Bombesin-like peptides and receptors in normal fetal baboon
lung: roles in lung growth and maturation
R. L.
Emanuel1,
J. S.
Torday2,
Q.
Mu3,
N.
Asokananthan3,
K. A.
Sikorski3, and
M. E.
Sunday3
1 Department of Medicine,
Children's Hospital and Harvard Medical School, and
2 Department of Pathology,
Children's Hospital, Brigham & Women's Hospital, and Harvard
Medical School, Boston, Massachusetts 02115; and
3 Department of Pediatrics,
Harbor-University of California, Los Angeles Medical Center, Torrance,
California 90502
 |
ABSTRACT |
Previously, we have shown that bombesin-like
peptide (BLP) promotes fetal lung development in rodents and humans but
mediates postnatal lung injury in hyperoxic baboons. The present study analyzed the normal ontogeny of BLP and BLP receptors as well as the
effects of BLP on cultured normal fetal baboon lungs. Transcripts encoding gastrin-releasing peptide (GRP), a pulmonary BLP, were detectable on gestational day 60 (ED60), peaked on approximately ED90, and then declined before term
(ED180). Numbers of BLP-immunopositive neuroendocrine cells peaked from
ED80 to ED125 and declined by ED160, preceding GRP-receptor mRNAs
detected from ED125 until birth. BLP (0.1-10 nM) stimulated type
II cell differentiation in organ cultures as assessed by
[3H]choline
incorporation into surfactant phospholipids, electron microscopy, and
increased surfactant protein (SP) A- and/or SP-C-immunopositive cells
and SP-A mRNA. BLP also induced neuroendocrine differentiation on ED60.
Cell proliferation was induced by GRP, peaking on ED90. Similarly,
blocking BLP degradation stimulated lung growth and maturation, which
was completely reversed by a BLP-specific antagonist. The dissociation
between GRP and
GRP-receptor gene
expression during ontogeny suggests that novel BLP receptors and/or
peptides might be implicated in these responses.
gastrin-releasing peptide; phyllolitorin; CD10/neutral
endopeptidase 24.11; surfactant phospholipid synthesis; deoxyribonucleic acid synthesis
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INTRODUCTION |
INVESTIGATION OF HORMONAL REGULATION of lung maturation
is of particular importance because it may reveal novel forms of
treatment for preventing or minimizing the severity of respiratory
distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). Lung
development, including morphogenesis, growth, and maturation, is a
complex process involving interactions between multiple cell types and hormones (35). During development, the first epithelial cells to
differentiate in both human and rodent fetal airways are the pulmonary
neuroendocrine cells (PNECs) (11, 42). Mammalian bombesin (BN)-like
peptide (BLP) was identified as the first neuropeptide immunoreactivity
localized to PNECs (46), with the highest levels in human fetal lungs
(39). Gastrin-releasing peptide (GRP) is the major endogenous pulmonary
BLP implicated in promoting growth and maturation during fetal lung
development in humans, rats, and mice (20, 36-38). In previous
studies (33, 36), peak GRP transcript levels were demonstrated to occur
in human and murine lungs during the canalicular period. Administration
of BN to fetal mice during this time of development in utero or in organ culture leads to increased cell proliferation and differentiation (36). A blocking anti-BLP monoclonal antibody (2A11) blocks type II
cell differentiation in fetal mice in utero (37) and in lung organ
culture (36), indicating that BLP and hence PNECs can play a direct
role in normal fetal lung development.
As a result of the collaborative program by the National Institutes of
Health, the baboon model of BPD established by Coalson et al. (8) and
Escobedo et al. (15) has been targeted as a tool in the
investigation of chronic lung disease of newborns with interrupted
gestation. Using this model, Sunday et al. (41) have recently
demonstrated that BLP mediates lung injury in the hyperoxic baboon
model of BPD. The present study was a comparative investigation of the
ontogeny of BLP and BLP receptors in the developing baboon lung. We
also compared the effects of BLP and other growth factors on cell
proliferation and type II cell differentiation in fetal baboon lung
organ cultures.
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METHODS |
Animals. Prematurely delivered baboons
were provided by the Bronchopulmonary Dysplasia Resource Core of the
Southwest Foundation for Biomedical Research (San Antonio, TX). Normal
fetal baboons were delivered by cesarean section at 60-180 days
gestation (full term ~180 days). Only one animal was available at
each of gestational ages 60, 80, and 90 days. Lung tissue was harvested
aseptically: one portion (right upper lobe) was flash-frozen and stored
at
70°C for RNA preparation, and another portion was filled
with medium, shipped at 4°C, and used ~24 h after harvest for
organ cultures.
RNA analyses. Total RNA was prepared
from frozen fetal lung tissue with the TRI REAGENT (Molecular Research
Center, Cincinnati, OH). Total RNA (10 µg/lane) was fractionated on
1% agarose-2.2 M formaldehyde denaturing gels and transferred to
nitrocellulose by standard capillary transfer (12). After being baked,
the blots were probed with cDNAs encoding human surfactant apoprotein (SP) A1 and SP-C. The cDNAs were obtained from non-small cell lung
carcinoma line H441 mRNA by RT-PCR with the following primer pairs: SP-A 5', CCCAGAGCCATGT, and 3',
TTCTCTCCACGCTCTCCA, and SP-C 5', AGCAAGATGGATGTGGGCAG, and
3', AGCTTAGACGTAGGCACT. The resulting predicted cDNA fragments of
275 bp for SP-A1 (16, 22, 28) and 494 bp for SP-C (24, 48) were gel
purified, eluted from agarose gel with Spin-X centrifuge tube filters
(Corning Costar, Corning, NY), and labeled with the random-primed
oligonucleotide method (Boehringer Mannheim, Indianapolis, IN). The
blots were hybridized at 42°C overnight in 50% formamide,
2.5× Denhardt's solution (0.2 g of Ficoll, 0.2 g of
polyvinylpyrrolidone, and 0.2 g of BSA fraction V in 1 liter of diethyl
pyrocarbonate-water), 0.8 M NaCl, 1 mM EDTA, 0.05 M sodium phosphate
buffer (pH 6.5), 200 µg of sonicated denatured salmon
sperm DNA, and 0.4 mg of tRNA; washed at 50°C in 1×
saline-sodium citrate-0.1% SDS; and exposed to Kodak Biomax film with
an intensifying screen.
