Serotonin-like immunoreactive cells in the pulmonary epithelium of ancient fish species

Summary The pulmonary mucosa of three species of ancient fish was studied immunohistochemically to show the distribution of serotonin, regarded as the main monoamine of mammalian bronchopulmonary paraneurons. Serotonin-like immunoreactive cells, dispersed through the airway epithelium as single cells, were found in all the fish species studied. They are presumably equivalent to the neuroendocrine cells reported in the lungs of mammalian and submammalian vertebrates. However, the precise role and the function of these cells remain unknown. Since the species studied belong to the most primitive extant groups of ancient fish, the present investigation suggests that serotonin is widely distributed in the lungs of the vertebrates. Several peptides, known to be specific cytochemical markers for the identification of the pulmonary neuroendocrine cells of mammals, are being investigated in the lungs of the fish species studied. They may help to trace the phylogeny of the pulmonary neuroendocrine cell system and to elucidate its function in lower vertebrates.     

Immunocytochemical Localization of Serotonin and Neuropeptides in the Neuroendocrine Paraneurons of Teleost and Lungfish Gills*

Neuroendocrine (NE) cells in the gills of six fish species were identified by serotonin and peptide immunohistochemistry. They occur either as single cells or as cell clusters within the filamental epithelium. Two populations of NE cells were identified that exhibit an immunoreactivity either for serotonin alone or for met-enkephalin and leu-enkephalin co-distributed in the same cells. We demonstrate for the first time the occurrence of open-type endocrine cells in the fish gill epithelium and confirm the presence of only serotonin-immunopositive cells of the closed type. The NE cells of the species studied do not react positively to other neuroendocrine markers occurring in the mammalian lung. Great differences probably exist in the expression of these epitopes on antigenic structures among various animal species. Although the physiological role of neuropeptides and serotonin in the fish gill NE cells is unknown, the presence of these molecular markers is of interest not only from an evolutionary aspect, but also in terms of a potential animal model to study the supposed functions of the airway neuroendocrine system in the vertebrates.     

Fine structure and serotonin immunohistochemistry of the neuroendocrine cells in the lungs of the bichirs Polypterus delhezi and P. ornatipinnis

Neuroendocrine cells (NE) occurring in the pulmonary epithelium of the fishes Polypterus delhezi and P. ornatipinnis are studied by electron microscopy and by immunostaining for serotonin which is often present in such cells in the mammalian lung. With the electron microscopy NE are found to occur single, resting upon the basement membrane and forming a narrow cytoplasmic extension towards the air lumen. They contain dense-cored vesicles of 80-165 nm which form exocytotic profiles at the level of the basal membrane. An immunoreactivity for serotonin is demonstrated for the first time in the NE of these species. The role of this mediator may involve a paracrine or endocrine function as postulated for the respiratory neuroendocrine mammalian cells. NE of the species studied are considered similar to those found within the wall of lung airways in mammals and submammalian vertebrates. Although much immunocytochemical investigations remain to be executed, they may also be included in the APUD (or DNES) cell system.     

An immimohistochemical study of regulatory peptides in lungs of amphibians

Summary The lungs of five species of European Anura and one species of Urodela (Triturus alpestris) have been studied by immunohistochemical methods to determine the occurrence, localization and distribution of serotonin, neuron-specific enolase, and eight regulatory peptides reported in the mammalian respiratory tract.Single and groups of serotonin-immunoreactive cells, corresponding to neuroendocrine cells of the mammalian lung, were identified in lungs of all amphibian species studied. Immunoreactivity for neuron-specific enolase was localized mainly in pulmonary nerves, nerve cell bodies and neuroendocrine cells. The localization and distribution of regulatory peptides varied among species. Bombesin and gastrin-releasing peptide immunoreactivities (predominant peptides in human lung) were localized mostly in submucosal nerves. Single bombesin-immunoreactive cells were found only in lungs of Urodela, i.e., Triturus alpestris. Occasional single cells, immunoreactive for somatostatin and leu-enkephalin were identified in lungs of Bombina variegata and a few cholecystokinin-immunoreactive cells in Hyla arborea. In all anuran species, numerous substance P-immunoreactive nerves were identified in submucosa, pulmonary septa and around blood vessels. No immunoreactive cells or nerves were demonstrated with antibodies against calcitonin and vasoactive intestinal peptide.The term pulmonary neuroendocrine (NE) cells (used here) does not imply neural origin or classical endocrine function for these cells, but rather indicates their potential involvement in neurohormonal regulation of pulmonary function (Cutz 1982)Supported by grant to E.C. from Medical Research Council of Canada (MT-7641)     

