Journal of Histochemistry and Cytochemistry, Vol. 47, 623-636, May 1999, Copyright © 1999, The Histochemical Society, Inc.


ARTICLE

Immunocytochemical Mapping of the Amidating Enzyme PAM in the Developing and Adult Mouse Lung

Laura Guembea, Ana C. Villaroa, and Anthony M. Trestonb
a Department of Cytology and Histology, University of Navarra, Pamplona, Spain
b Biomarkers and Prevention Research Branch, Division of Cancer Prevention and Control, National Cancer Institute, National Institutes of Health, Rockville, Maryland

Correspondence to: Laura Guembe, Dept. of Cytology and Histology, University of Navarra, 31080 Pamplona, Spain.


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 47:623–636, 1999)

Key Words: mouse lung, development, amidation, PAM, PHM, PAL, Clara cells, smooth muscle, macrophages


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Bioactive peptides are generated biosynthetically from larger precursors via a variety of posttranslational modifications (Sossin et al. 1989 ). Of all the possible chemical alterations (proteolysis, glycosylation, phosphorylation, and others), amidation of the peptide carboxy terminal amino acid is the best studied with regard to its biological significance. More than half of the known peptide hormones of mammals are amidated (Eipper et al. 1992b ). In addition to resistance to carboxypeptidase degradation, amidation frequently confers biological activity to the peptide. For example, amidated gastrin-releasing peptide (GRP) is 100- to 1000-fold more active than the nonamidated form (Moody et al. 1988 ). Likewise, it has been shown that bombesin requires {alpha}-amidation for optimal binding and biological activity (Mervic et al. 1991 ). On the contrary, amidation does not appear to be important for receptor binding for some other peptides (e.g., secretogranin) (Gafvelin et al. 1985 ).

The formation of an amidated peptide from its glycine-extended propeptide intermediate requires an enzyme complex denoted collectively as peptidyl-glycine {alpha}-amidating mono-oxygenase (PAM) (EC 1.14.17.3) (Eipper et al. 1983 ). Peptidylglycine {alpha}-hydroxylating mono-oxygenase (PHM) mediates the first step of the PAM pathway and uses molecular oxygen to form the peptidyl–{alpha}-hydroxyglycine intermediate. Peptidyl–{alpha}-hydroxyglycine {alpha}-amidating lyase (PAL), the second enzyme of the pathway, catalyzes the conversion of the peptidyl–{alpha}-hydroxyglycine derivative into the {alpha}-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 (Eipper et al. 1987 ). The amino terminal third of the 108-kD PAM precursor contains the PHM enzyme, whereas PAL enzyme is contained in the middle third of the PAM precursor (Glauder et al. 1990 ; Stoffers et al. 1991 ; Milgram et al. 1992 ). The carboxy terminal third of the molecule encodes a transmembrane domain and a hydrophilic cytoplasmic tail (Eipper et al. 1987 ).

The amidating enzymes have been localized in endocrine glands (May et al. 1990 ; Braas et al. 1992 ), endocrine cells of pancreas and digestive tract (Braas et al. 1992 ; Martinez et al. 1993a , Martinez et al. 1993b ), salivary glands (Braas et al. 1992 ), heart (Eipper et al. 1988 ; Ouafik et al. 1989 ; Braas et al. 1992 ), neurons, and glial cells (Rhodes et al. 1990 ; Schafer et al. 1992 ). Although several regulatory substances (Table 1), some of them amidated, are known to be involved in the maturation and function of mammalian lung, the presence of PAM in adult lung has been reported in only one species (human) (Saldise et al. 1996 ), and no studies have been made in developing lung. The aim of the present investigation was to study the distribution of PAM enzymes in developing and adult mouse lung and to determine co-localization between this enzyme and the regulatory peptides found in the mammalian pulmonary neuroendocrine system.


 
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Table 1. Antisera used to study the regulatory substances present in mouse lung


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 20–24 hr and were paraffin-embedded.

