|
|
|
|
 |
|||
|
|||
Volume 53 (6): 773-780, 2005
Copyright ©The Histochemical Society, Inc.
Identification of Adrenomedullin in Avian Type II Pneumocytes : Increased Expression after Exposure to Air Pollutants
Department of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore, Maryland (NC), and Cell and Cancer Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (AM,FC,EZ)
Correspondence to: Natalia Cuesta, PhD, Department of Microbiology and Immunology, School of Medicine, 660 W. Redwood Street, University of Maryland, Baltimore, MD 21201. E-mail: ncuesta{at}som.umaryland.edu
 |
Summary |
|---|
 Top Summary IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Adrenomedullin (AM) is a potent vasodilator peptide present in the lung of mammals where it is expressed mainly in the columnar epithelium and alveolar macrophages. AM increases the secretion of phosphatidylcholine by type II pneumocytes, which suggests a role as an autocrine modulator of surfactant secretion. In this study we show the expression of an AM-like protein in the lung of the pigeon, Columba livia. Using an antibody against its human ortholog, AM-like immunoreactivity was found to be associated with membranous structures of the multivesicular bodies of type II pneumocytes. We also studied the differential expression of AM-like peptide in the lung of pigeons exposed to polluted city air vs cleaner countryside conditions and found that AM-like expression was higher in city animals. Similar results were obtained in an experimental study in which pigeons were exposed to increasing concentrations of a single pollutant, ozone. Taken together, our findings support the implication of AM in the response of type II pneumocytes to air pollutants.
(J Histochem Cytochem 53:773–780, 2005)
Key Words: adrenomedullin • pigeon • air pollution • lung • surfactant • type II pneumocytes • ozone
|
Introduction |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
SINCE THE DISCOVERY of adrenomedullin (AM) in 1993 (Kitamura et al. 1993
), an increasing number of studies have detected its expression in almost every tissue of the body but most remarkably in heart, kidney, pancreas, intestines, and lung (Martínez et al. 1995; Richards et al. 1996; Cameron and Fleming 1998; Asada et al. 1999; Hinson et al. 2000; López and Cuesta 2002). As expected from a peptide with such a wide distribution, a broad range of biological actions is attributed to AM, including vasodilatation, regulation of hormone secretion, cell growth, and antimicrobial effects, among others (López and Martínez 2002).
In mammalian lung, AM is expressed in the columnar epithelium of bronchi and bronchioli, in some glands, endothelial cells, alveolar macrophages, neurons of the pulmonary parasympathetic nervous system, smooth muscle cells, and chondrocytes (Martínez et al. 1995). The AM receptor constituted by calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP2) is also abundantly expressed in mammalian lung (Martínez et al. 1997; Ono et al. 2000). With relation to its physiological role in lung, AM contributes to pulmonary vasodilatation and inhibition of bronchoconstriction (Yang et al. 1996). Increased levels of circulating AM have been reported in several respiratory pathologies such as asthma (Kanazawa et al. 1996; Kohno et al. 1996; Ceyhan et al. 2001).
It is also known that circulating AM is removed when it passes through the pulmonary vascular bed (Yoshibayashi et al. 1997), and an anti-inflammatory role has been suggested for AM in this organ because this peptide inhibits alveolar macrophage release of neutrophil chemoattractants in response to LPS (Kamoi et al. 1995).
AM increases the secretion of phosphatidylcholine, the predominant lipid component of pulmonary surfactant, in type II pneumocytes in culture (Okumura et al. 2000). Pulmonary surfactant is a complex mixture of lipids and proteins that reduces the surface tension in the alveolar air–liquid interface (Chander and Fisher 1990). There are two main surfactant reservoirs: the intracellular surfactant contained in the lamellar bodies of type II pneumocytes and the extracellular surfactant that lines the alveolar surface (Wright and Clements 1987; Risco et al. 1994; Haller et al. 1998). Defects in the synthesis and excretion of surfactant, as well as alterations in its components, result in a disturbance of the alveolar gas exchange rate, as has been reported in certain pathologies such as the respiratory distress syndrome (Hallman et al. 1982; van Golde et al. 1988).
