Department of 1Physiology and 3Hypertension and Renal Center of Excellence, Tulane University Health Sciences Center School of Medicine, New Orleans, Louisiana 70112; and Department of 2Pathology, University of North Carolina, Chapel Hill, North Carolina 27599
Submitted 5 March 2003 ; accepted in final form 18 June 2003
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ABSTRACT |
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sodium excretion; cGMP; gene disruption; gene duplication
Natriuretic peptides belong to a family of three homologous peptide hormones. ANP and brain natriuretic peptide (BNP) are released by the heart; and C-type natriuretic peptide (CNP) is produced in endothelial cells (10, 20, 46). All three natriuretic peptides are thought to exert important roles in the maintenance of blood pressure and cardiovascular homeostasis. Distinct natriuretic peptide receptors have been identified and characterized by molecular cloning (39, 41). These include natriuretic peptide receptor A, B, and C, also designated as NPRA, NPRB, and NPRC, respectively (11, 19, 33). Both ANP and BNP specifically bind to NPRA, whereas CNP binds to NPRB; nevertheless, all three natriuretic peptides show affinity to NPRC. The hormone binding to NPRA and NPRB results in the production of intracellular second messenger cGMP by guanylyl cyclase activity that resides in the intracellular domains of these receptors (22, 36, 43). NPRA is thought to be the primary ANP/BNP signaling molecule and has been suggested as the principal mediator of natriuretic peptide activities.
Previous experimental data established that ANP plays an important role in regulation of renal function by its vasodilatory and natriuretic responses and its ability to counteract the renin-angiotensin-aldosterone system in a tissue-specific manner (25). Attempts have been made to define physiological responses of ANP using several experimental approaches. It has been possible to correlate the effects of changes in blood hormone levels commensurate with those found in pathophysiological states by infusing the exogenous hormones (40). Cardiac appendectomy has been used to prevent ANP release; however, the problem in this setting is that the missing normal cardiac function results in a lack of physiological reflexes that are normally elicited by atria (42). Other studies used monoclonal antibodies against circulating ANP and agents that specifically inhibit the signaling pathway of NPRA by blocking cGMP production. Although two compounds, A-71915 and HS-1421, have been shown to diminish the effect of ANP by antagonizing NPRA, these compounds do not completely inhibit NPRA and may have nonspecific effects (9, 30). Gene-targeting strategies in mice provide novel approaches in the study of the physiological responses corresponding to gene dosage in vivo (15, 45).
Genetic mouse models with disruption of the ANP/NPRA system have provided strong support for a physiological role of this hormone-receptor system in the regulation of arterial pressure and other pathophysiological functions (13, 14, 21, 24, 31, 37, 44). Therefore, the genetic defects that reduce the activity of ANP and its receptor system can be considered as candidate contributors to essential hypertension and congestive heart failure (12, 14, 17, 18, 31, 44, 48). To examine the regulatory role of NPRA in kidney function and blood pressure homeostasis at the molecular level, we performed studies evaluating the changes in renal function using Npr1 (coding for NPRA) gene-disrupted and gene-duplicated mutant mouse models. We hypothesized that the quantitative genetic alterations in NPRA expression levels in vivo mediate the primary ANP signaling mechanism responsible for the natriuretic and hemodynamic responses to intravascular expansion. To test this hypothesis, we administered whole blood to null homozygous mutant (0-copy), wild-type (2-copy), and gene-duplicated (4-copy) mice to produce intravascular volume expansion not accompanied by hemodilution.