Immunocytochemistry
Sections (4 µm thick) were mounted on glass slides. After dewaxing, immunocytochemical staining was performed using the avidin–biotin method (Hsu et al. 1981 ). Endogenous peroxidase was blocked by treatment with 3% hydrogen peroxide in methanol for 30 min and sections were hydrated through alcohols and then placed in Tris-HCl-buffered saline (TBS: Tris buffer 0.05 M, NaCl 0.5 M, pH 7.36). Nonspecific background was blocked with normal swine or rabbit serum (1:20; University of Navarra) for 30 min and the sections were incubated overnight at 4C with the primary antiserum. Table 2 shows all the PAM antisera used in this study as well as their source and optimal dilution. To reveal neuroendocrine elements, antisera against regulatory substances described in mammalian respiratory tract (Table 1) were also applied.


 
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Table 2. Antisera against PAM enzymes used in the present study

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 avidin–biotin–peroxidase 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 (Shu et al. 1988 ). The sections were counterstained with hematoxylin, dehydrated, and mounted in DPX.

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 0.1–10 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.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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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Figure 1. Three serial sections of mouse lung stained with PHM(1) (A), PHM(3) (B), and CD (C) antisera. The three of them stain epithelial (arrowheads) and parenchymal cells (thin arrows). In addition, the PHM(3) and CD antisera label smooth muscle (thick arrows) of both bronchioles (b) and blood vessels (V). P-ad. Bars = 40 µm.


 
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Table 3. Immunocytochemical results obtained in different tissues with the PAM antisera that rendered immunostaining

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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Figure 2. (A) Panoramic view of a bronchus (B) in the hilus of mouse lung. Most of the epithelial cells show immunoreactivity for PAM. C, cartilage. Bar = 100 µm. (B) Detail of the rectangle in A. Positive immunostaining for PAM (arrows) can be observed throughout the cytoplasm of many epithelial cells. ABC, PHM(1) antiserum. P-ad. Bar = 20 µm.

Figure 3. (A) Low-power micrograph showing terminal bronchiole (b) displaying immunoreactivity for PAM. Bar = 100 µm. (B) Detail of one of these bronchioles (bottom). Most of the epithelial cells are intensely stained. ABC, PHM(1) antiserum. P-ad. Bar = 25 µm.

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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Figure 4. (A) Bronchial epithelium. Clara cells (thick arrows) are intensely immunoreactive for PAM, whereas ciliated cells (thin arrow) are negative. S, surface fluid. Bar = 10 µm. (B) Bronchiolar epithelium. Clara cells (thick arrows) are also immunoreactive, whereas other cells (thin arrows) remain unstained. ABC, PHM(1) antiserum. P-ad. Bar = 20 µm.

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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Figure 5. Serial paraffin sections stained with antibodies against CGRP (A,C) and PAM (B,D). Neuroepithelial bodies (arrows) positive for CGRP (A,C) lack PAM immunoreactivity (B,D). Arrowheads, PAM immunostaining. ABC; B,D, PHM(3) antiserum. A,B, E-19; C,D, P-ad. Bars: A,B = 25 µm; C,D = 20 µm.

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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Figure 6. Appearance and evolution of PAM immunoreactivity in bronchiolar epithelium. (A) Panoramic view of an E-16 lung. The bronchiolar epithelium (b) shows immunoreactivity for PAM. Bar = 100 µm. (B) Detail of the rectangle in A. The immunoreactivity is mainly located in the apical region of the epithelium (arrowheads), although some staining in the basal region (arrows) can be observed. (C) E-18. Some epithelial cells (arrows) show immunostaining throughout the whole cytoplasm. (D) P-6. Most of the bronchiolar epithelial cells show the entire cytoplasm intensely stained. ABC, PHM(3) antiserum. Bars = 20 µm.

