1 Departments of Pediatrics and 2 Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa 52242-1083
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ABSTRACT |
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Surfactant protein A (SP-A) is the most abundant of the surfactant-associated proteins. SP-A is involved in the formation of tubular myelin, the modulation of the surface tension-reducing properties of surfactant phospholipids, the metabolism of surfactant phospholipids, and local pulmonary host defense. We hypothesized that elimination of SP-A would alter the regulation of SP-B gene expression and the formation of tubular myelin. Midtrimester human fetal lung explants were cultured for 3-5 days in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to SP-A mRNA. After 3 days in culture, SP-A mRNA was undetectable in antisense ON-treated explants. After 5 days in culture, levels of SP-A protein were also decreased by antisense treatment. SP-B mRNA levels were not affected by the antisense SP-A ON treatment. However, there was decreased tubular myelin formation in the antisense SP-A ON-treated tissue. We conclude that selective elimination of SP-A mRNA and protein results in a decrease in tubular myelin formation in human fetal lung without affecting SP-B mRNA. We speculate that SP-A is critical to the formation of tubular myelin during human lung development and that the regulation of SP-B gene expression is independent of SP-A gene expression.
antisense oligonucleotide; fetal lung development; alveolar type II cell
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INTRODUCTION |
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SURFACTANT PROTEIN A (SP-A), a 35-kDa glycoprotein, is the most abundant of the surfactant proteins. SP-A is involved in the formation of tubular myelin, the modulation of the surface tension reducing properties of surfactant phospholipids, the regulation of the metabolism of surfactant phospholipids, and the innate immune defense of the lung (28). SP-A interacts with SP-B to facilitate the formation of tubular myelin in vitro (26). Tubular myelin is a membrane lattice of phospholipid bilayers found in the alveolar space as an intermediary structure in the formation of the phospholipid monolayer from lamellar bodies (30).
There are no known genetic diseases caused by SP-A deficiency; however, hereditary SP-B deficiency causes lethal respiratory failure in neonates (17). Humans with hereditary SP-B deficiency have both an aberrant form of SP-C (27) and a paucity of lamellar bodies (29). In transgenic mice homozygous for the SP-B knockout, there is also aberrant processing of SP-C and an absence of lamellar bodies (4). Because there is a relationship between SP-B and both SP-C and the formation of lamellar bodies and because SP-A is known to interact with SP-B to form tubular myelin (31), we wanted to investigate whether or not a relationship existed between SP-A and SP-B gene expression. Furthermore, SP-A, acting through its receptor, has been shown to increase transcription of both SP-B and SP-C in isolated rat type II cells (14, 25). Thus the possibility exists that the absence of SP-A could negatively affect SP-B expression.
In transgenic mice homozygous for the SP-A knockout, there is decreased tubular myelin formation without significant changes in the metabolism of either SP-B or saturated phosphatidylcholine (11, 13). However, in humans, especially during human fetal lung development, the role of SP-A has not yet been clearly defined. Because SP-A interacts with SP-B to organize surfactant phospholipids into tubular myelin (31), we hypothesized that selective elimination of SP-A in human fetal lung tissue would affect the formation of tubular myelin and potentially SP-B gene expression. To address this issue, we used antisense oligonucleotides (ON) to inhibit SP-A gene expression in human fetal lung explants undergoing alveolar type II cell differentiation in vitro.
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MATERIALS AND METHODS |
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Organ culture.
Human fetal lung tissue was obtained under a protocol approved by the
University of Iowa Human Subjects Review Committee. The explants were
prepared from lung tissue obtained from midtrimester abortuses
(15-20 wk) as previously described (21). The major airways were removed, and the distal lung tissue was minced into 1-mm3 pieces with a razor blade under sterile conditions.