In spite of the large amount of references related to the presence of AM in lung, little is known about the expression of this peptide in the lung of non-mammalian species. Moreover, although the function of AM in lung is well known, its physiological relationship with the surfactant system and the relevance of AM secretion in response to stress situations, such as the prolonged exposure to air pollutants, is still poorly understood. Air pollutants have been recognized to influence the structure and function of the surfactant system, resulting in increased synthesis of surfactant components but, at the same time, they reduce surfactant secretion (Müller et al. 1998). In avian species, it has been shown that air pollution causes a reduction in the number of lamellar bodies present in type II pneumocytes (Lorz and López 1997).
In the present study we focus on the distribution of AM-like peptide immunoreactivity in the lungs of animals raised in two different habitats: a polluted city and a non-contaminated village. In addition, to better understand the contribution of individual pollutants, we have designed an in vivo experimental model that allows for controlled exposure to different air pollutants. Using this model, we have studied the effects of ozone on AM expression. The animal species chosen was the common pigeon (Columba livia), because the clustered localization of type II pneumocytes covering the parabronchial surfaces in birds (López 1995; Klika et al. 1999) makes it easy to perform studies related to their physiology.
In this study we demonstrate the presence of an AM-like peptide in avian lung and describe the effects of air pollution on the expression of this peptide in type II pneumocytes.
|
Materials and Methods |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Animal Model
Eighteen specimens of Columba livia domestica were used for this study; six were collected from a farm located 50 km away from the closest industrial city, Guadalajara, Spain (referred to in the text as "countryside pigeons"). Another subset of six pigeons was captured in the city of Madrid, Spain ("city pigeons"). The six remaining specimens were used in the experimental model for ozone and were purchased from a farm in Valencia, far from the nearest city. All specimens were checked by a veterinary service before performing the experiment and were found to be healthy.
All protocols were approved by the Ethics and Animal Care Committee of the Universidad Autónoma de Madrid. Atmospheric levels of noxious agents in the city of Madrid during the month when the specimens for this comparative study were captured are shown in Table 1.
|
Ozone Exposure System
Because a polluted atmosphere consists of a mixture of air pollutants in different concentrations, the comparative study provides evidence of the effects of chronic exposure to pollution levels such as those found in an industrialized city. To further understand the effects of individual contaminants on the expression of AM-like peptide in the lung of the pigeon, we developed a model that allowed for the study of controlled exposures to single pollutants. For this model, we chose the open-top chambers that had been successfully used in studies of ozone exposure in vegetables (Gimeno et al. 1989
). The ozone exposure system comprised three open-top chambers, each consisting of a frame surrounded by clear polyethylene plastic and perforated at the bottom to allow air circulation (Heagle et al. 1989). Three different treatments were used in each chamber: non-filtered air (NF), air enriched with 60 µg/m3 O3 (30 ppb), and air enriched with 200 µg/m3 O3 (100 ppb). Ozone was pumped daily into the chambers from 8:00 AM to 4:00 PM during 5 consecutive days, representing a typical exposure to ozone for an urban environment. The ozone concentration inside the chambers was monitored continuously with an EPA-approved monitor. A representative daily average of the ozone concentrations in the chambers is depicted in Figure 1. Two pigeons per treatment were used. The animals were placed in cages in the middle of the chamber and were fed cracked corn and sterile water. After 5 days exposure, the lungs were processed as explained below.
|
For this study, the concentrations of ozone always remained within the range in which they could be found in a polluted city atmosphere. Animals breathing NF air were used as controls.
