1 Departments of Geriatric Medicine and 2 Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655; and 3 Division of Integrative Cell Biology, Department of Embryogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976, Japan
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ABSTRACT |
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Bronchial hyperresponsiveness and eosinophilia are major characteristics of asthma. Calcitonin gene-related peptide (CGRP) is a neuropeptide that has various biological actions. In the present study, we questioned whether CGRP might have pathophysiological roles in airway hyperresponsiveness and eosinophilia in asthma. To determine the exact roles of endogenous CGRP in vivo, we chose to study antigen-induced airway responses using CGRP gene-disrupted mice. After ovalbumin sensitization and antigen challenge, we assessed airway responsiveness and measured proinflammatory mediators. In the sensitized CGRP gene-disrupted mice, antigen-induced bronchial hyperresponsiveness was significantly attenuated compared with the sensitized wild-type mice. Antigen challenge induced eosinophil infiltration in bronchoalveolar lavage fluid, whereas no differences were observed between the wild-type and CGRP-mutant mice. Antigen-induced increases in cysteinyl leukotriene production in the lung were significantly reduced in the CGRP-disrupted mice. These findings suggest that CGRP could be involved in the antigen-induced airway hyperresponsiveness, but not eosinophil infiltration, in mice. The CGRP-mutant mice may provide appropriate models to study molecular mechanisms underlying CGRP-related diseases.
asthma; bronchial hyperreactivity; eosinophilia; leukotriene; knockout mouse
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INTRODUCTION |
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BRONCHIAL HYPERRESPONSIVENESS and inflammation, including eosinophilia, are major characteristics of asthma (11, 12, 23). Recent studies have shown that various mediators, including cytokines, eicosanoids, and adhesion molecules, are involved in the development of asthma. Genetic features are also potentially associated with the etiology of asthma. On the basis of the inheritance pattern, a number of genes could have substantial roles in the pathogenesis of bronchial asthma (43). However, the exact molecular mechanisms of bronchial asthma remain to be elucidated.
Calcitonin gene-related peptide (CGRP), a 37-amino acid neuropeptide,
has various biological actions, including responses to sensory stimuli,
cardiovascular regulation, and vasodilation (2, 3, 19,
20). CGRP belongs to the calcitonin family of peptides, which
includes calcitonin, amylin, and adrenomedullin. The calcitonin
receptor-like receptor functions as a CGRP receptor in the presence of
receptor activity-modifying protein 1 (RAMP1) (24). There
are two CGRP isoforms: -CGRP, which is present in the central and
peripheral nervous system (42), and
-CGRP, which is
expressed in specific neuronal sites (1). It has been shown that CGRP, a potent vasodilator (22), modulates
hypoxic pulmonary vasoconstriction (17). Recent studies
using genetically engineered mice have shown that CGRP-knockout mice
exhibit increased blood pressure and overactivation of the sympathetic
nervous system (38).
In the respiratory system, CGRP is synthesized by sensory C-fibers throughout the respiratory tree (47). CGRP is also found in neuroepithelial cells of the lung and coexists with tachykinins in many airway sensory nerves (20), and CGRP receptors have been found to densely populate lung vessels (17). In terms of its physiological role, it has been reported that CGRP potently constricts airway smooth muscle in humans (39) and guinea pigs (41). In addition, it has been shown that CGRP has a significant role in eosinophilia in allergic inflammation (7, 37). On the basis of these observations, it is assumed that CGRP might be involved in the pathogenesis of bronchial asthma.
In the present study, we questioned whether -CGRP might have
pathophysiological roles in airway hyperresponsiveness and eosinophil infiltration, which are hallmarks of bronchial asthma. To determine the
exact roles of
-CGRP in vivo, we chose to study the airway responsiveness and eosinophilia in
-CGRP gene-disrupted mice, which
have been recently established (38). After sensitization and antigen challenge, we assessed airway responsiveness and measured proinflammatory mediators.
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METHODS |
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Mice.
