MICROSCOPY RESEARCH AND TECHNIQUE 37:13–30 (1997) Neuroepithelial Bodies and Solitary Neuroendocrine Cells in the Lungs of Amphibia LUCYNA GONIAKOWSKA-WITALIŃSKA* Jagiellonian University, Department of Comparative Anatomy, Institute of Zoology, 30–060 Kraków, Poland KEY WORDS endocrine cells; ultrastructure; innervation; immunocytochemistry; lower vertebrates ABSTRACT In the lungs of 12 species of Amphibia investigated so far, solitary neuroendocrine (NE) cells, as well as groups of these cells called ‘‘neuroepithelial bodies’’ (NEB), are observed. They occur in the position strategic to monitoring gas composition, mainly in the ciliated epithelium of the apical part of the septa. A great diversity in the structure of NEB is observed. The NE cells and NEB in Amphibia are predominantly of the ‘‘closed type,’’ separated from the air space by a thin cytoplasmic layer of ciliated cells, goblet cells or pneumocytes. In two species, Bufo marinus and Ambystoma tigrinum, the ‘‘open type’’ of NEB occurs, where NEB communicate with the air space, by apical cells in Bufo and type II NE cells in Ambystoma. Both types of cells possess single atypical cilia with an 811 microtubule arrangement and microvilli on the free surface. Single and grouped NE cells are characterized by small dense core vesicles (DCV) dispersed in the cytoplasm. In Salamandra and Ambystoma the second type of NE cells with large DCV are observed. The DCV represent sites of storage of serotonin and several neuropeptides. The basal parts of NEB and of some solitary cells are invested by the intraepithelial sensory nerve terminals, both afferent and efferent morphologically. In the lungs of Amphibia, similarly to other vertebrates, NE cells, which act as endocrine/paracrine receptors, form epithelial endocrine systems. Microsc. Res. Tech. 37: 13–30, 1997. r 1997 Wiley-Liss, Inc. INTRODUCTION Solitary neuroendocrine cells (NE) have been observed in the respiratory epithelium of the airways and lungs of many vertebrates (Cutz and Conen, 1972; Fröhlich, 1949; Lauweryns and Peuskens, 1969; Lauweryns et al., 1972). A group of these cells connected by nerves was termed ‘‘neuroepithelial body’’ (NEB) by Lauweryns and Peuskens (1972). The investigations of NE cells have been mostly carried out on mammals (for review see Scheuermann, 1987; Sorokin and Hoyt, 1989). NE cells were also observed in all groups of vertebrates, in birds (Cook and King, 1969; Walsh and McLelland, 1974; Wasano and Yamamoto, 1979), reptiles (Pastor et al., 1987, 1989; Scheuermann et al., 1983; Wasano and Yamamoto, 1976), fish (Adriaensen et al., 1990; Zaccone et al., 1989a,b, 1994) and marsupials (Haller, 1992). The solitary NE cells as well as NEB were also found in the tailed and anuran amphibians (Adriaensen et al., 1994; Gomi et al., 1994; Goniakowska-Witalińska, 1980a, 1981, 1982; Goniakowska-Witalińska and Cutz, 1990; Goniakowska-Witalińska et al., 1990, 1992; Matsumura, 1985; Rogers and Haller, 1978, 1980; Scheuermann et al., 1989; Wasano and Yamamoto, 1978). The immunohistochemical and fluorescence investigations have shown that the NE cells contain chemical mediators such as serotonin (Adriaensen et al., 1994; Cutz et al., 1986; Gomi et al., 1994; Goniakowska-Witalińska et al., 1990, 1992; Rogers and Haller, 1978; Scheuerman et al., 1989), as well as several neuropeptide hormones r 1997 WILEY-LISS, INC. (Adriaensen et al., 1994; Bodegas et al., 1993; Cutz et al., 1986; Gomi et al., 1994; Goniakowska-Witalińska et al., 1992). Fujita (1977) and Fujita et al. (1988) indicated the functional relation of NE cells in the vertebrate airways to neurons and included these cells within the paraneuronic system. The reviewed studies on the endocrine epithelial cells in amphibians have particular significance for a better understanding of the trends in the evolution of these endocrine systems, from solitary NE cells to corpuscles (NEB) invested with the nerve endings. LIGHT MICROSCOPY—THE STRUCTURE OF AMPHIBIAN LUNGS The tailed amphibians (Urodela) are characterized by a simple saclike structure of the lung. The newts Triturus and Notophthalmus have a smooth internal surface in their lungs (Fig. 1). As in all Amphibia, this surface is mostly covered with the respiratory epithelium consisting of one type of pneumocyte. The ciliated epithelium is located exclusively along the pulmonary vein or in small patches along the pulmonary artery (Goniakowska-Witalińska, 1980a,b, 1994; Hightower et al., 1975). *Correspondence to: L. Goniakowska-Witalińska, Jagiellonian University, Department of Comparative Anatomy, Ingardena 6, 30–060 Kraków, Poland. E-mail: LWITAL@zuk.iz.uj.edu.pl Accepted 10 May 1995. 