Western Blotting
Tissue samples destined for Western blot analysis were frozen in liquid nitrogen immediately after dissection and maintained at –80C. Extracts were prepared by thawing the tissue on ice and homogenizing the samples in ice-cold homogenization buffer: 2x Tricine buffer with 8% SDS (Novex; San Diego, CA) containing 1 mM final concentration of the protease inhibitors pefablock (Centerchem; Stanford, CT), bestatin, and phosphoramidon (Sigma; St Louis, MO). The homogenate was centrifuged at 100,000 x g for 30 min, and the supernatant was collected. Homogenate supernatant protein content was measured using a BCA kit (BioRad Labs; Richmond, CA). Protein extracts were diluted to an approximate protein concentration of 1 µg/µl and were electrophoretically fractionated on a 12% Tricine SDS-PAGE gel (Novex) under reducing conditions (5% ß-mercaptoethanol). Proteins were transferred to a polyvinyldifluoride membrane (Immobilon PVDF; Millipore, Billerica, MA). The membrane was blocked overnight in 5% non-fat milk–PBS, incubated for 1 hr in a 1:1000 dilution of rabbit anti-AM (Martínez et al. 1995), and washed three times in 0.1% Igepal (Rhodia; Cranbury, NJ). The membrane was further exposed to biotinylated goat anti-rabbit immunoglobulins (1:200 dilution; Dakopatts, Glostrup, Denmark) for 1 hr and then exposed to avidin–biotinylated peroxidase complex (1:500; Dakopatts) for an additional hour. Peroxidase activity was revealed with the ECL + Plus chemiluminiscence kit (Amersham; Arlington Heights, IL) following manufacturer's instructions. The specificity control consisted of duplicate membranes incubated in antigen-preabsorbed (10 nmol/ml) antiserum.
Immunohistochemistry in Paraffin Sections
Pigeons were anesthetized with a mild dose of chloroform and then decapitated before proceeding to the dissection. The lungs were fixed by immersion in 4% buffered formalin for 24 hr and then embedded in paraffin and sectioned following routine procedures. The avidin–biotin histochemical staining procedure (Hsu et al. 1981) was used to localize AM in the lungs from city and countryside pigeons and in the experimental model for ozone. After deparaffinizing in xylene, tissue sections were rehydrated, and antigen retrieval was performed with a citrate buffer solution (0.01 M citric acid, pH 6) for 20–30 min in a microwave oven (maximum power). Briefly, slides were blocked with normal swine serum (1:20 dilution) and incubated overnight at 4C with the primary serum: rabbit anti-AM (1:500 dilution). Developing was performed using DAB (50 mg/100 ml) (Sigma) and H2O2 as a substrate. The sections were lightly counterstained with Harris hematoxylin, dehydrated, and mounted for analysis. Rat pancreas was used as a positive control for AM immunostaining. Substitution of the primary antibody by a prebleed serum was used as the negative control. To ensure the specificity of the immunostaining, liquid phase and solid phase absorption controls were performed with the synthetic antigen at a 10 nmol/ml concentration.
For the experiments in which we quantified the intensity of the immunostaining, reagents for avidin–biotin complexes were obtained from the Alkaline Phosphatase Substrate Kit (Vector Labs; Burlingame, CA), and Fast Red TR/Naphthol (Sigma) was used as the substrate. Experimental conditions for the immunolabeling, including batches of reagents and time of Red Naphthol development, were kept constant across all experiments.
Quantification of the Immunohistochemical Reaction
Three equivalent sections of the lung from each experimental pigeon were randomly selected and photographed with a Zeiss III photomicroscope (Zeiss; Oberkochen, Germany), using the same light intensity. Pictures were scanned with a minimal resolution of 300 ppi, and the immunostaining intensities were quantified by computerized densitometric analysis using Photoshop software (version 5.5; Adobe Systems, San Jose, CA) as described (Lehr et al. 1999; Zudaire et al. 2004). Color images were transformed into black and white, setting the gray level of all pixels between 0 and 255. The integrated optical density (IOD) was measured in each experimental group.
