduction of
82.5%. Application of 50 µM hexamethonium, another nicotinic ganglionic blocker, also reduced this Inic from 383.5 ± 35 pA (n = 6) to 15.5 ± 5 pA (n = 6, see Fig. 9B), a reduction of
97%. However, atropine (50 µM) only partially suppressed the nicotine-induced current responses in NEB cells (see Fig. 9B). Similar results were reported in petrosal neurons (50) and CB type I cells (43). In summary, these observations indicate expression of functional nAChR subunits in NEB cells and suggest the involvement of
3
2- and possibly
3
4-subunits. The nAChR receptors in NEB cells exhibit characteristics of ganglionic nAChR.
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MLA blocks nicotine-induced fast-desensitizing current and choline-induced current. It has been reported that in cultured rat hippocampal neurons, ACh elicited both a rapidly desensitizing and more slowly desensitizing response. -BTXN inhibited the fast-desensitizing current, suggesting the involvement of the nAChR
7-subunit (51). Whole cell patch-clamp studies on cultured human bronchial epithelial cells demonstrated the presence of fast-desensitizing currents activated by choline (a specific nAChR
7 agonist) and nicotine that were blocked reversibly by MLA and irreversibly by
-BTXN (45). In NEB cells, both ACh (50 µM) and nicotine (25 or 50 µM) induced currents with two components consisting of both fast- and slow-desensitizing phases, as discussed above. To test whether or not the
7-subunit may be responsible for the fast-desensitizing current in NEB cells, we applied 10 nM MLA. Interestingly, this drug reversibly inhibited the fast-desensitizing current, whereas the slow-desensitizing current was unaffected (n = 6, Fig. 7, A and B). After a 10-min washout of MLA, 50 µM mecamylamine was found to block both fast- and slow-desensitizing currents induced by 50 µM nicotine (n = 3, Fig. 7, C and D). Application of 100 nM
-BTXN irreversibly blocked nicotine-induced current with fast component in NEB cells (data not shown). In addition, 50 µM DH
E also blocked both fast- and slow-desensitizing currents induced by 50 µM nicotine (data not shown). These findings are similar to those reported for hippocampal neurons (51) and midbrain dopaminergic neurons (24). The amplitude of MLA-sensitive fast current was 298.8 ± 40.2 pA, and
was 4.8 ± 3.8s(n = 6). The amplitude of MLA-insensitive slow current (that was blocked by 50 µM mecamylamine) was 111.3 ± 7.8 pA, and
was 29.8 ± 3.8 s (n = 6). Choline (0.5 mM) induced a fast-desensitizing current in NEB cells (Fig. 8A). The mean amplitude of choline-induced current was 125.2 ± 8.7 pA (n = 11) at a holding potential of -60 mV, and
was 25.5 ± 7.6 s (n = 11). The choline-induced fast-desensitizing currents were reversibly blocked by 10 nM MLA (Fig. 8, B and C; n = 5). Together, these data suggest that the
7-subunit of nAChR is responsible for the fast-desensitizing current, whereas the
3
2- or
4
2-subunits of nAChR most likely contribute to the slow-desensitizing current. The resulting coexpression of the
7- and
2-subunits raises the possibility of a further heterogeneity among nAChR channels (e.g., homomeric
7 and heteromeric
7
2 channels) expressed in individual NEB cells. Indeed, coexpression of the
7- and
2-subunits was reported recently in rat hippocampal interneurons (28).
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A cumulative summary of inward currents in NEB cells induced by ACh, nicotine, DMPP, and choline is shown in Fig. 9A. These findings indicate that NEB cells express functional nAChR. The effects of blockade by selective antagonists on nicotine-induced current in NEB cells are shown in Fig. 9B. Nicotine-induced inward current was reduced 96.1 ± 2% (n = 12) by 50 µM mecamylamine, 95.3 ± 10% (n = 7) by 50 µM DHE, 86.5 ± 14.1% (n = 7) by 10 nM MLA, 93.5 ± 9% (n = 7) by 50 µM hexamethonium, and 65.7 ± 10% (n = 5) by 20 µM atropine.
