Departments of 1 Medicine, 2 Pathology, and 3 Obstetrics and Gynecology, Brigham and Women's Hospital, Boston, Massachusetts 02115; and 4 Department of Pathology, Washington University at St. Louis, St. Louis, Missouri 63110
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Peribronchial smooth muscle constriction causes airway stretch, an important mechanical force in developing lung. Little is known about factors influencing these spontaneously active muscle elements. We measured contractile activity of neurokinin (NK) receptors on fetal intrapulmonary smooth muscle by tracheal perfusion assay (n = 11). Injecting either capsaicin or the NK2 receptor agonist [NLE10]NKA resulted in significant (P < 0.05) bronchoconstriction. A specific NK2 receptor antagonist inhibited constriction caused by endogenous tachykinins released by capsaicin. We then examined NK2 receptor (n = 44) and NKA (n = 23) ontogeny in human lung. NKA immunostaining was identified in peribronchial nerves in samples with gestational age >12 wk. NK2 receptor protein was identified in peribronchial and perivascular smooth muscle. These results indicate that endogenous tachykinins released by the developing lung act via NK2 receptors to cause smooth muscle constriction. We speculate that tachykinins could modulate lung development.
tachykinins; mechanical transduction; immunohistochemistry
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE ONTOGENY OF THE TACHYKININS, an important family of peptides, has not been studied extensively. Specifically, the ontogeny of the neurokinin-2 (NK2) receptor has not been elucidated. We studied the ontogeny of the NK2 receptor. Our interest in this receptor stems from the fact that the most potent native ligand of the NK2 receptor is neurokinin A (NKA), an extremely effective bronchoconstrictor. Activity of this receptor-ligand system would likely be to affect airway tone, which could in turn alter the mechanical forces affecting the developing lung.
Mechanical forces are known to influence pulmonary organogenesis. The severe pulmonary abnormalities associated with clinical syndromes, such as congenital diaphragmatic hernia (6, 21, 49, 52, 54, 59) and alterations in the normal amount of lung fluid (including oligohydramnios and laryngeal atresia; see Refs. 8, 20, 45), are largely caused by disruption of the normal physical forces affecting the developing lung. Animal models using several different experimental approaches, including abrogating or minimizing the pressure differences during fetal breathing movements (3, 17, 26, 30), intrathoracic crowding during gestation as a result of intrathoracic balloon placement (18) or diaphragmatic hernia (5, 47), and changes in transpulmonary pressure as a result of altering the amount of lung fluid (3, 4, 22, 25, 43), have demonstrated that normal pulmonary organogenesis depends on complex interactions among distinct types of physical forces.
The mechanisms transducing the effects of mechanical forces on the developing respiratory system have not yet been completely elucidated. It is possible that components of the lung regulate some of these factors. Peribronchial smooth muscle is ideally located to modulate the physical forces acting on the developing lung.
Peribronchial smooth muscle begins to differentiate from the
primitive mesenchyme at gestational week 6 in humans
(37). Investigations using confocal microscopy have
demonstrated that the peribronchial muscle is covered by a network of
nerves, and neural extensions to the smooth muscle can be visualized by
gestational week 7.5 (55). Immunohistochemical
analyses of fetal rat lung have demonstrated an early muscle protein,
-actin, in the clefts of branching airways (29, 41,
42). This location of immature peribronchial smooth muscle makes
it a plausible candidate to modulate airway stretch during
morphogenesis. Regular spontaneous peribronchial smooth muscle
contractions have been observed in organ cultures from first-trimester
human fetal lung (40). Although the amplitude and
frequency of these contractions varied in response to smooth muscle
agonists and relaxants, including isoproterenol and carbechol, possible
responses to tachykinins were not evaluated (40). The
potent contractile activity of the tachykinins in adult airway smooth
muscle suggested to us that developing airway smooth muscle might
constrict in response to endogenously released tachykinins.
Tachykinins are endogenous bronchoconstrictors with greater potency [that is, producing 50% of their maximal effects (ED50) at lower doses] than methacholine (1). This family of neuropeptides has diverse functions, including bronchoconstriction and vasodilation (9, 46). Two of the most common tachykinins, substance P and NKA, are released by unmyelinated sensory C fibers. These nerves also contain other neuropeptides, such as calcitonin gene-related peptide (CGRP). The neuropeptides released by C fiber nerves act via specific receptors: substance P is the most potent ligand of the NK1 tachykinin receptor, NKA is the most potent ligand of the NK2 receptor, and CGRP has two types of specific receptors (9, 10). Because NKA is a more potent agonist of the NK2 receptor in human bronchial tissue than either substance P or CGRP (9), tachykinin-related effects on airway stretch are likely mediated by the NKA-NK2 receptor system. NKA functions are well characterized in the adult lung and include bronchoconstriction and vasodilation (9, 46). In addition, NKA is a growth factor and chemoattractant for cultured human lung fibroblasts (19). It is therefore plausible that NKA could modulate airway development and/or repair. Although the maturing bronchial smooth muscle of the human neonate can constrict after NKA stimulation (14), little is known regarding possible actions of the tachykinins in the more primitive developing lung.