Electron Microscopy
Small pieces of 1 mm3 of the lungs from countryside and city pigeons were fixed in 4% paraformaldehyde plus 0.1% glutaraldehyde in PBS at 4C for 4 hr. They were then washed overnight in 0.25 M sucrose in PBS at 4C. The samples were all dehydrated and embedded in epoxy resin, TAAB-812 (TAAB Labs; Aldermaston, Berkshire, UK). The single immunogold staining technique was applied to ultrathin sections (60–80 nm) as follows: after an incubation in 3% H2O2 for 5 min at room temperature followed by three washes in bidistilled water, the sections were exposed to normal goat serum for 30 min. The primary antibody for AM, diluted 1:100 in 0.1% BSA-TBS, was added overnight at 4C. The sections were then rinsed in 1% BSA-TBS and 0.5% Tween-20 prior to incubation with the secondary antibody, goat anti-rabbit IgG 20-nm gold colloidal particles (E-Y Labs; San Mateo, CA) for 45 min (1:30 dilution). They were then rinsed and double stained with 5% aqueous uranyl acetate (15 min) and lead citrate (5 min) and examined in a JEOL JEM-1010 electron microscope (JEOL; Tokyo, Japan). Electronographies were obtained with a Multiscan GATAN camera and digitalized with Digital Micrograph 3.1 software (Gatan; Pleasanton, CA).
Statistical Analysis
Staining intensities for AM were expressed as mean IOD ± SEM. Differences between countryside and city pigeons and among the three experimental groups exposed to ozone were compared using Student's t-test. Statistically significant differences were indicated by p<0.05. Data were analyzed with SPSS 6.1.3 (SPSS; Chicago, IL).
|
Results |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
AM-like Immunoreactivity in the Lung of Columba livia
The expression of AM in the lung of the pigeon (Columba livia) has been studied by both Western blot and immunohistochemical methods.
A clear AM-like immunoreactive band with a molecular weight of 18 kDa, which presumably represents the AM-like precursor, was detected in lysates from all tested organs of the pigeon, with the lung showing the strongest immunoreactivity (Figure 2). This band was no longer visible after preabsorption of the antiserum with its antigen, demonstrating specificity of detection (data not shown). The antibody did not cross-react with any other proteins in the lysates.
|
To identify the cell type responsible for the production of AM in avian lung, immunohistochemical methods were used. The histology of avian lung, having type I pneumocytes restricted to capillary airways and type II pneumocytes covering the atrial and parabronchial surfaces, makes it easy to localize both cell types in paraffin sections (for a detailed description of the histology of avian lung, see López 1995
). As shown in Figure 3, AM-like immunoreactivity was found mainly in the cytoplasm of type II pneumocytes (Figures 3A, 3E, and 3F), but there was also immunoreactivity in mesobronchial cells (Figure 3B), chondrocytes, and atrial macrophages (results not shown). The specificity of the staining was proven by absorption controls, which resulted in a total quenching of the immunolabeling (Figure 3C).
|
Electron microscopy immunostaining was applied to investigate the subcellular localization of the AM-like peptide: colloidal gold particles were located mainly in lamellar structures of the multivesicular system or associated with lamellar bodies (Figure 3D) and occasionally in vacuoles of type II pneumocytes (data not shown). No gold particles were found outside this location with the exception of pulmonary macrophages, where some gold particles were also found in the cytoplasm (data not shown).
AM-like Peptide Production in Response to Air Pollutants
To further understand the role of AM in pulmonary type II cells, we designed two sets of experiments, seeking for differences in the production and/or distribution of AM after exposure to air pollutants. The first approach consisted of a comparative study between pigeons raised in the polluted atmosphere of a large city and their counterparts raised in the countryside. When comparing the intensity of the immunostaining in these two sets of animals (Figures 3E and 3F), the immunolabeling for AM was stronger in city pigeons than in countryside pigeons. Densitometric analysis showed that the AM IOD ratio in countryside pigeons was significantly lower than the ratio found in the group of city pigeons (Figure 4).
|
AM-like Peptide Production in Response to Ozone
In agreement with the previous results in the comparative study, the intensity of AM-like immunostaining was significantly higher in the cytoplasm of type II pneumocytes from animals exposed to higher ozone concentrations (Figure 5). These differences are statistically significant between the control group and the two groups exposed to ozone (p<0.05), with no perceptible difference in the intensity of the immunostaining between the two subsets of animals exposed to ozone.
|
|
Discussion |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
This study presents the first immunohistochemical description of an AM-like protein in the pulmonary surfactant system of a non-mammalian vertebrate species. The antibody used was raised against the human peptide but has been used successfully in previous studies in non-mammalian species including birds (López et al. 1999a
,b), supporting that AM is well preserved throughout the evolution of vertebrates. The specificity of the immunostaining has been demonstrated by Western blots and absorption controls.