RT-PCR. RT-PCR was required to detect
the low levels of mRNAs encoding GRP and GRP receptor (GRPR) in the
available small tissue samples. cDNA was prepared, and semiquantiative
RT-PCRs were carried out as previously described (40, 47) with 30 and
35 cycles of PCR for GRP and GRPR, respectively, and 18 cycles for 18S
rRNA, each cycle including denaturation (0.5 min at 93°C), annealing (1.0 min at 55°C for GRP and 18S rRNA and 50°C for
GRPR), and extension (3 min at 72°C). Synthetic
oligodeoxynucleotide pairs were designed to span at least one intron
corresponding to the conserved sequences of human GRP (yielding a
194-bp product) (32), human GRPR (yielding a 388-bp product) (1), and
human 18S rRNA (yielding a 567-bp product) (GenBank). Southern blots of
the PCR products were probed with a corresponding end-labeled internal
oligonucleotide specific for GRP, GRPR, or 18S rRNA. Primers were
synthesized by Oligos Etc. (Wilsonville, OR). The sequences of the
primers used were GRP 5', AAGCAGCAGCTGAGAGAGTA, GRP 3',
GAGAGTCTACCAACTTTGCC, and GRP probe, GAAGTTGCTGCTATCCTCTG; GRPR
5', CTCCCCGTGAACGATGACTGG, GRPR 3', ATCTTCATCAGGGCATGGGAC, and GRPR probe, AGGTTTGGAACGTTTCGC; and 18S 5',
TAGCTCTTTCTCGATTCCGT, 18S 3', TTCACCTACGGAAACCTTGT, and
18S probe, GTTATTGCTCAATCTCGGGTG. Total RNAs from human lung carcinoma
cell lines were used as positive controls for RT-PCR: H128 (small cell
lung carcinoma) for GRP, H720 (pulmonary carcinoid) for GRPR, and H441
(non-small cell lung carcinoma) for SP-A and SP-C. The human lung
carcinoma cell lines were cultured as previously described (6). In
compliance with guidelines for submission to the
American Journal of Physiology, it
should be noted that specific testing for mycoplasma was not carried
out in these short-term primary cultures.
In situ hybridization. Nonisotopic in
situ hybridization was carried out to localize mRNAs in tissue
sections. Three-millimeter-thick sections of fetal baboon lung were
fixed for 3-4 h in 4% paraformaldehyde, and then transferred into
70% ethanol at 4°C overnight before being routinely processed into
paraffin. A baboon GRPR cDNA was obtained by PCR with primers
corresponding to human GRPR cDNA sequence (1). To prepare a template
for cRNA probes, the resulting 388-bp GRPR cDNA was subcloned into
Bluescript SK+ and its identity was verified by dideoxy sequencing.
cRNA probes were labeled with digoxigenin (Dig)-UTP with the Dig RNA
labeling kit (Boehringer Mannheim) according to the manufacturer's
specifications. The remainder of the protocol was carried out as
previously described (34) with the following modifications:
hybridization was carried out at 65°C overnight with 25 ng
Dig-labeled cRNA/µl hybridization buffer prepared according to the
manufacturer's specifications (Boehringer Mannheim). After being
washed, the slides were developed for 4 h at 20°C with 1 µl/slide
of anti-Dig antibody (alkaline phosphatase conjugated; Boehringer
Mannheim), 5 µl normal goat serum, 1.5 µl 0.3% Triton X-100, and
0.5 ml 0.1 M Trizma base-0.15 M NaCl, pH 7.5 (TS). After a wash in TS
for 5 min, 500 µl of nitro blue tetrazolium (NBT)
color solution was applied to each slide from a mixture of 2.5 ml of
TS-0.05 M MgCl2, 11 µl of 100 mg/ml of NBT, 9 µl of 5-bromo-4-chloro-3-indolyl phosphate (BCIP;
Sigma, St. Louis, MO) and 2 drops (90 µl) of 50 mM
levamisole. The NBT solution was incubated for 2-4 h in a dark
humid chamber, the reaction was stopped with Tris-EDTA, and the
sections were coated with aqueous mounting medium before placement of
glass coverslips and viewing with a light microscope.
Immunoperoxidase and morphometric
analyses. Three-millimeter-thick sections of fetal
baboon lung were fixed for 18-24 h in 4% paraformaldehyde before
being routinely processed into paraffin. Three-micrometer paraffin
sections were dewaxed, and immunoperoxidase analyses carried out with a
mouse monoclonal anti-BN antibody (2A11) and the avidin-biotin complex
immunoperoxidase technique with diaminobenzidine as the substrate as
previously described (41). All of the BLP-immunopositive neuroendocrine
cells were counted on every slide in which there was comparable
representation of proximal cartilaginous airways and terminal
bronchioles near the pleural surface. The number of BLP-positive cells
was normalized by expressing the value as a function of the total
length of intrapulmonary bronchial plus bronchiolar (columnar)
epithelium present on each slide. Immunostaining for SP-B and proSP-C
was carried out with 1:500 dilutions of antisera generously provided by
Dr. Jeffrey Whitsett (Children's Hospital Medical Center, Cincinnati,
OH) with the avidin-biotin complex immunoperoxidase technique as
described (41).
Lung organ cultures. Fetal lung was
harvested at the time of cesarean section and collected at 4°C in
Waymouth medium (GIBCO BRL, Life Technologies, Frederick, MD)
supplemented with 5% fetal calf serum (Atlanta Biologicals, Atlanta,
GA). After overnight shipping at 4°C, the tissue was chopped into
0.5-mm cubes and cultured in six-well plates with the same medium with
and without added BLP or other agents. Cultures were grown for 48 h or
6 days at 37°C in 5% CO2-air
on a rocking platform at 3 oscillations/min (36). Tissue viability was
checked by histological analyses, satisfactory baseline incorporation
of [3H]choline and
[3H]thymidine, and
significant positive control responses.
Determination of [3H]DNA,
saturated
[3H]phosphatidylcholine, DNA,
and protein contents.
On the day of harvest (after 44 h), the tissue was incubated with
[3H]thymidine (4 µCi/ml) or
[3H]choline (16 µCi/ml; NEN-Dupont, Boston, MA) for 4 h at 37°C in 5%
CO2-air on a rocking platform
before being harvested and analyzed for
3H-labeled DNA, DNA content,
3H-labeled saturated
phosphatidylcholine (Sat PC), and protein content.
[3H]thymidine
incorporation into acid-precipitable counts was carried out in
quadruplicate as previously described (36). DNA was assayed after
trichloroacetic acid precipitation with the method of Burton (5). For
determination of the rate of surfactant phospholipid synthesis, lipids
were extracted from cell homogenates with chloroform-methanol, and
[3H]choline
incorporation into Sat PC was determined as previously described (43).
Protein was determined with the method of Bradford (2) with BSA
(Bio-Rad, Hercules, CA) as a standard. Experimental values were
normalized by defining the mean of the control groups as baseline and
expressing the values as percent change above or below this.
Statistical analyses. Numerical data
were analyzed with unpaired Student's
t-test, with values expressed as
means ± SE.
 |
RESULTS |
Ontogeny of GRP and GRPR gene expression. GRP
transcripts were detected by semiquantitative RT-PCR analysis as early
as ED60, peaked on ED90, and declined to low levels during the rest of gestation and to undetectable levels in adult animals (Fig.
1A). In
most cases, when the lung tissue was subdivided, GRP transcripts were
more abundant in the distal than in the proximal region of the
developing lung (Fig. 1A). In one of two samples on
ED125 and the one subdivided ED140 lung, there was approximately
twofold more GRP proximally compared with distally, likely representing the relative predominance of conducting airways in proximal tissue versus those in the distal lung, which at this stage includes predominantly primitive alveoli; GRP was expressed in PNECs in bronchi
and bronchioles but not in normal alveoli (see below). These kinetics of GRP gene expression
preceded peak GRPR gene expression, which was first clearly
detected on ED125, peaked on ED160, and decreased to undetectable
levels in adult animals (Fig. 1A). We were unable to
detect mRNA for the BN-related peptide neuromedin B (NMB) or for the
other two cloned mammalian BLP receptors, the NMB receptor (NMBR) and
BN-receptor subtype 3 (BRS-3), with 30-35 cycles for RT-PCR
despite good detection of positive control mRNAs in baboon brain (for
NMB), esophagus (for NMBR), and testis (for BRS-3) (data not shown).
