Comparative histological overview of the chemical coding of the pulmonary neuroepithelial endocrine system in health and disease.

Pulmonary neuroepithelial endocrine cells have been shown to contain serotonin-immunoreactivity in almost every species studied. Regulatory peptides, of which at least ten have been reported so far, were mostly only demonstrated in a number of the investigated species or in a subpopulation of neuroepithelial endocrine cells. Calcitonin gene-related peptide, calcitonin, bombesin/gastrin-releasing peptide, enkephalin, somatostatin, substance P, cholecystokinin and polypeptide YY were found in normal lung tissues, whereas ACTH and several other bioactive substances should be regarded as ectopic. The human pulmonary neuroepithelial endocrine system seems to harbour the largest spectrum of bioactive mediators. The distribution patterns of bioactive substances in various subpopulations of solitary neuroepithelial endocrine cells or neuroepithelial bodies and in different cells of a single neuroepithelial body reveal a great complexity. Therefore, further research is needed to elucidate the chemical coding of this system.     

Localization of serotonin, cholecystokinin and somatostatin immunoreactivity in the lower respiratory tract of embryonic, foetal and postnatal sheep.

The lower respiratory tract of the sheep was studied by light-microscopical immunocytochemistry for serotonin, cholecystokinin, somatostatin, bombesin and calcitonin during different periods of lung development; embryonic, foetal and postnatal. At embryonic period only intraepithelial serotonin-containing cells as solitary neuroendocrine cells (NEC) and neuroepithelial bodies (NEB) were found. At foetal stages, immunoreactive cells to serotonin, cholecystokinin and somatostatin were observed in airway epithelium, as solitary NEC and NEBs, and in autonomic intrapulmonary ganglia as single or clusters of small intensely-fluorescent (SIF) cells. In postnatal sheep, serotonin- and cholecystokinin-containing cells were found within airway mucosa as solitary NECs and NEBs. No immunoreactive cells were observed with antiserum to bombesin and calcitonin. Quantitative studies showed that serotonin was the predominant substance, and that solitary neuroendocrine cells were more numerous in distal conducting airways and at foetal stages.     

Immunoreactivity for calcitonin gene-related peptide in neuroepithelial bodies of the newborn cat

Summary Immunoreactivity for calcitonin gene-related peptide is demonstrated for the first time in neuroepithelial bodies in the lung of newborn cats after Bouin fixation and embedding in paraffin. The intense staining clearly identifies these bodies at the level of bronchioli and alveoli. Occasionally, single neuroepithelial endocrine cells, displaying immunoreactivity for calcitonin gene-related peptide are observed. In the kitten lung, identification and localization of neuroepithelial bodies after immunocytochemical staining for calcitonin gene-related peptide are superior to the analysis based on other techniques, i.e., the argyrophilic reaction, periodic acid Schiff-lead hematoxylin method, and immunocytochemical staining for serotonin. The serial-section technique revealed that in neuroepithelial bodies of the newborn kitten lung, immunoreactivity for calcitonin gene-related peptide coexists with immunoreactivity for serotonin in individual cells. The functional significance of the calcitonin gene-related peptide in neuroepithelial bodies remains to be elucidated.     

Regulatory peptides in the gastrointestinal tract of Alligator mississipiensis

Summary The gastrointestinal tract of the alligator Alligator mississipiensis has been investigated for the presence of immunoreactivity to fourteen regulatory peptides all known to occur in the mammalian gut system.Mucosal endocrine cells reacting specifically with the antisera to neurotensin, C-terminal gastrin, somatostatin, bombesin, secretin, pancreatic glucagon and enteroglucagon were detectable, the distribution of these cells being, in general, similar to the mammalian pattern. Peripheral nerve cell bodies and nerve fibres were detected with the antisera to vasoactive intestinal polypeptide, substance P, bombesin and somatostatin again with a distribution similar to that seen in mammals.No immunoreactivity was observed with the available antisera to glicentin, motilin, gastric inhibitory polypeptide, gastrin 34, cholecystokinin 9–20 and met-enkephalin.     

Evidence for the coexistence of serotonin and calcitonin gene-related peptide at the subcellular level in neuroepithelial bodies in the lung of a marsupial,Isoodon macrourus

Summary The coexistence of serotonin and calcitonin gene-related peptide (CGRP) in neuroepithelial bodies of the bandicoot, Isoodon macrourus, has been examined using immunocytochemistry at the light- and electronmicroscope levels. The avidin-biotin technique of antigen localisation was used initially to identify serotonin-like and CGRP-like immunoreactivity (-LI). Serotonin-LI and CGRP-LI were found in neuroepithelial cells in the lungs of 30-day-old bandicoots. CGRP-LI could also be demonstrated in nerve fibres associated with some neuroepithelial bodies. The protein A-gold technique of antigen localisation was used to label neuroepithelial cells and nerve fibres at the subcellular level. Serotonin-LI and CGRP-LI were observed in the same dense-cored vesicles of most neuroepithelial cells; however, some neuroepithelial cells were shown to possess serotonin-LI without CGRP-LI. Nerve fibres immediately adjacent to neuroepithelial bodies exhibited mainly CGRP-LI. These results show that serotonin-LI and CGRP-LI are present in neuroepithelial cells of the bandicoot in the same secretory vesicles. This pattern of co-localisation may reflect co-ordinated or synergistic actions of these two neuroactive substances.     

Ultrastructural localization of serotonin in the intrapulmonary neuroepithelial bodies of neonatal rabbits by use of immunoelectron microscopy

Summary The ultrastructural localization of serotonin in neuroepithelial bodies (NEB) of neonatal rabbits was examined by use of the recently developed immunogold-staining method. In the granulated epithelial cells of NEB serotoninimmunoreactive material could be demonstrated only in dense-cored vesicles (DCV) of the first type. No immunoreactivity was observed in DCV of the second type. The present findings support the previously made suggestion that the corpuscular NEB cells contain two distinctive DCV types which react differently with the immunogold-staining procedure for identifying serotonin. NEB, known as intrapulmonary sources of serotonin (and peptide substances), may play a prominent role in lung physiology.     

Immunocytochemical survey of the neuropeithelial endocrine system in the respiratory tract of the Tokyo salamander,Hynobius neubulosus tokyoensis Tago

The epithelial lining of the respiratory tract of urodeles has been shown to harbor an innervated system of neuroepithelial endocrine (NEE) cells. Even between phylogenetically closely related species, large differences have been reported in the appearance and chemical coding of the NEE system. Although urodeles are well suited for the purpose, none of the prior studies have provided an immunocytochemical survey of the NEE system in all parts of the respiratory tract. In the present study, many bioactive substances and a general marker were immunocytochemically demonstrated in serial sections of the entire respiratory tract of the Tokyo salamander, Hynobius nebulosus tokyoensis, a species in which neuroepithelial bodies (NEBs) were previously characterized at the electron microscopic level. In the current study, serotonin-immunoreactive solitary NEE cells were observed in variable numbers in the larynx, in all parts of the trachea, and in areas of the lungs covered with ciliomucous epithelium. Serotonin-containing NEBs, however, were detected in small cranial areas of the lung only. Solitary NEE cells were seen in the trachea and lungs of H. nebulosus tokyoensis by immunocytochemical staining for somatostatin, calcitonin, calcitonin gene-related peptide, and bombesin, but the number, localization, and appearance of the labeled NEE cells differed considerably. Only calcitonin-like immunoreactivity was also noted in some NEB-like cell clusters in the cranial parts of the lungs. Unlike many other vertebrates, neuron specific enolase was found to be a poor marker for the NEE system in the salamander species used in this investigation. It may be concluded that the NEE system of H. nebulosus tokyoensis contains at least five different bioactive substances. The different markers, however, demonstrate the presence of NEE cells with obvious differences in respect to appearance and topographical distribution. The necessity is emphasized of reliable methods for adequate sampling of all regions of the respiratory tract in comparative histological studies of the NEE system.     

An immunocytochemical study of endocrine pancreas of snakes

Summary The pancreas from eleven species of snakes representing both advanced and primitive families has been investigated for the presence of eleven regulatory peptides reported to occur in the mammalian endocrine pancreas. Of the eleven peptides studied, insulin, pancreatic glucagon and somatostatin were present in endocrine cells within the islets of all the species investigated. The neuropeptide, vasoactive intestinal polypeptide, was located within nerve terminals innervating the islets in the Boidinae, Colubrinae, Elaphidae and Crotalidae but absent from the Natricinae investigated.No immunoreactivity was demonstrable with the antisera to substance P, met-enkephalin, C-terminal gastrin, bombesin, glicentin and gastric inhibitory polypeptide. Pancreatic polypeptide-like immunoreactivity was demonstrable only in the boid snakes and exclusively stained by a C-terminal specific antiserum.     

Bombesin-like peptides in small cell lung cancer: biochemical characterization and secretion from a cell line

High intracellular levels of BN-like peptides are present in tumors and cell lines of small cell carcinoma of the lung (SCCL) as well as the putative precursor cells of this tumor, the pulmonary endocrine cell. In cell line NCI-H209 the density of bombesin-like peptides was 8.9 +/- 1.1 pmol/mg total protein. Gel filtration chromatography of an extract of these cells revealed one major peak of immunoreactivity which coeluted with synthetic bombesin (1620 daltons). Also, high pressure liquid chromatography revealed one major peak of immunoreactivity was present which eluted before synthetic peptide. Therefore, SCCL bombesin-like peptides may be of similar size but are more hydrophilic than synthetic peptide. Cells maintained in culture continuously release bombesin-like peptides into the growth medium. Also, high concentrations of K+ stimulated the secretion of immunoreactive bombesin from cell lines in a Ca++-dependent manner. These SCCL bombesin-like peptides may function as important regulatory agents in the malignant lung.     

Regulatory peptides in the pancreas of two species of elasmobranchs and in the Brockmann bodies of four teleost species

Summary Immunohistochemistry was used to localize regulatory peptides in endocrine cells and nerve fibres in the pancreas of two species of elasmobranchs (starry ray,Raja radiata and spiny dogfish,Squalus acanthias), and in the Brockmann bodies of four teleost species (goldfish,Carassius auratus, brown troutSalmo trutta, rainbow trout,Oncorhynchus mykiss and cod,Gadus morhua). In the elasmobranchs, the classical pancreatic hormones somatostatin, glucagon and insulin were present in endocrine cells of the islets. In addition, endocrine cells were labelled with antisera to enkephalins, FMRF-amide, gastrin/cholecystokinin-(CCK)/caerulein, neurotensin, neuropeptide Y (NPY), and peptide YY (PYY). Nerve fibres were demonstrated with antisera against bombesin, galanin and vasoactive intestinal polypeptide (VIP). These nerve fibres innervated the walls of blood vessels, in the exocrine as well as the endocrine tissue. In the four teleost species immunoreactivity to somatostatin, insulin and glucagon was intense in the Brockmann bodies. Cells were labelled with antisera to enkephalin, neurotensin, FMRFamide, gastrin/CCK/ caerulein, NPY, PYY and VIP. Only a few nerve fibres were found with antisera against dopamine--hydroxylase (DBH, cod), enkephalin (met-enkephalin-Arg-Phe, cod), bombesin (cod), gastrin/CCK/caerulein (cod) and VIP. Galanin-like-immunoreactive fibres were numerous in the Brockmann bodies of all teleosts examined. Immunoreactivity to calcitonin gene-related peptide (CGRP), substance P, tyrosine hydroxylase (TH), and phenyl-N-methyl transferase (PNMT) could not be found in any of the species studied.     

The nervous system of the chicken proventriculus: an immunocytochemical and ultrastructural study

The proventriculus constitutes the glandular region of the chicken stomach. This organ is innervated by two parasympathetic networks, the myenteric and submucous plexus, and here we present a systematic study of this system by immunohistochemistry and electron microscopy. All the neurons and fibres were positive for the neural markers, protein gene product 9.5 and the amidating enzymes. Immunoreactivities for the constitutive neuronal isoform of the enzyme nitric oxide synthase and the vasoactive intestinal peptide were present in neuronal bodies suggesting an intrinsic origin for the similarly immunoreactive fibres found in the proventriculus. On the other hand, immunoreactivity to gastric inhibitory peptide was only found in varicose fibres making contact with the blood vessels and the glandular epithelium, but never in the neuronal somas, suggesting that this substance may be provided by an extrinsic nervous system whose neuronal bodies are located elsewhere. Electron microscopy revealed frequent neuromuscular and neuroepithelial connections in the muscle layers, the wall of the blood vessels and the epithelium. In addition, synapsis-like structures were identified in the proximity of cells belonging to the diffuse endocrine system, providing a new example of neuroendocrine contacts. No positivity was found for antibodies against other neural substances including somatostatin, peptide histidine–isoleucine, peptide tyrosine–tyrosine, neuropeptide tyrosine, bombesin, met-enkephalin, serotonin, substance P, galanin, calcitonin gene-related peptide and S-100 protein.     

Calcitonin and calcitonin gene-related peptide immunoreactivity and colocalization in newborn cat lung.

Calcitonin (CT) and calcitonin gene-related peptide (CGRP) are generated by alternate RNA processing from alpha and beta CT/CGRP genes. In this report, an immunocytochemical investigation was undertaken on the occurrence and distribution of immunoreactive CT as well as its colocalization with CGRP in newborn cat bronchopulmonary endocrine cells. A widespread distribution of solitary endocrine cells and neuroepithelial bodies immunostained for CT was recorded within the lung. In all animals studied, CT immunoreactivity represents a subpopulation of CGRP positive cells, while the intrapulmonary nerve fibers contain only CGRP. To the best of our knowledge, this is the first time that CT and its colocalization with CGRP have been demonstrated immunocytochemically in the cat lung. Our results indicate, that different molecular processing of both CT/CGRP genes may be represented by different patterns in the cellular immunoreactivity of the synthetized peptides.     

The distribution of putative neurotransmitters in the nervous system of the dipteran Chironomus tentans insect larva: An immunohistochemical study using antisera to 5-hydroxytryptamine,tyrosine hydroxylase,methionine-enkephalin,proctolin and bombesin

Using the indirect immunofluorescence technique, the localization and distribution of transmitters, transmitter-related enzymes and neuropeptides was studied in the larvae of the dipteran species Chironomus tentans. Immunoreactivity could be seen for 5-hydroxytryptamine, tyrosine hydroxylase (the rate-limiting enzyme in the catecholamine synthesis), and the neuropeptides methionine-enkephalin (met-enk), proctolin and bombesin. The immunoreactivity was confined both to cell bodies as well as to nerve fibers within ganglia and along the alimentary canal. Furthermore, tyrosine hydroxylase immunoreactivity could also be seen in epithelial cells locally distributed along a short, middle part of the alimentary tract. These latter cells were regarded as endocrine-like cells. No immunoreactivity could be found with certainty for the enzyme phenylethanolamine-N-methyltransferase (PNMT) nor for the peptides vasoactive intestinal polypeptide (VIP), dynorphin, substance P, somatostatin, thyrotropin releasing hormone (TRH), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), neurotensin, galanin and cholecystokinin (CCK).     

Gastrin-releasing peptide (GRP,mammalian bombesin) in the pathogenesis of lung cancer

Established human lung cancer exhibits a complex pattern of genetic changes as well as several distinct autocrine growth factor loops for regulatory peptides. The best studied example is that of gastrin-releasing peptide (GRP), the mammalian homolog of the amphibian bombesin. It is produced by up to 70% of small cell lung cancers and 10–20% of non-small cell lung cancers. GRP stimulates the growth of normal bronchial epithelium as well as that of small cell lung cancer, and its blockade with the use of antibodies or synthetic antagonists inhibits the growth of these tumors. Study of its molecular biology has revealed a complex pattern of mRNA processing which has lead to the recent isolation of a novel family of peptides termed gastrin-releasing peptide gene-associated peptides (GGAPs), present in normal and malignant human tissues. Additional efforts have been directed at characterizing the GRP receptor as well as its intracellular signaling pathways which have been reported both as G protein phospholipase C coupled events as well as activation of a membrane associated tyrosine kinase. In view of its expression in normal bronchial epithelium and its mitogenic effects on this tissue, GRP appears to play a central role in the early events of pulmonary carcinogenesis.     

Bombesin-like peptides and receptors in human tumor cell lines

Human cancer cell lines were assayed for bombesin-like peptides and receptors. Acid extracts derived from small cell lung cancer, but not other types of cancer had high levels of immunoreactive bombesin. Regardless of patient treatment, site of tumor origin (bone marrow, lymph node, or pleural effusion) or culture conditions, small cell lung cancer cell lines had high levels of bombesin-like peptides. Thus, bombesin levels in small cell lung, but not other types of human cancer, are routinely elevated. Also, small cell lung cancer lines in contrast to other cell lines have a high density of binding sites for a radiolabeled bombesin analogue. The presence of high concentrations of bombesin-like peptides and receptors suggests that bombesin may function as an important regulatory agent in human small cell lung cancer.     

The presence of immunoreactive vertebrate bioactive peptide substances in hemocytes of the freshwater snailViviparus ater (gastropoda,prosobranchia)

1. Using an immunocytochemical procedure a wide range of immunoreactive vertebrate bioactive peptides (BAPs) has been found in hemocytes of Viviparus ater: bombesin, calcitonin, CCK-8, CCK-39, GH, glucagon, insulin, oxytocin, neurotensin, secretin, serotonin, somatostatin, substance P, vasopressin, and VIP. 2. No immunostaining was observed for antigastrin and antithyroglobulin antibodies. 3. The presence of BAP-like molecules in hemocytes suggests a correlation between hemocyte and APUD cells and is evidence of a relationship between the neuroendocrine and the immune systems.     

Recent advances and contraversies on the role of pulmonary neuroepithelial bodies as airway sensors

Pulmonary neuroepithelial bodies are polymodal sensors widely distributed within the airway mucosa of mammals and other species. Neuroepithelial body cells store and most likely release serotonin and peptides as transmitters. Neuroepithelial bodies have a complex innervation that includes vagal sensory afferent fibers and dorsal root ganglion fibers. Neuroepithelial body cells respond to a number of intraluminal airway stimuli, including hypoxia, hypercarbia, and mechanical stretch. This article reviews recent findings in the cellular and molecular biology of neuroepithelial body cells and their potential role as airway sensors involved in the control of respiration, particularly during the perinatal period. Alternate hypotheses and areas of controversy regarding potential function as mechanosensory receptors involved in pulmonary reflexes are discussed.     

An immunocytochemical and ultrastructural study of the larval anterior intestine of the frog Rana temporaria, with especial reference to endocrine cells

Endocrine cells of the larval intestine of Rana temporaria tadpoles have been identified by argyrophilic, immunocytochemical and electron-microscopical techniques. Scarce endocrine cells have been found in both the short non-absorptive zone immediately following the stomach, and in the rest of the anterior intestine. Endocrine cells are frequently seen to extend a cytoplasmic process towards the lumen. Immunoreactivity for serotonin, somatostatin, bombesin and cholecystokinin-8 has been detected. According to the ultrastructural traits of the endocrine granules, three larval intestinal endocrine populations have been differentiated.