ARTICLE |
Correspondence to: Laura Guembe, Dept. of Cytology and Histology, University of Navarra, 31080 Pamplona, Spain.
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Summary |
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The enzyme PAM is required for activation of many peptide hormones. In adult mouse lung, immunostaining for PAM was located in Clara cells, which constitute most of the epithelial cells of the mouse bronchial/bronchiolar tree. Immunoreactivity appeared for the first time in the epithelium on gestational Day 16, being slight and mostly restricted to the apical cytoplasm. As the lung developed, the labeling became gradually stronger and extended throughout the cell. Smooth muscle of airways and blood vessels, and some parenchymal cells, probably macrophages, also showed PAM immunoreactivity. Of the two enzymatically active domains of PAM, only PHM and not PAL immunoreactivity was found at all stages studied. The early appearance of PAM in developing mouse lung, as well as its presence in a variety of tissues, probably indicates a complex role of this enzyme in pulmonary development and function. (J Histochem Cytochem 47:623636, 1999)
Key Words: mouse lung, development, amidation, PAM, PHM, PAL, Clara cells, smooth muscle, macrophages
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Introduction |
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Bioactive peptides are generated biosynthetically from larger precursors via a variety of posttranslational modifications (-amidation for optimal binding and biological activity (
The formation of an amidated peptide from its glycine-extended propeptide intermediate requires an enzyme complex denoted collectively as peptidyl-glycine -amidating mono-oxygenase (PAM) (EC 1.14.17.3) (
-hydroxylating mono-oxygenase (PHM) mediates the first step of the PAM pathway and uses molecular oxygen to form the peptidyl
-hydroxyglycine intermediate. Peptidyl
-hydroxyglycine
-amidating lyase (PAL), the second enzyme of the pathway, catalyzes the conversion of the peptidyl
-hydroxyglycine derivative into the
-amidated product. Both enzymes are encoded by the same gene (PAM gene), which has been cloned and appears to be highly conserved among different species (
The amidating enzymes have been localized in endocrine glands (
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Materials and Methods |
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Lungs of 86 Swiss mice at different points in fetal and postnatal development were studied. The mean time of gestation in this species is approximately 19 days. Lungs were obtained on gestational (E) Days 14, 15, 16, 17, 18, and 19 and on postnatal (P) Days 0, 1, 2, and 6, the age at which mouse lung is considered to be mature. The lungs of pregnant females (adult lung, P-ad) were also studied.
The pregnant mice were anesthetized with 12.5% urethane (1 ml/100 g). The abdomen was opened and the fetuses were rapidly removed and chilled in ice. The newborn mice were anesthetized with ice. The chests of fetuses, newborn, and adult mice were opened and lungs were removed, except in the case of the small E-14 fetuses, which were immersed in toto in the fixative. All samples were fixed in Bouin's fixative for 2024 hr and were paraffin-embedded.
Immunocytochemistry
Sections (4 µm thick) were mounted on glass slides. After dewaxing, immunocytochemical staining was performed using the avidinbiotin method (
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The sections were then incubated for 30 min with biotinylated swine anti-rabbit (K353; Dakopatts, Glostrup, Denmark) or rabbit anti-mouse (K354; Dakopatts) immunoglobulins and then with avidinbiotinperoxidase complexes (K355; Dakopatts) for 30 min. After each incubation, sections were rinsed in TBS. The bound antibodies were visualized with 3-3'-diaminobenzidine tetrahydrochloride (D-5637; Sigma, St Louis, MO) in sodium acetate/acetic acid 0.1 M, pH 5.6, containing 2.5% nickel ammonium sulfate, 0.2% ß-D-glucose, 0.04% ammonium chloride, and 0.001% glucose oxidase (
Specificity Controls
Absorption controls were performed when the corresponding antigens were available. Antisera to PHM and CGRP which rendered immunolabeling were preincubated with their respective synthetic antigens at concentrations of 0.110 nmol of peptide per ml of optimally diluted antiserum before application to tissue sections. In both cases, preabsorption of the antisera with the peptide abolished immunoreaction.
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Results |
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In mouse, immunoreactivity for PAM was localized in airway epithelial cells, smooth muscle of airway and blood vessel walls, and cells of pulmonary parenchyma (Table 3). Immunostaining was obtained with some of the antisera raised against PHM enzyme and the hydrophilic cytoplasmic domain (CD), although with variations in tissue localization (Figure 1). No immunoreaction was observed with the antiserum against the PAL domain.