For comparison to known parameters of lung maturation, levels of SP-A
and SP-C mRNAs were determined by Northern blot analysis (Fig.
1B). SP-A mRNA was first detected as a 2.2-kb transcript
late in gestation, peaked on approximately ED160, and was expressed
throughout adulthood. SP-C mRNA expression was first detected as a
1.0-kb transcript on ED90, peaked from ED160 to ED175, and continued to
be expressed abundantly during adulthood.


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Fig. 1.
Ontogeny of gastrin-releasing peptide (GRP) and GRP-receptor (GRPR)
transcripts compared with surfactant protein (SP) A and SP-C in fetal
baboon lungs. A: lungs were harvested
from fetal baboons [embryonic day
60 (E60) to E175] and adult animals. Total RNA
was prepared, and RT-PCR was carried out with GRP, GRPR, and 18S rRNA
primers as described in METHODS. P,
proximal; D, distal; T, term; AD, adult; +Con, positive control. RNAs
obtained from human cell lines (small cell lung carcinoma H128 and
pulmonary carcinoid cell line H720) were used as positive controls for
GRP and
GRPR gene expression, respectively.
Nos. on right, size of transcripts.
B: total RNA prepared from baboon
lungs (E60 to adult animals) was used for Northern blot analyses as
described in METHODS. RNA used for
SP-A-positive control was obtained from cell line H441. 18S rRNA was
estimated from ethidium bromide photograph of Northern gel. Nos. on
right, size of SP-A and SP-C bands;
nos. on left, location of RNA
markers.
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In situ hybridization analyses with antisense cRNA probes localized
GRPR transcripts primarily to airway epithelium (Fig. 2A,
black arrows), to undifferentiated (loose) mesenchymal cells surrounding developing blood vessels and airways (Fig. 2,
A and C, yellow asterisks) and throughout
the alveolar interstitium (Fig. 2, A,
between white arrows, and C, alv), and
to alveolar macrophages (data not shown). In contrast, there was no
mRNA signal with the corresponding sense control slides in immediately
serial sections (Fig. 2, B and
D).

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Fig. 2.
In situ hybridization analyses for GRPR and GRP-containing cells in
fetal baboon lungs. A and
C: in situ hybridization for GRPR was
carried out with 3-µm sections of E160 fetal baboon lung with a
homologous GRPR antisense cRNA probe. Note localization of GRPR mRNAs
in loose mesenchyme (yellow asterisks) surrounding developing blood
vessels (bv) and airways (AW). There was also expression of GRPR in
airway epithelium (black arrows) and in interstitium of developing
alveoli (alv; between white arrows), consistent with previous reports
(21, 23, 44, 45). Original magnification, ×25 in
A and ×100 in
C. B
and D: negative control for in situ
hybridization was carried out with immediately serial sections of E160
fetal baboon lung with homologous GRPR sense cRNA probe. No
hybridization signal was observed with the GRPR sense cRNA probe.
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BLP-immunopositive cells were detectable between ED60 and ED140 (Fig.
3). At ED60, single BLP-positive cells were
localized only to large proximal airways that had begun to branch (data not shown). By ED80-90, BLP-positive PNECs occurred predominantly in small clusters of two to four cells within the basement membrane of
the conducting airway epithelium (Fig. 3,
A and
B). A few larger PNEC clusters
containing 8-14 PNECs were also present, usually at airway
bifurcations (Fig. 3C). By ED125,
most PNEC clusters were in bronchioles (Fig.
3D, arrows). The same cells
immunostained for both BLP and protein gene product 9.5 immunoreactivity (data not shown). GRP in situ hybridization on thin
serial tissue sections colocalized GRP mRNA in over half of the
BLP-immunopositive PNEC clusters (Fig.
3E, thin arrow). GRP mRNA was
additionally present in longer stretches of airway epithelium that was
devoid of BLP or protein gene product 9.5 immunostaining (Fig.
3E, double-headed arrow). The
corresponding GRP sense control section was negative (Fig.
3F).


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Fig. 3.
Immunohistochemical analyses for bombesin-like peptide (BLP) and
GRP-positive cells in normal fetal baboon lungs.
A-C:
immunoperoxidase staining was carried out on E80-90 fetal baboon
lung with anti-bombesin antibody 2A11 (10) as described in
METHODS. Most BLP-positive cells
occurred in small clusters of 2 (A)-4
(B) cells. Infrequent (<10%)
larger clusters of ~8-14 cells were also present on E80-90,
some of which protruded into airway lumen (AW), mostly at airway
bifurcations (C). Serial sections
incubated with irrelevant control IgG1 MOPC21 were devoid of signal
(data not shown). Original magnification, ×200.
D-F:
by E125, most BLP-positive clusters
(D, arrows) occurred immediately
adjacent to GRP mRNA-positive cell clusters
(E, small arrow) in thin serial
sections. Occasional BLP-positive cell clusters did not correspond to
GRP mRNA-positive cells in serial sections
(D:
top arrow). In addition, many
epithelial cells containing GRP mRNA did not contain detectable BLP
immunoreactivity (E, double-headed
arrow). There was no GRP mRNA signal in mesenchyme. No hybridization
signal was observed with GRP sense cRNA probe
(F). Original magnification,
×50. G: epithelial cells
positively immunostained for BLP were quantitated and normalized, then
plotted vs. gestational age. Two different methods for normalization
were carried out: 1) for total
amount of airway epithelium present (on E60-90; this included all
primitive airways lined by columnar to cuboidal epithelium, in which it
was not possible to distinguish between primitive alveoli and future
conducting airways) and 2) for total
area of lung tissue present (noninflated). PNEC, pulmonary
neuroendocrine cell.
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Morphometric analysis of BLP-immunopositive cells in fetal lungs
demonstrated the maximal number of BLP-positive PNECs per square
centimeter of lung tissue at approximately ED80-90 (Fig. 3G), corresponding to the time of
peak GRP gene expression (Fig. 1A). However, when the number of
PNECs was normalized for the total length of airway epithelium per
slide, the relative number of PNECs peaked on approximately ED125 (Fig.
3G). By ED160, the number of PNECs
detected immunocytochemically declined to near zero.
Role of BLP in type II cell differentiation in fetal
baboon lung. The role of BLP in fetal baboon lung
maturation was explored with a simple in vitro system with lung tissue
collected at various gestational ages and grown in culture for 48 h.
Lung organ maturation, defined as type II pneumocyte differentiation
with surfactant phospholipid synthesis, was measured as the amount of
[3H]choline
incorporation into Sat PC.
[3H]choline uptake was
optimally stimulated after 48 h in culture with 1 nM BN or GRP (Fig.
4, A and
B, respectively).
There was significant stimulation of choline uptake by GRP as early as
ED60 (one animal harvested; data not shown). The maximal effect of BN
was on E140 and of GRP on ED90 (31 ± 7 and 240 ± 45% increase over baseline, respectively; P < 0.001; Fig. 4, A and
B, respectively). The BN-related
peptide NMB (not present in normal lung; Table 1) had minimal effects on lung maturation
(<15%) at all gestational ages (data not shown).