These observations led us to speculate that NK2 receptor-mediated actions of NKA might contribute to the mechanical forces in developing lung. We hypothesized that the NK2 receptor-NKA receptor-ligand system would be functional in the developing lung. To test this hypothesis, we examined the capacity of the developing lung to constrict to exogenous tachykinin agonists and to endogenous tachykinins released by capsaicin. Next, we investigated the expression of the NK2 receptor gene during gestational periods correlating with the pseudoglandular and canalicular stages of pulmonary organogenesis. Finally, we examined the cellular localization and ontogeny of NKA and NK2 receptor protein in developing human lung. Our investigations indicate that NKA and NK2 receptors are both expressed and functional during these critical periods of pulmonary development.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sample acquisition and specimen processing.
Tissue samples of normal adult and first- and second-trimester human
lung were obtained from discarded surgical specimens. The protocols for
this study were approved in advance by the Brigham and Women's
Hospital Human Research Committee. Each tissue sample was partitioned,
as size allowed, for RNA and immunohistochemical analyses. Samples for
immunohistochemistry (n = 37) were fixed in 4%
paraformaldehyde and then processed into paraffin-embedded tissue
blocks. Samples for RNA analysis (n = 22) were frozen
in liquid N2 and kept at 70°C until the RNA was
extracted. Samples evaluated by the tracheal perfusion assay
(n = 11) were immersed in ice-cold buffer (137 mM NaCl,
1.8 mM CaCl2, 1.05 mM MgCl2, 1 g/l dextrose,
0.6 mM NaHCO3, 0.13 mM NaH2PO4, and
0.896 Na2HPO4, pH 7.4) until placed in the
perfusion chamber. Samples evaluated by both tracheal perfusion and RNA
analysis (n = 9) were frozen in liquid N2
at the completion of the tracheal perfusion assay.
Tracheal perfusion assay.
Eleven lung samples were evaluated by tracheal perfusion assay. Because
of size constraints of the perfusion equipment and limits of
sensitivity of the pressure transducer, only samples of at least 20 wk
gestation were evaluated in this assay. Tracheal perfusion was
performed as previously described (32, 33, 39). Briefly,
the lungs were dissected en bloc and placed in ice-cold low-potassium
perfusion buffer (137 mM NaCl, 1.8 mM CaCl2, 1.05 mM
MgCl2, 1 g/l dextrose, 0.6 mM NaHCO3, 0.13 mM
NaH2PO4, and 0.896 mM
Na2HPO4, pH 7.4). A tracheal catheter was
placed, and the lungs were suspended in a 37°C, 100% humidity,
Plexiglas perfusion chamber. Perfusion buffer (low-potassium perfusion
buffer with 2.68 mM KCl added) was warmed to 45°C and pumped at 5 ml/min through a bubble trap before being cooled to 37°C and
administered to the lungs via the tracheal cannula. Perfusate exited
the fully expanded lungs via small holes placed in the pleura. The
openings in the pleura served to remove contributions of visceral
pleural pressure to the airway opening pressure (Pao). The "back
pressure" resulting from continuous-flow tracheal perfusion
represents Pao and was recorded from a side tap at the tracheal cannula
with a pressure transducer (P23Db; Statham Instruments, Oxnard, CA). Prior investigations demonstrated that, in isolated lung preparations with continuous flow, changes in Pao directly reflect changes in the
contractile state of the lung (32, 33, 39). The lungs were
allowed to equilibrate in the perfusion chamber for 15 min. After this,
contractile agonists except [NLE10]NKA (NLE-10) were
diluted in 100-µl volumes and injected in the tracheal cannula (Table
1). The diluent for all agonists except NLE-10 was perfusion buffer. The NLE-10 solution was prepared by
dissolving 1 mg of NLE-10 in 300 µl of DMSO (Sigma, St. Louis, MO)
and then diluting this in 700 µl of perfusion buffer. NLE-10 solution
(74.8 µl; 10 µmol) was injected in the tracheal cannula. Tracheal
injection of either diluent did not affect Pao. All neuropeptides were
obtained from Peninsula Laboratories (Belmont, CA). Because of the
limited number of samples available for the tracheal perfusion studies,
doses for the peptide agonists and SR-48968 were chosen from published
studies examining human and animal model airway responses. Doses used
in the tracheal perfusion assays were those that caused
bronchoconstriction in adult guinea pig samples studied with tracheal
perfusion assay (31, 32) and those demonstrated to be
effective bronchoconstricting doses in other models (13, 48,
63). At the completion of the assays, the intact capacity of the
pulmonary smooth muscle to respond to agonists was demonstrated by
administering 6.1 mg (0.025 mol) of the nonspecific smooth muscle
agonist BaCl2.
|
RNA extraction and analysis. Total RNA was prepared from frozen tissue using the Stratagene RNA isolation kit (Stratagene, La Jolla, CA). RNA integrity was evaluated by examination of 18S and 28S RNA bands after electrophoresis using ethidium bromide-stained 1.5% agarose formaldehyde gels.
RT preparation and PCR analysis. cDNA was prepared using RT from 22 samples with gestational ages between 10 and 23 wk. Each RT reaction consisted of 1 µg of total RNA, Moloney murine leukemia virus RT (2 units; Bethesda Research Laboratories, Gaithersburg, MD), RNasin (5 units; Sigma), and random hexamers [1 unit, 5'-pd(N)6; Pharmacia LKB Biotechnology, Piscataway, NJ] in a total volume of 10 µl of 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, and 3 mM MgCl2 for 60 min at 42°C. Each PCR contained 3 µl of the RT solution. Negative controls consisted of substituting an equal volume of diethyl pyrocarbonate-treated water for the volume of RNA in the corresponding reaction.