The minced tissue was placed on a piece of lens paper that rested on a
metal grid inside of a 35-mm culture dish containing 1 ml of serum-free Waymouth MB 752/1 medium (Gibco Laboratories, Grand Island, NY) with
added penicillin G (100 U/ml), streptomycin (100 µg/ml), and
amphotericin (0.25 µg/ml). The explants were incubated at 37°C in a
humidified atmosphere of 5% CO2 and 95% air for 3-6 days with the media changed daily. Starting tissue (human fetal lung
tissue before culture) and the harvested explants were frozen in liquid
nitrogen and stored at 70°C or fixed in 2.5% glutaraldehyde until
subsequent analysis. Explants used for the assessment of tubular myelin
were cultured for 6 days in the presence of 1 mM dibutyryl cAMP to
stimulate the secretion of lamellar bodies into the lumen
(18). All experiments were conducted with explants prepared from individual fetuses.
Antisense ON. An 18-mer antisense phosphorothioate ON of the following sequence (5'-GAGGGTGAGGGCCAGAGG-3'), targeted against the human SP-A mRNA (8) and complimentary to a region 10 nucleotides downstream from the initiation codon region, was synthesized by Oligos Etc. (Wilsonville, OR). A sense ON (5'-CCTCTGGCCCTCACCCTC-3') similarly modified and a carrier-only condition were used as controls. Lipofectamine (Gibco BRL) was used a carrier of the ON, and we modified the manufacturer's protocol for transfection of cells as follows. First, 45 nmol of ON were added to 100 µl of Opti-MEM I (Gibco BRL) to create solution A. Then, 20 µl of Lipofectamine was added to 100 µl of Opti-MEM I to create solution B. Solutions A and B were mixed together and then incubated at room temperature for 15 min to form DNA-liposome complexes. The resulting complexes were added to the 35-mm culture dishes that contained the explants along with serum-free Waymouth medium to reach a final total volume of 500 µl. The final concentration of ON was 90 µM. The media were changed daily at which time fresh ON was added as described above. The explants were harvested after 3-6 days. Cytotoxicity was assessed by measuring the release of lactate dehydrogenase (LDH; LDH assay, LDL-20 kit, Sigma) into the media.
Northern blot analysis of SP-A and SP-B mRNA. Total RNA was isolated by a single step acid-phenol-chloroform extraction method (5). Ten micrograms of total RNA per condition were separated by gel electrophoresis (1.2% agarose), transferred by capillary action to a nylon membrane (S&S Nytran, Schleicher & Schuell, Keene, NH), baked 30 min, ultraviolet cross-linked (UV Stratalinker 1800, Stratagene, La Jolla, CA), and prehybridized as previously described (6).
SP-A and SP-B cDNA probes (kindly supplied by J. Whitsett, Department of Pediatrics, University of Cincinnati) were radiolabeled with [Western blot immunoanalysis of SP-A. Starting tissue and harvested explants were homogenized in phosphate-buffered saline (PBS) with 1 mM phenylmethylsulfonyl fluoride, leupeptin (20 µg/ml), soybean trypsin inhibitor (5 µg/ml), and 5 mM EDTA. Samples were centrifuged (600 × g) for 5 min, and supernatant protein (75 µg per lane) was separated by electrophoresis on a 12.5% SDS-polyacrylamide gel, transferred to an Immobilon-P membrane (Millipore, Bedford, MA), and blocked as described (21). The membrane was incubated for 1 h at room temperature with guinea pig polyclonal anti-human SP-A antibodies (1:1,000 dilution), rinsed with double-distilled water and then incubated with sheep anti-guinea pig IgG conjugated to alkaline phosphatase (1:2,000 dilution, Boehringer Mannheim, Indianapolis, IN) for 1 h at room temperature, and then washed it as previously described (22). The immunoreactive SP-A bands were then detected by incubating the membrane at room temperature for 30 min in a solution containing 100 mM Tris (pH 9.5), 100 mM NaCl, 5 mM MgCl2, 5-bromo-4-chloro-3-indoyl phosphate (165 µg/ml), and nitro blue tetrazolium (330 µg/ml). The membrane was rinsed in distilled water, dried, and photographed. The relative amount of immunoreactive SP-A present in each sample was quantitated by densitometry (AMBIS Radioanalytic and Visual Imaging System, Ambis). The densitometric data from each blot were normalized to the control condition with the control value set equal to one for each experiment.