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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Figure 7. PAM immunoreactivity in smooth muscle of airways (A–C,E) and blood vessels (D). (A) Panoramic view of bronchial wall, tangentially sectioned. In addition to the epithelium, PAM immunostaining can be observed in the smooth muscle (M). B, bronchial lumen. An identical pattern of immunostaining with PHM(3) antiserum is shown in Figure 9A. Bar = 100 µm. (B) Detail of the bronchial smooth muscle, longitudinally sectioned. Many myocytes show PAM immunoreactivity. (C) A similar tangential section of a bronchiole. Bronchiolar smooth muscle cells (M) may also show PAM immunoreactivity. E, epithelium. (D) Pulmonary blood vessel (V) showing PAM immunoreactivity in some cross-sectioned smooth muscle cells (arrows). Bars = 20 µm. (E) Tangentially sectioned bronchial wall immunostained with the PHM(1) antiserum. Whereas epithelium (E) shows immunoreactivity, smooth muscle (M) remains unstained. ABC; A,B, CD antiserum; C,D, PHM(3) antiserum. P-ad. Bar = 25 µm.

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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Figure 8. (A) P-ad. Frequent parenchymal cells (arrows), showing a variety of shapes, display immunoreactivity for PAM. Bar = 20 µm. (B) Detail of some of these cells. Bar = 10 µm. (C) P-6. Immunoreactivity in parenchymal cells (arrows) appears at this stage. Note the lesser numbers of positive cells and amount of immunoreactivity compared with adult lungs. Bar = 20 µm. (D) Detail of P-6 parenchymal cells. ABC, PHM(1) antiserum. Bar = 10 µm.

In the three cell types—epithelium, smooth muscle, and parenchymal cells—preabsorption of the antiserum with its antigen abolished immunolabeling (Figure 9).



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Figure 9. Absorption controls. The immunoreactivity for PAM (A) disappears when the antiserum is preabsorbed with the corresponding antigen (B) in the three immunoreactive tissues found in mouse lung: epithelium (arrowheads), smooth muscle (M), and parenchymal cells (arrows). ABC, PHM(3) antiserum. P-ad. Bars = 25 µm.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Saldise et al. 1996 ). PAM immunostaining was found in airway epithelium, smooth muscle, and alveolar macrophages, as it occurs in the mouse lung but, in addition, endothelium, neurons, Schwann cells, and chondrocytes were also immunostained with some of the antisera used in the present study.

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 (Saldise et al. 1996 ) using the same antiserum used in this study.

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 (Saldise et al. 1996 ), but not in mouse lung (only PHM).

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 (Saldise et al. 1996 ) and in other mammalian cells, such as glucagon/GLP-1-containing endocrine cells of rat colon (Martinez et al. 1993a ). Because it has been reported that PHM is able to complete the amidation process under certain conditions (Katopodis et al. 1990 ), a similar process might occur in pulmonary cells lacking PAL immunoreactivity. We must consider that tissue-specific expression of alternative mRNA splice forms and posttranslational proteolysis have also been extensively demonstrated, showing the existence of different PAM isoforms, some of them lacking the PAL domain (Stoffers et al. 1989 , Stoffers et al. 1991 ; Eipper et al. 1992a ; Milgram et al. 1992 ). Such diversity of isoforms could explain some of the differences in the distribution pattern of PHM and PAL immunoreactivities. More difficult to explain is the presence of immunostaining for both PHM and CD domains in mouse lung, because an alternative mRNA splice form containing both PAM and CD domains but lacking the PAL domain has never been described. Therefore, the absence of PAL immunostaining in mouse lung could more reasonably be due to the low sensitivity of the PAL antibody used in the present study. This and other possibilities may be adressed using in situ hybridization and generation of new antibodies.

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 cells—at least most of them—are 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 (Saldise et al. 1996 ).

Endocrine Cells
The presence of PAM enzymes has been described in endocrine organs producing amidated peptides (Birnbaum et al. 1989 ; Markosian et al. 1989 ; Braas et al. 1992 ), but little is known about the presence of these enzymes in the diffuse neuroendocrine system (Martinez et al. 1993a , Martinez et al. 1993b ).

