Expression of C-type natriuretic peptide during development of rat lung

Kuniaki Nakanishi1, Fumiko Tajima2, Hiroshi Itoh3, Yasuko Nakata2, Norio Hama3, Osamu Nakagawa3, Kazuwa Nakao3, Toshiaki Kawai4, Chikao Torikata4, Tatsuko Suga5, Kunio Takishima5, Takashi Aurues1, and Tomosumi Ikeda1

1 Division of Environmental Medicine, National Defense Medical College Research Institute, and Departments of 4 Pathology and 5 Biochemistry, National Defense Medical College, Tokorozawa 359-8513; 2 Biochemical Division, Aeromedical Laboratory, Japan Air Self-Defense Force, Tachikawa 190-0003; and 3 Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan


    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

C-type natriuretic peptide (CNP), recently found to be secreted from vascular endothelial cells, is now viewed as a novel endothelium-derived relaxing peptide. However, the distribution and expression of CNP during cardiopulmonary development is unclear. To follow changes in the expression of CNP during lung development, we examined rat embryos and neonates using Northern blot analysis and in situ hybridization for CNP mRNA and radioimmunoassay and immunohistochemistry for CNP protein. A substantial expression of CNP mRNA was first detected on postnatal day 2, and it thereafter remained fairly steady. The level of CNP protein also increased rapidly after postnatal day 1, reaching a settled level on postnatal day 4. CNP protein and mRNA were detected in the endothelium and smooth muscle cells of blood vessels and in bronchial airway and alveolar epithelia. Immunoreactivity for CNP protein in the endothelium of blood vessels increased to an intense level after the saccular stage. These results suggest that the changes in CNP levels may be related to the occurrence of pulmonary vasodilation after birth.

cardiopulmonary system


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AT BIRTH, pulmonary arterial pressure and pulmonary vascular resistance decrease rapidly and pulmonary blood flow increases 8- to 10-fold. Although the mechanisms underlying these pulmonary circulatory changes are not completely understood, the establishment of an air-liquid interface, the initiation of ventilation, and an increase in oxygen tension appear to contribute to postnatal adaptation (4, 5, 7). In addition, vasoactive substances, including bradykinin and prostacyclin, may modulate birth-related changes in pulmonary vascular tone (3, 27). Recent studies (2, 19, 25) have shown that in the late-gestation ovine fetus, endogenous endothelium-derived relaxing factor (nitric oxide) may modulate basal pulmonary vascular tone and thus contribute to the normal fall in pulmonary vascular resistance that occurs at birth.

It is generally accepted that mammalian cardiac tissues synthesize and secrete atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) (14, 17, 22, 26). ANP and BNP have potent diuretic, natriuretic, and vasorelaxant properties, and they appear to play important roles as cardiac hormones in fluid-electrolyte homeostasis (13, 14, 16, 17, 22, 24, 26). Furthermore, changes in the cardiac ventricular expression of ANP can be seen as an adaptation to hemodynamic requirements during development (18, 31). Moreover, ANP when given by intrapulmonary infusion in fetal lambs causes sustained increases in pulmonary blood flow (1). In contrast, the cardiac ventricular expression of BNP mRNA was barely detectable in the normal midgestational human fetus, and expression of C-type natriuretic peptide (CNP) mRNA was not detectable in the cardiac ventricles of the normal midgestational human fetus (30). In fact, the expression and distribution of BNP and CNP in the developing cardiopulmonary system has yet to be properly characterized.