14 Figs. 1, 2. lungs. L. GONIAKOWSKA-WITALIŃSKA Schematic representations of the structure of amphibian Fig. 1. The lung of the newts, with smooth internal surface lined by ciliated epithelium (CE) with goblet (dotted) cells along the pulmonary artery (AR) and vein (V) and by respiratory epithelium (EP) on the remaining surface. The lung wall is composed of three layers: internal epithelium (EP), connective tissue (CT) with numerous capillaries (C), and external epithelium (ET). A, air space. The solitary NE cells and the NEB are dispersed in the ciliated epithelium along the pulmonary vein. In some species solitary NE cells may occur along the pulmonary artery and between pneumocytes. The internal surface of the lungs of salamanders, e.g., Salamandra, Megalobatracus, Hynobius and Ambystoma, is corrugated by low septa of Ist, IInd and IIIrd orders. The apical part of the Ist and IInd order septa are lined by ciliated epithelium with goblet cells, while the IIIrd order septa and remaining internal surface are lined by a network of capillaries covered with pneumocytes (Goniakowska-Witalińska, 1978, 1982; Goniakowska-Witalińska et al., 1992; Hashimoto et al., 1983; Matsumura, 1985; Meban, 1979). The internal air space of the lungs of anuran amphibians is partitioned into ‘‘alveolar chambers’’ by long septa deeply protruding toward the lung lumen (Fig. 2). Such chambers differ in size. The apical parts of the Ist, IInd and IIIrd order septa are headlike and are filled with veins and smooth muscle bundles (Ist and IInd order septa) or exclusively with smooth myocytes in the IIIrd order septa. The ciliated epithelium with goblet cells is located mainly on the apical part of the Ist and IInd order septa. The remaining internal surfaces are covered with one type of pneumocyte (Dierichs, 1975; Goniakowska-Witalińska, 1981, 1986, 1995; Gonia- Fig. 2. The lungs of anurans possess numerous septa of Ist (1), IInd (2) and IIIrd (3) order covered with ciliated epithelium (CE). The apical parts of the septa filled with smooth muscle cells (SM) and veins (V) surround the central air space (A). AR, artery; LW, the lung wall. The NE cells and NEB are located in the ciliated epithelium which covers the apical part of septa. In some species NEB may also occur on IIIrd order septa under pneumocytes. Solitary NE cells are dispersed in the lateral part of septa and covered by pneumocytes. kowska-Witalińska and Cutz, 1990; GoniakowskaWitalińska et al., 1990; Meban, 1973). Localization of NE Cells The localization of NE cells in the epithelium is possible in semithin epon sections stained with methylene blue and Azur II because their cytoplasm is clearer than that of the surrounding cells. Generally, it should be pointed out that the NE cells in the lungs of amphibians, both solitary as well as those within the NEB, occur mainly in association with the ciliated epithelium along the entire lung. However, the greatest concentration of the NE cells and NEB occurs in the proximal region of the lungs. They are, therefore, located usually in the ciliated epithelium along pulmonary vein in the tailed amphibians (Fig. 1) or in the apical and lateral parts of the Ist and IInd order septa in anurans. Nevertheless, in some anuran species the NEB are also found on the IIIrd order septa, where they occur under pneumocytes (Fig. 2). In the tailed amphibians, such as Triturus alpestris and Salamandra salamandra, there are only solitary NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS NE cells which are located near the lumen of the lung, as in the newt, or are deeply embedded in ciliated epithelium, as in the salamander (GoniakowskaWitalińska, 1980a, 1982). In Hynobius nebulosus and Hynobius nebulosus tokyoensis mostly solitary NE cells occur, although some also form NEB covered with a thick layer of mucous cells (Matsumura, 1985) or ciliated epithelium (Gomi et al., 1994). Fully aquatic Ambystoma mexicanum possesses solitary and clustered NE cells, mainly in the ciliated epithelium. However, in this species some solitary NE cells are also observed between pneumocytes (Scheuermann et al., 1989). Ambystoma tigrinum is a terrestrial amphibian; in its lungs the NEB are associated with small patches of ciliated epithelium and are covered with thin cytoplasmic protrusions of goblet cells. The solitary NE cells are found in this species only between pneumocytes (Goniakowska-Witalińska et al., 1992). In anurans, e.g., in the frog Rana nigromaculata, the NEB are situated deeply under a thick layer of the ciliated epithelium (Wasano and Yamamoto, 1978). In such species as Rana lessonae, Bufo bufo and R. temporaria, besides solitary NE cells there are NE cells grouped in single-layered, round NEB. Such NEB contain circa 20–25 cells covered with a thin layer of flat ciliated cells (Fig. 3). In Bombina variegata and B. orientalis the NEB are organized with circa 25–100 cells that form three layers. Such NEB are covered with a thin layer of ciliated cells or pneumocytes (Bodegas et al., 1993; Cutz et al., 1986; Goniakowska-Witalińska and Cutz, 1990; and own unpublished data). In the tree frog Hyla arborea the dome-shaped NEB occur on the Ist and IInd order septa. The NEB are always located in the vicinity of the patches of ciliated epithelium but are covered exclusively with flat pneumocytes (Fig. 4) (Goniakowska-Witalińska, 1981). In two species of toads, Bufo marinus and B. viridis, characteristic spherical protrusions on the main septa are observed (Goniakowska-Witalińska and Cutz, 1990; Rogers and Haller, 1980). The protrusions are filled with connective tissue only, as in B. marinus, or with either smooth muscle cells or connective tissue, as in B. viridis. The NEB are always situated in the apical epithelium of such protrusions (Figs. 5a,b). In the several investigated species a capillary (Figs. 3, 4) or a large vein was observed in the connective tissue under the NEB (Figs. 5a,b). SCANNING ELECTRON MICROSCOPY Although lungs of several species of Amphibia were investigated, NEB were observed with SEM only in a few species. The NEB were seen with SEM only when they were protruded toward the lung lumen. The extent of the protrusion was different in various species: 1) slightly protruded, as in Bombina orientalis (Fig. 6) and Hynobius nebulosus, 2) dome-shaped, as in Hyla arborea (Figs. 7a,b), or 3) located on the special septal protrusions, as in Bufo marinus and B. viridis (Goniakowska-Witalińska, 1981; Goniakowska-Witalińska and Cutz, 1990; Goniakowska-Witalińska et al., 1990; Matsumura, 1985; Rogers and Haller, 1978). The NEB located in Bombina orientalis on the Ist and IInd order septa are covered tightly with the flat 15 ciliated epithelium. On the contrary, those located on the IIIrd order septa are slightly protruded into the lung lumen and are covered with pneumocytes and single ciliated cells (Fig. 6) (Goniakowska-Witalińska et al., 1990). The NEB in Rana temporaria are considerably protruded toward the lung lumen and are tightly covered with ciliated epithelium (own unpublished data). In the lungs of the tree frog Hyla arborea the ciliated epithelium is reduced considerably; on the Ist and IInd order septa only small irregular patches of ciliated cells are found (Fig. 7a). In their vicinity the dome-shaped protrusions (90 µm in width and 60 µm in height) are situated. They are covered exclusively by modified pneumocytes with numerous long microvilli (Fig. 7b) (Goniakowska-Witalińska, 1981, 1986). In Bufo marinus (Rogers and Haller, 1980) and Bufo viridis (Goniakowska-Witalińska et al., 1990) the NEB are located in some parts of the apical epithelium covering special protrusions of 200–300 µm width and 150–180 µm height. In the salamander Hynobius nebulosus the NEB underlying the thick layer of mucous cells are also slightly protruded to the lung lumen (Matsumura, 1985). TRANSMISSION ELECTRON MICROSCOPY The solitary cells and the NEB in the investigated species of amphibians are of the ‘‘closed’’ type, i.e. they are separated from the lung lumen by the covering cells of varying thickness. Sometimes the covering cytoplasmic sheet is very thin (approx. 0.1 µm) (Fig. 8). Only in two species of Amphibia, Bufo and Ambystoma, some NEB are in contact with the air space via one cell: apical cell in Bufo marinus (Rogers and Haller, 1980) and neuroendocrine type II cell in Ambystoma tigrinum (Goniakowska-Witalińska et al., 1992). Both cells are equipped with a single modified cilium with an 811 microtubule arrangement and several microvilli on the free surface. In Ambystoma the NEB consist of three to five cells (Fig. 8), while in the other species NE cells form NEB as a one-layered round group of 20–25 cells (Bufo bufo, Rana temporaria) or as a three to four layered corpuscle which consists of approx. 