-CGRP-null mice were established as previously reported
(38). Briefly, the mouse CT/
-CGRP genomic
DNA was cloned from a BALB/c mouse genomic library in EMBL3 using
synthetic oligonucleotide probes derived from the mouse
CT/
-CGRP cDNA sequence. A 7.0-kb fragment containing
exons 3-5 of the mouse CT/
-CGRP gene was subcloned
into pBluescript (Stratagene). A targeting vector was constructed by
replacing the 1.6-kb XbaI-XbaI fragment
encompassing exon 5, which is specific for
-CGRP, with
the neomycin resistance gene, and flanking the thymidine kinase gene.
This plasmid was linearized with NotI and introduced into
129/Sv-derived SM-1 ES cells by electroporation; then the cells were
selected in medium containing G418 and ganciclovir. Homologous
recombinants were identified by PCR and Southern blot analysis.
Targeted ES cell clones were injected into C57BL/6 mouse blastocysts to
generate chimeric mice. Male chimeras were then cross bred with C57BL/6 females, and germline transmission was achieved. Mice heterozygous for
-CGRP-mutant allele with the genetic background of the 129/Sv × C57BL/6 hybrid were mated. Offspring were genotyped at 4 wk of age.
For genotyping, genomic DNAs were isolated from biopsied tail and
subjected to PCR amplification. The animals were maintained on a 7:20-h
light-dark cycle at 23°C. Mice were fed a standard laboratory diet
and water ad libitum. Mutant mice
(
-CGRP
/
) and their littermate controls
(
-CGRP+/+) were used in the present study.
Sensitization and antigen challenge. Mice were sensitized with an intraperitoneal injection of 0.5 ml of a solution containing 0.1 mg of ovalbumin (OA) complexed with aluminum hydroxide (2 mg/ml). On day 8, the mice were boosted with the same mixture. On day 12, these sensitized mice were challenged for 30 min with 1% OA in saline aerosol generated with an ultrasonic nebulizer (Ultra-Neb100, DeVilbiss, Somerset, PA). Control mice received an intraperitoneal injection of saline and saline aerosols in the same manner. Three days after the aerosol challenge, we measured bronchial responsiveness or performed bronchoalveolar lavage (BAL).
Animal preparation. Animals were anesthetized with pentobarbital sodium (25 mg/kg ip) and ketamine hydrochloride (25 mg/kg ip) in combination and then paralyzed with pancuronium bromide (0.3 mg/kg ip). Anesthesia and paralysis were maintained by supplemental administration of 10% of the initial dose every hour. After tracheostomy, a metal endotracheal tube (1 mm ID, 8 mm long) was inserted in the trachea. Animals were mechanically ventilated (model 683, Harvard Apparatus, South Natick, MA) with tidal volumes of 10 ml/kg and frequencies of 2.5 Hz. The thorax was widely opened by means of a midline sternotomy, and a positive end-expiratory pressure of 2 cmH2O was applied by placing the expired line under water. During the experiments, O2 gas was continuously supplied to the ventilatory system. Under these ventilatory conditions, arterial pH, PO2, and PCO2 were 7.35-7.45, 100-180 mmHg, and 30-45 mmHg, respectively, at the end of experiments (Compact 3 blood gas analyzer, AVL Medical Systems). A heating pad was used to maintain the body temperature of animals.
Tracheal pressure was measured with a piezoresistive microtransducer (model 8510B-2, Endevco, San Juan Capistrano, CA) placed in the lateral port of the tracheal cannula. Tracheal flow was measured by means of a Fleisch pneumotachograph (Metabo, Lausanne, Switzerland). All signals were amplified, filtered at a cutoff frequency of 100 Hz, and converted from analog to digital with a converter (model DT2801-A, Data Translation, Marlborough, MA). The signals were sampled at a rate of 200 Hz and stored on an IBM-AT-compatible computer. Lung resistance (RL) and elastance (EL) were measured as previously described (27, 31, 32).Airway responsiveness to methacholine administration. At the start of the protocol, two deep inhalations (3 times tidal volume) were delivered to standardize volume history. All animals were then challenged with saline aerosol for 2 min. Aerosols were generated by an ultrasonic nebulizer and delivered through the inspiratory line into the trachea. Measurements of 10-s duration were sampled during tidal ventilation 1 min after administration of saline aerosol. This represented the baseline measurement. Then each dose of methacholine (MCh) aerosol was administered for 2 min in a dose-response manner (0.625-20 mg/ml). Airway responsiveness was assessed using the concentration of MCh required to increase RL to 200% of baseline values (30).