Synthetic oligodeoxynucleotides were purchased from Ransom Hill Bioscience (Ramona, CA). All primer pairs yielded products that spanned at least one intron to permit distinction between cDNA and any contaminating genomic DNA. Amplification of primers forAnalysis of PCR products.
The PCR products obtained from amplifying the fetal lung cDNA samples
using the NK2 receptor primers were subjected to
restriction enzyme digest analysis. AvaII and
HaeIII digests were performed according to the
manufacturer's instructions (New England BioLabs, Beverly, MA).
Restriction enzyme digest products were analyzed on 1.5%
agarose gels to determine their size and then were transferred to nylon
membranes and hybridized to an end-labeled cDNA probe specific for
adult NK2 receptor, as described below. This probe was
obtained from amplification of normal adult lung cDNA using the
NK2 receptor primers, which resulted in the expected 689-bp product. This 689-bp product, corresponding to the region of interest in the NK2 receptor, was cloned using a TA cloning kit
(Invitrogen, Carlsbad, CA), and its sequence was confirmed by direct
cycle sequencing. The 689-bp product was then labeled with -ATP
using a Boehringer Mannheim kit (Boehringer Mannheim). The probe (100 µl) was incubated with the Southern blot at 65°C for 2 h, and then the blot was washed first in 1× SSC (150 mM NaCl and 15 mM sodium
citrate) with 0.1% SDS (Fisher Chemicals, Springfield, NJ) for 15 min
at 65°C followed by 1× SSC at room temperature for 10 min. After the
stringency washes, the blot was placed on film (Kodak, Rochester, NY)
overnight at
80°C for autogradiography.
Immunohistochemical analysis.
Immunohistochemical analyses were performed in 37 samples with
gestational ages between 10 and 23 wk using a modified avidin-biotin complex (ABC) technique (23). Rabbit polyclonal antibodies
included affinity-purified anti-NKA used at 1:200 dilution (Accurate
Chemical and Scientific, Westbury, NY), antisera to the neuronal and
neuroendocrine cell marker protein gene product 9.5 (PGP9.5) used at
1:1,000 (Ultraclone, Isle of Wight, UK), antisera to the Clara cell
marker CC10 used at 1:2,500 (generous gift from Dr. Gurmukh Singh,
University of Pittsburgh, Pittsburgh, PA), and antisera to the
epithelial cell marker cytokeratin used at 1:300 (DAKO, Carpinteria,
CA). Murine monoclonal IgG1 antibodies included anti-NK2
receptor (supplied by Krause) used at 1:50, the muscle marker
anti-desmin used at 1:100 (DAKO), the neuroendocrine cell marker
anti-chromogranin A used at 1:500 (Boehringer Mannheim), and the muscle
marker anti--actin (clone HHF35) used at 1:30 (Enzo Diagnostics,
Farmingdale, NY) dilution. Negative controls included substituting the
primary antibody with the irrelevant murine IgG1 MOPC-21 (Sigma).
Preabsorbance of the diluted primary antibody against 10-50 µg
of specific peptide overnight at 4°C was used as an additional
negative control for the NKA and NK2 receptor antibodies.
Morphometry. The immunostaining results were evaluated by an experienced reader (K. J. Haley) using a semiquantitative analysis. Cell types were identified by colocalizing immunostaining for the markers described above in serial slides having an average thickness of 3-5 µm. The results of this analysis were confirmed by a Brigham and Women's Hospital staff pathologist (M. E. Sunday).
Statistics. The results of the tracheal perfusion assays are reported as means ± SE. Data were tested for normalcy, and one-way ANOVA was used to evaluate the differences in the change in the Pao. Differences were regarded as statistically significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tracheal perfusion assay.
We found that the NK2 receptor agonist NLE-10 was a highly
effective contractile stimulus in our samples of midtrimester human lung. Indeed, 100 nmol of NLE-10 were more effective than either the NK1 receptor agonist
[Sar9,Met(O2)11]- substance P
(Sar-9) or 10 µmol of methacholine. Comparison of the
contractile activity of the neurokinin receptors with the cholinergic
receptors in human fetal lung was accomplished in this system by
evaluating the changes in Pao after smooth muscle agonists
were administered via tracheal perfusion. The use of changes in
Pao to indicate the contractile state of the lung has been validated in
prior investigations (32, 33, 39). The agonists used in
the comparison studies (Fig. 1) included
perfusion buffer, methacholine, the NK1 receptor agonist
Sar-9 (13, 63), the NK2 receptor agonist
NLE-10 (48), the diluent for NLE-10, and
BaCl2. We found that NLE-10 caused the greatest amount of bronchoconstriction in the samples with Pao after buffer
(n = 10; 0.05 ± 0.158 cmH2O) and Pao
after NLE-10 (n = 8; 4.5 ± 1.79 cmH2O; P < 0.05). All samples demonstrated
significantly increased Pao after BaCl2 (data not shown).
|
|
NKA.