We have shown that AM-like immunoreactivity is expressed in the lung of Columba livia mainly by type II pneumocytes, atrial macrophages, and mesobronchial epithelial cells. This distribution is similar to the pattern described for mammals (Martínez et al. 1995), with the exception of type II pneumocytes. In rat type II pneumocytes, only AM mRNA has been detected (Okumura et al. 2000). The presence of the peptide has not been demonstrated by immunocytochemical methods, probably because this peptide is rapidly secreted, making the concentration of AM in the cytoplasm too low to allow for detection with these methods. In pigeons, however, the amount of intracellular AM-like peptide seems to be sufficient for immunohistochemical detection. If confirmed in other avian species, this difference could be relevant from a phylogenetic point of view.
At the subcellular level, we have detected the presence of AM-like immunoreactivity in membrane structures of the multivesicular system and in lamellar bodies of type II pneumocytes. This observation implies that AM may be stored and secreted together with the components of pulmonary surfactant, as do other molecules such as vitamin E (Rüstow et al. 1993) or lysozyme (Beers et al. 1994). We have also detected immunogold particles linked to the AM antibody in vacuoles of type II pneumocytes. This fact suggests that AM could be secreted to the extracellular milieu by an alternative mechanism independent of the excretion of lamellar bodies. Its presence in atrial macrophages and mesobronchial cells suggests a role as a protective peptide, participating in the prevention of local infection, as some authors have previously proposed (Martínez et al. 1995,1997; Allaker et al. 1999). In this sense, the simultaneous secretion of AM and pulmonary surfactant may add an important physiological value to the surfactant components, namely, antimicrobial activity. Such localization might also point to a possible role in the regulation of surfactant secretion. It has been previously demonstrated that AM increases phosphatidylcholine secretion (the main component of pulmonary surfactant) in rat type II pneumocytes (Okumura et al. 2000). These results, together with the subcellular localization of AM in lamellar bodies, suggest a possible role for this peptide as an autocrine/paracrine modulator of surfactant secretion.
Our results show that AM-like immunoreactivity is significantly elevated after either chronic or acute exposure to air pollutants. This observation suggests that AM expression may be activated by lung injury. It has been shown previously that pulmonary AM levels are significantly elevated in response to stress situations such as hypoxia and acute restrain (Khan et al. 1999; Matsui et al. 2004). Under such conditions, pulmonary surfactant components present alterations in their synthesis and excretion rates (Hallman et al. 1982; van Golde et al. 1988). Previous studies showed that the relative amount of lamellar bodies is reduced in city pigeons when compared with countryside birds (Lorz and López 1997). These results, together with our recent findings of a relative increase in AM-like peptide levels in type II pneumocytes of city pigeons, allow us to hypothesize that AM synthesis might be activated to maintain constant levels of this peptide in the atrial space.
The relaxation of the vascular bed is another physiological role attributed to AM (Nossaman et al. 1995). An increase in AM production in response to air pollutants, as well as the production of other vasodilatory peptides, would allow some nutrients to more easily reach the damaged cells.