100 cells (Bufo viridis, B. marinus, Bombina and Hyla). The shape of the latter NEB is pyramidal, as in Bufo, or fan-shaped (reversed pyramid), as in Hyla and Bombina (Fig. 9a) (Bodegas, 1993; Cutz et al., 1986; Goniakowska-Witalińska, 1981; Goniakowska-Witalińska and Cutz, 1990; GoniakowskaWitalińska et al., 1990, 1992; Rogers and Haller, 1978, 1980). The solitary NE cells, as well as NEB in some species, remain in direct contact with the basal lamina, although in some other species they may be separated from it in some areas by a thin cytoplasmic layer of the surrounding cells: pneumocytes, and ciliated or goblet cells (Figs. 8, 9a). NE cells are round or oval in shape, 10–20 µm in diameter and tightly adhering to each other. Only in Bombina variegata they are separated by some thin cytoplasmic protrusions of the ciliated cells that cover them. The NE cells are connected to the neighbouring cells and between each other by the desmosomes in which long tonofilaments are anchored in several spe- 16 L. GONIAKOWSKA-WITALIŃSKA Figs. 3–5. Light microscopy. Localization of neuroepithelial bodies at the apical part of main septa in the lungs of anurans. Figures 3 and 5a,b reprinted from Goniakowska-Witalińska, L. and Cutz, E. In Ultrastructure of neuroendocrine cells in the lungs of three anuran species. J. Morphol., 203:1–9, 1990. with permission from the publisher. A, air space; C, capillary; CT, connective tissue; SM, smooth muscle cells; V, vein. Fig. 4. Hyla arborea. NEB protruding into the lung lumen and covered with flat pneumocytes. 31,200. Fig. 5. Bufo viridis. Special protrusions filled with smooth muscle cells (a) and connective tissue (b). NEB are often located in the apical part of protrusion under ciliated epithelium between the open arrowheads. 3150. Fig. 3. Bufo bufo. Flat one-layer NEB covered with ciliated epithelium. 31,400. cies. In most cases NEB are accompanied by the capillary blood vessels located in the underlying connective tissue (Figs. 3, 4) or located just under the surface of basal lamina. The cytoplasm of the NE cells is lighter than that of the surrounding cells. Their characteristic large nuclei have numerous deep invaginations of nuclear membrane and show patches of condensed chromatin (Figs. 8, 9a). The cytoplasm contains numerous elongated mitochondria, a centrosome, scarce lysosomes, and multivesicular bodies. RER and free ribosomes as well as Golgi complexes and numerous long actin filaments are mostly grouped in the basal part of the cell (Figs. 10a–c). In some species, among NE cells grouped in NEB a single cilium between NE cells was observed. The NE cells are also characterized by numerous dense core vesicles (DCV). They show an electron dense interior and less dense periphery between the limiting membrane and the dense centre. Such numerous DCV, which are polymorphic in shape, occur throughout the entire cytoplasm, with some prevalence observed in the basal cell region. The size of these vesicles vary in NE cells of the investigated species of amphibians (Table 1), but in the most species only one type of NE cell occurs, NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS 17 Fig. 6. SEM. NEB on the IIIrd order septum in the lung of Bombina orientalis covered with pneumocytes and scarce ciliated cells. From Goniakowska-Witalińska et al., 1990. Reprinted with permission from the publisher, Plenum Press, New York. 33,600. with small dense core vesicles (30–200 nm in diameter). Only in the lungs of Salamandra salamandra and Ambystoma tigrinum the second type of NE cells, with larger DCV, was observed (Table 1) (Figs. 10b, 11, 12, 13a,b). In some species of anuran amphibians and in Ambystoma tigrinum, scarce, large DCV are seen in the NE cells. The diameter of such large DCV range from 210 to 862 nm. Small DCV are often located near the Golgi complexes (Fig. 10c), suggesting their formation within this organelle. Both in solitary cells as well as in the grouped ones, DCV in different secretory phases are often observed in the vicinity of the basal cell membrane. Some coated vesicles are also observed in this region of cell. In some species the solitary NE cells form several invaginations of the cell membrane, although similar invaginations were observed in NEB in the shape of reversed piramid (which are in contact with basal lamina in relatively small area). Sometimes cytoplasmic processes enlarge the secretory surface of the cell (Fig. 10a). In Hyla arborea and Bombina orientalis, some NE cells in their basal part possess 1.5 µm long, thin cytoplasmic protrusions which penetrate to the connective tissue. In one investigated species—Bombina orientalis— the