Immunohistochemistry for SP-A.
Two explants per condition, from three different experiments, were
sectioned and stained after being cultured for 5 days . After
harvesting, the tissue was frozen immediately in liquid nitrogen and
stored at 70°C. The frozen tissue was mounted in optimal cutting
temperature compound, and 7-micron sections were prepared with a
cryostat and thaw mounted on glass slides. Sections were fixed for 10 min at room temperature in freshly prepared 10% formalin in PBS. The
sections were rinsed twice for 10 min per rinse in PBS. We quenched
endogenous peroxidase activity by incubating sections in 0.3%
H2O2 in methanol for 30 min followed by rinsing
them twice in PBS for 10 min. The sections were stained using an avidin
biotinylated complex kit (Vectastain Elite kit, Vector Labs,
Burlingame, CA). Nonspecific binding sites were blocked by incubating
the sections with 2% normal goat serum in PBS for 20 min at room
temperature followed by a second blocking step using 2% normal goat
serum and 0.25% BSA for 20 min. The sections were rinsed in PBS and
then incubated for 1 h in a humidified chamber at room temperature
with a rabbit polyclonal anti-human SP-A antibody (Chemicon, Temecula,
CA) at a dilution of 1:1,000 in PBS. The tissue sections were washed
two times in PBS, 5 min per rinse, then incubated for 30 min in
biotinylated secondary antibody, and then rinsed two times in PBS, 5 min per rinse. The sections were then incubated for 45 min in
avidin-peroxidase reagent. After being rinsed two times in PBS, 5 min
per rinse, the sections were incubated in diaminobenzidine (700 µg/ml) for 1-3 min. Sections were rinsed in PBS for 5 min,
rinsed quickly in distilled water, and then dehydrated and mounted with
glass coverslips. In some experiments, the sections were counterstained
with hematoxylin for 30 s. Negative staining controls were
incubated with secondary antibody alone and were performed for all
experimental conditions. Sections were viewed and photographed with a
Nikon FX photomicroscope.
Electron microscopy. Fetal lung explants from control, antisense ON, and sense ON condition were cultured for 6 days in the presence of dibutyryl cAMP (1 mM) to increase the secretion of lamellar bodies into the lumen (18). The harvested explants were fixed overnight in 2.5% glutaraldehyde. Staining was performed en bloc with 1% osmium tetroxide and 2.5% uranyl acetate; the tissue was dehydrated, embedded in Spurr compound (Polysciences, Warrington, PA), sectioned, and poststained with uranyl acetate and lead citrate. Sections were photographed with an electron microscope operated at 75 kV (Hitachi H700, Tokyo, Japan).
Statistical analysis. All data are presented as the means ± SE. The effects of antisense and sense SP-A ON on SP-A mRNA, and SP-A protein levels were evaluated by one-way analysis of variance (ANOVA). The assessment of significant differences among multiple comparisons was performed using the Student-Newman-Keuls Test (SigmaStat) to compare differences among all possible experimental conditions in a pairwise manner. Significance was defined as P < 0.05. The unpaired Student's t-test was used for experiments not involving multiple comparisons.
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RESULTS |
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Effect of antisense SP-A inhibition on steady-state levels of SP-A
and SP-B mRNA.
We used an antisense SP-A ON to selectively mediate the degradation of
SP-A mRNA in cultured fetal lung tissue. Using data derived from our
previous work in treating human fetal lung explants with antisense
epidermal growth factor (EGF) receptor ON (12), we
performed all of the experiments using phosphorothioate ON at a
concentration of 90 µM. We cultured explants for 3 days in the presence or absence of either sense or antisense SP-A ON and measured the level of SP-A and SP-B mRNA present by Northern blot. Steady-state levels of SP-A and SP-B mRNA were increased in the control, Lipofectamine, and sense-cultured conditions compared with the
undifferentiated start tissue (Fig. 1).