As we pointed out previously, studying the effects of combined exposures of pollutants is essential for health effect predictions but describing the effects of single pollutants on the pulmonary surfactant system is necessary as well. For this purpose, we chose an experimental model to study the effects of a common oxidant, ozone, present in urban atmosphere. In the majority of studies involving ozone as a single atmospheric pollutant, the concentration used is much higher than that found in polluted air (Balis et al. 1991; Putman et al. 1995,1997; Currie 1998). The concentrations of ozone analyzed in our study were situated within the range of "permitted" levels established by the European Community Legislation and provide a better understanding of the possible noxious effects to pulmonary cells of a polluted environment. Under these experimental conditions we could detect statistically significant changes in AM-like immunoreactivity following short-term exposure to ozone concentrations as low as 30 ppb. In agreement with the comparative study between countryside and city pigeons, AM-like peptide expression is augmented after exposure to a single air pollutant. As some studies have reported, there is an increase in AM secretion after oxidative stress in endothelial cells (Chun et al. 2000; Saito et al. 2001), suggesting that AM could be a protective peptide when an oxidative injury occurs. This antioxidant effect, if proven in type II pneumocytes, would explain the augmented AM-like immunoreactivity after ozone exposure.
To summarize, our study demonstrates the presence of an AM-like peptide in type II pneumocytes of the avian lung. Long-term exposure to poor air quality, even at levels within current standards, results in increased AM-like immunoreactivity in type II pneumocytes. AM increased expression and secretion in response to air pollutants might suggest a protective role for this peptide in the pulmonary epithelium and a potential contribution to surfactant secretion regulation.
|
Acknowledgments |
|---|
Supported partially by grants from Comunidad Autónoma de Madrid and Caja de Madrid Foundation to NC.
We are grateful to the Atmospheric Pollution Department of the Madrid Council and to the Electron Microscopy Service of the Universidad Autónoma de Madrid for their help. We wish to thank Dr. M.J. Sanz and Centro de Estudios Ambientales del Mediterráneo (Valencia, Spain) for letting us use the open top chambers located in their installations.
This study is dedicated to the memory of the late Prof. José López Diez del Corral, under whose supervision it was performed at the Department of Biology, Universidad Autónoma de Madrid.
|
Footnotes |
|---|
Received for publication August 11, 2004; accepted December 9, 2004
|
Literature Cited |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Allaker RP, Zihni C, Kapas S (1999) An investigation into the antimicrobial effects of adrenomedullin on members of the skin, oral, respiratory tract and gut microflora. FEMS Immunol Med Microbiol 23:289–293[CrossRef][Medline]
Asada Y, Hara S, Marutsuka K, Kitamura K, Tsuji T, Sakata J, Sato Y, et al. (1999) Novel distribution of adrenomedullin-immunoreactive cells in human tissues. Histochem Cell Biol 112:185–191[CrossRef][Medline]
Balis JU, Paterson JF, Lundh JM, Haller EM, Shelley SA, Montgomery MR (1991) Ozone stress initiates acute perturbations of secreted surfactant membranes. Am J Pathol 138:847–857[Abstract]
Beers MF, Kim CY, Dodia C, Fisher AB (1994) Synthesis of type II cell lamellar body lysozyme-15 kD protein (lbl-15) by perfused rat lung. Am J Respir Cell Mol Biol 11:240–248[Abstract]
Cameron VA, Fleming AM (1998) Novel sites of adrenomedullin gene expression in mouse and rat tissues. Endocrinology 139:2253–2264