BAL fluid.
BAL was performed [5 times with 1 ml of phosphate-buffered saline
(PBS)] in each group. In each animal, 90% (4.5 ml) of the total
injected volume was consistently recovered. After BAL fluid (BALF) was
centrifuged at 450 g for 10 min, the total and differential cell counts of the BALF were determined from the cell fraction (29, 34, 35). The supernatant was stored at 70°C until assays were performed. The concentration of protein was measured by
Lowry's method, with bovine serum albumin as a standard.
Assay of IgE in BALF. IgE levels in the BALF were determined using ELISA kits (Amersham Pharmacia Biotech, Piscataway, NJ). The detection limit of the ELISA assays for IgE was 10 ng/ml.
Assay of thromboxane and leukotriene in BALF. Thromboxane (Tx) A2 (TxA2, measured as TxB2) and leukotriene (LT) C4/D4/E4 were determined using enzyme immunoassay (EIA) kits (Amersham Pharmacia Biotech). The detection limits of the EIA assays for TxB2 and LTC4/D4/E4 were 3.6 and 10 pg/ml, respectively.
Assay of endothelin-1 in BALF. Endothelin-1 (ET-1) levels in the BALF were determined using EIA kits (IBL, Fujioka, Japan). The detection limit of the EIA assays for ET-1 was 0.78 pg/ml.
Assessment of CGRP immunoreactivity. In each group, the lungs of the mouse were removed intact and fixed with 10% formalin. After fixation, the tissue blocks obtained from midsagittal slices of the lungs were embedded in paraffin. Blocks were cut 4 µm thick by using a microtome. The preparations were processed for immunostaining by means of the avidin-biotin-peroxidase complex method. The slides were deparaffinized in a xylene bath and dehydrated in ethanol. Endogenous peroxidase activities were blocked by treatment with 0.6% H2O2 in 100% methanol for 30 min at room temperature. The primary antibody, rabbit anti-CGRP (rat) IgG (Peninsula Laboratories, San Carlos, CA), was diluted 1:200 in 10% FCS-PBS and added to the preparations overnight in a cold room. After six washes with PBS, the preparations were exposed to biotin-bound goat antiserum against rabbit IgG as the second antibody for 60 min at room temperature (Histofine, Nichirei, Tokyo, Japan). After the slides were washed with PBS, the tissues were incubated in horseradish peroxidase-bound streptavidin for 45 min (Histofine). The peroxidase reaction was performed with 3,3'-diaminobenzidine tetrahydrochloride as a chromogen (Vector Laboratories, Burlingame, CA).
Sections were screened and graded using the immunoreactivity score by two observers who were blind to the status of the specimen (5, 21). Visual assessments of the density of CGRP immunoreactivity were graded from none (score = 0) to abundant (score = 4). To examine the location of CGRP immunoreactivity, we analyzed large airways (airway diameter >0.2 mm), small airways (airway diameter <0.1 mm), and lung parenchyma. The scores represent the density of CGRP-immunoreactive cells and tissues that might include nerves, ganglia, and neuroepithelial bodies (NEBs) (5). In terms of the reproducibility of this assessment, inter- or intraobserver variances were not significant.Materials and chemicals. Materials and chemicals were obtained from Sigma Chemical (St. Louis, MO) unless otherwise specified.
Data analysis. Comparisons of data among the experimental groups were carried out with ANOVA (Scheffé's test). Values are means ± SE. P < 0.05 was taken as significant.
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RESULTS |
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Airway responsiveness to MCh administration.
There were no significant differences in baseline RL and
EL among each group. MCh dose-response curves for
RL and EL are demonstrated in Figs. 1 and
2,
respectively.
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Assessment of the BALF.
Antigen exposure increased protein amount in BALF, although there was
no difference between the wild-type and mutant mice (Fig.