Immunostaining in 23 lung samples with gestational ages ranging from 10 to 23 wk identified peribronchial immunopositive cells in all but one
sample. The sole negative sample was of 12 wk gestational age. However,
three additional samples with gestational age 11-12 wk
demonstrated immunopositive cells in the airway peribronchial nerves.
Representative immunostaining for NKA in lung with gestational age
19-22 wk is shown in Fig. 3,
A and C. The immunostaining colocalized with the
neural marker PGP9.5, consistent with peribronchial nerve immunopositivity (Fig. 3, B and D). The
immunostaining was predominantly identified in the airway-associated
nerves of primitive airways lined with cuboidal epithelium in samples
with <18 wk gestation (n = 11); in older samples, NKA
was identified in the peribronchial/peribronchiolar nerves of airways
lined with either columnar or cuboidal epithelium (n = 12). The profusion of immunostaining increased with gestational age.
|
NK2 receptor.
RT-PCR analysis was used to identify the mRNA for the NK2
receptor. A single PCR product of the size expected for the
NK2 receptor was detected in 19 of 22 samples. Restriction
digest analysis with HaeIII and AvaII was used to
evaluate the identity of these PCR products. The uncut PCR product size
obtained from fetal lung was identical to that obtained from adult
lung, 689 bp, and contained restriction sites for both
HaeIII and AvaII. The fragments resulting from
restriction enzyme digest were of the sizes expected for the
NK2 receptor (231 and 458 bp for AvaII and 524 and 165 bp for HaeIII). The uncut PCR products and the restriction digest fragments bound the radiolabeled NK2
receptor probe (Fig. 4). Expression of
mRNA for actin was detected in all samples (Fig.
5, A and B). In
specimens that had not been used in the tracheal perfusion assay,
NK2 receptor mRNA was identified in 19 of 22 samples from
gestational ages 10 to 22 wk (Fig. 5A). All of the samples
without detectable NK2 receptor mRNA were between 16 and 18 wk gestation. The pattern of NK2 receptor mRNA was the same
with and without normalization for actin mRNA expression. In separate
experiments, nine of the samples used in the tracheal perfusion assay
were also evaluated for the presence of mRNA for NK2
receptor, and all nine demonstrated abundant expression of mRNA for the
NK2 receptor (Fig. 5B). Because the samples used in the tracheal perfusion assay were evaluated separately from the
other samples, the densitometry is not directly comparable between
these experiments. The samples used in the tracheal perfusion assays
were at least 18 wk gestational age and demonstrated abundant NK2 receptor mRNA.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we report that the NKA-NK2 receptor-ligand system is present during pulmonary organogenesis and transduces pulmonary constriction in tracheally perfused lungs more effectively than the cholinergic system in developing lung. We found that the mRNA and protein for the NK2 receptor and the protein for its most potent native ligand, NKA, are expressed during the pseudoglandular and canalicular stages of lung development. NK2 receptor protein expression was demonstrated throughout gestational ages 10-23 wk. The protein for NKA was abundant in all samples of >12 wk gestation, and samples of at least 22 wk gestational age demonstrated immunopositive cells in both the peribronchial and perivascular nerves. This is similar to the distribution reported by other investigators in human adult lung (9, 46, 53) where NKA has been identified in C fiber nerves (9, 46). The tracheal perfusion assay demonstrated greater contractile responses to the NK2 receptor agonist than the NK1 receptor agonist. In addition, a greater contractile response was observed after 100 nmol of the NK2 receptor agonist than after 10 µmol of methacholine. The potent contractile effects of the NK2 agonist led us to examine the NKA-NK2 receptor system.
NK2 receptor protein, the preferred receptor for NKA, was identified in all samples of at least 10 wk gestational age and was located predominantly in the peribronchial and perivascular smooth muscle. The localization to airway smooth muscle is similar to that reported for adult human lung (7, 38). In our samples of developing lung, two unanticipated cell types were also positive for NK2 receptor protein. In samples of at least 18 wk gestation, NK2 receptor protein localized to rare (<5% in all samples) airway epithelial cells in cartilaginous airways. In addition, isolated loose mesenchymal cells were positive for NK2 receptor, with this staining being most prominent in the samples of <16 wk gestational age. The mesenchymal and epithelial NK2 receptor immunostaining suggests that mediators acting via the NK2 receptor, such as NKA, might contribute to the growth and/or maturation of these cell types. NKA has been demonstrated to be a growth factor and chemoattractant for cultured human bronchial epithelial cells (28) and human pulmonary fibroblasts (19). Our findings suggest that NKA might have similar roles in vivo.
Tracheal perfusion assays were used to define the functional capacity of the tachykinin receptors present in our samples. Because of the limited number of samples available for study, we chose to use substantial doses of agonists that had been previously demonstrated to be effective in causing bronchoconstriction in the tracheal perfusion model (31-33). In our studies, tracheal perfusion assay of capsaicin-challenged lungs demonstrated that the midgestational (i.e., canalicular stage) developing lung can release endogenous neuropeptides that activate neurokinin receptors on smooth muscle to cause bronchoconstriction. Furthermore, pretreatment with a specific NK2 receptor antagonist prevented the response to capsaicin, demonstrating that the effects of endogenous tachykinins in the immature lung are mediated predominantly through the NK2 receptor. The endogenous ligand that most closely matches the mediator identified by the tracheal perfusion studies is NKA. Thus the tracheal perfusion data imply that the presence of the NKA-NK2 receptor-ligand system is functional during lung development. The presence of both the NK2 receptor and NKA immunostaining as early as 11 wk gestation suggests that this system may be active even before 20 wk.