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 (Martinez et al. 1993a , Martinez et al. 1993b ; Steel et al. 1994 ), this is an unexpected finding. Similar results have been reported in human fetal lung (canalicular period, spontaneous miscarriage), in which GRP-positive cells lacked PAM immunoreactivity (Saldise et al. 1996 ). The lack of co-localization of PAM and amidated peptides has also been previously reported in endocrine cells of other organs, such as human pancreas (Martinez et al. 1993b ) and rat small intestine (Martinez et al. 1993a ). In rat stomach, only a subset of gastrin cells were also positive for PAM antisera (Martinez et al. 1993a ), suggesting that only the more mature cells contain the amidating enzymes. Some possibilities have been suggested to explain the lack of PAM immunoreactivity in amidated peptide-producing cells, which could apply to mouse lung endocrine cells. The presence of an amidating enzyme different from PAM has been suggested (Martinez et al. 1993a , Martinez et al. 1993b ), but all attempts to identify other amidating enzymes have been unsuccessful (Martinez and Treston 1996 ). Another possibility could be the existence of tissue-specific isoforms of PAM that may not be detected by the antibodies used (Braas et al. 1989 ). A third possibility includes different functions for amidated and nonamidated forms of peptide hormones (Seva et al. 1994 ).

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) (Giaid et al. 1990 ; Rozengurt et al. 1990 ; Springall et al. 1991 ). Although ET itself is not amidated, the presence of PAM enzymes could be related to the amidation of other peptides derived from the ET precursor, which contain predicted amidation sites (Cuttitta 1993 ). The biological actions of these peptides remain unknown. In addition, immunoreactivity for adrenomedullin, another amidated peptide, has recently been described in human airway epithelium (Martinez et al. 1995 ). These authors indicated that distribution of adrenomedullin was coincident with that of PAM in human lung (Saldise et al. 1993 , Saldise et al. 1996 ). Finally, it has been suggested that enzymes required for processing peptide hormone precursors, such as PAM, may be a common feature of cells containing secretory granules, even though they may not produce any endogenous prohormone substrate (Dickinson et al. 1993 ). Further biochemical and cytochemical studies are needed to clarify this point.

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 (Saldise et al. 1996 ). Some other observations support this hypothesis. First, PAM has also been reported in lung exocrine glands (Saldise et al. 1996 ), in salivary glands (von Zastrow et al. 1986 ; Braas et al. 1992 ), and in saliva (von Zastrow et al. 1986 ; Kato et al. 1992 ). Furthermore, PHM and PAL enzymatic activities can be found in human bronchoalveolar lavage fluid (Scott et al. 1993 ), and cultured cells expressing PAM secrete this enzyme (Maltese and Eipper 1992 ).

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 (May and Braas 1991 ) and in airways and blood vessels of human lung (Saldise et al. 1996 ). The presence of PAM in these cells suggests the production of amidated regulatory peptides that could be involved, through an auto- and/or a paracrine mechanism, in the control of broncho- and vasomotor tone. In fact, ET (Giaid et al. 1993 ; Seldeslagh and Lauweryns 1993a , Seldeslagh and Lauweryns 1993b ) and adrenomedullin (Martinez et al. 1995 ) have also been found in pulmonary smooth muscle cells.

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 (Saldise et al. 1996 ). The presence of PAM in these cells could be related to the reported production of IGF-I (Room et al. 1988 ), adrenomedullin (Martinez et al. 1995 ), or ET (Ehrenreich et al. 1990 ). Similar to ET, IGF-I is not an amidated peptide but its precursor contains a biologically active amidated peptide (Siegfried et al. 1992 ).

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 (Ouafik et al. 1987 ; Scharfmann et al. 1988 ; Maltese et al. 1989 ; Zhou and Thorn 1990 ; Kapuscinski and Shulkes 1995 ) and rat heart (Ouafik et al. 1989 ) have been studied in relation to the presence of the PAM enzyme during fetal and/or neonatal stages. In both organs, peaks of the enzyme at different points in development and a decrease of PAM in the adult period have been described. On the contrary, in mouse lung, as already stated, the strongest PAM immunostaining was found in adult lungs. These results indicate a different pattern of PAM expression in developing organs, which supports the existence of a tissue-specific regulation of PAM expression during development (Maltese et al. 1989 ; Ouafik et al. 1989 ).

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.