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Fig. 4.
Role of BLP in fetal baboon lung maturation. Lungs from various
gestational ages were cultured with and without bombesin (BN;
A) or GRP
(B) before being collected for
measurement of
[3H]choline
incorporation into saturated phosphatidylcholine (Sat PC). NEG,
negative control. Each determination for a given animal was carried out
in quadruplicate (see METHODS).
Values are means ± SE representing pooled data for 1, 7, 4, and 2 animals at 90, 125, 140, and 160 days of gestation, respectively.
Typical average values for absolute
[3H]choline
incorporation in negative control cultures (48 h) were 139 ± 5 (80 days), 3,523 ± 306 (90 days), 4,884 ± 264 (125 days), and 6,371 ± 911 (140 days) counts · min 1 · µg
protein 1. P
values in Figs. 4-7, 10, and 11 were derived from a comparison
between NEG and each of the treatment groups in the same experiments.
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Another amphibian BN-related peptide,
[Leu8]- phyllolitorin
(L8PL), that King et al. (21) previously found to stimulate branching morphogenesis in murine embryonic lung buds also stimulated lung maturation in 48-h cultures at gestational ages ED125 and ED140, with
the maximal effect observed with 10 nM on ED125 and 0.1 nM on ED140 (55 ± 2 and 49 ± 6% increase over baseline, respectively; P < 0.001) as shown in Fig.
5A. The
CD10/neutral endopeptidase (NEP) 24.11 inhibitor Sch-32615, which
blocks hydrolysis of BLP and thus potentiates tissue levels of
endogenous BLP (31), increased [3H]choline
incorporation at all gestational ages tested, with the maximal effect
seen with 5 nM in ED90 cultures (46 ± 2% increase over baseline;
P < 0.05; Fig.
5B). The specific BLP-receptor
antagonist [Leu13-
(CH2NH)Leu14]BN
(L13BN) alone inhibited lung maturation maximally at 100 nM in ED125
cultures (
31 ± 3% compared with negative controls;
P < 0.001; Fig.
5B). At ED140, the combination of
L13BN and Sch-32615 resulted in net inhibition of
[3H]choline
incorporation (~28 ± 5% compared with baseline;
P < 0.001; Fig.
5B).

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Fig. 5.
Effect of BLP-related peptides on fetal baboon lung maturation. Lungs
collected at various gestational ages were cultured with and without
BN-related peptide
[Leu8]phyllotorin
(L8PL; A) or CD10/neutral
endopeptidase (NEP) inhibitor Sch-32615 and BN-receptor antagonist
[Leu13- (CH2NH)Leu14]BN
(L13BN; B) before being harvested
for measurement of
[3H]choline
incorporation into Sat PC (see
METHODS). Values are means ± SE
representing pooled data for 1, 7, and 4 animals at 90, 125, and 140 days of gestation, respectively.
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Dexamethasone (Dex), used as the positive control for choline uptake
assays, increased choline uptake in lung explants at all gestational
ages tested. The maximal effect of Dex was observed with 10 nM in
cultures of ED140 lung (121 ± 14%;
P < 0.001 compared with baseline;
Fig.
6A). The
combination of low-dose BN (0.1 nM) and low-dose Dex (0.1 nM)
resulted in the inhibition of the Dex effect at both ED125 and
ED140 (Fig. 6B).

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Fig. 6.
Effect of dexamethasone (Dex) with and without BN on lung maturation.
Lungs from various gestational ages were cultured for 48 h with varying
doses of Dex (A) or low-dose (1 nM)
Dex plus low-dose BN (B) before
being harvested for measurement of
[3H]choline
incorporation into Sat PC (see
METHODS). Values are means ± SE
for 1, 7, 4, and 2 animals at 90, 125, 140, and 160 days of gestation,
respectively.
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Epidermal growth factor (EGF), a second positive control for choline
uptake assays, stimulated choline uptake in culture only on ED125, with
10 nM resulting in a maximal effect (39 ± 4% increase over
baseline; P < 0.001) as
shown in Fig.
7A. In
combination, low doses of EGF (0.1 nM) and BN (0.1 nM) synergistically
increased [3H]choline
incorporation in ED140 explants by 27 ± 9% over baseline (P < 0.001) as indicated in Fig.
7B.

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Fig. 7.
Effect of epidermal growth factor (EGF) with and without BN on fetal
lung maturation. Lungs collected at various gestational ages were
cultured for 48 h with varying doses of EGF
(A) or low-dose EGF (0.1 nM) plus
low-dose BN (B) before being
harvested for measurement of
[3H]choline
incorporation into Sat PC (see
METHODS). Values are means ± SE
for 7, 4, and 2 animals at 125, 140, and 160 days of gestation,
respectively.
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Additional effects of BLP on cell
differentiation. To provide additional supporting
evidence for the effects of BLP (BN or GRP) on cell differentiation in
the fetal baboon lung, immunoperoxidase analyses were carried out after
6 days of culture with and without added BLP or Dex. Although 2-day
cultures were optimal for the choline uptake assay, 6-day cultures were
necessary to elicit a differentiated phenotype as detected by
immunoperoxidase analyses. Increased PNEC differentiation was observed
only in the youngest fetal lung available, of which we were able to
obtain just one animal. Results from this one ED60 lung are given in
Fig. 8. The most striking observation was
an increased number of PNECs in the presence of GRP (peak at 0.1 nM;
Fig. 8, A and
C) or Dex (1 nM; Fig.
8C) compared with negative control
cultures (Fig. 8, B and
C). The total number of BLP-positive
PNECs was quantitated and normalized for the total area of tissue
present on each slide (with proximal and distal portions of the lung
equally represented in every group) and for the total number of PNECs
per airway (which was significantly increased only for 0.1 nM GRP; Fig.
8C). There was no effect of either
GRP or Dex on the number of immunostainable PNECs between ED80 and
ED160.



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Fig. 8.
BN induced increased differentiation of PNECs and type II cells in E60
fetal baboon lung. A and
B: 6-day lung organ cultures from E60
baboon lung incubated with GRP (0.1 nM;
A) or medium alone (negative
control; B) demonstrated marked
induction of many strongly BLP-immunopositive cells (arrows). In
contrast, only a few weakly BLP-positive cells were observed in
negative control group (arrow). C:
morphometric analyses were carried out with 2 different methods: no. of
BLP-positive PNECs/unit length (in mm) airway epithelium (determined
for each airway; means ± SE) and total no. of PNECs/slide
normalized for area of tissue present. BLP-positive cells were also
positive for protein gene product 9.5 (data not shown).
D: ultrastructural analyses of the
same organ cultures of E60 lung were carried out to determine
percentage of alveolar epithelial cells containing lamellar bodies and
no. of lamellar bodies/lamellar body-positive (%LB+) cells. Twelve
×2,000 electron photomicrographs of randomly selected primitive
alveoli were analyzed for each group. Results are means ± SE.
* P < 0.001 compared with
negative control.
|
|
Electron microscopy was also carried out on the same lung cultures, and
morphometric analyses were carried out for early type II cell
differentiation, defined as the proportion of lamellar body-positive
cells per primitive alveolus (Fig.
8D). Both GRP (1 nM) and Dex (1 nM)
induced a significant increase in the relative number of lamellar
body-positive cells on ED60 (Fig.