The greater potency (a lower ED50) of NKA compared with methacholine for causing bronchoconstriction in intact lungs is similar to that previously observed in isolated human neonatal bronchi (14). In isolated bronchial rings from neonates (age 1-60 days postnatal), NKA demonstrated the lowest ED50 compared with carbachol, histamine, or KCl (14). In contrast, a greater maximal degree of constriction was observed from complete carbachol dose-response curves compared with partial dose-response curves for NKA (14). Our data indicate that canalicular stage peribronchial smooth muscle in the intact lung is more responsive to tachykinin stimulation. The greater potency (lower ED50) of NLE-10 compared with cholinergic stimulation observed in the present study is similar to that observed in adults (1).
There are at least two plausible mechanisms by which the NKA-NK2 receptor system could contribute to lung development. The first is by modulating the mechanical forces affecting the airway. Stimulation of the NK2 receptor via endogenous tachykinins results in peribronchial smooth muscle constriction, which alters airway stretch. Therefore, it is feasible that the NKA-NK2 receptor system could modify the mechanical forces impacting on the immature lung by altering airway stretch. The second mechanism would be by direct proliferative and/or maturational effects of NKA on the cells of the developing airway wall.
Several investigators have examined the effects of periodic stretch on pulmonary cells (11, 35, 36, 50, 51, 57, 61, 62). These effects include stimulating proliferation (11, 35), causing the release of growth factors capable of acting in a paracrine fashion on surrounding cells (36, 51, 61), enhancing responses to growth factors (58) and mediators such as parathyroid hormone-related peptide (57), altering the secretion of components of the extracellular matrix such as proteoglycans (61), and increasing the secretion of surfactant apoproteins (50). The effects of stretch depend on the maturational stage of the exposed cells; fetal rabbit type II epithelial cells demonstrated increased [3H]choline uptake after stretch, but these effects were decreased by exposure to fibroblast-conditioned medium, which promotes differentiation (51). Thus stretch has been demonstrated to be a mechanical force that modulates the lung through several mechanisms. Therefore, factors that alter stretch have the potential to affect lung development.
In addition to effects secondary to changes in airway stretch, the NKA-NK2 receptor system could have direct effects on the developing lung. NKA has been demonstrated to be a growth factor and chemoattractant for human pulmonary fibroblasts (19), human bronchial epithelial cells (28), and guinea pig tracheal epithelial cells (28, 60). NKA also induces rat aortic smooth muscle cell proliferation in culture (24, 44). Our present observation of the location of the NK2 receptor in the epithelium and smooth muscle of human fetal lung suggests that such in vitro effects may also be present in vivo.
Our findings extend prior investigations regarding potential roles of the tachykinin receptors during lung development. Adult NK1 receptor knockout mice have unremarkable pulmonary histology at baseline but have diminished inflammatory responses during immune complex-mediated alveolitis (12). These findings suggest that the NK1 receptor itself is not absolutely required for development of normal pulmonary morphology. These findings are consistent with our findings of a lower degree of contractile effect of an NK1 receptor agonist in tracheally perfused lung. The greater potency of the NK2 receptor suggests that it may be relevant to pulmonary development. The importance of the NK2 receptor for murine lung growth and/or maturation is not known. Many critical physiological processes employ redundant pathways that allow compensation in the absence of any one component. Although it is possible that the NKA-NK2 receptor system may have important roles in lung development, it is also possible that the absence of the NKA-NK2 receptor system could be compensated for by other factors. It is clear that endogenously produced tachykinins and the neurokinin receptors are present and functional in immature lung. These mediators are the most potent known components of a system producing mechanical forces that are crucial to normal lung development.
In conclusion, this investigation describes the ontogeny of the NK2 receptor and its most potent native ligand, NKA, in human lung. We have demonstrated that both NKA and NK2 receptors are present during critical phases of pulmonary organogenesis and that the NK2 receptor transduces peribronchial smooth muscle constriction after at least 20 wk gestation. This study extends the results of other investigators by finding that the NK2 receptor is the primary tachykinin receptor involved in peribronchial smooth muscle constriction in developing human lung. Because the tachykinins are present and their receptors are functional during pulmonary organogenesis, they may modulate lung development and/or maturation.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: C. M. Lilly, Respiratory and Critical Care Medicine, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.
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.
Received 29 June 1999; accepted in final form 17 January 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Advenier, C,
Naline E,
Drapeau G,
and
Regoli D.
Relative potencies of neurokinins in guinea pig trachea and human bronchus.
Eur J Pharmacol
139:
133-137,
1987[ISI][Medline].
2.
Advenier, C,
Rouissi N,
Nguyen QT,
Emonds-Alt X,
Breliere JC,
Neliat G,
Naline E,
and
Regoli D.
Neurokinin A (NK2) receptor revisited with SR 48968, a potent non-peptide antagonist.
Biochem Biophys Res Commun
184:
1418-1424,
1992[ISI][Medline].
3.
Adzick, NS,
Harrison MR,
Glick PL,
Villa RL,
and
Finkbeiner W.