CNP is a novel natriuretic peptide with 22 amino acids; it was originally isolated from porcine brain and its homology to ANP and BNP is striking (26, 27). Although the major production site for both ANP and BNP is the heart, CNP is not distributed in the heart but instead mainly in the brain, suggesting a principal role as a neuropeptide (26). Recently, CNP has been found to be synthesized and released from endothelial cells, and it has been suggested that it plays a paracrine role in the control of vascular tone and vascular remodeling as a novel endothelium-derived relaxing peptide (15). This suggestion and the known changes that occur in the cardiopulmonary system during gestation and at birth led us to try to determine whether CNP is associated with particular pulmonary regions and tissues at particular developmental stages. To this end, we examined rat embryonic and neonatal lungs using Northern blot analysis and in situ hybridization for CNP mRNA and radioimmunoassay (RIA) and immunohistochemistry for CNP protein.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Animals. Adult Wistar rats were used and were fed commercial rat chow and water ad libitum. Proestrus females were introduced into a male rat's cage. Embryonic age was dated from the midnight before the finding of a vaginal plug, this day being designated as day 0 of gestation. Neonatal age was determined from the time of birth. Birth normally occurred between 21.5 and 22.5 days after conception. Tissues were obtained from embryos at 13, 15, 18, 19, 20, 21, and 22 days gestation after their removal from pregnant rats anesthetized with an intramuscular injection of ketamine hydrochloride (60 mg/kg body wt). In addition, tissues were obtained from 0-, 1-, 2-, 4-, 8-, 16-, 24-, and 32-day-old postnatal rats after decapitation. At autopsy, the lungs were removed, then weighed, frozen in liquid nitrogen, and stored at -80°C until used. The lungs so obtained were subjected to RNA and peptide extractions. Other lungs from three to five rats at each embryonic or neonatal age were fixed with periodic acid-lysine-paraformaldehyde (PLP) solution for in situ hybridization, immunohistochemistry, and immunoelectrography. This experimental study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health [DHEW Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20892].

Total RNA extraction and Northern blot analysis. Total RNA was isolated from lung tissues with acid guanidinium isothiocyanate-phenol-chloroform extraction and ethanol precipitation (6). After extraction, 20 mg of total RNA were electrophoresed on a 1.5% agarose gel containing 8% formaldehyde. The electrophoresed RNA was transferred onto a nylon membrane by capillary action. Rat CNP cDNA was used as a digoxigenin-labeled 51-base oligonucleotide probe (antisense) in the in situ hybridization method described in In situ hybridization. After hybridization with the digoxigenin-labeled oligonucleotide probe in 0.1% N-sarkosyl, 0.02% (vol/vol) sodium dodecyl sulfate (SDS), 1% blocking solution, 5× saline-sodium citrate (SSC; 1× SSC is 150 mM NaCl and 15 mM sodium citrate), 0.1 mg/ml of poly(A), and 5 µg/ml of poly(dA) for 16 h at 45°C, the membrane was washed three times in 1× SSC with 0.1% SDS for 20 min at 42°C. The blots were detected immunoluminescently and then exposed to X-ray film at room temperature for 2 h. Control RNA was obtained from the adult brain and pituitary gland.

In situ hybridization. In situ hybridization was performed essentially as previously described (9). The 51-mer oligonucleotide antisense probe (corresponding to nucleotides 432-482) for CNP used was 5'-GCT CAT GGA GCC GAT CCG GTC CAG CTT GAG GCC AAA GCA GCC TTT GGA CAA-3'. The corresponding sense probe used for control purposes was 5'-TTG TCC AAA GGC TGC TTT GGC CTC AAG CTG GAC CGG ATC GGC TCC ATG AGC-3'. These probes were synthesized with an automated DNA synthesizer, labeled with digoxigenin with a 3'-end oligonucleotide tailing kit (Boehringer Mannheim, Postfach, Germany), and purified. Briefly, the PLP-fixed frozen sections were treated with 0.2 N HCl for 20 min, incubated in 2× SSC for 10 min at 37°C, and then incubated in 1 µg/ml of proteinase K for 10 min at 37°C. Endogenous peroxidase activities were blocked by soaking sections in 75% methanol containing 0.3% hydrogen peroxide for 45 min. Sections were subsequently postfixed in 4% paraformaldehyde for 5 min and incubated in 10 mmol/l of dithiothreitol for 10 min to prevent nonspecific binding due to oxidation of the tissue. Hybridization was carried out for 3 h at 42°C in 50% (vol/vol) deionized formamide, 5× Denhardt's solution, 5% (wt /vol) dextran sulfate, 2× SSC, 0.3 mg/ml of salmon sperm DNA, 5 mM EDTA, and 10 ng/ml of digoxigenin-labeled probe. After a final stringency wash at 55°C for 20 min, hybridization was detected immunologically (Genpoint, DAKO, Glostrup, Denmark). Adult brain served as a control.