Steady-state levels of SP-A mRNA decreased significantly after 3 days
in culture with the antisense SP-A ON, compared with the control,
Lipofectamine, or sense ON conditions (Fig. 1, ANOVA, P < 0.002, Student-Newman-Keuls test, P < 0.05 for
antisense ON compared with all other conditions, n = 3 experiments). However, SP-B gene expression was not affected by
exposure to the antisense SP-A ON, whereas SP-A mRNA was clearly decreased with exposure (Fig. 1), confirming the specific effect of the
antisense SP-A ON in fetal lung tissue. After a total of 5 days of
exposure to the antisense SP-A ON, there still was a complete lack of
SP-A mRNA by Northern blot analysis in the explants (data not shown,
n = 3). This finding was consistent with the 3-day
antisense data as seen in Fig. 1. Thus 5 days in culture was chosen as
the time point for evaluating SP-A protein levels by both immunoblot
and immunohistochemistry to allow adequate time for degradation of any
preexisting SP-A protein.
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Effect of inhibiting SP-A mRNA on SP-A content.
Immunoreactive SP-A protein was measured in human fetal lung explants
that were cultured for 5 days with antisense SP-A ON. There was a
significant 85% reduction in SP-A protein in tissue exposed to
antisense SP-A ON compared with control (Fig.
2, n = 3 experiments,
ANOVA, P = 0.04, *Student-Newman-Keuls test, P < 0.05 for antisense condition vs. control, sense or
Lipofectamine conditions). This decrease was consistent with the SP-A
mRNA data (Fig. 1). SP-A content was not significantly decreased with 5 days of exposure to the sense SP-A ON or to Lipofectamine (Fig. 2).
There was an apparent 18% reduction in SP-A content in the vehicle
(Lipofectamine) condition; however, this decrease was not statistically
significant.
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Effect of inhibiting SP-A mRNA on immunostaining for SP-A in human
fetal lung.
Immunohistochemical staining for SP-A was performed on frozen sections
of cultured fetal lung tissue to localize and observe the effects on
SP-A of using antisense SP-A ON to selectively inhibit SP-A mRNA. As
expected, no SP-A immunostaining was detected in epithelial cells
lining the lumina of prealveolar ducts from undifferentiated fetal lung
tissue (start tissue) before culture (Fig.
3A). SP-A immunostaining was
clearly observed in distal pulmonary epithelium lining the ductal
lumina of control fetal lung explants that had been cultured for 5 days
(Fig. 3B). However, in the explants cultured for 5 days with
antisense SP-A ON, there was a clear reduction in the degree of SP-A
immunostaining detected in the distal pulmonary epithelial cells that
lined the ducts (Fig. 3D). This attenuation of
immunostaining for SP-A was not seen in explants cultured with either
the sense SP-A ON (Fig. 3C) or Lipofectamine alone (Fig.
3E). Negative staining controls using PBS instead of the
primary antibody along with the secondary antibody also resulted in an
absence of immunostaining for SP-A (Fig. 3F). This decrease
in SP-A immunostaining in explants exposed to antisense SP-A ON was
replicated in two additional experiments using different starting
tissue. In the sense and antisense SP-A ON-treated explants, there was
decreased cellularity of the connective tissue compared with the
control and Lipofectamine conditions with the prealveolar ducts
remaining intact (Fig. 3).
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Effect of inhibiting SP-A on tubular myelin.
To determine the effects of selectively inhibiting SP-A on tubular
myelin, we performed electron microscopy of human fetal lung tissue
that had been cultured for 6 days in the presence or absence of
antisense SP-A ON (90 µM). All the explants were cultured with
dibutyryl cAMP (1 mM) to enhance secretion of lamellar bodies into the
ductal lumina. Neither the sense nor the antisense SP-A ON prevented
the normal differentiation of the distal pulmonary epithelial cells
into type II pneumocytes. As seen in electron micrographs from both the
sense (Fig. 4A) and antisense
conditions (Fig. 4B), alveolar type II cells that contained
lamellar bodies and have microvilli lining their apical surface were
present. However, at higher magnification, there was a clear decrease
in tubular myelin formation among the secreted lamellar bodies within the ductal lumen of explants exposed to antisense SP-A ON (Fig. 4D). In the control condition, at similar magnification,
secreted lamellar bodies within the ductal lumen were frequently
observed unwinding to form tubular myelin (Fig. 4C). Tubular
myelin was rarely seen in cultured explants exposed to antisense SP-A
ON from two additional experiments from separate fetuses (6 sections examined per explant).