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DISCUSSION |
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The two major subtypes of nAChR in the brain are composed of 4
2- and
7-subunits. The nAChR formed by
7-subunits are known to exhibit several unique properties. For example, homomeric
7 channels are more permeable to Ca2+ and desensitize more rapidly than channels formed by other nAChR subunits (3). It has been suggested that ACh and choline might function as local "cytotransmitters" and modulate cellular functions (45). The
7-subunit of nAChR is a good candidate for mediating long-lasting, "hormonal" functions of ACh, since it can be activated by choline long after cleavage of ACh by acetylcholinesterase, and the resulting change in intracellular Ca2+ could result in a variety of metabolic effects (45). In the central nervous system (CNS),
7-subunits are predominantly presynaptic, suggesting that they modulate synaptic transmission in addition to their function in signal transduction (49). In embryonic muscle,
7 nAChR appear before the formation of synapses, and therefore, they may be involved in muscle development (19). Multiple functional subtypes of
7-containing nAChR have been reported in rat intracardiac ganglia, superior cervical ganglion neurons (14), and chick sympathetic neurons (46), suggesting the possibility for heteromeric
7-containing nAChR (28). Chronic exposure to nicotine related to the use of tobacco is known to upregulate the number of high-affinity
4
2 nicotine binding sites in the CNS and in heterologous expression systems (10). These findings implicate
4
2 nAChR in nicotine addiction (6, 10). The expression of postsynaptic or presynaptic nAChR
3
2-,
4-, and
7-subunits in NEB cells could indicate that ACh functions as an excitatory transmitter modulating the responses of NEB. There is now substantial evidence indicating that NEB function as airway chemoreceptors, possibly involved in the control of breathing (16, 20, 47). NEB possess complex innervation, although there is a considerable species variation. Recent immunohistochemical studies using a combination of confocal microscopy, vagotomy procedures, and neural tracing techniques revealed at least three distinct neural components innervating NEB in rat lungs (2, 8, 9, 42). The major component was represented by vagal afferents that originated in the nodose ganglion (8). The second component comprised CGRP-immunoreactive nerve fibers, originating in the spinal ganglia (8), whereas the third component exhibited immunoreactivity for nitric oxide synthase, with nerve fibers originating within the peribronchial ganglia (9). The evidence for cholinergic mechanisms in NEB and their possible involvement in hypoxia chemotransduction is, at present, mostly indirect. Previous ultrastructural studies on NEB in neonatal rabbit reported the presence of a sparse population of efferent-like (motor) nerve fibers containing small agranular vesicles (29, 30). Because these intracorpuscular nerve endings survived after supranodose vagotomy, their origin was considered to be that of side branches of sensory nerve fibers rather than being the endings of separate motor nerve fibers. Histochemical studies demonstrated high levels of acetylcholinesterase in NEB cells of rabbit fetal/neonatal lungs (15). Recent immunohistochemical studies identified vesicular ACh transporter immunoreactivity in nerve fibers in contact with NEB in rat lungs (1). Together, these data support the notion of a cholinergic efferent-like component of NEB innervation involved in axon-like reflex affecting local neuroregulatory mechanisms (29, 30).
The potential role of nAChR in pulmonary pathophysiology is strongly linked to effects of smoking. Of particular relevance to the function of NEB are perinatal pulmonary disorders related to the effects of maternal smoking (35, 38). Recent studies have implicated cholinergic mechanisms in pulmonary development. Chronic exposure to nicotine in utero was reported to significantly increase the number of alveolar type II cells and NEB cells and also to increase expression of 7 nAChR in monkey fetal lungs (39). Thus NEB cells may act as a modulator of cell proliferation under conditions of chronic nicotine exposure. This could be critical during lung development since the release of 5-HT and peptides from NEB cells could affect adjacent bronchovascular structures by targeting airway and/or vascular smooth muscle cells and associated nerve endings (16). Epidemiological studies have identified a close relationship between maternal smoking and sudden infant death syndrome (SIDS) (32). Although the precise mechanism is not known, nicotine, a major component of cigarette smoke, may increase the vulnerability of infants to SIDS via its action on peripheral chemoreceptors. Increased size and number of NEB have been reported in the lungs of SIDS victims born to smoking mothers (17). By analogy with known effects of nicotine on CB (26), the responses of hyper-plastic NEB to acute hypoxia may be blunted, making the infants of smoking mothers more susceptible to SIDS. The present study demonstrates expression and functional characterization of nAChR in NEB cells of neonatal hamster lungs, providing a useful model to further study the role of these receptors in NEB function as airway chemoreceptors under normal and pathological conditions.
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DISCLOSURES |
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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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REFERENCES |
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