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MATERIALS AND METHODS |
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The breeding of gene-duplicated 3-copy (++/+) heterozygous mice generated progenies consisting of 2-, 3-, and 4-copy animals. The Npr1 gene-duplicated mice were genotyped using upstream (5'-CCT CTA GAT GCA TAC ATG TCG C-3') and downstream (5'-GGT CAA GTT AAG TGT ATT TTT TTC CC-3') primers. The PCR reaction from tail DNA included 10 mM Tris · HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 0.4 mM each of dNTPs, 2.5 U of Taq DNA polymerase, and 40 nM primers. The PCR was performed by the use of a 30-s denaturation step at 94°C, a 30-s annealing step at 60°C, and a 30-s extension step at 72°C, respectively, for 35 cycles as previously described (32) with modifications. The amplified genomic fragments were separated on nondenaturing acrylamide gels and corresponded to 108- and 124-bp bands from 2- and 4-copy genotypes, respectively, whereas the heterozygous (3-copy) animals contained both 108- and 124-bp amplified genomic fragments (Fig. 1B).
Animal preparation. The mice were housed under a 12: 12-h light-dark cycle at 25°C and fed regular chow (Purina Laboratory) and tap water ad libitum. In the present experiments, 16- to 24-wk-old male mice weighing 3035 g were used. Animals were anesthetized with Inactin (100 mg/kg ip thiobutabarbital sodium salt). A supplemental dosage of anesthetic (5 mg/kg im ketamine) was administered as required. The animals were placed on a servo-controlled surgical table that maintained body temperature at 37°C, and a tracheotomy was performed. The animals were allowed to breathe humidified 95% O2-5% CO2 by placing the exterior end of the tracheal cannula inside a small plastic chamber. The right jugular vein was catheterized with PE-10 tubing for fluid infusion. After catheterization, 0.9% NaCl containing 10% Inutest (Laevasom-Gesellschoft, Ling, Austria), 3% PAH (Sigma), and 1% BSA was infused at a rate of 2.5 µl/min. The left carotid artery was cannulated (PE-10 tubing connected to PE-50 tubing) for measurements of arterial pressure. Blood pressures were recorded on a pressure transducer connected to a Grass polygraph (Grass Instrument, Quincy, MA). Blood pressures were determined continuously throughout the duration of the experiment. The bladder was catheterized with PE-50 tubing via a supra pubic incision for urinary collections as previously described (6). After equilibration for 45 min, eight consecutive 20-min urinary collections were obtained. At the end of the experiment, blood was collected from the carotid artery into hematocrit tubes and chilled tubes containing 5 µl of 0.2 M sodium-EDTA and was immediately centrifuged at 4°C. Plasma was removed and stored at 80°C until used to assay ANP, total protein content, Inutest, and PAH concentrations.
Volume expansion. Npr1 homozygous mutant (0-copy; n = 9),
wild-type (2-copy; n = 9), and gene-duplicated (4-copy; n =
9) mice received whole blood obtained from age-matched 1-copy (n =
18) or 3-copy (n = 9) male donor mice. The donor animals were
anesthetized with Inactin (100 mg/kg ip), and the whole blood was drawn by
cardiac puncture using a heparinized syringe. Hemodynamic and kidney functions
were determined before (060 min), during (6080 min), and after
(80160 min) blood volume expansion. Before the volume expansion period
(060 min), three initial consecutive 20-min urine collections and blood
pressure measurements were performed, after which the blood volume expansion
was carried out beginning at 60 min. Fresh whole blood was infused into 0-,
2-, and 4-copy mice over a 20-min volume expansion period (6080 min) to
expand the circulating blood volume by an estimated 15% (450 µl, 1.5%
of body wt). Five consecutive 20-min recovery (60160 min) blood
pressure measurements and urinary collections were performed during and after
the blood infusion. One hundred microliters of blood were collected at 60 and
100 min, and total blood was collected at 160 min after the completion of the
experiment.