  Acknowledgments

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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Birnbaum RS, Bortelsen AH, Ross BA (1989) Glucocorticoid regulation of amidating enzymes in a neoplastic C-cell line. Mol Cell Endocrinol 61:109-116[Medline]

Braas KM, Harakall SA, Ouafik L, Eipper BA, May W (1992) Expression of peptidylglycine alpha-amidating monooxygenase: an in situ hibridization and immunocytochemical study. Endocrinology 130:2778-2788[Abstract]

Braas KM, Stoffers DA, Eipper BA, May V (1989) Tissue specific expression of rat peptidylglycine {alpha}-amidating monooxygenase activity and mRNA. Mol Endocrinol 3:1387-1398[Abstract]

Cuttitta F (1993) Peptide amidation: signature of bioactivity. Anat Rec 236:87-93[Medline]

Dickinson CJ, Takeuchi T, Guo YJ, Stadler BT, Yamada Y (1993) Expression and processing of prohormones in nonendocrine cells. Am J Physiol 264:G553-G560[Abstract/Free Full Text]

Ehrenreich H, Anderson RW, Fox CH, Riekmann P, Hoffman GS, Coligan JE, Kehrl JH, Fauci AS (1990) Endothelins, peptides with potent vasoactive properties are produced by human macrophages. J Exp Med 172:1741-1748[Abstract]

Eipper BA, Green CB, Campbell TA, Stoffers DA, Keutmann HT, Mains RE, Ouafik L (1992a) Alternative splicing and endoproteolytic processing generate tissue-specific forms of pituitary peptidylglycine alpha-amidating monooxygenase (PAM). J Biol Chem 267:4008-4015[Abstract/Free Full Text]

Eipper BA, Mains RE, Glembloski CC (1983) Identification in pituitary tissue of a peptide {alpha}-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper and ascorbic acid. Proc Natl Acad Sci USA 80:5144-5148[Abstract]

Eipper BA, May V, Braas KM (1988) Membrane-associated peptidylglycine alpha-amidating monooxygenase in the heart. J Biol Chem 263:8371-8379[Abstract/Free Full Text]

Eipper BA, Park LP, Dickerson MI, Keutmann HT, Thiele EA, Rodriguez H, Schofield PR, Mains RE (1987) Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis. Mol Endocrinol 1:777-790[Abstract]

Eipper BA, Stoffers DA, Mains RE (1992b) The biosynthesis of neuropeptides: peptide {alpha}-amidation. Annu Rev Neurosci 15:57-85[Medline]

Gafvelin G, Carlquist M, Mutt V (1985) A proform of secretin with high secretin-like bioactivity. FEBS Lett 184:347-350[Medline]

Giaid A, Polak JM, Gaitonde V, Hamid QA, Moscoso G, Legon S, Uwanogho D, Roncalle M, Shinni O, Sawamura T (1990) Distribution of endothelin-like immunoreactivity and m-RNA in the developing and adult human lung. Am J Respir Cell Mol Biol 4:50-58

Giaid A, Yanagisawa M, Langleben D, Michel RP, Levy R, Shennie H, Kimura S, Masaki T, Duguid WP, Path FRC, Stewart DJ (1993) Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 333:214-221[Abstract/Free Full Text]

Glauder J, Ragg H, Rauch J, Engels FW (1990) Human peptidylglycine {alpha}-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells. Biochem Biophys Res Commun 169:551-558[Medline]

Hsu SM, Raine L, Fanger H (1981) 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[Abstract]

Kapuscinski M, Shulkes A (1995) Peptide amidating activity and gastrin processing in the developing sheep pancreas. J Endocrinol 145:137-142[Abstract]

Kato T, Hanaoka K, Ochiai O, Chikuma T, Takeda K, Yajima R, Kumegawa M (1992) Secretion of peptidylglycine {alpha}-amidating monooxygenase (PAM) from rat salivary glands. Neurochem Int 21:153-158[Medline]

Katopodis AG, Ping D, May SW (1990) A novel enzyme from bovine neurointermediate pituitary catalizes dealkylation of alpha-hydroxyglycine alpha-amidating monooxygenase in peptide amidation. Biochemistry 29:6115-6120[Medline]