Experimental pulmonary hypoplasia and oligohydramnios: relative contributions of lung fluid and fetal breathing movements.
J Pediatr Surg
19:
658-665,
1984[ISI][Medline].
4.
Alcorn, D,
Adamson TM,
Lambert TF,
Maloney JE,
Ritchie BC,
and
Robinson PM.
Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung.
J Anat
123:
649-660,
1977[ISI][Medline].
5.
Alfonso, LF,
Vilanova J,
Aldazabal P,
Lopez de Torre B,
and
Tovar JA.
Lung growth and maturation in the rat model of experimentally induced congenital diaphragmatic hernia.
Eur J Pediatr Surg
3:
6-11,
1993[ISI][Medline].
6.
Areechon, W,
and
Eid L.
Hypoplasia of lung with congenital diaphragmatic hernia.
Br Med J
5325:
230-233,
1963.
7.
Bai, TR,
Zhou D,
Weir T,
Walker B,
Hegele R,
Hayashi S,
McKay K,
Bondy GP,
and
Fong T.
Substance P (NK1)- and neurokinin A (NK2)-receptor gene expression in inflammatory airway diseases.
Am J Physiol Lung Cell Mol Physiol
269:
L309-L317,
1995
8.
Bain, AD,
and
Scott JS.
Renal agenesis and severe urinary tract dysplasia: a review of 50 cases, with particular reference to the associated anomalies.
Br Med J
5176:
841-846,
1960.
9.
Barnes, PJ,
Baraniuk JN,
and
Belvisi MG.
Neuropeptides in the respiratory tract. Part I.
Am Rev Respir Dis
144:
1187-1198,
1991[ISI][Medline].
10.
Barnes, PJ,
Baraniuk JN,
and
Belvisi MG.
Neuropeptides in the respiratory tract. Part II.
Am Rev Respir Dis
144:
1391-1399,
1991[ISI][Medline].
11.
Bishop, JE,
Mitchell JJ,
Absher PM,
Baldor L,
Geller HA,
Woodcock-Mitchell J,
Hamblin MJ,
Vacek P,
and
Low RB.
Cyclic mechanical deformation stimulates human lung fibroblast proliferation and autocrine growth factor activity.
Am J Respir Cell Mol Biol
9:
126-133,
1993[ISI][Medline].
12.
Bozic, CR,
Lu B,
Hopken UE,
Gerard C,
and
Gerard NP.
Neurogenic amplification of immune complex inflammation.
Science
273:
1722-1725,
1996
13.
Drapeau, G,
D'Orleans-Juste P,
Dion S,
Rhaleb N-E,
Rouissi N-E,
and
Regoli D.
Selective agonists for substance P and neurokinin receptors.
Neuropeptides
10:
43-54,
1987[ISI][Medline].
14.
Fayon, M,
Ben-Jebria A,
Elleau C,
Carles D,
Demarquez JL,
Savineau JP,
and
Marthan R.
Human airway smooth muscle responsiveness in neonatal lung specimens.
Am J Physiol Lung Cell Mol Physiol
267:
L180-L186,
1994
15.
Gonzalo, JA,
Jia GQ,
Aguirre V,
Friend D,
Coyle AJ,
Jenkins NA,
Lin GS,
Katz H,
Lichtman A,
Copeland N,
Kopf M,
and
Gutierrez-Ramos JC.
Mouse eotaxin expression parallels eosinophil accumulation during lung allergic inflammation but it is not restricted to a Th2-type response.
Immunity
4:
1-14,
1996[ISI][Medline].
16.
Haley, KJ,
Patidar K,
Zhang F,
Emanuel RL,
and
Sunday ME.
Tumor necrosis factor induces a partial neuroendocrine cell phenotype in undifferentiated small cell lung carcinoma cell lines.
Am J Physiol Lung Cell Mol Physiol
275:
L311-L321,
1998
17.
Harding, R,
Hooper SB,
and
Han VK.
Abolition of fetal breathing movements by spinal cord transection leads to reductions in fetal lung liquid volume, lung growth, and IGF-II gene expression.
Pediatr Res
34:
148-153,
1993[Abstract].
18.
Harrison, MR,
Jester JA,
and
Ross NA.
Correction of congenital diaphragmatic hernia in utero. I. The model: intrathoracic balloon produces fatal pulmonary hypoplasia.
Surgery
88:
174-182,
1980[ISI][Medline].
19.
Harrison, NK,
Dawes KE,
Kwon OJ,
Barnes PJ,
Laurent GJ,
and
Chung KF.
Effects of neuropeptides on human lung fibroblast proliferation and chemotaxis.
Am J Physiol Lung Cell Mol Physiol
268:
L278-L283,
1995
20.
Hedrick, MH,
Ferro MM,
Filly RA,
Flake AW,
Harrison MR,
and
Adzick NS.
Congenital high airway obstruction syndrome (CHAOS): a potential for perinatal intervention.
J Pediatr Surg
29:
271-274,
1994[ISI][Medline].
21.
Hermann, RE,
and
Barber DH.
Congenital diaphragmatic hernia in the child beyond infancy.
Clevel Clin Q
30:
73-80,
1963.
22.
Higuchi, M,
Kato T,
Yoshino H,
Matsuda K,
Gotoh K,
Hirano H,
Koyama K,
and
Maki M.
The influence of experimentally produced oligohydramnios on lung growth.
J Dev Physiol
16:
223-227,
1991[Medline].
23.
Hsu, S-M,
Raine L,
and
Fanger H.
Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures.
J Histochem Cytochem
29:
577-580,
1981[Abstract].
24.
Hultgardh-Nilsson, A,
Larsson SH,
Jin P,
Sejersen T,
and
Ringertz NR.
Neurokinin A induces expression of the c-fos, c-jun, and c-myc genes in rat smooth muscle cells.
Eur J Biochem
194:
527-532,
1990[Abstract].
25.
Jakubowska, AE,
Billings K,
Johns DP,
Hooper SB,
and
Harding R.
Respiratory function in lambs after prolonged oligohydramnios during late gestation.
Pediatr Res
34:
611-617,
1993[Abstract].
26.
Joe, P,
Wallen LD,
Chapin CJ,
Lee CH,
Allen L,
Han VK,
Dobbs LG,
Hawgood S,
and
Kitterman JA.
Effects of mechanical factors on growth and maturation of the lung in fetal sheep.
Am J Physiol Lung Cell Mol Physiol
272:
L95-L105,
1997
27.
Kamikawa, Y,
and
Shimo Y.
SR 48968, a novel non-peptide tachykinin NK-2-receptor antagonist, selectively inhibits the non-cholinergically mediated neurogenic contraction of guinea-pig isolated bronchial muscle.
J Pharm Pharmacol
45:
1037-1041,
1993[ISI][Medline].
28.
Kim, JS,
Rabe KF,
Magnussen H,
Green JM,
and
White SR.
Migration and proliferation of guinea pig and human airway epithelial cells in response to tachykinins.
Am J Physiol Lung Cell Mol Physiol
269:
L119-L126,
1995
29.
Leslie, KO,
Mitchell JJ,
Woodcock-Mitchell JL,
and
Low RB.
Alpha smooth muscle actin expression in developing and adult human lung.
Differentiation
44:
143-149,
1990[ISI][Medline].
30.
Liggins, GC,
Vilos GA,
Campos GA,
Kitterman JA,
and
Lee CH.
The effect of spinal cord transection on lung development in fetal sheep.
J Dev Physiol
3:
267-274,
1981[Medline].
31.
Lilly, CM,
Besson G,
Israel E,
Rodger IW,
and
Drazen JM.
Capsaicin-induced airway obstruction in tracheally perfused guinea pig lungs.
Am J Respir Crit Care Med
149:
1175-1179,
1994[Abstract].
32.
Lilly, CM,
Hall AE,
Rodger IW,
Kobzik L,
Haley KJ,
and
Drazen JM.
Substance P-induced histamine release in tracheally perfused guinea pig lungs.
J Appl Physiol
78:
1234-1241,
1995
33.
Lilly, CM,
Martins MA,
and
Drazen JM.
Peptidase modulation of vasoactive intestinal peptide pulmonary relaxation in tracheal superfused guinea pig lungs.
J Clin Invest
91:
235-243,
1993[ISI][Medline].
34.
Lilly, CM,
Nakamura H,
Kesselman H,
Nagler-Anderson C,
Asano K,
Garcia-Zepeda EA,
Rothenberg ME,
Drazen JM,
and
Luster AD.
Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids.
J Clin Invest
99:
1767-1773,
1997
35.
Liu, M,
Skinner SJ,
Xu J,
Han RN,
Tanswell AK,
and
Post M.
Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch.
Am J Physiol Lung Cell Mol Physiol
263:
L376-L383,
1992
36.
Liu, M,
Xu J,
Tanswell AK,
and
Post M.
Stretch-induced growth-promoting activities stimulate fetal rat lung epithelial cell proliferation.
Exp Lung Res
19:
505-517,
1993[ISI][Medline].
37.
Loosli, CG,
and
Hung KS.
Development of pulmonary innervation.
In: Development of the Lung, edited by Lenfant C,
and Hodson WA.. New York: Dekker, 1977, p. 269-306.
38.
Mapp, CE,
Miotto D,
Saetta M,
Krause JE,
Boyd N,
Braccioni F,
Cunco P,
Ciaccia A,
Geppetti P,
and
Fabbri LM.
Distribution of neurokinin 1 and 2 (NK-1, NK-2) receptors is not changed in the central airways of smokers with or without chronic airflow limitation (Abstract).
Am J Respir Crit Care Med
159:
A808,
1999[ISI].
39.
Martins, MA,
Shore SA,
Gerard NP,
Gerard C,
and
Drazen JM.
Peptidase modulation of the pulmonary effects of tachykinins in tracheal superfused guinea pig lungs.
J Clin Invest
85:
170-176,
1990[ISI][Medline].
40.
McCray, PB, Jr.
Spontaneous contractility of human fetal airway smooth muscle.
Am J Respir Cell Mol Biol
8:
573-580,
1993[ISI][Medline].
41.
McHugh, KM.
Molecular analysis of smooth muscle development in the mouse.
Dev Dyn
204:
278-290,
1995[ISI][Medline].
42.
Mitchell, JJ,
Reynolds SE,
Leslie KO,
Low RB,
and
Woodcock-Mitchell J.
Smooth muscle cell markers in developing rat lung.
Am J Respir Cell Mol Biol
3:
515-523,
1990[ISI][Medline].