3). Total cell counts and cell fractions
in BALF are shown in Table 1, indicating
the increases in the total cell number in the OA-sensitized groups. OA
challenge induced eosinophil infiltration, whereas no differences in
the fraction and number of BALF eosinophils were observed between the
wild-type and -CGRP
/
mice.
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Measurements of thromboxane, leukotriene, and ET-1 in BALF.
There were no significant differences in BALF TxB2 among
the groups: 0.140 ± 0.047 and 0.135 ± 0.076 ng in
saline-treated -CGRP+/+ and
-CGRP
/
, respectively, and 0.423 ± 0.204 and 0.498 ± 0.262 ng in OA-treated
-CGRP+/+ and
-CGRP
/
, respectively.
|
Assessment of CGRP immunoreactivity.
Figure 6 demonstrates the
immunohistochemistry of CGRP in large airways. In the OA-sensitized
wild-type mouse, significant immunoreactivity for CGRP was observed in
the airway epithelium and submucosa, while the immunostaining was
modest in the saline-treated wild-type animal. On the other hand, there
was little CGRP immunoreactivity in saline- or OA-treated
-CGRP
/
mice. In small airways and lung
parenchyma, little CGRP immunoreactivity was observed in each
experimental group (Fig. 7). Table
2 summarizes the visual assessment of
CGRP immunoreactivity in each group. In airway epithelium, submucosa,
and smooth muscle of large airways, the scores were significantly
higher in OA-treated CGRP+/+ mice than in any
other group, although there were marked differences between
-CGRP+/+ and
-CGRP
/
mice. Meanwhile, the scores were
much lower in peripheral airways and lung parenchyma than in large
airways in the wild-type mice.
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DISCUSSION |
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The results of the present experiments show that antigen-induced
bronchial hyperresponsiveness was significantly reduced in CGRP-deficient mice. Meanwhile, eosinophil infiltration elicited by
antigen challenge was unaffected by disruption of the CGRP gene.
Antigen-induced increases in BALF
LTC4/D4/E4 were significantly attenuated in -CGRP-disrupted mice. These findings suggest that CGRP
could be involved in the antigen-induced airway hyperresponsiveness, but not eosinophil infiltration, in mice.
CGRP has pleiotropic and pathophysiological effects on various cells
and organs (3, 44). CGRP exerts trophic effects on
skeletal muscle and vascular smooth muscle (3). CGRP also modulates some macrophage functions, including antigen presentation (13, 36). In the respiratory system, CGRP is synthesized
by sensory C-fibers in the respiratory tree (47). However,
the pathophysiological roles of CGRP in the lung have not been
determined. Palmer et al. (39) demonstrated that CGRP
potently constricts human airway smooth muscle. On the other hand,
recent studies have reported that CGRP acts as a potent inhibitor of
responses elicited by bronchoconstrictive stimuli (4, 33).
Regarding eosinophil chemotaxis, Numao and Agrawal (37)
reported that neuropeptides, including CGRP, may play a significant
role in eosinophil infiltration by priming cells in allergic
inflammation. Meanwhile, Teixeira et al. (46) demonstrated
that CGRP has little effect on eosinophil accumulation in guinea pig
skin. In the present study, we hypothesized that CGRP could play a
significant role in the underlying mechanism of asthma. To test this
hypothesis, we studied the allergic pulmonary responses using -CGRP
gene-disrupted mice, which have been recently established by Oh-hashi
et al. (38).
Allergen-induced airway hyperresponsiveness was significantly
attenuated in the -CGRP-deficient mice, suggesting that the existence of CGRP per se might be associated with bronchial
hyperresponsiveness, which is a major trait of asthma (11, 12,
23). To our knowledge, this is the first report to use mutant
mice to study whether the CGRP gene and endogenous CGRP could be
involved in the airway hyperresponsiveness. Recently, using a
pharmacological approach, Dakhama et al. (6) found that
exogenous administration of CGRP to sensitized and challenged mice
results in the normalization of airway responsiveness. However, the
exact mechanism to explain the involvement of CGRP in airway
hyperresponsiveness remains to be clarified. In the present study, the
molecular and pathophysiological mechanisms underlying airway
hyperresponsiveness were further examined using CGRP-mutant mice.