8D) but not on ED140 (data not
shown). There was no difference in the number of lamellar bodies per
lamellar body-positive cell on either ED60 or ED140 (data not shown).
We also evaluated the effect of BLP on immunostaining for SP-A (Fig.
9, A and
B) and SP-C (Fig. 9,
C and
D). BLP (1 nM) induced a marked
increase in immunostaining for SP-A (Fig.
9B) and SP-C (Fig.
9D) compared with the same lungs
cultured with medium alone (Fig. 9, A
and C). The results in Fig. 9 are
from one representative experiment of five separate experiments
conducted at different times (each from a different ED125 fetal
baboon), all with similar results. There was also increased SP-A and
SP-C immunostaining from ED60 to ED90, but by ED140, the baseline
levels were high and no appreciable further increase was apparent.


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Fig. 9.
BLP led to increased surfactant protein (SP) and gene expression in
vitro. Organ cultures of E125 fetal lung were treated for 6 days with
medium alone (A and
C) or medium containing 1.0 nM BN
(B and
D). Sections were immunostained for
SP-A (A and
B) or proSP-C
(C and
D). Representative sections from 1 of 5 separate experiments are shown. Sections from BN-treated cultures
demonstrated increased immunostaining for SP-A and proSP-C in cells
lining primitive alveoli and also in alveolar macrophages. Original
magnification, ×100. E:
semiquantitative RT-PCR analyses for SP-A and 18S rRNA in E125 lung
organ cultures treated with medium alone (lane
1), 1.0 nM BN (lane
2), 10.0 nM BN (lane
3), medium alone (lane
4), and 10 nM Dex (lane
5).
|
|
To determine whether the observed increase in SP-A and SP-C
immunostaining was in part due to transcriptional regulation, RNA was
prepared from three of five experiments of the same 6-day ED125 lung
organ cultures. Compared with the negative control lung (Fig.
9E, lanes
1 and 4), there was
a marked induction of SP-A mRNA in the presence of BLP (10 nM;
lane 3) or Dex (10 nM; lane 5). In other experiments, the
effective dose of BLP was between 1 and 10 nM BN or 1 and 10 nM L8PL
(data not shown). There was no consistent effect of BLP or Dex on
levels of SP-C mRNA in these cultures (data not shown).
Role of BLP in cell proliferation in fetal baboon
lung. Lung growth was determined with
[3H]thymidine
incorporation into DNA, normalized for the total DNA content of the
tissue samples. In organ cultures, BN and GRP induced cell
proliferation on ED140, with a maximal effect at 0.01 nM (53 ± 36%
increase over baseline; P < 0.05) as
given in Fig. 10, A and
B, respectively. However, the peak
effect elicited by GRP occurred in the one ED90 lung available, at
which time 0.1 nM GRP stimulated increased thymidine uptake (64 ± 7% over baseline; P < 0.001; Fig.
10B). In contrast, high-dose Dex
(100 nM) treatment of the lung explants from the same animals resulted
in inhibition of DNA synthesis by >70% on ED125 and ED160, but there
was stimulation of thymidine uptake with low-dose Dex (1 nM) on ED140
(36 ± 4% increase over baseline;
P < 0.001) as given in Fig.
10C. EGF, the positive control,
stimulated thymidine uptake on ED140 and ED160, the maximal effect
occurring on ED140 with 10 nM (52 ± 12% increase over baseline;
P < 0.001) as summarized in Fig.
10D. Proliferating cell nuclear
antigen immunostaining of tissue sections indicated a widespread
induction of both epithelial and mesenchymal cell proliferation (data
not shown), consistent with earlier observations by Sunday et al. (36)
in fetal mouse lung.

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Fig. 10.
Effect of BLP on lung cell proliferation. Lungs from various
gestational ages were cultured for 48 h with BN
(A) or GRP
(B) before being harvested for
measurement of
[3H]thymidine
incorporation into DNA as described in
METHODS. For controls, effects of Dex
(C) and EGF
(D) were also determined. Due to
small size of available tissue sample, the one E90 sample was cultured
only with GRP for these determinations. Values are means ± SE for
1, 5, 3, and 2 animals at 90, 125, 140, and 160 days of gestation,
respectively. Proliferating cell nuclear antigen immunostaining was
increased in both epithelial cells and mesenchymal cells in tissue
sections from 6-day explants (data not shown).
|
|
Finally, combinatorial effects between low doses of BN and Dex or EGF
on cell proliferation were analyzed on ED125 and ED140 as shown in Fig.
11. Dex alone (1 nM) slightly inhibited
growth on ED125 and modestly increased thymidine uptake on ED140 (Fig. 11A), consistent with observations
in Fig. 10 from a different set of animals. The combination of low-dose
BN plus Dex was synergistic in inhibiting or augmenting thymidine
incorporation on ED125 or ED140, respectively (Fig.
11A). BN alone at 0.1 nM or EGF
alone at 0.1 nM had only marginal effects on thymidine uptake in these cultures (Fig. 11B). However, there
was synergism between BN and EGF for either inhibition of proliferation
on ED125 or stimulation on ED140 (Fig.
11B).

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Fig. 11.
Effects of BN, Dex, and EGF on cell proliferation. Lungs collected on
E125 and E140 were cultured for 48 h with BN and Dex
(A) or BN and EGF
(B) before being harvested for
measurement of
[3H]thymidine
incorporation into DNA (see
METHODS). Values are means ± SE
for 7, 4, and 2 animals at 125, 140, and 160 days of gestation,
respectively.
|
|
 |
DISCUSSION |
In our previous studies, we observed that BLP promotes
fetal lung development in rodents and humans but mediates postnatal lung injury in hyperoxic baboons (20, 21, 36-38, 41). The present
study demonstrates that BLP induces cell proliferation and type II cell
differentiation in organ cultures of normal fetal baboon lung. Lung
organ maturation was evaluated with multiple approaches:
[3H]choline
incorporation into Sat PC, the rate-limiting step in surfactant
phospholipid synthesis; electron microscopy for lamellar bodies;
immunostaining for SP-A and SP-C; and mRNA levels for SP-A. Cell
proliferation was assessed with
[3H]thymidine
incorporation into DNA. We correlate these effects with the kinetics of
gene expression for GRP, GRPR, SP-A, and SP-C.
GRP gene expression was detectable in
the one ED60 baboon lung available for study, then peaked at
midgestation (ED80-90) and became undetectable after ED140 (Fig.
1). GRP expression was more abundant in distal than in proximal lung
tissue, consistent with the major localization of BLP-immunopositive
neuroendocrine cells in small bronchioles scattered throughout the
distal parenchyma. These kinetics of
GRP gene expression, peaking at
midgestation (Fig. 2), match the time of a peak number of
BLP-immunopositive cells per square centimeter of lung tissue. These
results are essentially identical to previous reports on fetal lung
from humans (33) and rhesus monkeys (23). Thus baboons demonstrate
transient developmental expression of the
GRP gene during the canalicular phase
of lung development, the same as all other species studied to date (23,
33, 36, 37). It should also be noted that GRP gene expression from ED60 to ED125
precedes that of SP-A and SP-C, two markers of type II cell
differentiation, both of which peak closer to birth (approximately
ED160). Our results on the ontogeny of SP-A mRNA are consistent with
observations from another laboratory (22).