lamellar bodies characteristic for pneumocytes are observed in the basal parts of the NE cells (Figs. 9a–c). In the pneumocytes the lamellar bodies represent the site of storage of surface-active material (Fig. 9c) which is next secreted to the lung lumen. The function of these bodies in the basal part of NE cells is unknown. In Salamandra both types of NE cells are solitary and are located deeply in the ciliated epithelium (Figs. 13a,b) (Goniakowska-Witalińska, 1982). However, unlike NE cells in Salamandra, the type II of NE cells in Ambystoma tigrinum possesses also large DCV. The type II NE cells in Ambystoma (Figs. 10b, 11, 12) is an unusual cell, which is not observed in the other species of amphibians. These large solitary type II cells (up to 20 µm in length) occur only in some of the NEB, which consist of three to five cells containing small DCV. The type II cells are connected with the type I NE cells by desmosomes. Type II NE cells, which are spread over from the basal membrane to the air space of the lung, 18 L. GONIAKOWSKA-WITALIŃSKA Fig. 7. SEM. Localization of the NEB in the lung of Hyla arborea. From Goniakowska-Witalińska, 1981. Reprinted with permission from the publisher, Springer Verlag, Germany. a: The NEB (arrows) on the main septum in the vicinity of patches of ciliated cells. 3320. b: The dome-shaped NEB covered with pneumocytes with longer microvilli. 32,200. are probably mature cells. On their free surface microvilli and an atypical cilium are observed (Fig. 12). The apical cytoplasm contains several small (50–70 nm in diameter) electron lucent vesicles and scarce DCV. In the lung of Ambystoma tigrinum the solitary NE cells and those in the NEB containing small DCV are very often covered by very thin cytoplasmic layer of surrounding cells. The observations of several NE cells with disrupted covering layer suggest that the NE cells are secreted to the air space and their contents released to the lung lumen. Innervation The TEM observations show that the basal part of the NEB is invested by several intraepithelial sensory nerves which form ‘‘nerve plexus’’ (Fig. 9a). In the connective tissue near NEB several unmyelinated axon profiles are also observed (Fig. 11). The nerve fibres are NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS 19 Fig. 8. TEM. The NEB in the lung of Ambystoma tigrinum consisting of three NE cells between goblet cells (G). Afferent and efferent nerve endings (arrows) between NE cells are observed. From GoniakowskaWitalińska et al., 1992. A, air space; CT, connective tissue. 35,000. of various types and their number and ultrastructure are different in the investigated species. The axoplasm possesses glycogen particles, neurotubules and neurofilaments, mitochondria and various types of vesicles. The nerve fibres are morphologically of afferent and efferent types (Figs. 8, 9a,b, 14a–c). These terms are used for nerve fibres that conduct to and from the central nervous system, respectively. The morphologically efferent nerve fibres and terminals (Fig. 9a,b, 14b,c) are characterized by various numbers of small lucent vesicles 40–100 nm in diameter, occasional larger vesicles and relatively scarce dense core vesicles (90–110 nm). In Bufo viridis the efferent nerve fibres possess also concentric lamellated bodies. The morphologically afferent nerve fibres and terminals (Figs. 9b, 14a,c) contain abundant mitochondria with long cristae, glycogen particles and a few small clear vesicles 40–100 nm in diameter. The solitary NE cells in Amphibia are not innervated, similarly as in mammals. The only exception to this rule is Triturus alpestris, which possesses exclusively solitary NE cells innervated in their basal or apical parts by morphologically efferent nerve endings (Goniakowska-Witalińska, 1980a). Afferent and efferent nerve fibres were observed in most of the investigated amphibian species, though in different composition. In such species as Bombina orientalis, B. variegata and Ambystoma tigrinum, the nerve terminals are found in basal part and also between cells and in the apical part of the NEB near the air space (Figs. 8, 9a, 11). In the lungs of Hyla arborea the nerve profiles in the basal part of NEB are predominantly of afferent type. Several sections show about eight to 10 basally located profiles of afferent type and two profiles of efferent type. In contrast in Bufo viridis, Bombina variegata and Bufo bufo, the nerve fibres are predominantly of efferent type (Goniakowska-Witalińska, 1981; Goniakowska-Witalińska and Cutz, 1990; Goniakowska-Witalińska et al., 1990). In Bombina orientalis the numbers of afferent and efferent nerve fibres are similar. 