Determination of plasma and urinary inulin (Inutest) and PAH. Ten microliters of plasma were mixed with 110 µl of 3.2% TCA, centrifuged at 3,000 rpm for 30 min, and the supernatant was collected. Each 5 µl of collected urine were diluted with 9.995 ml of water. For Inutest measurements, 25 µl of TCA-precipitated plasma supernatant, diluted urine samples, or Inutest standards were added to a 96-well plate. Two hundred fifty microliters of 0.1% anthrone were added to each well, and samples were incubated at 60°C for 10 min. The plates were counted at 620-nm wavelength with a multiscan plate reader (Lab System, Franklin, MA). Similarly, for PAH measurement, 50-µl samples and PAH standards were added to a 96-well plate. Simultaneously, water (150 µl), 0.2 N HCl (40 µl), 0.1% sodium nitrite (20 µl), 0.5% ammonium sulfamate (20 µl), and 0.1% N-(naphthyl) ethylenediamine (20 µl) were added to each well. After each addition, the plate was shaken and then counted at 540 nm. Glomerular filtration rate (GFR) was calculated as urine-to-plasma Inutest concentration ratio times urinary flow and was factored per gram kidney weight. Renal plasma flow (RPF) was calculated as urine-to-plasma PAH concentration ratio times urinary flow and was factored per gram kidney weight. The plasma protein concentrations of donor and recipient mice were determined in a 96-well plate using a Bio-Rad protein assay kit (Hercules, CA).
Analytic procedures. The hematocrit of each blood sample from donor or recipient mice was measured after centrifugation with Adams Autocrit. Urinary volumes were determined by gravimetry. The sodium and potassium concentrations of all samples were determined by flame photometry (model 443, Instrumentation Laboratory). Fractional sodium excretion was calculated by dividing the urine/plasma sodium concentration ratio by the urine/plasma Inutest ratio. Similarly, fractional potassium excretion was calculated by dividing the urine/plasma potassium concentration ratio by the urine/plama Inutest ratio.
Urinary cGMP assay. Ten microliters of acetylated urine samples were mixed with 90 µl of 0.05 M sodium acetate buffer, pH 6.2, and cGMP in the samples was determined with a radioimmunoassay kit (Peninsula, Belmont, CA) as previously described (35).
Plasma ANP assay. A plasma sample (400 µl) was applied to Sep-Pak C-18 column that was prewashed with methanol and 0.1% trifluoroacetic acid (TFA). The column was washed with 5 ml of 0.1% TFA and eluted with a 3-ml mixture of methanol:water:TFA (80:19.9:0.1) as previously described (34, 44). The eluates were brought to dryness in a speed vac centrifuge. The residues were dissolved into 100 mM Tris buffer, pH 7.5, containing 2.5 mM EDTA, 1 mM PMSF, 0.02% sodium azide, and 0.1% BSA. ANP was quantitated using ANP antibodies (Peninsula; 10,000x dilution) and 125I-ANP as previously described (38). One hundred-microliter plasma samples were mixed with 100 µl antibody at 4°C for 48 h, after which 100 µl 125I-ANP (15,000 cpm) were added and samples were incubated further for 24 h at 4°C. On day 4, 100 µl (500-fold dilution) goat anti-rabbit immunoglobulin serum and 10 µl (500-fold dilution) normal rabbit serum were added and mixed. After centrifugation at 1,500 g for 30 min at 4°C, the supernatant was aspirated and radioactivity in the pellet was counted with a gamma counter.
Statistical analyses. The results are presented as means ± SE. Statistical analyses were performed by factorial one-way ANOVA followed by the Student-Newman-Keuls post hoc test for multiple comparisons (GraphPad Instat Software, GraphPad Software, San Diego, CA). For all tests, statistical significance was set at P < 0.05.
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RESULTS |
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Figure 3 shows that after volume expansion, the plasma ANP concentrations (pmol/ml) were significantly higher in 0-copy mice (2.5 ± 0.2; P < 0.01) compared with 2-copy (1.5 ± 0.2) and 4-copy (0.7 ± 0.1) mice, respectively.
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Plasma protein concentrations and hematocrits. The plasma protein concentrations were not significantly different among either donor (1- and 3-copy) or recipient (0-, 2-, and 4-copy) mice (Fig. 4A). Similarly, there were no significant differences in hematocrits in the donor mice. However, after volume expansion, the hematocrits in recipient 2- and 4-copy mice were significantly higher compared with recipient 0-copy mice (Fig. 4B).