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In adults, most of the epithelial cells of both bronchi (Figure 2) and bronchioles (Figure 3) showed immunoreactivity for PAM, with the proportion of positive epithelial cells higher in distal than in proximal airways.
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The identification of the PAM-immunostained cells was not always evident in paraffin sections. Both Clara and ciliated cells could be recognized in the sections immunostained for PAM on the basis of morphological criteria. Clara cells, clearly identifiable because of their characteristic dome-shaped apex, were intensely stained both in bronchi (Figure 4A) and bronchioles (Figure 4B). On the contrary, when their identification was certain (Figure 4A) the ciliated cells were seen to be negative.
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The identification of neuroendocrine cells required, on the contrary, immunocytochemical techniques using antibodies against regulatory substances (Table 1). Only CGRP-positive cells were found in mouse lung in our study (Figure 5A and Figure 5C). Using serial reversed-face sections, we observed a total lack of co-localization between CGRP, which is generally considered an amidated peptide, and PAM immunoreactivities (Figure 5) with the antisera used (Table 2). Both CGRP-positive isolated endocrine cells and neuroepithelial bodies (Figure 5A and Figure 5C) lacked PAM immunostaining throughout development (Figure 5B) and in adulthood (Figure 5D).
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In developing lungs, PAM immunoreactivity was first detected in airway epithelial cells at E-16 (Figure 6A and Figure 6B). At this gestational age, the immunostaining, although slight, was observed in most bronchial epithelial cells and intrapulmonary epithelial tubules. Immunolabeling was mainly located in the apical region of epithelial cells and showed a granular aspect (Figure 6B). A lesser number of positive granules could also be observed scattered in the rest of the cytoplasm.
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As development continued, the immunolabeling increased and extended towards the base of epithelial cells (Figure 6). At E-18 and E-19 (Figure 6C), some epithelial cells showed the whole cytoplasm immunostained, whereas in others the immunoreactivity was still restricted to the apical region. After birth, almost all the positive epithelial cells showed immunostaining throughout the cytoplasm (Figure 6D). This developmental pattern was similar in the epithelial cells of bronchi and bronchioles.
Although this study focused on epithelial cells, other tissues also exhibited immunostaining for the PAM enzyme.
Most of the smooth muscle cells of the bronchi (Figure 7A and Figure 7B) and bronchioles (Figure 7C) were PAM-immunoreactive. In blood vessels (Figure 7D), variable numbers of positive cells, usually less frequent than in airways, were observed. As in the case of the epithelium, only some of the antisera against the PHM domain and the cytoplasmic tail rendered labeling. However, not all the antisera that stained epithelial cells were positive in smooth muscle (Table 3; Figure 7E).
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In adult lungs, cells in the pulmonary parenchyma were also positive for PAM (Figure 8A and Figure 8B), the immunoreactivity extending throughout the cytoplasm. Neither fetal nor P-0 to P-2 lungs showed positive parenchymal cells. At P-6, some positive parenchymal cells were observed (Figure 8C and Figure 8D), but in lesser numbers and less intensely stained than in adults.
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In the three cell typesepithelium, smooth muscle, and parenchymal cellspreabsorption of the antiserum with its antigen abolished immunolabeling (Figure 9).
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Discussion |
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This study shows the distribution of the amidating enzyme PAM in developing and adult mouse lung. PAM immunoreactivity was localized in diverse cell types: epithelial cells and smooth muscle (both in fetal and postnatal lungs) and pulmonary parenchymal cells, probably macrophages (only in postnatal lungs). Epithelium was the first tissue in which immunostaining was detected, on E-16 fetuses.
In the respiratory tract, PAM immunoreactivity has been reported only in human lung (
Immunoreaction was obtained in the mouse lung with antisera raised against the PHM domain and the hydrophilic cytoplasmic tail but not with the PAL antiserum. These results are, again, only partially coincident with those in human lung, in which, in addition to PHM immunoreactivity, PAL immunostaining has also been detected (
According to immunocytochemical studies on the presence of PAM in mammalian tissues, three patterns of PAM labeling have been found: immunoreactivity to both PHM and PAL, or immunostaining only for PHM or PAL. Because PHM and PAL are derived from the same mRNA precursor, it would be expected to find both enzymes in the same cells, i.e., the first pattern of staining seems more logical. This pattern has been found in several organs, including normal human lung and some lung epithelial tumors (
Considering that mouse and bovine PAL2 sequences are identical, the lack of PAL immunoreactivity in mouse lung is an intriguing result. The lack of PAL immunoreactivity has also been reported in most human lung epithelial tumors (
Our immunocytochemical results indicate that Clara cells of both bronchi and bronchioles of the mouse respiratory tract are immunoreactive for PAM, whereas CGRP-positive endocrine cells and the ciliated cellsat least most of themare negative. In a similar manner, Clara cells of human lung were shown to be positive and GRP endocrine cells were negative. On the contrary, the many ciliated cells present in human airways were immunoreactive for PAM (
Endocrine Cells
The presence of PAM enzymes has been described in endocrine organs producing amidated peptides (
In mouse lung, although CGRP is an amidated peptide, endocrine CGRP-positive cells have been found to be negative for PAM with the antisera used in all stages studied. Because PAM has been demonstrated in endocrine cells producing amidated peptides (
Clara Cells
The presence of amidating enzymes in Clara cells, which constitute most of the epithelial cells of mouse airways, is difficult to explain because the existence of amidated factors in these cells is as yet unknown. Perhaps the presence of PAM in them may be an indicator of the production of amidated peptides, still unknown, by this cell type. A possible explanation for the presence of PAM immunoreactivity in airway epithelial cells is provided by the fact that mammalian epithelial cells produce endothelin (ET) (
Classically, Clara cells have been described as exocrine cells secreting towards the lumen of airways. During mouse lung fetal development, immunoreactivity for PAM appears first in the apical cytoplasmic region of Clara cells. Later, the labeling is observed throughout the cell but remains especially evident in the apical region. Similarly, PAM immunoreactivity in adult human airways has been described in the apical region of epithelial cells, and the possible secretion of amidating enzymes to airway lumen has been suggested (
On the other hand, the presence of PAM immunoreactivity in the base of the Clara cells could be explained as merely a consequence of physical expansion of the increasing amount of PAM, which would be exported later to the apex of the cell. However, we must consider that the existence of basal PAM could also reflect other specific functions taking place in the basal cytoplasm.
Smooth Muscle
PAM immunostaining was clearly observed in smooth muscle cells in neonatal lungs (P-0). The fact that not all the PAM antisera that stained epithelial cells in mouse lung gave immunoreaction in smooth muscle suggests the presence of different isoforms in both cell types. The presence of PAM in smooth muscle cells has been previously reported in hypophysial arteries (
Parenchymal Cells
PAM-immunoreactive cells present in the pulmonary parenchyma of mouse are suggested to be macrophages because they were absent in fetal animals, appearing only in postnatal lungs (P-6). In addition, the localization of PAM in macrophages, although alveolar, has also been reported in human lung (
Development
To our knowledge, this is the first description of the appearance and evolution of PAM immunoreactivity in developing lung. In mouse lung, PAM immunoreactivity in epithelial cells appeared in the fetal period (E-16), rose while development continued, and reached the strongest immunostaining in mature lungs (from P-6 onwards). During development, the early apical immunolabeling of the epithelium, probably in relation to the amidation of secretory products, extended towards the base of the cells. As indicated, PAM in the basal cytoplasm could also be involved in the processing of hypothetical peptides secreted into the basal extracellular space.
It is difficult to date the appearance of PAM immunoreactivity in smooth muscle cells, because in mouse fetuses mesenchymal cells gradually differentiate into smooth muscle cells.
Little is known about the presence of PAM in developing organs. To our knowledge, only rat and sheep pancreas (
The early appearance of PAM in mouse developing lung, as well as its presence in a variety of fetal and adult tissues, probably indicates a complex role of this enzyme in pulmonary function.
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Acknowledgments |
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Supported by the Spanish Ministry of Education and Science (DGCYT project no. PB93-0711) and the University of Navarra (PIUNA), and by a grant from the Departamento de Educación y Cultura del Gobierno de Navarra, Spain (LG).
We would like to thank Dr B.A. Eipper (Johns Hopkins University School of Medicine; Baltimore, MD) for some of the antisera against PAM and for her advice and review of the manuscript, and Prof J.M. Polak (Hammersmith Hospital; London, UK) for the antisera against regulatory substances. We thank Dr M.A. Burrell and Dr L.M. Montuenga for critically reading the manuscript, and I. Ordoqui and A. Urbiola for technical assistance.
Received for publication August 17, 1998; accepted December 8, 1998.
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