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Cytotoxicity. The presence of LDH in the media from fetal lung explants cultured for 3 days in the presence of 90 µM antisense SP-A phosphorothioate ON was measured to assess possible toxicity. The LDH data (IU/l) obtained were normalized to the control condition, with the control level set equal to one for each experiment. There were no significant differences in LDH levels between the Lipofectamine (1.5 ± 0.1) sense SP-A ON (2.0 ± 0.5) and antisense SP-A ON (1.8 ± 0.6) conditions (means ± SE, n = 3 experiments, P = 0.7).
Reversibility of antisense depletion of SP-A mRNA.
To further ensure that the antisense-induced decrease in SP-A mRNA was
not due to toxicity, we cultured fetal lung explants with antisense
SP-A ON for 3 days and then cultured the same explants in media without
antisense ON for an additional 2-8 days. We compared the level of
SP-A mRNA isolated from these explants to both day 3 control
and day 3 antisense-exposed explants. Again there was a
clear decrease in the level of SP-A mRNA after 3 days of exposure to
antisense SP-A ON compared with control explants (Fig.
5, lane 2 vs. lane
1). In explants that had been exposed to antisense SP-A ON for 3 days and then cultured for an additional 4-8 days without
antisense ON, the level of SP-A mRNA began to rise back towards the
control baseline as the inhibitory effect of the antisense SP-A ON was
reversed over time (Fig. 5, lane 5 vs. lane 2).
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DISCUSSION |
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SP-A is the most abundant of the surfactant proteins. SP-A aids in the formation of tubular myelin, facilitates the surface tension-reducing properties of surfactant phospholipids, regulates the recycling and secretion of surfactant phospholipids, and contributes to pulmonary host defense (28). Transgenic mice lacking SP-A have decreased tubular myelin. However, in these same transgenic animals, SP-B and saturated phosphatidylcholine are not significantly affected (11, 13). Transgenic mice lacking SP-A are found to be more susceptible to infection with either Pseudomonas aeruginosa (15) or group B streptococcus (16), whereas transgenic mice that overexpress SP-A have enhanced resistance to surfactant inactivation from protein inhibitors (7).
In premature baboons with bronchopulmonary dysplasia, levels of SP-A are significantly reduced (1), leading to an increased risk of infection in primates with chronic lung injury. In patients with bacterial pneumonia there is also a significant decrease in the level of SP-A in lung lavage fluid (2). Furthermore, ventilated patients who go on to develop acute respiratory distress syndrome have much lower levels of SP-A than ventilated patients who remain in a more stable condition (9). Thus, in humans, the level of SP-A appears to be associated with lung injury, but whether the low level is a consequence or a cause of the injury remains unclear.
To elucidate the role of SP-A during human fetal lung development, we blocked the synthesis of SP-A in human tissue using cultured fetal lung explants as an in vitro model of fetal lung maturation. It has previously been shown in this model that undifferentiated midtrimester human fetal lung tissue will spontaneously differentiate into alveolar type II cells capable of producing SP-A after 3-4 days in culture (22). In the present experiments, we inhibited expression of the SP-A gene by using antisense ON to selectively mediate the degradation of SP-A mRNA in cultured human fetal lung explants. This, in turn, resulted in a selective decrease in SP-A protein content and allowed us to observe the effects of eliminating SP-A in a model of human fetal lung development.