Ceyhan BB, Karakurt S, Hekim N (2001) Plasma adrenomedullin levels in asthmatic patients. J Asthma 38:221–227[CrossRef][Medline]
Chander A, Fisher AB (1990) Regulation of lung surfactant secretion. Am J Physiol 258:L241–L253
Chun TH, Itoh H, Saito T, Yamahara K, Doi K, Mori Y, Ogawa Y, et al. (2000) Oxidative stress augments secretion of endothelium-derived relaxing peptides, C-type natriuretic peptide and adrenomedullin. J Hypertens 18:575–580[Medline]
Currie WD (1998) Breathing and pulmonary surfactant function in mice 24 h after ozone exposure. Eur Respir J 12:288–293[Abstract]
Gimeno BS, Selleras JM, Bermejo V, Ochoa MJ, Tarruel A (1989) Efectos del ozono sobre plantas de sandía en el delta del Ebro: sintomatología. Phytoma España 12:19–28
Haller T, Ortmayr J, Friedrich F, Dietl P (1998) Dynamics of surfactant release in alveolar type II cells. Proc Natl Acad Sci USA 95:1579–1584
Hallman M, Spragg R, Harrell JH, Moser KM, Gluck L (1982) Evidence of lung surfactant abnormality in respiratory failure. Study of bronchoalveolar lavage phospholipids, surface activity and plasma inositol. J Clin Invest 70:673–683
Heagle AS, Philbeck RB, Ferrell RE, Heck WW (1989) Design and performance of a large, field exposure chamber to measure effects or air quality on plants. J Environ Qual 18:361–368
Hinson JP, Kapas S, Smith DM (2000) Adrenomedullin, a multifunctional regulatory peptide. Endocr Rev 21:138–167
Hsu SM, Raine L, Fanger M (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. A comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 29:577–580[Abstract]
Kamoi H, Kanazawa H, Hirata K, Kurihara N, Yano Y, Otani S (1995) Adrenomedullin inhibits the secretion of cytokine-induced neutrophil chemoattractant, a member of the interleukin-8 family, from rat alveolar macrophages. Biochem Biophys Res Commun 211:1031–1035[CrossRef][Medline]
Kanazawa H, Kawaguchi T, Fujii T, Shoji S, Hirata K, Kudoh S, Kurihara N, et al. (1996) Potentiation of the bronchoprotective effects of vasoactive intestinal peptide, isoprenaline, and theophylline against histamine challenge in anaesthetised guinea pigs by adrenomedullin. Thorax 51:1199–1202[Abstract]
Khan S, Michaud D, Moody TW, Anisman H, Merali Z (1999) Effects of acute restraint stress on endogenous adrenomedullin levels. Neuroreport 10:2829–2833[Medline]
Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T (1993) Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192:553–560[CrossRef][Medline]
Klika E, Scheuermann DW, De Groodt-Lasseel MHA, Bazantova I, Switka A (1999) A scanning and transmission electron microscopy study of the parabronchial unit in quail (Coturnix coturnix) and town pigeons (Columba livia). Scanning 21:273–284[Medline]
Kohno M, Hanehira T, Hirata K, Kawaguchi T, Okishio K, Kano H, Kanazawa H, et al. (1996) An accelerated increase of plasma adrenomedullin in acute asthma. Metabolism 45:1323–1325[CrossRef][Medline]
Lehr HA, van der Loos CM, Teeling P, Gown AM (1999) Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis. J Histochem Cytochem 47:119–126
López J (1995) Anatomy and histology of the lung and air sacs of birds. In Pastor LM, ed. Histology, Ultrastructure and Immunohistochemistry of the Respiratory Organs in Non-Mammalian Vertebrates. Murcia, Spain, Servicio de Publicaciones de la Universidad de Murcia, 178–233
López J, Cuesta N (2002) Adrenomedullin as a pancreatic hormone. Microsc Res Tech 57:61–75[CrossRef][Medline]
López J, Cuesta N, Cuttitta F, Martínez A (1999a) Adrenomedullin in non-mammalian vertebrate pancreas: an immunocytochemical study. Gen Comp Endocrinol 115:309–322[CrossRef][Medline]
López J, Cuesta N, Martínez A, Montuenga L, Cuttitta F (1999b) Proadrenomedullin N-terminal 20 peptide (PAMP) immunoreactivity in vertebrate juxtaglomerular granular cells identified by both light and electron microscopy. Gen Comp Endocrinol 116:192–203[CrossRef][Medline]