Maltese YJ, Eipper BA (1992) Developmental expression of peptidylglycine alpha-amidating monooxygenase (PAM) in primary cultures of neonatal cardiocytes. A model for studying regulation of PAM expression in the rat heart. Mol Endocrinol 6:1998-2008[Abstract]

Maltese JY, Giraud P, Kowalski C, Ouafik LH, Salers P, Pelen F, Oliver C (1989) Ontogenetic expression of peptidyl-glycine alpha-amidating monooxygenase mRNA in the rat pancreas. Biochem Biophys Res Commun 158:244-250[Medline]

Markosian KA, Paitian NA, Abramian KS, Nalbandian RM (1989) Peptidylglycine alpha-amidating monooxygenase from bovine heart atrium secretory granules and adrenal chromaffin granules. Biokhimiia 54:2046-2053[Medline]

Martínez A, Burrell MA, Kuijk M, Montuenga LM, Treston A, Cuttitta F, Polak JM (1993a) Localization of amidating enzymes (PAM) in rat gastrointestinal tract. J Histochem Cytochem 41:1617-1622[Abstract/Free Full Text]

Martínez A, Montuenga LM, Springall DR, Treston A, Cuttitta F, Polak JM (1993b) Immunocytochemical localization of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in human endocrine pancreas. J Histochem Cytochem 41:375-380[Abstract/Free Full Text]

Martínez A, Miller MJ, Unsworth EJ, Siegfried JM, Cuttitta F (1995) Expression of adrenomedullin in normal lung and in pulmonary tumors. Endocrinology 136:4099-4105[Abstract]

Martínez A, Treston AM (1996) Where does amidation takes place? Mol Cell Endocrinol 125:113-117

May V, Braas KM (1991) Peptidylglycine alpha-amidating monooxygenase (PAM) expression in endothelial cells. Proc 73rd Annu Meet Endocrine Soc 1339

May V, Ouafik JH, Eipper BA, Braas KM (1990) Immunocytochemical and in situ hybridization studies on peptidylglycine {alpha}-amidating monooxygenase in pituitary gland. Endocrinology 127:358-364[Abstract]

Mervic M, Moody TW, Komoriya A (1991) A structure function study of C-terminal extensions of bombesin. Peptides 12:1149-1151[Medline]

Milgram SL, Johnson R, Mains RE (1992) Expression of individual forms of peptidyl-glycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules. J Cell Biol 117:717-728[Abstract]

Moody TN, O'Donohue TL, Chrinwall BM, Cuttitta F, Linden CD, Geta RC, Wolf SS (1988) Neurochemistry and pharmacology of bombesin-like peptides. In Negrovilla A, Conn PM, eds. Peptide Hormones: Effects and Mechanisms of Action. Boca Raton, FL, CRC Press, 3-40

Ouafik L, Giraud P, Salers P, Dutour A, Castanas E, Boudouresque F, Oliver C (1987) Evidence for high peptide {alpha}-amidating activity in the pancreas from neonatal rats. Proc Natl Acad Sci USA 84:261-264[Abstract]

Ouafik L, May V, Keutmann HT, Eipper BA (1989) Developmental regulation of peptidylglycine alpha-amidating monooxygenase (PAM) in rat heart atrium and ventricle. Tissue-specific changes in distribution of PAM activity, mRNA levels, and protein forms. J Biol Chem 264:5839-5845[Abstract/Free Full Text]

Rhodes CH, Xu RY, Angeletti RH (1990) Peptidylglycine alpha-amidating monooxygenase (PAM) in Schwann cells and glia as well as neurons. J Histochem Cytochem 38:1301-1311[Abstract]

Room WN, Basser C, Fells GA, Nukiwa T, Trapnell BC, Crystal RG (1988) Alveolar macrophages release an insulin-like growth factor I-type molecule. J Clin Invest 82:1685-1690[Medline]

Rozengurt N, Springall DR, Polak JM (1990) Localization of endothelin-like immunoreactivity in airway epithelium of rats and mice. J Pathol 160:5-8[Medline]