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Mean arterial blood pressures before, during, and after whole blood volume expansion. The mean arterial pressures during all conditions were 3040 mmHg higher in Npr1 homozygous null mutant (0-copy) mice than in wild-type (2-copy) mice (Fig. 5). In contrast, the mean arterial pressures were 1520 mmHg lower in Npr1 gene-duplicated (4-copy) mice than in wild-type (2-copy) control animals. Before volume expansion (060 min), the mean arterial pressures (mmHg) were significantly higher in 0-copy mice (129 ± 4; P < 0.001) and significantly lower in 4-copy mice (77 ± 2; P < 0.01) compared with 2-copy wild-type mice (92 ± 3). During volume expansion (6080 min), the mean arterial pressures increased and remained at significantly higher levels in 0-copy mice (140 ± 4; P < 0.001) but remained at significantly lower levels in 4-copy mice (90 ± 3; P < 0.01) compared with 2-copy wild-type mice (108 ± 3). Even after volume expansion (80160 min), the mean arterial pressures remained at significantly higher levels in 0-copy mice (136 ± 4; P < 0.001) and at significantly lower levels in 4-copy mice (81 ± 3; P < 0.01) compared with 2-copy wild-type animals (95 ± 4).
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GFR and RPF. As shown in Fig. 6A, before volume expansion, the baseline (060 min) GFR (ml · min1 · g kidney wt1) was significantly lower in 0-copy mice (0.49 ± 0.03; P < 0.05) and significantly higher in 4-copy mice (0.81 ± 0.04; P < 0.05) compared with 2-copy wild-type counterparts (0.63 ± 0.03). During the volume expansion period, GFR increased in all groups, which was maintained at a significantly lower level in 0-copy mice (0.58 ± 0.04; P < 0.01) and at a significantly higher level in 4-copy mice (1.19 ± 0.12; P < 0.01) compared with 2-copy wild-type animals (0.82 ± 0.09). Nevertheless, after volume expansion, GFR still remained at a significantly lower level in 0-copy mice (0.49 ± 0.05; P < 0.05) and at a significantly higher level in 4-copy mice (0.87 ± 0.08; P < 0.05) compared with 2-copy wild-type counterparts (0.68 ± 0.06).
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Similarly, as shown in Fig. 6B, the baseline RPF (ml · min1 · g kidney wt1) was significantly lower in 0-copy mice (2.24 ± 0.41; P < 0.05) and significantly higher in 4-copy mice (4.00 ± 0.49; P < 0.05) compared with 2-copy wild-type animals (2.96 ± 0.17). During volume expansion, the RPF remained at a significantly lower level in 0-copy mice (2.72 ± 0.37; P < 0.01) and increased significantly in 4-copy mice (5.78 ± 0.50; P < 0.01) compared with 2-copy wild-type control mice (4.36 ± 0.41). Again, after volume expansion, the RPF was lower in 0-copy mice (2.29 ± 0.30; P < 0.05) and remained significantly elevated in 4-copy mice (4.48 ± 0.47; P < 0.05) compared with 2-copy wild-type animals (3.10 ± 0.34).
Urinary flow and urinary sodium and potassium excretion. During volume expansion, urinary flow (µl · min1 · g kidney wt1) in 2-copy animals increased from 4.9 ± 1.0 to 14.4 ± 1.8 and sodium excretion rate (µeq · min1 · g kidney wt1) from 1.15 ± 0.22 to 3.11 ± 0.60 (Fig. 7, A and B). In contrast, 0-copy animals exhibited only a small change in urinary flow (3.4 ± 0.2 to 5.0 ± 1.0; P < 0.001) and sodium excretion (0.69 ± 0.21 to 1.10 ± 0.18; P < 0.001) despite the greater increases in arterial pressures. Interestingly, 4-copy mice showed significantly higher urinary flow (6.5 ± 0.6 to 24.0 ± 2.5; P < 0.01) and sodium excretion (1.86 ± 0.23 to 4.75 ± 0.60; P < 0.01) compared with 2-copy control animals (Fig. 7, A and B). Even after volume expansion, both 2- and 4-copy mice had significantly higher urinary flows and sodium excretion rates compared with 0-copy mice. In contrast, urinary potassium excretion rates were not significantly different in any of the Npr1 genotypes before, during, and after volume expansion and were not increased by blood volume expansion (Fig. 7C).