Evolutionary significance of the phylogenetic distribution of the mammalian hypothalamic releasing hormones

The hypophysiotrophic hormones isolated from the mammalian hypothalamus are distributed throughout the nervous system of vertebrate species. Although their role in regulating pituitary hormone secretion in mammals is clear, a similar function in lower species has not been established. Thyrotropin-releasing hormone is unable to stimulate thyroid function in amphibia and fish, despite being present in the hypothalamus and brain of these species of high concentration. The tripeptide is also found in high concentration in frog skin, a tissue derived from (or programed by) primitive neuroectoderm that is also a rich source of other peptides structurally related to neural peptides located in mammalian brain and gut. Luteinizing hormone-releasing hormone (LHRH) is able to activate gonadotropin secretion in submammalian species but there is evidence that the LHRH material present in avian, reptilian, and piscine brain is not identical to the mammalian decapeptide. An LHRH-like material present in frog sympathetic ganglia appears to function as a neurotransmitter in this location. Somatostatin is present in high concentrations in the hypothalamus, brain, pancreas, and gastrointestinal tract of all vertebrates and chromatographically is identical to the mammalian material, suggesting that this peptide is an "ancient" molecule with an important role in neuronal pancreatic and digestive function. The hypothalamic releasing hormones are part of a family of neural peptides that have a widespread anatomic and phylogenetic distribution and form a diffuse neuroendocrine system. It an material, suggesting that this peptide is an "ancient" molecule with an important role in neuronal pancreatic and digestive function. The hypothalamic releasing hormones are part of a family of neural peptides that have a widespread anatomic and phylogenetic distribution and form a diffuse neuroendocrine system. It an material, suggesting that this peptide is an "ancient" molecule with an important role in neuronal pancreatic and digestive function. The hypothalamic releasing hormones are part of a family of neural peptides that have a widespread anatomic and phylogenetic distribution and form a diffuse neuroendocrine system. It appears likely that the releasing hormones initially arose with a neurocrine or paracrine function, and that only later in evolution did they acquire the role of regulating adenohypophysial secretion.     

Effect of calcium channel blockers on serotonin uptake

We previously demonstrated that verapamil inhibits serotonin uptake by bovine pulmonary arterial endothelial cells by a mechanism not involving alterations in calcium fluxes. In this study, we determine whether verapamil inhibition of serotonin uptake occurs in other pulmonary cell types (bovine pulmonary artery smooth muscle cells), in cells from other organs and species (rat epididymal endothelial cells), and in intact organs (isolated rat lungs). We also compare the effects of verapamil with those of nifedipine and diltiazem. At concentrations of 10(-6) M or greater, verapamil is an inhibitor of serotonin uptake by cultured cells and isolated lungs. Nifedipine and diltiazem are weak inhibitors of serotonin uptake by cultured bovine cells only at suprapharmacologic doses and have no effect on serotonin uptake by isolated lungs. Surprisingly, nifedipine stimulates serotonin uptake by rat epididymal endothelial cells. We conclude that inhibition of serotonin uptake by verapamil is a generalized phenomenon, occurring in a variety of cell types, in intact organs, and in different species that does not occur consistently with other calcium channel blockers.     

Serotonin-like immunoreactivity in the epidermal club cells of teleost fishes

The present immunocytochemical study concerns the distribution of serotonin in the epidermis of three species of teleost fish. Serotonin-like immunoreactivity was found in the club cells of Heteropneustes fossilis and Carapus acus but not in those from the sea eel Conger conger. This study is the first immunocytochemical identification of serotonin in the club cells of teleost epidermis. By comparing data from the literature (Zaccone et al. 1986, 1987, 1988) regarding the occurrence of serotonin and GRP/bombesin in the exocrine sacciform gland cells of piscine skin, it is worthy mentioning here that the serotonin contained in the club cells of the species studied may have the ability to affect the pheromonal or other possible functions of these cells. The presence of serotonin in these systems has been correlated with the capacity of the exocrine glands of fish skin to secrete, ectopically, amine messengers in contrast to those produced eutopically i.e. in the neuron-paraneuron system in some vertebrates (Fujita et al. 1988).     

Pulmonary neuroendocrine cells in physiology and pathology

Pulmonary neuroendocrine cells are scant and widespread within the pulmonary epithelium. The function they play is not fully known, more studies are needed to clearly define it. They have been implicated however, as either the culprit or victim of many pulmonary diseases. That is the reason, why so many scientists take interest in the pulmonary neuroendocrine system. This paper reviews current information regarding pulmonary neuroendocrine cells, their origin, morphology, ontogeny, role, neuroendocrine cell markers, dysplasia and hyperplasia of pulmonary neuroendocrine cells in various conditions, diffuse idiopathic pulmonary neuroendocrine cell hyperplasia, typical carcinoid, atypical carcinoid, small-cell lung carcinoma, large-cell neuroendocrine carcinoma and the unusual spectrum of pulmonary neuroendocrine tumours.     

Localization of hematopoietic tissue in sturgeon

Morphological and autoradiographic studies on various hemopoietic tissues in sturgeon are presented. The classic hemopoietic organs characteristic of lower vertebrates, such as the kidney and spleen, are studied, as well as unique hemopoietic structures described only in the evolutionarily most ancient fish species (hemopoietic tissues of cartilaginous skull capsules and epicardium). The intensity of cell divisions in hemopoietic foci has been characterized by autoradiography. The results obtained provide a basis for the revision of traditional views about the phylogeny of hemopoiesis. They provide evidence that the osteogenic gravitation of hemopoietic tissue shows up in evolution alongside the appearance of the inner skeleton.     

Study on Localization of Hemopoietic Tissue in Sturgeon

Morphological and autoradiographic studies on various hemopoietic tissues in sturgeon are presented. The classic hemopoietic organs characteristic of lower vertebrates, such as the kidney and spleen, are studied as well as unique hemopoietic structures described only in the evolutionarily most ancient fish species (hemopoietic tissues of cartilaginous skull capsules and epicardium). The intensity of cell divisions in hemopoietic foci has been characterized by autoradiography. The results obtained provide a basis for the revision of traditional views about the phylogeny of hemopoiesis. They provide evidence that the osteogenic gravitation of hemopoietic tissue shows up in evolution alongside the appearance of the inner skeleton.     

Apparent normal lung architecture in protein tyrosine phosphatase-sigma-deficient mice

Protein tyrosine phosphatase-sigma (PTP-sigma) is a member of the mammalian LAR family of phosphatases, which is characterized by a cell adhesion-like ectodomain, a single transmembrane segment, and two tandemly repeated intracellular catalytic domains. The expression of PTP-sigma is developmentally regulated in epithelial, neuronal, and neuroendocrine tissues. We previously showed that PTP-sigma is strongly expressed within the fetal, but not adult, rat lung and is localized to the Clara cells and type II pneumocytes. In view of the developmentally regulated pulmonary expression of PTP-sigma, we performed a detailed histological and ultrastructural study of the lungs of PTP-sigma knockout mice we have generated. Our findings indicate no apparent structural abnormalities in the lungs of PTP-sigma-/- mice, including airway and alveolar epithelium. In addition, pulmonary neuroendocrine cells also appear normal, in contrast to pituitary, pancreatic, and gastrointestinal endocrine cells, in the knockout mice, suggesting different developmental regulation of these neuroendocrine cells. These observations suggest compensation for the absence of PTP-sigma during development by related family member phosphatases, such as LAR.     

Serotonin-like immunoreactivity in the epidermal club cells of teleost fishes

Summary The present immunocytochemical study concerns the distribution of serotonin in the epidermis of three species of teleost fish. Serotonin-like immunoreactivity was found in the club cells of Heteropneustes fossilis and Carapus acus but not in those from the sea eel Conger conger. This study is the first immunocytochemical identification of serotonin in the club cells of teleost epidermis. By comparing data from the literature (Zaccone et al. 1986, 1987, 1988) regarding the occurrence of serotonin and GRP/bombesin in the exocrine sacciform gland cells of piscine skin, it is worthy mentioning here that the serotonin contained in the club cells of the species studied may have the ability to affect the pheromonal or other possible functions of these cells. The presence of serotonin in these systems has been correlated with the capacity of the exocrine glands of fish skin to secrete, ectopically, amine messengers in contrast to those produced eutopically i.e. in the neuron-paraneuron system in some vertebrates (Fujita et al. 1988).     

Somatostatin family of peptides and its receptors in fish

Somatostatin (SRIF or SS) is a phylogenetically ancient, multigene family of peptides. SRIF-14 is conserved with identical primary structure in species of all classes of vertebrates. The presence of multiple SRIF genes has been demonstrated in a number of fish species and could extend to tetrapods. Three distinct SRIF genes have been identified in goldfish. One of these genes, which encodes [Pro2]SRIF-14, is also present in sturgeon and African lungfish, and is closely associated with amphibian [Pro2,Met13]SRIF-14 gene and mammalian cortistatin gene. The post-translational processing of SRIF precursors could result in multiple forms of mature SRIF peptides, with differential abundance and tissue- or cell type-specific patterns. The main neuroendocrine role of SRIF-14 peptide that has been determined in fish is the inhibition of pituitary growth hormone secretion. The functions of SRIF-14 variant or larger forms of SRIF peptide and the regulation of SRIF gene expression remain to be explored. Type 1 and type 2 SRIF receptors have been identified from goldfish and a type 3 SRIF receptor has been identified from an electric fish. Fish SRIF receptors display considerable homology with mammalian counterparts in terms of primary structure and negative coupling to adenylate cyclase. Although additional types of receptors remain to be determined, identification of the multiple gene family of SRIF peptides and multiple types of SRIF receptors opens a new avenue for the study of physiological roles of SRIF, and the molecular and cellular mechanisms of SRIF action in fish.     

The thymus in fish: Development and possible function in the immune response

With the exception of Agnatha, fish possess the functional equivalent of the thymus gland found in higher vertebrates. As in other vertebrates, this gland originates from the pharyngeal pouches and ontogenically is the first lymphoid organ to be infiltrated with lymphoid cells. Histology of the structure may differ from one species to another but the cellular component is basically similar. The (paired) gland is surrounded by an epithelial capsule. Within the gland a framework of reticulo-epithelial cells supports the lymphocytes. The age-related involution process, which characterizes the thymus of higher vertebrates, does not necessarily occur in fish. Nevertheless, thymus growth and function may be modulated by those factors that induce its involution such as aging, season, sexual maturity, and stress. The major role played by the thymus in the immune response of higher vertebrates is presumed to occur in fish. Thymus-derived cell dependent immune reactions have been demonstrated in fish. The cells that mediate these functions are designated as T-like cells. So far, cell surface markers equivalent to those of mammalian T lymphocytes have not been characterized. The T lymphocyte specificities are supposed to be acquired within or via the thymic microenvironment. Unfortunately, there is limited data concerned with the cytological and physiological basis of the maturation of thymus-derived cells. Direct involvement of the fish thymus in defense mechanisms has not been investigated extensively. The gland appears to be weakly protected because of its superficial location and is easily exposed to pathogens. Neoplasia is the main pathologic condition reported in the thymus of fish, with little else having been published regarding thymic pathology.     

Orthologous nature of mammaliam insulin genes

Summary Investigation of 7 insulin sequences from a bony fish, a bird, and 5 mammalian species showed that guinea pig and coypu insulin, that have a strongly divergent primary and quarternary structure, are not the result of gene duplication in an ancient vertebrate or invertebrate ancestor but that they diverged from the other mammals after divergence of the mammals from the other vertebrates. After this divergence both insulins underwent evolution at a highly increased rate.     

Invertebrate and fish cytokines

Cytokine-like molecules are well described in invertebrates, although most recent studies have revealed that there is analogy, rather than homology, between invertebrate and vertebrate cytokine-like activities. Cytokines certainly appeared early in the evolution of vertebrates, dating back some 400 millions years. Here, evidence will be reviewed and updated of the presence of these molecules in jawed fish and in particular, in bony fish, which represent the oldest group displaying true functionality of immune system as known in modern vertebrates. Many studies during the last ten years have confirmed the presence of functional homologues of mammalian cytokines in fish. In this review, particular attention will be focussed on IL-1beta, a very ancient defence cytokine recently sequenced in two species, rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio). Original data on the partial peptide sequence of IL-1beta in the mediterranean sea bass Dicentrarchus labrax are also presented.     

Distribution patterns of the paraneuronal endocrine cells in the skin, gills and the airways of fishes as determined by immunohistochemical and histological methods

Summary The neuro-endocrine cells of fish skin and respiratory surfaces, and their bioactive secretion as far as is known, are reviewed, and compared with similar elements in tetrapods, particularly amphibians. In the skin of teleost fish, immunohistochemistry has shown that Merkel cells react for serotonin, neuron-specific enolase and enkephalins. The pharmacology is not established in dipnoans or lampreys. In some teleosts, neuromasts react for substance P and leu-enkephalins; substance P is also reported from some ampullary organs (electroreceptors). Taste buds of teleosts may react for enkephalin and substance P. Basal cells of taste buds react for serotonin and neuron-specific enolase. Some unicellular skin glands of teleosts express bioactive compounds, including serotonin and some peptides; this ectopic expression is paralleled in amphibian skin glands. The dipnoan Protopterus has innervated pulmonary neuro-endocrine cells in the pneumatic duct region with dense-cored vesicles. In Polypterus and Amia the lungs have serotonin-positive neuro-endocrine cells that are apparently not innervated. In fish gills, a closed type of neuro-endocrine cell reacts for serotonin, an open type for enkephalins and some calcium-binding proteins (calbindin, calmodulin and S-100 protein). The functions of neuro-endocrine cells in fishes await investigation, but it is assumed they are regulatory.     

Changes in goldfish retinal ganglion cells during axonal regeneration

Recent work suggests that mammalian retinal ganglion cells may become more like developing ganglion cells in form while regenerating through a peripheral nerve graft. We have injected Lucifer Yellow into regenerating ganglion cells of goldfish to look for similar changes. Within three weeks of injury, we saw dye-coupling to nearby cells, which is a common developmental feature in many species. Dendrites and axons, which in most mature ganglion cells are smooth, became varicose and hairy, like those examined in mammalian development. Secondary axons arose later, not only as side-branches of the primary axon but also from the soma, as in mammalian development and regeneration. Since, in fish, these responses are clearly an intrinsic part of functional regeneration, their equivalence in fish and mammals strengthens the view that a similar regenerative competence may exist in the retinal ganglion cells of all vertebrates.     

New insights into ancient seasonal life timers

Organisms must adapt to seasonal changes in the environment and time their physiology accordingly. In vertebrates, the annual change in photoperiod is often critical for entraining the neuroendocrine pathways, which drive seasonal metabolic and reproductive cycles. These cycles depend on thyroid hormone (TH), reflecting its ancestral role in metabolic control. Recent studies reveal that - in mammals and birds - TH effects are mediated by the hypothalamus. Photoperiodic manipulations alter hypothalamic TH availability by regulating the expression of TH deiodinases (DIO). In non-mammalian vertebrates, light acts through extraretinal, 'deep brain' photoreceptors, and the eyes are not involved in seasonal photoperiodic responses. In mammals, extraretinal photoreceptors have been lost, and the nocturnal melatonin signal generated from the pineal gland has been co-opted to provide the photoperiodic message. Pineal function is phased to the light-dark cycle by retinal input, and photoperiodic changes in melatonin secretion control neuroendocrine pathway function. New evidence indicates that these comparatively divergent photosensensory mechanisms re-converge in the pars tuberalis of the pituitary, lying beneath the hypothalamus. In all vertebrates studied, the pars tuberalis secretes thyrotrophin in a light- or melatonin-sensitive manner, to act on neighbouring hypothalamic DIO expressing cells. Hence, an ancient and fundamentally conserved brain thyroid signalling system governs seasonal biology in vertebrates.     

APUD cells the human pulmonary neuroendocrine system

Literature data are presented on the anatomy of the diffuse neuroendocrine system (DNES) of the human lungs. The following subjects are discussed: physiological effects of the biologically active substances synthesized, stored, and secreted by the pulmonary neuroendocrine cells; their immunomodulating function; and the role of the DNES in the pathogenesis of bronchial asthma and other bronchopulmonary diseases.