Antisense ON strategies to decrease or inhibit levels of a specific
protein have been used successfully in tissue culture. In embryonic
chick heart explants, Potts et al. (19) used
phosphoramidate-modified ON (1 µM) to inhibit transforming growth
factor-3. In cultured embryonic mouse lungs, Seth et al.
(20) used unmodified ON (30 µM) to inhibit EGF. Souza et
al. (23) used phosphorothioate-modified ON (10 µM) to
inhibit the platelet-derived growth factor receptor in cultured
embryonic rat lungs. We previously reported (12) that we needed to use a slightly higher concentration (90 µM) of the
phosphorothioate-modified ON to inhibit EGF receptor in human fetal
lung explants than others have used, probably because of the 1-mm cubic
thickness of the human fetal lung explants compared with the embryonic
chick, mouse, and rat explants.
We found that exposure to antisense SP-A ON significantly decreased SP-A mRNA and protein in cultured human fetal lung explants compared with the control, sense ON, and vehicle conditions. Tubular myelin was deficient in human fetal lung tissue lacking SP-A. This observation was completely consistent with a similar finding in the SP-A knockout mouse, in which tubular myelin figures were decreased (13). We also found no effect on either SP-B gene expression or the morphological differentiation of distal pulmonary epithelium into alveolar type II cells. These observations, seen in human fetal lung tissue deficient in SP-A, were again consistent with similar findings in the SP-A knockout mouse (13). These data clearly support a role for SP-A in the formation of tubular myelin within human fetal lung tissue.
There was some variability in the degree to which SP-A was eliminated from epithelial cells within the explants. This was most likely due to the thickness of the human fetal lung explants used for culture. The effect of antisense ON on SP-A gene expression in fetal lung explants is due to its specific effect on SP-A itself and not due to a global effect of the phosphorothioate ON on overall gene expression, as seen by a lack of an effect on SP-B mRNA levels. In explants exposed to phosphorothioate ON, there was a mild decrease in the cellularity of the connective tissue, whereas the epithelial cells lining the prealveolar ducts remained intact. This was not a result of cytotoxicity since there was no increase in the release of LDH into the media in the antisense ON-treated explants. Also, there was no evidence of structural cytotoxicity, because the ON did not prevent the normal differentiation of the distal pulmonary epithelial cells into lamellar body-containing alveolar type II cells. The decreased connective-tissue cellularity observed was most likely a nonspecific (sequence independent) effect of the phosphorothioate backbone. This same finding was previously observed in human fetal lung explants exposed to different ON sequences that had the same phosphorothioate backbone (12). Phosphorothioate compounds have been found to interact nonspecifically with a number of cellular proteins (24), in part, because of their polyanionic nature, which mimics the charge density of heparin (10, 32). Sequence-independent binding of phosphorothioate ON to both laminin and fibronectin, extracellular matrix proteins possessing heparin-binding domains, has been reported (3). Thus by potentially decreasing the availability of heparin binding sites on these important extracellular proteins in the lung, the phosphorothioate ON appears to inhibit the normal migration and attachment of cells within the connective tissue in our explants.
We have demonstrated that antisense ON can be used to selectively attenuate SP-A gene and protein expression in fetal lung explants during human type II cell differentiation in vitro. We found that the presence of SP-A is critical for optimizing the formation of tubular myelin in human fetal lung tissue, but the absence of SP-A does not prevent the morphological differentiation of alveolar type II cells in vitro. Thus SP-A does not appear to play a critical regulatory role during human fetal lung development in vitro.
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ACKNOWLEDGEMENTS |
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We thank Kelli Goss for technical assistance.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-52055 (J. M. Klein) and HL-50050 (J. M. Snyder). We also acknowledge the assistance of the University of Iowa's Central Electron Microscopic Research Facility.
Address for reprint requests and other correspondence: J. M. Klein, Dept. of Pediatrics, Univ. of Iowa, Iowa City, IA 52242-1083 (E-mail: jonathan-klein{at}uiowa.edu).
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.
10.1152/ajplung.00410.2000
Received 14 November 2000; accepted in final form 27 March 2001.
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