Extraction of tissues and determination of CNP protein. Tissue samples were boiled for 5 min in 10 volumes of 0.1 M acetic acid containing 0.1% Triton X-100 to avoid intrinsic proteolysis, then homogenized with a polytron homogenizer (Kinematica, Lucerne, Switzerland). The homogenate was centrifuged at 15,000 g for 30 min at 4°C, and the supernatant was stored at -80°C until subjected to radioimmunoassay (RIA) (11) to determine the concentration of CNP protein. Cross-reactivity in the RIA between CNP protein and rat ANP or BNP protein was not detectable. The minimum detection limit for CNP protein was 30 fmol/tube in the nonextracted method.

Immunohistochemistry. The indirect immunoperoxidase method was applied to PLP-fixed frozen sections (10). The frozen sections, 4 mm thick, were prepared with 3-aminopropyltrimethoxysilane-coated glass slides. Endogenous peroxidase activities were blocked by soaking sections in 75% methanol containing 0.3% hydrogen peroxide for 45 min. Primary rabbit polyclonal antibodies against CNP protein (Peninsula Laboratories, Belmont, CA) diluted 1:50 were reacted at room temperature for 1 h followed by a 10-min rinse in 0.01 mol/l of phosphate-buffered saline (PBS), pH 7.2. Horseradish peroxidase-labeled secondary antibody to rabbit immunoglobulins (Chemicon International, Temecula, CA) diluted 1:250 was then incubated at room temperature for 30 min. After the second 10-min rinse in PBS, the peroxidase coloring reaction was performed by immersing sections in 0.05 mol/l of Tris · HCl buffer, pH 7.6, containing 20 mg/dl of diaminobenzidine, 65 mg of sodium azide, and 0.003% hydrogen peroxide for 10 min. The nuclei were counterstained with 0.2% Meyer's hematoxylin. For the positive control, adult brain was used. For the negative control, the incubation step with the primary antibody was omitted. Immunoperoxidase staining was absent from samples incubated with antiserum preabsorbed with CNP protein.

Immunoelectron microscopy. Immunostaining for CNP protein was performed by the indirect antibody method as previously described (21). After immersion in 10% nonimmunized goat serum, 6-mm-thick PLP-fixed frozen sections were incubated for 24 h at 4°C with anti-CNP protein antibody (Peninsula Laboratories) diluted 1:50 and then overnight with the horseradish peroxidase-labeled Fab fragment of the rabbit Ig secondary antibody (Amersham International) diluted 1:50. After fixation in 1% glutaraldehyde, the specimens were reacted with 0.03% 3,3'-diaminobenzidine tetrahydrochloride, postfixed in 1% osmium tetroxide for 1 h, and embedded in Epon. Ultrathin sections were observed with an electron microscope without being stained. For the negative control, the primary antibody was preabsorbed with CNP protein or released by nonimmunized rabbit serum or PBS.

Statistical analysis. The results are expressed as means ± SE. Fisher's protected least significant difference test was applied to the data when significant F-ratios were obtained in an analysis of variance. Differences were considered significant at P < 0.05.


    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Northern blot analysis of CNP mRNA. Northern blot analysis with the rat CNP 51-base oligonucleotide probe (antisense) identified a main band of ~1.2 kb in fetal and neonatal rat lungs (Fig. 1A) as well as in adult brain and pituitary gland (data not shown). A substantial expression of CNP mRNA in the lungs was first detected on postnatal day 2, and it thereafter remained at a fairly steady level (Fig. 1B). However, it may also have been present in small amounts on days 19 and 20 of gestation.