One of the possible mechanisms is that CGRP and CGRP gene expression might affect airway inflammation, including eosinophilia, after antigen challenge. Airway eosinophilia is one of the common features in asthmatic patients and could be involved in bronchial hyperresponsiveness (11, 23). In the present study, however, no significant difference in BALF eosinophil counts was observed between the wild-type and CGRP-deficient mice. These results suggest that disruption of the CGRP gene has little effect on antigen-induced airway eosinophilia in mice. Although the sequence of rodent CGRP contains the tetrapeptide eosinophil chemotactic factor, the effect of CGRP on airway eosinophil infiltration is not remarkable in the present model. Therefore, CGRP-dependent airway hyperresponsiveness might not be mediated by eosinophilia.
Possibly, immunization provoked by antigen challenge might be affected by modulation of the CGRP gene. However, increased IgE levels after antigen challenge were observed in both groups, whereas there were no significant differences in measured IgE levels between the wild-type and CGRP-deficient groups. Alveolar protein leakage or airway mucus secretion assessed by BALF protein was consistent with the results of IgE measurement in this study. These findings indicate that modulation of the CGRP gene might not affect the mechanism of IgE production.
Recently, it has been shown that bronchial asthma is related to the generation of various potent mediators, including thromboxane, leukotriene, and ET-1 (28, 45, 49). These mediators are reported to be involved in airway hyperresponsiveness (28). Cysteinyl leukotrienes (LTC4, LTD4, and LTE4) are reported to be among the most important targets for treating bronchial asthma. It has been shown that administration of cysteinyl leukotriene antagonist reduces antigen-induced airway hyperresponsiveness (10, 49) and the increases in airway smooth muscle after antigen exposure (49). Irvin et al. (14) demonstrated that antigen-induced airway hyperresponsiveness is significantly decreased in 5-lipoxygenase-deficient mice, suggesting the important role of leukotrienes in development of airway hyperresponsiveness. The potential sources of cysteinyl leukotrienes in the lung include alveolar macrophages, eosinophils, basophils, mast cells, and platelets (40). The proinflammatory activities of cysteinyl leukotrienes, including bronchoconstriction, mucus secretion, and plasma exudation, are mediated via the interaction with its receptor, the CysLT1 receptor (9). In humans, it has been recently demonstrated that the CysLT1 receptor is expressed in lung smooth muscle, lung macrophages, and peripheral blood leukocytes, while the identification of the CysLT1 receptor is consistent with the anti-inflammatory actions of CysLT1 receptor antagonists (9).
Potentially, genetic disruption of the CGRP gene may modulate the production levels of various potent mediators. We therefore measured possible mediators in the BALF and found that the production of cysteinyl leukotrienes was enhanced in the sensitized wild-type mice. In contrast, the level of cysteinyl leukotrienes was significantly reduced in the sensitized CGRP-deficient mice. There were no significant differences in thromboxane or ET-1 in each group. These observations indicate that CGRP gene disruption might inhibit the production of cysteinyl leukotrienes, which could be associated with reduced airway hyperresponsiveness. Meanwhile, after antigen challenge of wild-type and CGRP-deficient mice, there were no significant differences in the number of alveolar macrophages or eosinophils, i.e., potential sources of cysteinyl leukotrienes. One of the possible mechanisms to explain this observation is that CGRP might be involved in activation of the 5-lipoxygenase pathway.
In the present study, we used mutant mice deficient in -CGRP but not
-CGRP. Therefore, these mutant mice should express
-CGRP. Because
the
-CGRP antibody used in this study cross-reacts with
-CGRP
(79%), the CGRP immunoreactivity represents
-CGRP and, similarly,
-CGRP. The very small amounts of CGRP immunoreactivity in the mutant
mice may indicate
-CGRP expression in the lung. It has been
previously reported that
-CGRP concentrations are approximately four
times greater than
-CGRP concentrations in the rat lung, whereas in
the intestine,
-CGRP concentrations are up to seven times greater
than
-CGRP concentrations (26). Presumably, it seems
that
-CGRP expression in the lung might not be affected by
disruption of the
-CGRP gene in mice.