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As shown in Fig. 8, before volume expansion, the percent baseline fractional sodium excretions remained at a significantly lower level in 0-copy mice (0.76 ± 0.14%) and were not significantly different in 4-copy (1.51 ± 0.14%) mice compared with 2-copy (1.32 ± 0.13%) wild-type mice. However, during volume expansion, fractional sodium excretions were significantly elevated in 4-copy mice (3.65 ± 0.22%; P < 0.01) and remained significantly lower in 0-copy mice (1.23 ± 0.16%; P < 0.01) compared with 2-copy wild-type animals (2.45 ± 0.16%). After volume expansion, fractional sodium excretions were still significantly higher in 2-copy (1.64 ± 0.16%) and 4-copy (1.90 ± 0.18%) mice compared with 0-copy mice (1.14 ± 0.15%). No significant differences in fractional potassium excretions were observed among the three groups of Npr1 mice (data not shown).
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DISCUSSION |
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A number of factors influence the kidney's ability to excrete sodium and water in response to blood volume expansion (2, 3, 23, 27, 28). Activation of natriuretic systems such as natriuretic peptides (ANP, BNP) and nitric oxide enhances the pressure-natriuresis relationship and reduces arterial pressures. It has also been suggested that chloride-mediated feedback control of NPRA occurs in the kidney and probably plays a role in the regulation of ANP-mediated natriuresis (26). Initial studies have shown that ANP suppresses renin and decreases blood pressures (5, 24, 29). Our earlier findings with Npr1 gene-disrupted mice demonstrated that, at birth, the absence of NPRA allows greater renin and ANG II levels and increased renin mRNA expression compared with 2-copy control mice (44). However, at 316 wk of age, both circulating and kidney renin and ANG II levels were decreased dramatically in 0-copy mice compared with 2-copy control animals. This decrease in the renin activity in adult 0-copy mice could be due to progressive elevation in arterial pressures leading to inhibition of renin synthesis and release from the kidney juxtaglomerular cells. However, aldosterone levels in 0-copy mice were dissociated from the circulating ANG II levels and were elevated. It is also possible that the renin in 0-copy mice might be suppressed as a consequence of aldosterone-mediated sodium retention with associated fluid retention (44). These and other various systems could be responsible for the differences in basal values among the three groups.
Previous studies suggested that increased levels of ANP released into the plasma in response to blood volume expansion in rats were mainly responsible for the natriuretic and diuretic responses (3, 4, 40). Furthermore, Paul et al. (40) showed that both ANP and acute blood volume expansion act on the kidney through a similar saturable mechanism. However, direct evidence demonstrating the quantitative contribution of ANP working through NPRA to the natriuresis and diuresis resulting from an isohemic, isooncotic blood volume expansion has not been obtained. In a study using Npr1 homozygous null mutant and wild-type mice, it was demonstrated that infusion of ANP, while causing substantial natriuresis and diuresis in wild-type mice, did not cause significant increases in sodium excretion or urinary flow in NPRA-deficient mice (16). Furthermore, urinary flow and sodium excretion rapidly increased in response to volume expansion with albumin containing Ringer solution in wild-type (2-copy) mice compared with homozygous null mutant (0-copy) mice. In the present study, we examined the quantitative contribution and possible mechanisms mediating the responses of NPRA by determining the RPF, GFR, urinary flow, and sodium and potassium excretion patterns following blood volume expansion in 0-, 2-, and 4-copy mice in a Npr1 gene dose-dependent manner. By using whole blood, hemodilution did not occur and plasma protein levels were not reduced. Thus other natriuretic mechanisms related to plasma dilution, such as decreases in colloid osmotic pressure or decreases in hematocrit, were not activated with this protocol. Although the blood volume expansion stimulated the release of ANP in all three Npr1 genotypes of mice, significant functional responses occurred only in 2- and 4-copy mice but not in 0-copy animals. These results demonstrate that the ANP/NPRA axis is primarily responsible for mediating the renal hemodynamic and sodium excretion responses to intravascular blood volume expansion. Furthermore, the sodium excretion responses appear to be due to the combined contribution of increases in filtered load as well as reductions in tubular fractional reabsorption. The associated changes in urinary cGMP excretion rates are consistent with the activation of tubular NPRA leading to increased formation of cGMP, which might pass into tubular fluid and be excreted in the urine. In addition, it should be noted that the increase in urinary flow could be due, in part, to an inhibition of antidiuretic hormone levels via the low-pressure volume receptors stimulated by intravascular volume expansion.