Treatment of fetal baboon lung explants with exogenous BLP results in
increased parameters of both maturation and growth. Maturation or type
II cell differentiation, evaluated with
[3H]choline
incorporation into Sat SPC, was stimulated by BN or GRP (collectively
termed BLP as shown in Table 1), with maximal effect occurring on
approximately ED90. GRP-induced maturational effects were also observed
in ED60 and ED80 lungs. These observations are especially intriguing
considering the fact that GRPR transcripts were extremely low in the
same ED60-90 lung specimens. GRPR
gene expression was absent or only marginally detectable in fetal
baboon lungs before ED125, attaining maximal levels on ED160 and
becoming undetectable after birth. These data suggest the possible
presence of a novel mammalian BLP receptor in the midgestation baboon
lung mediating BLP effects during the canalicular period. We did not detect transcripts encoding NMBR or BRS-3 in these normal baboon fetal
lung samples using the given RT-PCR conditions. It is unlikely that RNA
degradation was a factor in reducing detectable levels of these mRNAs
because all tissue was snap-frozen at the time of harvest and no RNA
degradation was evident on ethidium gels.
It is also interesting that there was a lack of GRP effects in cultures
of ED125-160 lungs despite significant effects of the amphibian
peptide BN, suggesting that there might be a novel mammalian BN-related
peptide better capable of interacting with the GRPR expressed during
this time period. We did not detect transcripts encoding NMB, the only
other cloned mammalian BN-related peptide, in the normal fetal baboon
lung. There remains the possibility that there could be another
bioactive peptide such as a mammalian phyllolitorin acting at the GRPR
(21), especially considering the immunological cross-reactivity of
phyllolitorins with anti-BN antibodies. Phyllolitorins are able to
elicit potent responses in the mammalian lung (14, 21). This
possibility is even more feasible considering our observation that
fetal baboon lung maturation was significantly stimulated from ED125 to
ED140 by the amphibian peptide L8PL, with a maximal effect occurring
with 10 nM L8PL on ED140.
BLP induced differentiation of multiple epithelial cell types in the
fetal lung. In the earliest lung sample collected (ED60), BLP induced
differentiation of single PNECs, supporting the concept that this
peptide can act as an autocrine differentiation factor. In addition to
stimulation of choline uptake, BLP (both BN and L8PL) induced other
parameters of type II cell differentiation. There was increased
immunostaining for SP-A (in Clara cells and type II cells) and SP-C
(type II cell specific) as well as increased mRNA encoding SP-A on
ED125. These data suggest that upregulation of SP-C was occurring
primarily at the posttranslational level and/or that increased SP-C
mRNA levels are not sustained for the full 6 days in culture. Divergent
regulation of SP-A and
SP-C genes is a frequent observation
(20). The timing of BLP-induced cell differentiation according to
different assays appears to depend on the status of endogenous cell
differentiation in lung tissue at different gestational ages. Thus
differentiation of PNECs normally precedes differentiation of all other
epithelial cell types in the lung, and
SP-C gene expression precedes that of
SP-A in vivo. BLP appears to function
more to induce the expression of several differentiated phenotypic
features rather than simply enhancing the level of established features.
In previous experiments with murine and human fetal lungs, King et al.
(20) and Sunday et al. (38) observed increased cell proliferation and
differentiation using the CD10/NEP inhibitor Sch-32615. CD10/NEP
hydrolyzes BLP, leading to a loss of BLP bioactivity (4, 31). In the
present study, specific CD10/NEP inhibition by low-dose Sch-32615 (5 nM) stimulated thymidine and choline uptake in fetal baboon lungs.
These effects were neutralized by the specific BN-receptor antagonist
L13BN, similar to observations in human and murine fetal lungs (20,
38). Treatment with L13BN alone resulted in decreased baseline choline
incorporation at 125 days gestation, suggesting that the GRPR is likely
to play a role at this time in ontogeny. These observations indicate
that endogenous BLP or closely related peptides such as potential
mammalian phyllolitorins are required for the observed potentiation of
cell proliferation and type II cell differentiation resulting from CD10/NEP inhibition. In murine experiments with in utero inhibition of
CD10/NEP, King et al. (20) observed increased mRNA encoding SP-A,
similar to the present data on BLP-treated fetal baboon lung.
Consistent with previous work in primates and rodents (17, 25, 30), the
positive controls EGF and Dex augmented baboon lung maturation. It was
previously reported (23) that BN can induce DNA synthesis in rhesus
monkey fetal lung cultures at midgestation. We similarly observed
significant stimulation of thymidine incorporation with low-dose GRP
(<1 nM) in the ED90 baboon lung; 1 nM GRP inhibited growth at this
gestational age in the baboon, likely due to receptor downregulation by
the higher dose. In contrast, at later ages of gestation, BN and/or GRP
either inhibited growth (on ED125 and ED160) or had marginal effects on
stimulating proliferation (on ED140). Such diverse effects argue for
the presence of additional BLP receptors in addition to the GRPR
functioning in these systems. Alternatively, signal transduction
between ED125 and ED160 could favor cell differentiation over
proliferation. It should be noted that observed small effects on lung
growth are of questionable clinical significance, even the inhibitory
effects of higher doses of Dex (18, 19), because this appears to be
reversible inhibition (27). It is lung immaturity rather than lung size
per se that appears to present the major clinical problem in preterm
infants with RDS as evidenced by the dramatic improvement in infant
survival with the advent of exogenous surfactant replacement
therapy (7).
The synergistic effect of low-dose BLP with low doses of either EGF or
Dex suggests that all three factors act via distinct receptors to
stimulate the same common final pathway. In contrast to the cloned
seven-transmembrane-spanning, G protein-coupled BLP receptors (9), the
EGF receptor is a protein tyrosine kinase (13), whereas glucocorticoid
receptors are present in the cytoplasm and translocate to the nucleus
after ligand binding (29). It appears that EGF (30) and Dex (26) induce
type II pneumocyte differentiation by stimulating production of
fibroblast-derived factors, which, in turn, trigger type II cell
differentiation. Consistent with the hypothesis that BLP similarly acts
via a fibroblast intermediary, we localized GRPR transcripts primarily
to interstitial fibroblasts in the developing alveoli (Fig. 2). In a
separate study of fetal rat lungs, Brimhall et al. (3) have recently reported that fibroblasts are required for the effect of BLP on type II
cell maturation. It is likely that a similar mechanism of action is
operating in the developing baboon lung.
The present study demonstrates that pulmonary BLP plays an important
role in promoting growth and maturation of the fetal baboon lung,
especially during the canalicular period at midgestation. This is
important because baboons provide the only animal model of RDS that is
clinically and pathologically similar to acute RDS in human infants
followed by BPD (8, 15). Postnatally, BLP appears to act as a
proinflammatory mediator in the hyperoxic baboon model of BPD in which
anti-BLP-blocking monoclonal antibodies greatly diminish the severity
of chronic lung disease (41). In conclusion, these observations
indicate that BLP functions to promote lung embryogenesis during stages
preceding clinical viability but could, in fact, be harmful in
premature infants when sustained postnatally.
 |
ACKNOWLEDGEMENTS |
We thank the National Institutes of Health for support of the R-10
Collaborative Program in Bronchopulmonary Dysplasia headed by Dr.