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Fig. 1.   Northern blot analysis for C-type natriuretic peptide (CNP) mRNA from lungs of fetal rats at 18-22 days gestation and from lungs of postnatal rats at 0, 1, 2, 4, 8, 16, 24, and 32 days of age. A: signal for CNP mRNA was identified at 1.2 kb. A substantial expression of CNP mRNA was first clearly detectable on postnatal day 2. 18S, 18S rRNA. B: summary data for quantitative densitometry in 3 independent experiments. Values are means ± SE of CNP mRNA abundance corrected for 18S rRNA.

In situ hybridization of CNP mRNA. CNP mRNA was detected in the endothelium and smooth muscle cells of pulmonary blood vessels and in airway and alveolar epithelia (Table 1). At the pseudoglandular stage (15-18 days gestation), the lung consisted of loose mesenchymal tissue surrounding buds of airway epithelium. The formation of blood vessels was apparent in the central regions. At the early pseudoglandular stage (15 days gestation), CNP mRNA was first observed in the buds of airway epithelium and in the interstitium (Fig. 2, A and B). No significant staining was seen in blood vessels. At the late pseudoglandular stage (18 days gestation), a significant level of CNP mRNA was observed in the buds of airway epithelium, in bronchial airway epithelium, and in the endothelium of the pulmonary artery, but it was not detected in the smooth muscle cells of the pulmonary artery (Fig. 2C).

                              
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Table 1.   Localization of CNP during development in rat lung



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Fig. 2.   Localization of CNP mRNA during development of rat lung. In rat lung at 15 days gestation, hybridization with a CNP antisense oligonucleotide probe (A) revealed localization of CNP mRNA in epithelium of some airways (aw), whereas hybridization with a CNP sense probe (B) revealed no localization. At 18 days gestation (C), CNP mRNA is abundant in buds of airway epithelium (bu) and bronchial airway epithelium (br) as well as in endothelium of blood vessels (v). At 21 days gestation (D), CNP mRNA is abundant in bronchial airway and alveolar (a) epithelia and in smooth muscle cells and endothelium of blood vessels. On postnatal day 1 (E), CNP mRNA is localized in endothelium of blood vessels (arrows), but it decreased in bronchial airway and alveolar epithelia. On postnatal day 32 (F), CNP mRNA is localized in endothelium of blood vessels (arrows), but it was less abundant in bronchial airway epithelium. Bars, 100 µm.

At the canalicular stage (19-21 days gestation), the respiratory portion of the lung was apparent. The terminal ends of the buds of airway epithelium branched to form the respiratory bronchioles. At 21 days gestation, terminal sacs could be seen in the peripheral regions. Vessel formation was increased in the mesenchymal tissue. The number of airways showing CNP mRNA-reactive epithelium (bronchial and bronchiolar) increased in the late canalicular stage (Fig. 2D). Hybridizing staining was also noted in the endothelium and smooth muscle cells of blood vessels, particularly in the smooth muscle cells.

In the saccular and alveolar stages (newborn to 4 days and 8 days postnatal, respectively), the number of airways showing CNP mRNA-reactive epithelium decreased gradually (Fig. 2E), whereas CNP mRNA reactivity of the endothelium increased gradually. At the mature stage (32 days postnatal), CNP mRNA was observed in the endothelium of blood vessels, whereas it was rarely observed (and then only weakly) in the bronchial airway and alveolar epithelia (Fig. 2F).

CNP levels in the lung. CNP protein was not detected by RIA in the lungs of any of four rats at 18 days gestation (Fig. 3). Although CNP protein was detected in the lungs of only some rats during embryonic development, it was detected in the lungs of all rats after postnatal day 2. The level of CNP protein in the lung increased rapidly after postnatal day 1 and reached a settled level on postnatal day 4.


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Fig. 3.   Plots of CNP protein concentration during development of rat lung. CNP levels in rat lung increased rapidly after postnatal day 1, reaching a settled level on postnatal day 4. Open bar, levels under minimum detection limit for CNP protein.