The finding that the absence of NPRA almost completely prevented the sodium excretory responses to blood volume expansion is somewhat surprising in that there are multiple systems that respond to volume expansion. An important part of the experimental design was to minimize nonspecific responses that could be associated with hemodilution, reductions in plasma colloid osmotic pressure, and other compositional changes in the blood that could directly affect sodium reabsorption and/or GFR. In addition, the blood volume infusion period was extended over a 20-min period to minimize reflexogenic alterations in sympathetic tone and volume expansion-mediated inhibition of vasopressin release. In this setting, it was possible to demonstrate the critical role that NPRA exerts in mediating the sodium excretory responses to a selective stimulus associated with blood volume expansion, which is presumably due to increases in right atrial pressure caused by the blood infusion.
Earlier studies suggested that the ANP/NPRA system plays an important role
in blood pressure homeostasis by direct natriuretic, diuretic, and
vasodilatory actions on the kidneys
(4,
25). ANP-deficient genetic
strains of mice demonstrated that a defect in ANP synthesis can cause
hypertension in homozygous null mutant mice with no circulating or cardiac ANP
(14). Therefore, genetic
defects that reduce the activity of the natriuretic peptide system can be
considered as candidate contributors to essential hypertension. Mice lacking
the ANP gene function and kept on a high-salt diet (8% NaCl) showed
hypertension with increased arterial pressures of 22 mmHg, which
suggested that genetically reduced production of ANP can lead to
salt-sensitive hypertension
(14). Npr1 gene-deficient mice
used in the present study exhibited a higher mean arterial pressure on
high-salt diet compared with animals kept on medium- or low-salt diets
(32). The absence of NPRA
expression in 0-copy mice provoked salt-sensitive increases in blood
pressures, whereas an increased expression of NPRA in 4-copy mice was able to
lower the blood pressures and protected against high dietary salt intake.
These previous studies provide evidence that ANP can be considered a major
system contributing an important role in the regulation of blood volume and
altered blood pressure. However, it should be acknowledged that Lopez et al.
(21) failed to show
salt-sensitive hypertension in their Npr1 gene-deficient mice. Thus it is
likely that ANP/NPRA axis induces hemodynamic and excretory parameters
including sodium excretion, which reduces the intravascular fluid volume and
is thus responsible for the decreased blood pressures observed in
gene-duplicated (4-copy) mice. Our data demonstrate that ANP/NPRA axis serves
an important mediator in acute natriuresis and diuresis after blood volume
expansion.
In conclusion, our present results demonstrate that both GFR and RPF were significantly lower in 0-copy and higher in 4-copy mice compared with 2-copy wild-type animals before, during, and after volume expansion. The data show that ANP responses to volume expansion led to the significantly lesser excretion of sodium and water in 0-copy mice and significantly greater excretory responses along with reduced tubular reabsorption in 4-copy mice compared with 2-copy (wild type) mice. Similarly, during the volume expansion, urinary cGMP concentration was significantly lower in 0-copy mice and greater in 4-copy mice compared with 2-copy control animals. Thus the higher cGMP concentrations in urinary samples were increased corresponding to Npr1 gene copy numbers. Our findings establish that NPRA is critical in mediating the natriuresis, diuresis, and renal hemodynamic responses to acute blood volume expansion.
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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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