Jacqueline Coalson, without which this work would not have been
possible. Key R-10 support staff include Vicki Winters and the
pathology staff at University of Texas Health Sciences Center at San
Antonio and the Neonatal Intensive Care Unit and animal production
staffs at the Southwest Foundation for Biomedical Research.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant U10-HL-52638 (to M. E. Sunday) and Resource Grant HL-52636.
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: M. E. Sunday,
Children's Hospital, Dept. of Pathology, 320 Longwood Ave, Boston, MA
02115 (E-mail: sunday{at}a1.tch.harvard.edu).
Received 5 February 1999; accepted in final form 17 May 1999.
 |
REFERENCES |
1.
Benya, R.,
T. Kusui,
T. Pradhan,
J. Battey,
and
R. Jensen.
Expression and characterization of cloned human bombesin receptors.
Mol. Pharmacol.
47:
10-20,
1995[Abstract].
2.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
3.
Brimhall, B. B.,
K. A. Sikorski,
K. J. Haley,
J. S. Torday,
A. Shahsafaei,
and
M. E. Sunday.
Syntaxin 1A is transiently expressed in fetal lung mesenchymal cells: potential developmental roles.
Am. J. Physiol.
277 (Lung Cell. Mol. Physiol. 21):
L401-L411,
1999[Abstract/Free Full Text].
4.
Bunnett, N. W.,
R. Kobayashi,
M. S. Orloff,
J. R. Reeve,
A. J. Turner,
and
J. H. Walsh.
Catabolism of gastrin releasing peptide and substance P by gastric membrane-bound peptidases.
Peptides
6:
277-283,
1985[Medline].
5.
Burton, K.
A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid.
Biochemistry
62:
315-322,
1956.
6.
Carney, D. N.,
A. F. Gazdar,
G. Bepler,
J. G. Guccio,
P. J. Marangos,
T. W. Moody,
M. H. Zweig,
and
J. D. Minna.
Establishment and identification of small cell lung cancer cell lines having classic and variant features.
Cancer Res.
45:
2913-2923,
1985[Abstract].
7.
Clements, J. A.,
and
M. E. Avery.
Lung surfactant and neonatal respiratory distress syndrome.
Am. J. Respir. Crit. Care Med.
157:
S59-S66,
1998[Medline].
8.
Coalson, J. J.,
T. J. Kuehl,
M. B. Escobedo,
J. L. Hilliard,
F. Smith,
K. Meredith,
D. M. Null,
W. Walsh,
D. Johnson,
and
J. L. Robotham.
A baboon model of bronchopulmonary dysplasia. II. Pathologic features.
Exp. Mol. Pathol.
37:
335-350,
1982[Medline].
9.
Corjay, M. H.,
D. J. Dobrzanski,
J. M. Way,
J. Viallet,
H. Shapira,
P. Worland,
E. A. Sausville,
and
J. F. Battey.
Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells.
J. Biol. Chem.
266:
18771-18779,
1991[Abstract/Free Full Text].
10.
Cuttitta, F.,
D. N. Carney,
J. Mulshine,
T. W. Moody,
J. Fedorko,
A. Fischler,
and
J. D. Minna.
Bombesin-like peptides can function as autocrine growth factors in human small cell cancer.
Nature
316:
823-826,
1985[Medline].
11.
Cutz, E.
Cytomorphology and differentiation of airway epithelium in developing human lung.
In: Lung Carcinomas: Current Problems in Tumor Pathology, edited by E. M. McDowell. Edinburgh, UK: Churchill Livingstone, 1987, p. 1-41.
12.
Davis, L. G.,
M. D. Dibner,
and
J. F. Battey.
Basic Methods in Molecular Biology. New York: Elsevier, 1986, p. 1-388.
13.
Di Fiore, P. P.,
O. Segatto,
W. G. Taylor,
S. A. Aaronson,
and
J. H. Pierce.
EGF receptor and erbB-2 tyrosine kinase domains confer cell specificity for mitogenic signaling.
Science
248:
79-83,
1990[Medline].
14.
Erspamer, G. F.,
G. Mazzanti,
G. Farruggia,
T. Nakajima,
and
N. Yanaihara.
Parallel bioassay of litorin and phyllolitorins on smooth muscle preparations.
Peptides
5:
765-768,
1984[Medline].
15.
Escobedo, M. B.,
J. L. Hilliard,
F. Smith,
K. Meredith,
W. Walsh,
D. Johnson,
J. J. Coalson,
T. J. Kuehl,
D. M. Null,
and
J. L. Robotham.
A baboon model of bronchopulmonary dysplasia. I. Clinical features.
Exp. Mol. Pathol.
37:
323-334,
1982[Medline].
16.
Floros, J.,
S. DiAngelo,
M. Koptides,
A. M. Karinch,
P. K. Rogan,
H. Nielsen,
R. G. Spragg,
K. Watterberg,
and
G. Deiter.
Human SP-A locus: allele frequencies and linkage disequilibrium between the two surfactant protein A genes.
Am. J. Respir. Cell Mol. Biol.
15:
489-498,
1996[Abstract].
17.
Goetzman, B.,
L. Read,
C. Plopper,
A. Tarantal,
C. George-Nascimento,
T. Merrit,
J. Whitsett,
and
D. Styne.
Prenatal exposure to epidermal growth factor attenuates respiratory distress syndrome in rhesus infants.
Pediatr. Res.
35:
30-36,
1994[Abstract].
18.
Johnson, J. W. C.,
W. Mitzner,
J. C. Beck,
W. T. London,
D. L. Sly,
P. A. Lee,
V. A. Khouzami,
and
R. L. Cavalieri.
Long-term effects of betamethasone on fetal development.
Am. J. Obstet. Gynecol.
141:
1053-1064,
1981[Medline].
19.
Johnson, J. W. C.,
W. Mitzner,
W. T. London,
A. E. Palmer,
and
R. Scott.
Betamethasone and the rhesus fetus: multisystemic effects.
Am. J. Obstet. Gynecol.
133:
677-684,
1979[Medline].
20.
King, K. A.,
J. Hua,
J. S. Torday,
J. M. Drazen,
S. A. Graham,
M. A. Shipp,
and
M. E. Sunday.
CD10/neutral endopeptidase regulates fetal lung growth and maturation in utero by potentiating endogenous bombesin-like peptides.
J. Clin. Invest.
91:
1969-1973,
1993[Medline].
21.
King, K. A.,
J. S. Torday,
and
M. E. Sunday.
Bombesin and [leu8]-phyllolitorin promote fetal mouse lung branching morphogenesis via a specific receptor-mediated mechanism.
Proc. Natl. Acad. Sci. USA
92:
4357-4361,
1995[Abstract].
22.
Lach, E.,
E. B. Haddad,
and
J. P. Gies.
Contractile effect of bombesin on guinea pig lung in vitro: involvement of gastrin-releasing peptide-preferring receptors.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L80-L86,
1993[Abstract/Free Full Text].
23.
Li, K.,
S. R. Nagalla,
and
E. R. Spindel.
A rhesus monkey model to characterize the role of gastrin-releasing peptide (GRP) in lung development.
J. Clin. Invest.
94:
1605-1615,
1994[Medline].
24.