López J, Martínez A (2002) Cell and molecular biology of the multifunctional peptide, adrenomedullin. Int Rev Cytol 221:1–92[Medline]
Lorz C, López J (1997) Incidence of air pollution in the pulmonary surfactant system of the pigeon (Columba livia). Anat Rec 249:206–212[CrossRef][Medline]
Martínez A, Miller MJ, Catt KJ, Cuttitta F (1997) Adrenomedullin receptor expression in human lung and in pulmonary tumors. J Histochem Cytochem 45:159–164
Martínez A, Miller MJ, Unsworth EJ, Siegfried JM, Cuttitta F (1995) Expression of adrenomedullin in normal human lung and in pulmonary tumors. Endocrinology 136:4099–4105[Abstract]
Matsui H, Shimosawa T, Itakura K, Guanqun X, Ando K, Fujita T (2004) Adrenomedullin can protect against pulmonary vascular remodeling induced by hypoxia. Circulation 109:2246–2251
Müller B, Seifart C, Barth PJ (1998) Effect of air pollutants on the pulmonary surfactant system. Eur J Clin Invest 28:762–777[CrossRef][Medline]
Nossaman BD, Feng CJ, Cheng DY, Dewitt BJ, Coy DH, Murphy WA, Kadowitz PJ (1995) Comparative effects of adrenomedullin, an adrenomedullin analog, and CGRP in the pulmonary vascular bed of the cat and rat. Life Sci 56:PL63–66[CrossRef][Medline]
Okumura M, Kai H, Arimori K, Iwakiri T, Hidaka M, Shiramoto S, Isohama Y, et al. (2000) Adrenomedullin increases phosphatidylcholine secretion in rat type II pneumocytes. Eur J Pharmacol 403:189–194[CrossRef][Medline]
Ono Y, Okano I, Kojima M, Okada K, Kangawa K (2000) Decreased gene expression of adrenomedullin receptor in mouse lungs during sepsis. Biochem Biophys Res Commun 271:197–202[CrossRef][Medline]
Putman E, Boere AJ, van Bree L, van Golde LM, Haagsman HP (1995) Pulmonary surfactant subtype metabolism is altered after short-term exposure. Toxicol Appl Pharmacol 134:132–138[CrossRef][Medline]
Putman E, Liese W, Voorhout WF, van Bree L, van Golde LM, Haagsman HP (1997) Short-term ozone exposure affects the surface activity of pulmonary surfactant. Toxicol Appl Pharmacol 142:288–296[CrossRef][Medline]
Richards AM, Nicholls MG, Lewis L, Lainchbury JG (1996) Adrenomedullin. Clin Sci 91:3–16[Medline]
Risco C, Romero C, Bosch MA, da Silva PP (1994) Type II pneumocytes revisited: intracellular membranous systems, surface characteristics and lamellar body secretion. Lab Invest 70:407–417[Medline]
Rüstow B, Haupt R, Stevens P, Kunze D (1993) Type II pneumocytes secrete vitamin E together with surfactant lipids. Am J Physiol 265:L133–139
Saito T, Itoh H, Chun TH, Fukunaga Y, Yamashita J, Doi K, Tanaka T, et al. (2001) Coordinate regulation of endothelin and adrenomedullin secretion by oxidative stress in endothelial cells. Am J Physiol Heart Circ Physiol 281:H1364–371
van Golde LM, Batenburg JJ, Robertson B (1988) The pulmonary surfactant system: biochemical aspects and functional significance. Physiol Rev 68:374–455
Wright JR, Clements JA (1987) Metabolism and turnover of lung surfactant. Am Rev Respir Dis 136:426–444[Medline]
Yang BC, Lippton H, Gumusel B, Hyman A, Mehta JL (1996) Adrenomedullin dilates rat pulmonary artery rings during hypoxia: role of nitric oxide and vasodilator prostaglandins. J Cardiovasc Pharmacol 28:458–462[CrossRef][Medline]
Yoshibayashi M, Kamiya T, Kitamura K, Saito Y, Kangawa K, Nishikimi T, Matsuoka H, et al. (1997) Plasma levels of adrenomedullin in primary and secondary pulmonary hypertension in patients <20 years of age. Am J Cardiol 79:1556–1558[CrossRef][Medline]
Zudaire E, Simpson SJ, Illa I, Montuenga LM (2004) Dietary influences over proliferating cell nuclear antigen expression in the locust midgut. J Exp Biol 207:2255–2265
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact |