Lectin histochemistry of epidermal glandular cells in the earthworm Lumbricus terrestris (Annelida Oligochaeta).

Carbohydrate residues were localized in the glandular cells of the epidermis of Lumbricus terrestris by lectin histochemistry. The following biotinylated lectins were used: ConA, PNA, WGA, UEA-I. Each lectin has a specific binding pattern in the epidermal glandular cells. The ConA binding is evident in the orthochromatic mucous cells; PNA in the metachromatic mucous cells; WGA in the neuroendocrine-like cells; UEA-I in the cuticle. The epidermal glandular cells possess specific sites for the different lectins in relation to their functional characteristics. Therefore, these sugar residues indicate different behaviours of the cells in epidermal functions related to ion transport, receptor-secretory processes and defence.

Neuronal nitric oxide synthase (nNOS) expression in the epithelial neuroendocrine cell system and nerve fibers in the gill of the catfish, Heteropneustes fossilis.

We studied immunohistochemically the localization of neuronal nitric oxide synthase (nNOS) in gills of an Indian catfish species, Heteropneustes fossilis. It is shown that most of the epithelial neuroendocrine cells that are present in gill filaments and lamellae stained positively. Co-localization of nNOS and endothelin was also shown in neuroendocrine cells. A dense plexus of nNOS-containing nerve fibers was present beneath the gill epithelium, associated with efferent filament arteries and the basal side of neuroendocrine cells. nNOS immunopositive neurons were not found in gill areas. nNOS immunopositive neuroendocrine cells appeared to differ from neuroepithelial cells in gills of various teleost species, which are considered as oxygen-sensitive receptors and are present in the distal halves of gill filaments. Other types of neuroendocrine cells have been identified previously in other areas of gills using antibodies to serotonin and endothelin peptides. These cell types are likely to be involved in chemical regulation of the physiology of gill cells. In relation to the function of the other cell types, our data on nNOS localization suggest that NO is a wide-spread transmitter in the gill of the Indian catfish. It may play a role both in the local regulation of vascular tone and in inhibitory innervation of the gill.

Immunohistochemical localization of bioactive substances in the epidermis of the earthworm, Lumbricus terrestris (Annelida, oligochaeta).

Serotonin (5HT), endothelin-big (ET-big) and neuron-specific enolase (NSE) are present in some epithelial paraneuronal cells of vertebrates. In the invertebrates, we show for the first time, by immunofluorescence and immunoperoxidase methods, the immunoreactions to antibodies raised to ET, 5-HT and NSE in the epidermis of the Lumbricus terrestris. It is assumed that the reactive cells are small granular cells that may have a paracrine action in the mechanisms of cell proliferation and secretory processes. The function of these bioactive substances in the epidermis of the earthworm awaits investigation. Phylogenetically it is very interesting that paraneuronal-like type of cells appear in such an ancient species of invertebrate which uses the epidermis for various functional activities.

Paraneurons in the gills and airways of fishes.

This chapter describes the distributional patterns of the neuroendocrine cells in the respiratory surfaces of fishes and their bioactive secretions which are compared with similar elements in higher vertebrates. The neuroendocrine cells in the airways of fishes differentiate as solitary and clustered cells, but the clusters are not converted into neuroepithelial bodies which are reported in terrestrial vertebrates. The dipnoan fish Protopterus has innervated neuroendocrine cells in the pneumatic duct region. In Polypterus and Amia the lungs have neuroendocrine cells that are apparently not innervated. Two types of neuroendocrine cells are found in the gill of teleost fishes. These cells are very different by their location, structure and immunohistochemistry. Advanced studies on functional morphology of neuroendocrine cells in fish airways are still necessary to increase our understanding of their multifunctional role in the gill area.

Localization of immunoreactive endothelin in the neuroendocrine cells of fish gill.

Immunohistochemical tests have demonstrated for the first time the presence of endothelins in the neuroendocrine cells of fish gill. We have sought co-localization of endothelins with serotonin and neuropeptides which are regarded as neuroendocrine markers of pulmonary diffuse neuroendocrine systems in higher vertebrates. Regarding their endocrine and paracrine activities in mammals, endothelins are considered as peptide hormones and growth factors regulating respiratory function. The roles of endothelins in the gill await investigation based on the multifunctional organization of this organ.

Enkephalin immunoreactivity in the paraneurons of the tiger salamander (Ambystoma tigrinum) tongue.

Immunohistochemical tests have demonstrated the presence of leu-5-enkephalin and other bioactive compounds (serotonin and neuron-specific enolase) in the basal cells of lingual taste buds in Ambystoma tigrinum; there was also a weak reaction for met-5-enkephalin. Similar reactions were obtained from particular cells dispersed within the lingual epithelium, which are provisionally identified as Merkel cells.

Cytokeratin type distribution in the skin and gill epithelia of the Indian freshwater catfish, Heteropneustes fossilis as detected by immunohistochemistry.

Based on the general cross-reactivity of the cytokeratins in vertebrates, we describe the immunoreactivity for some mammalian cytokeratins of both the epidermis and gill of H. fossilis. The following monoclonal antibodies, commercially supplied, were tested: K8.13, KL1, AE1 and AE3, which have a wide spectrum of specificity, and LDS-68, M 20, K8.60, KS-B17.2, K4.62, which are more narrowly specific. The reaction of the epithelial cells of the skin to K8.13 was negative in the basal layer, weakly positive in the layers above but strongly positive in some cells of the superficial layer. KL1 was negative in the basal layer, positive in the outer layers. AE1 was strongly positive in the basal layer, negative in the superficial cells. AE3 gave a general but weak reaction in the epithelial cells. K8.60 was negative for the epithelial cells, but reacted positively in the club cells. Club cells also reacted to K8.13 and AE1, and were strongly positive to KL1 and AE3. Goblet mucous cells were negative to all antibodies tested. In the gills, K8.13 labelled cells of both filament and lamellar epithelia. The reaction to AE3 was similar but less intense. KL1 was positive in the basal layer of the filament epithelium but not elsewhere, and K8.60 was negative throughout. AE1 and KS- B17.2 specifically labelled small cells scattered in the filament and lamellar epithelia, which are tentatively identified as neuroendocrine cells.

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

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.

Ectopic expression of bioactive peptides and serotonin in the sacciform gland cells of teleost skin.

Immunohistochemical methods identified serotonin, and the peptides bombesin and caerulein, in the skin of the teleosts Lepadogaster candollei and Mastacembelus erytrotaenia. In both species, the secretory content of epidermal sacciform cells reacted positively for all three substances. These results are compared with studies on the skin glands of amphibians, which also contain multiple active compounds, and on various neuroendocrine cells of fish. The precise functions of the secretions are not known.

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).

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

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.

Localization of calmodulin positive immunoreactivity in the surface epidermis of the brown trout, Salmo trutta.

Calmodulin is a Ca2+-dependent modulatory protein which is required in the general regulation of a large number of key processes of cellular metabolism. In the present study, the localization of calmodulin positive immunoreactivity in the epidermis of the brown trout, Salmo trutta was investigated using a specific mouse monoclonal antibody to calmodulin of IgG2 class. The immunoreaction was found only in the superficial epithelial cells that constitute the main histological site for the production of calmodulin positive substances. Because of its distribution, this protein might have a physiological significance in the activation of the microvillar skeleton and in the control of the permeability of the skin epithelium.

Occurrence of gastrin-releasing peptide-like and bombesin-like immunoreactivities in the epidermal sacciform gland cells of the clingfish Lepadogaster candollei.

Gastrin-releasing peptide (GRP)-like and bombesin (BOM)-like immunoreactivities were localized in the exocrine sacciform gland cells and in the surface epidermis of the clingfish Lepadogaster candollei with the use of previously characterized antibodies. The GRP-like and BOM-like peptides show resemblance to those immunohistochemically found in the dermal secretory products of the amphibian skin. The immunoreactive peptides tested in this study are unusual bioactive components of secretory sources of exocrine glands since these peptides were first demonstrated in cells of the neuroendocrine system. The results suggest that such bioactive compounds may exert regulatory activity in the absorptive processes of the skin epithelium and in influencing secretion release from exocrine complexes.

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae. A light- and electron-microscopic study.

The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone which was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.