Lin, J. T.,
D. H. Coy,
S. A. Mantey,
and
R. T. Jensen.
Comparison of the peptide structural requirements for high affinity interaction with bombesin receptors.
Eur. J. Pharmacol.
294:
55-69,
1995[Medline].
25.
Plopper, C. G.,
J. A. St. George,
L. C. Read,
S. J. Nishio,
A. J. Weir,
L. Edwards,
A. F. Tarantal,
K. E. Pinkerton,
T. A. Merritt,
J. A. Whitsett,
C. George-Nascimento,
and
D. Styne.
Acceleration of alveolar type II cell differentiation in fetal rhesus monkey lung by administration of EGF.
Am. J. Physiol.
262 (Lung Cell. Mol. Physiol. 6):
L313-L321,
1992[Abstract/Free Full Text].
26.
Post, M.,
A. Barsoumian,
and
B. T. Smith.
The cellular mechanism of glucocorticoid acceleration of fetal lung maturation: fibroblast-pneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity.
J. Biol. Chem.
261:
2179-2184,
1986[Abstract/Free Full Text].
27.
Salokorpi, T.,
N. Sajaniemi,
H. Hallback,
A. Kari,
H. Rita,
and
L. von Wendt.
Randomized study of the effect of antenatal dexamethasone on growth and development of premature children at the corrected age of 2 years.
Acta Paediatr.
86:
294-298,
1997[Medline].
28.
Schindler, M. B.,
D. J. Bohn,
A. C. Bryan,
E. Cutz,
and
M. Rabinovitch.
Increased respiratory system resistance and bronchial smooth muscle hypertrophy in children with acute postoperative pulmonary hypertension.
Am. J. Respir. Crit. Care Med.
152:
1347-1352,
1995[Abstract].
29.
Schmid, W.,
T. J. Cole,
J. A. Blendy,
and
G. Schutz.
Molecular genetic analysis of glucocorticoid signalling in development.
J. Steroid Biochem. Mol. Biol.
53:
33-35,
1995[Medline].
30.
Sen, N.,
and
M. H. Cake.
Enhancement of disaturated phosphatidylcholine synthesis by epidermal growth factor in cultured fetal lung cells involves a fibroblast-epithelial cell interaction.
Am. J. Respir. Cell Mol. Biol.
5:
337-343,
1991[Medline].
31.
Shipp, M. A.,
G. E. Tarr,
C.-Y. Chen,
S. N. Switzer,
L. B. Hersh,
H. Stein,
M. E. Sunday,
and
E. L. Reinherz.
CD10/NEP hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung.
Proc. Natl. Acad. Sci. USA
88:
10662-10666,
1991[Abstract].
32.
Spindel, E. R.,
W. W. Chin,
J. Price,
L. H. Rees,
G. M. Besser,
and
J. F. Habener.
Cloning and characterization of cDNAs encoding human gastrin-releasing peptide.
Proc. Natl. Acad. Sci. USA
81:
5699-5703,
1984[Abstract].
33.
Spindel, E. R.,
M. E. Sunday,
H. Hofler,
H. J. Wolfe,
J. F. Habener,
and
W. W. Chin.
Transient elevation of mRNAs encoding gastrin-releasing peptide (GRP), a putative pulmonary growth factor, in human fetal lung.
J. Clin. Invest.
80:
1172-1179,
1987[Medline].
34.
Sunday, M. E.
Cell-specific localization of neuropeptide gene expression: gastrin-releasing peptide (GRP; mammalian bombesin).
Methods Neurosci.
5:
123-136,
1991.
35.
Sunday, M. E.
Neuropeptides and lung development.
In: Lung Growth and Development, edited by J. A. McDonald. New York: Dekker, 1997, vol. 100, p. 401-494. (Lung Biol. Health Dis. Ser.)
36.
Sunday, M. E.,
J. Hua,
H. B. Dai,
A. Nusrat,
and
J. S. Torday.
Bombesin increases fetal lung growth and maturation in utero and in organ culture.
Am. J. Respir. Cell Mol. Biol.
3:
199-205,
1990[Medline].
37.
Sunday, M. E.,
J. Hua,
B. Reyes,
H. Masui,
and
J. S. Torday.
Anti-bombesin antibodies modulate fetal mouse lung growth and maturation in utero and in organ cultures.
Anat. Rec.
236:
25-32,
1993[Medline].
38.
Sunday, M. E.,
J. Hua,
J. Torday,
B. Reyes,
and
M. A. Shipp.
CD10/neutral endopeptidase 24.11 in developing human fetal lung: patterns of expression and modulation of peptide-mediated proliferation.
J. Clin. Invest.
90:
2517-2525,
1992[Medline].
39.
Sunday, M. E.,
L. M. Kaplan,
E. Motoyama,
W. W. Chin,
and
E. R. Spindel.
Biology of disease: gastrin-releasing peptide (mammalian bombesin) gene expression in health and disease.
Lab. Invest.
59:
5-24,
1988[Medline].
40.
Sunday, M. E.,
and
C. G. Willett.
Induction and spontaneous regression of intense pulmonary neuroendocrine cell differentiation in a model of preneoplastic lung injury.
Cancer Res.
52, Suppl.:
2677s-2686s,
1992[Abstract].
41.
Sunday, M. E.,
B. A. Yoder,
F. Cuttitta,
K. J. Haley,
and
R. L. Emanuel.
Bombesin-like peptide mediates lung injury in a baboon model of bronchopulmonary dysplasia.
J. Clin. Invest.
102:
584-594,
1998[Abstract/Free Full Text].
42.
Ten Have-Opbroek, A. A. W.
Lung development in the mouse embryo.
Exp. Lung Res.
17:
111-130,
1991[Medline].
43.
Torday, J. S.
Dihydrotestosterone inhibits fibroblast-pneumonocyte factor mediated synthesis of saturated phosphatidylcholine by fetal rat lung cells.
Biochim. Biophys. Acta
835:
23-28,
1985[Medline].
44.
Wada, E.,
J. Battey,
and
S. Wray.
Bombesin receptor gene expression in rat embryos: transient GRPR gene expression in the posterior pituitary.
Mol. Cell. Neurosci.
4:
13-24,
1993.
45.
Wang, D.,
H. Yeger,
and
E. Cutz.
Expression of GRP receptor gene in developing lung.
Am. J. Respir. Cell Mol. Biol.
14:
409-416,
1996[Abstract].
46.
Wharton, J.,
J. M. Polak,
S. R. Bloom,
M. A. Ghatei,
E. Solcia,
M. R. Brown,
and
A. G. E. Pearse.
Bombesin-like immunoreactivity in the lung.
Nature
273:
769-770,
1978[Medline].
47.
Willett, C. G.,
D. I. Smith,
V. Shridhar,
M. H. Wang,
R. L. Emanuel,
K. Patidar,
S. A. Graham,
F. Zhang,
V. Hatch,
D. Sugarbaker,
and
M. E. Sunday.
Differential screening of a human chromosome 3 library identifies hepatocyte growth factor-like/macrophage stimulating protein and its receptor in injured lung: possible implications for neuroendocrine cell survival.
J. Clin. Invest.
99:
2979-2991,
1997[Abstract/Free Full Text].
48.
Zetter, B. R.
Angiogenesis and tumor metastasis.
Annu. Rev. Med.
49:
407-424,
1998[Medline].
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