Immunohistochemistry. CNP protein was expressed in the endothelium and smooth muscle cells of blood vessels and in airway and alveolar epithelia (Table 1). At 15 days gestation, immunoreactivity for CNP protein could be detected, weakly, in the buds of airway epithelium and in some cells in the interstitium, the origin of which remains unknown (Fig. 4A), but it was not detected in the endothelium of the main pulmonary vessels or aorta. At 18 days gestation, weakly immunoreactive endothelium was first detected in pulmonary arteries and veins and aorta (Fig. 4B).


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Fig. 4.   Localization of CNP protein during development of rat lung. At 15 days gestation (A), CNP protein is detectable in epithelium of some airway epithelium. At 18 days gestation (B), CNP protein is first detected in endothelium of pulmonary vessel as well as in buds of airway epithelium and bronchial airway epithelium. At 20 days gestation (C), CNP protein is seen in bronchial airway and alveolar epithelia and endothelium of pulmonary vessel. On postnatal day 0 (D), CNP protein is intensely visualized in endothelium of pulmonary vessel but only weakly visualized in bronchial airway and alveolar epithelia. On postnatal day 4 (E), CNP protein is detected in smooth muscle cells (arrows) of pulmonary vessel as well as in endothelium (arrowheads) and bronchial airway and alveolar epithelia. On postnatal day 32 (F), CNP protein is intensely visualized in endothelium (arrows) of blood vessel but only weakly visualized in bronchial airway and alveolar epithelia. Bars, 100 µm.

At the canalicular stage, immunoreactivity for CNP protein was weakly visualized in bronchial airway and alveolar epithelia and in the endothelium of blood vessels in the central regions (Fig. 4C). However, it was not seen in smooth muscle cells of blood vessels.

At the saccular stage, immunoreactivity for CNP protein was intensely visualized in the endothelium of blood vessels, but it was weak in bronchial airway and alveolar epithelia (Fig. 4D). On postnatal day 4, it was detected in some smooth muscle cells of large blood vessels (Fig. 4E).

At the alveolar and mature stages, immunoreactivity for CNP protein was intense in the endothelium of blood vessels, particularly large blood vessels, but it was detected only weakly in some airway epithelium and a few alveolar epithelium (Fig. 4F).

Immunoelectron microscopy. In airway epithelial cells, CNP protein was distributed throughout the cytoplasm, particularly on the rough endoplasmic reticulum of Clara-type epithelium (nonciliated bronchiolar epithelium), but it was not distributed in ciliated bronchiolar epithelium (Fig. 5A). CNP protein was also distributed on the plasma membrane and in the cytoplasm of the endothelial cells of blood vessels (Fig. 5B).


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Fig. 5.   Immunoelectron micrographs showing distribution of CNP protein. In airway epithelium at 21 days gestation (A), CNP protein is distributed throughout cytoplasm, particularly on rough endoplasmic reticulum (arrows) and plasma membrane (arrowheads) in Clara-type epithelium (cl; nonciliated bronchiolar epithelium), whereas it is not distributed in ciliated bronchiolar epithelium (B). In endothelium of pulmonary vein at 21 days gestation (B), CNP protein is distributed in cytoplasm and on plasma membrane (arrowheads). Bars, 1 µm.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that CNP mRNA and protein are expressed in a specific and characteristic pattern during development of the rat lung. The levels of CNP mRNA and protein (as detected by Northern blot analysis and RIA, respectively) were seen to increase rapidly in the lung in the immediate postnatal period. These observed maturational changes in CNP may well be related to the pulmonary vasodilation that occurs after birth. In addition, we have shown by in situ hybridization and immunohistochemistry, respectively, that CNP mRNA and protein are localized in bronchial airway and alveolar epithelia and smooth muscle cells of blood vessels as well as in the endothelium during late gestation and on postnatal days 0-32. These findings provide strong support for the notion that the above tissues may all be capable of transcribing the CNP gene and synthesizing CNP protein.

The physiological function of lung CNP is still unclear. Like many other hormones and autacoids, CNP may be involved in the modulation of a wide spectrum of activities related in some way to the control of blood pressure and to body fluid homeostasis (27). Recently, Suga and colleagues (28, 29) reported that CNP was synthesized in cultured endothelial cells and in the bovine thoracic aorta, and they suggested a possible paracrine role as a novel endothelium-derived relaxing peptide in the control of vascular tone and vascular remodeling. In our study, CNP was found to be synthesized by the lung, and the rapid postnatal increase in the CNP mRNA level of the whole rat lung was accompanied by an increased level of CNP protein (as measured by RIA). Furthermore, CNP mRNA signals (as detected by in situ hybridization) were expressed continuously in the endothelium of blood vessels from the pseudoglandular stage to the mature growth stage. Moreover, the immunoreactivity for CNP protein was visualized intensely in the endothelium of blood vessels at and after the saccular stage. In contrast, the number of airways showing CNP mRNA- and protein-reactive epithelium was found to be greatest during the canalicular stage, and it then decreased gradually. We consider these results to be consistent with the idea that CNP activity of the endothelium of the blood vessels in the lung is already induced at the time of birth and that thereafter CNP may act in a paracrine fashion as a pulmonary vasodilator.

When we looked at the airway localization of CNP mRNA, we found that the intensity of staining and the number of reactive cells varied both spatially and temporally during lung development. CNP mRNA signals in the airway epithelium increased during late gestation, peaked at 21 days gestation, then decreased gradually after birth. In contrast, immunoreactivity for CNP protein in the airway epithelium was seen to some extent from the pseudoglandular stage to the mature growth stage. By immunoelectron microscopy, expression of CNP protein was observed mainly in the cytoplasm of Clara-type epithelium (nonciliated bronchiolar epithelium), which exists in the bronchiolar region. Although the exact physiological role played by CNP in the fetal airway epithelium is unknown, these results are consistent with CNP being involved in a paracrine fashion in the control of airway function, especially of the bronchiolar region, from the very early stage of lung development.

Several studies (8, 20, 23) have demonstrated that CNP increases the intracellular level of cGMP in cultured vascular smooth muscle cells and acts as a potent inhibitor of the proliferation of smooth muscle cells. Thus it appears that CNP is one of the hormones with regulatory effects on the growth of smooth muscle tissue. Our results with in situ hybridization and immunohistochemistry showed that in smooth muscle cells of blood vessels, CNP mRNA was present from the pseudoglandular stage to the canalicular stage and CNP protein was present at the saccular stage. These results suggest that CNP may have an autocrine role in smooth muscle cells.

By means of immunoelectron microscopy, ANP and BNP proteins have been found to be localized in granules in myocytes (12). In contrast, CNP protein was found to be distributed throughout the cytoplasm in endothelium and airway epithelium in the present study; we did not find CNP-containing vesicles. Therefore, these results support the idea that CNP protein may be released constitutively but not by exocytosis via vesicles (31).

In conclusion, the present study has revealed that maturational changes occur in the CNP content of the rat lung. CNP may act in a paracrine fashion to modulate vasomotor tone in the lung after birth. However, at present, this idea is speculative, and further experiments will be necessary to elucidate the exact physiological role played by CNP, perhaps as a local mediator, during the development of the lung. An insight into this role might be obtained by examining the effect of intrauterine closure of the ductus arteriosus (for example, by indomethacin) on CNP expression.


    ACKNOWLEDGEMENTS

We are indebted to S. Tominaga and M. Uenoyama for technical assistance and to Dr. R. Timms for correcting the English version of the manuscript.


    FOOTNOTES

This research was supported in part by a grant from the Kawano Foundation of Saitama Prefecture.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: K. Nakanishi, Division of Environmental Medicine, National Defense Medical College Research Institute, Tokorozawa 359-8513, Japan (E-mail: nknsknak{at}ndmc.ac.jp).

Received 9 November 1998; accepted in final form 16 June 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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