Saldise L, Martínez A, Montuenga LM, Treston A, Springall DR, Polak JM, Vázquez JJ (1996) Distribution of peptidyl-glycine {alpha}-amidating monooxygenase (PAM) enzymes in normal human lung and in lung epithelial tumors. J Histochem Cytochem 44:3-12[Abstract/Free Full Text]

Saldise L, Martínez A, Montuenga LM, Vázquez, JJ (1993) Enzimas amidantes (PAM) de péptidos reguladores en el pulmón humano. Proc VII Congr Nacional Histología 195–196

Schafer MK, Stoffers DA, Eipper BA, Watson SJ (1992) Expression of peptidylglycine {alpha}-amidating monooxygenase (EC 1.14.17.3) in the rat central nervous system. J Neurosci 2:222-234

Scharfmann R, Leduque P, Aratan–Spire S, Dubois P, Basmaciogullari A, Czernichow P (1988) Persistence of peptidylglycine alpha-amidating monooxygenase activity and elevated thyrotropin-releasing hormone concentrations in fetal rat islets in culture. Endocrinology 123:1329-1334[Abstract]

Scott F, Cuttitta F, Treston AM, Avis Y, Gupta P, Ruckeschel L, Kelly K, Piantadosi S, Tockman N, Mulshine J (1993) Prospective trial evaluating immunocytochemical-based sputum techniques for early lung cancer detection: assays for promotion factors in the bronchial lavage. J Cell Biochem Suppl 17F:175-183

Seldeslagh KA, Lauweryns JM (1993a) Sarafotoxin in the bronchopulmonary tract: immunohistochemical occurrence and colocalization with endothelins. Histochemistry 100:257-263[Medline]

Seldeslagh KA, Lauweryns JM (1993b) Endothelin in normal lung tissue of newborn mammals: immunocytochemical distribution and co-localization with serotonin and calcitonin gene-related peptide. J Histochem Cytochem 41:1495-1502[Abstract/Free Full Text]

Seva C, Dickinson CJ, Yamada T (1994) Growth-promoting effects of glycine-extended progastrin. Science 265:410-412[Medline]

Shu S, Ju G, Fan L (1988) The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci Lett 85:169-171[Medline]

Siegfried JM, Kasprzyk PG, Treston AM, Mulshine JL, Quinn KA, Cuttitta F (1992) A mitogenic peptide amide encoded within the E-peptide domain of the insulin-like growth factor-IB prohormone. Proc Natl Acad Sci USA 89:8107-8111[Abstract]

Sossin WS, Fisher JM, Scheller RH (1989) Cellular and molecu-lar biology of neuropeptide processing and packaging. Neuron 2:1407-1417[Medline]

Springall DR, Howarth PH, Couniban H, Djukanovic R, Holgate ST, Polak JM (1991) Endothelin immunoreactivity of airway epithelium in asthmatic patients. Lancet 337:697-701[Medline]

Steel JH, Matínez A, Springall DR, Treston AM, Cuttitta F, Polak JM (1994) Peptidylglycine-amidating monooxygenase (PAM) immunoreactivity and messenger RNA in human pituitary and increased expression in pituitary tumours. Cell Tissue Res 276:197-207[Medline]

Stoffers DA, Green CB, Eipper BA (1989) Alternative mRNA splicing generates multiple forms of peptidyl-glycine {alpha}-amidating monooxygenase in rat atrium. Proc Natl Acad Sci USA 86:735-739[Abstract]

Stoffers DA, Ouafik L, Eipper BA (1991) Characterization of novel mRNAs encoding enzymes involved in peptide alpha-amidation. J Biol Chem 266:1701-1707[Abstract/Free Full Text]

von Zastrow M, Tritton TR, Castle JD (1986) Exocrine secretion granules contain peptide amidation activity. Proc Natl Acad Sci USA 83:3297-3301[Abstract]

Zhou A, Thorn N (1990) Evidence for presence of peptide {alpha}-amidating activity in pancreatic islets from newborn rats. Biochem J 267:253-256[Medline]