In the wild-type mice, we observed substantial CGRP immunoreactivity in the epithelium and submucosal tissues in large airways but not in small airways or parenchyma. Presumably, CGRP-immunoreactive cells and tissues include nerve fibers in submucosal tissues, whereas airway epithelium contains nerves and NEBs. Terada et al. (47) reported that nerve plexuses of CGRP-immunoreactive fibers are located in the basal part of the rat tracheal epithelium. These CGRP-immunoreactive intraepithelial nerves lack myelin and Schwann sheaths and run through the bases of the epithelial cells (47). In this study, the CGRP immunoreactivity in large airway epithelium was remarkable, and it was enhanced by antigen challenge. These observations suggest that the epithelium of central airways, including nerves and NEBs, may have a significant role in antigen-induced airway hyperresponsiveness. Meanwhile, it is assumed that the contribution of peripheral airways and parenchyma to CGRP-related airway physiology is small.
Recently, Dakhama et al. (6) showed that CGRP expression was diminished in airway epithelium and submucosal nerve plexuses only after the third OA challenge, although CGRP depletion did not occur after the single antigen exposure. In our study, however, the single antigen challenge enhanced CGRP immunoreactivity in large airways in the wild-type mice, whereas little CGRP immunoreactivity was observed in CGRP-deficient mice in the absence or presence of antigen challenge. The present findings suggest that endogenous CGRP per se may be related to the development of antigen-induced airway hyperresponsiveness.
Genetic features, including single-nucleotide polymorphism, are
potentially associated with the etiology of asthma. On the basis of the
inheritance pattern, a number of genes could have substantial roles in
the pathogenesis of bronchial asthma (43). Murine models
of asthma have been recently used to investigate individual genes
associated with airway hyperresponsiveness (8, 15, 16, 18, 31,
48). Because CGRP is one of the potent mediators possibly
involved in bronchial asthma (39), genes regulating the
function of CGRP, calcitonin receptor-like receptor, and RAMP1 could be
targets to study the pathogenesis of asthma. Consistently, the present
observations suggest that -CGRP and the
-CGRP gene play
significant roles in the molecular mechanism underlying bronchial
asthma, indicating that the
-CGRP gene could be a target for
single-nucleotide polymorphism research. The
-CGRP-mutant mice used
in this study may contribute to the study of the genetic roles of
-CGRP in bronchial asthma and may provide novel insights into the
pathophysiological roles of
-CGRP and the
-CGRP gene in vivo.
In summary, reduction of antigen-induced airway hyperresponsiveness was
detected in -CGRP-deficient mice. Meanwhile, eosinophilic infiltration associated with antigen exposure was not altered by
disruption of the
-CGRP gene. Antigen-induced increases in cysteinyl
leukotriene production were significantly reduced in
-CGRP-disrupted
mice. Disruption of the
-CGRP gene might inhibit production of cysteinyl leukotrienes, which could be associated with reduced airway hyperresponsiveness. Antigen challenge enhanced CGRP immunoreactivity in the wild-type mice, whereas little CGRP immunoreactivity in epithelium or submucosa was observed in
-CGRP-deficient mice. These findings suggest that endogenous CGRP
may be involved in development of antigen-induced airway
hyperresponsiveness. Taken together, CGRP and CGRP gene expression
might be involved in the pathogenesis of bronchial asthma by acting as
a mediator. The CGRP-mutant mice may provide appropriate models to
study molecular and pathophysiological mechanisms underlying diseases
related to CGRP.
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ACKNOWLEDGEMENTS |
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We thank Y. Tateno (University of Tokyo) for technical assistance.
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FOOTNOTES |
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This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan, and an AstraZeneca research grant.
Address for reprint requests and other correspondence: T. Nagase, Dept. of Geriatric Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan (E-mail: takahide-tky{at}umin.ac.jp).
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.
June 21, 2002;10.1152/ajplung.00130.2002
Received 14 May 2002; accepted in final form 17 June 2002.
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