43.
Nakayama, DK,
Glick PL,
Harrison MR,
Villa RL,
and
Noall R.
Experimental pulmonary hypoplasia due to oligohydramnios and its reversal by relieving thoracic compression.
J Pediatr Surg
18:
347-353,
1983[ISI][Medline].
44.
Nilsson, J,
von Euler AM,
and
Dalsgaard CJ.
Stimulation of connective tissue cell growth by substance P and substance K.
Nature
315:
61-63,
1985[ISI][Medline].
45.
Perlman, M,
Williams J,
and
Hirsch M.
Neonatal pulmonary hypoplasia after prolonged leakage of amniotic fluid.
Arch Dis Child
51:
349-353,
1976[Abstract].
46.
Piedimonte, G.
Tachykinin peptides, receptors, and peptidases in airway disease.
Exp Lung Res
21:
809-834,
1995[ISI][Medline].
47.
Pringle, KC,
Turner JW,
Schofield JC,
and
Soper RT.
Creation and repair of diaphragmatic hernia in the fetal lamb: lung development and morphology.
J Pediatr Surg
19:
131-140,
1984[ISI][Medline].
48.
Regoli, D,
Rhaleb NE,
Dion S,
Tousignant C,
Rouissi N,
Jukic D,
and
Drapeau G.
Neurokinin A. A pharmacological study.
Pharmacol Res
22:
1-14,
1990[ISI][Medline].
49.
Salepcioglu, A,
Barlas O,
and
Ergun R.
The congenital diaphragmatic hernia and its surgical treatment. A review of ten cases.
Am J Gastroenterol
41:
19-27,
1964[ISI].
50.
Sanchez-Esteban, J,
Tsai SW,
Sang J,
Qin J,
Torday JS,
and
Rubin LP.
Effects of mechanical forces on lung-specific gene expression.
Am J Med Sci
316:
200-204,
1998[ISI][Medline].
51.
Scott, JE,
Yang SY,
Stanik E,
and
Anderson JE.
Influence of strain on [3H]thymidine incorporation, surfactant-related phospholipid synthesis, and cAMP levels in fetal type II alveolar cells.
Am J Respir Cell Mol Biol
8:
258-265,
1993[ISI][Medline].
52.
Shane, RA.
Congenital diaphragmatic herniae.
J Okla State Med Assoc
58:
209-213,
1965.
53.
Sheldrick, RL,
Rabe KF,
Fischer A,
Magnussen H,
and
Coleman RA.
Further evidence that tachykinin-induced contraction of human isolated bronchus is mediated only by NK2-receptors.
Neuropeptides
29:
281-292,
1995[ISI][Medline].
54.
Snyder, WH, Jr,
and
Greaney EM, Jr.
Congenital diaphragmatic hernia; 77 consecutitve cases.
Surgery
57:
576-588,
1965[ISI].
55.
Sparrow, MP,
Weichselbaum M,
and
McCray PB, Jr.
Development of the innervation and airway smooth muscle in human fetal lung.
Am J Respir Cell Mol Biol
20:
550-560,
1999
56.
Strigas, J,
and
Burcher E.
Autoradiographic localization of tachykinin NK2 and NK1 receptors in the guinea-pig lung, using selective radioligands.
Eur J Pharmacol
311:
177-186,
1996[ISI][Medline].
57.
Torday, JS,
Sanchez-Esteban J,
and
Rubin LP.
Paracrine mediators of mechanotransduction in lung development.
Am J Med Sci
316:
205-208,
1998[ISI][Medline].
58.
Torday, JS,
Sanchez-Esteban J,
and
Rubin LP.
The role of keratinocyte growth factor in stretch-activated fetal lung development (Abstract).
Pediatr Res
43:
300A,
1998.
59.
Tuqan, NA.
Some observations on congenital diaphragmatic hernias.
J Pathol Bacteriol
89:
370-372,
1965[ISI].
60.
White, SR,
Garland A,
Gitter B,
Rodger I,
Alger LE,
Necheles J,
Nawrocki AR,
and
Solway J.
Proliferation of guinea pig tracheal epithelial cells in coculture with rat dorsal root ganglion neural cells.
Am J Physiol Lung Cell Mol Physiol
268:
L957-L965,
1995
61.
Xu, J,
Liu M,
Liu J,
Caniggia I,
and
Post M.
Mechanical strain induces constitutive and regulated secretion of glycosaminoglycans and proteoglycans in fetal lung cells.
J Cell Sci
109:
1605-1613,
1996
62.
Xu, J,
Liu M,
Tanswell AK,
and
Post M.
Mesenchymal determination of mechanical strain-induced fetal lung cell proliferation.
Am J Physiol Lung Cell Mol Physiol
275:
L545-L550,
1998
63.
Yiamouyiannis, CA,
Stengel PW,
Cockerham SL,
and
Silbaugh SA.
Pulmonary actions of the neurokinin1-specific agonist [Sar9, Met(O2)11]-substance P.
Neuropeptides
28:
35-42,
1995[ISI][Medline].
64.
Zeng, XP,
Lavielle S,
and
Burcher E.
Evidence for tachykinin NK-2 receptors in guinea-pig airways from binding and functional studies, using [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10).
Neuropeptides
26:
1-9,
1994[ISI][Medline].