20 L. GONIAKOWSKA-WITALIŃSKA Fig. 9. TEM. Bombina orientalis. a,b from Goniakowska-Witalińska et al., 1990. Reprinted with permission from the publisher a: NEB consisting of numerous NE cells under ciliated epithelium. Several intracorpuscular nerve endings (filled arrows) and ‘‘nerve plexus’’ in the basal part of the NEB are seen. Some NE cells possess lamellar bodies (open arrows). A, air space; CT, connective tissue. 34,500. b: The basal part of NE cell with lamellar bodies and three nerve fibers (stars): the left one is typically efferent, the upper right one is typically afferent. The one in the middle shows morphological features of both afferent fibre (in the lower region) and efferent fibre (in the upper region). Such ambivalent nerve fibers are observed to split into separate endings of different types. Note the synapse (arrow) between NE cells and efferent nerve ending. N, nucleus. 324,500. c: Two pneumocytes with lamellar bodies in different stages of maturation. A, air space. 346,000. The NE cells in Ambystoma mexicanum are innervated exclusively by efferent cholinergic nerves; however, the reciprocal synapses of these nerves were found (Scheuermann et al., 1989). In other species of tailed amphibian Hynobius nebulosus the NE cells are innervated by cholinergic and adrenergic nerve fibres (Matsumura, 1985). The adrenergic axons characterized by numerous neurotubules and dense core vesicles 80 nm in diameter were observed also in Bufo marinus (Rogers and Haller, 1978, 1980). In the last species NEB are innervated in 60% by the fibres containing agranular vesicles, 20% are innervated by adrenergic nerve fibres and the remaining 20% by both types of nerve fibres (Rogers and Haller, 1978). The peptidergic nerves were not observed in the vicinity of NEB in Amphibia. The serial sections of the intraepithelial nerves in Ambystoma tigrinum and Bombina orientalis reveal the situation previously demonstrated in the NEB of rabbit and cat (Lauweryns and Van Lommel, 1987; Van Lommel and Lauweryns, 1993) that both afferent and efferent types of terminals can be formed by the same nerve fibre which branch at some distance from the endings (Fig. 14c) (own unpublished data and Goniakowska-Witalińska et al., 1992). On Figure 9b are seen three types of nerve fibres with the efferent synapse on left side and afferent one in the upper right corner. Centrally located is the nerve profile with both afferent and efferent morphological features. Such nerves in B. orientalis split into separate efferent and afferent nerve endings. The synaptic junctions between NE cells and nerve endings were observed in several species. The extracellular space between synaptic membranes is about 20 nm wide and the length of the synaptic zone ranges from 0.3 to 0.5 µm. The synaptic grid on the NE cell side is also observed (Fig. 14c). The synaptic junctions are located on efferent (Fig. 9b) and afferent (Fig. 14c) nerve endings. In the two species Bufo marinus and NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS Figure 9 Ambystoma mexicanum, the reciprocal synapses were demonstrated (Rogers and Haller, 1978; Scheuermann et al., 1989). Development The NE cells were observed exclusively in the developing larval lung of Salamandra salamandra (Goniakowska-Witalińska, 1982). In the early stages of urodelan development (Ist and IInd), lungs play a hydrostatic role and the NE cells are absent. At the stage III, when the lungs begin to act as a respiratory organ and the gills are reduced, a well developed respiratory surface and a thin air-blood barrier is observed. At this stage just before metamorphosis, two types of solitary NE cells appear in the ciliated epithelium (Figs. 13a,b): type I, with typical small DCV and some larger ones, and type II, with large DCV (Table 1). 21 (Continued.) Both types of NE cells are large, solitary, dispersed in the basal part of the ciliated epithelium and free of innervation. Some of these cells are situated on the basal lamina and extend very closely to the epithelial surface. Nevertheless, no contact of these cells with the air space was observed at this developmental stage. HISTOCHEMISTRY The histochemical composition of NE cells was investigated in 13 species of anuran and urodele amphibians by the fluorescence (Rogers and Haller, 1978; Scheuermann et al., 1989) or immunocytochemical methods (Adriaensen et al., 1994; Bodegas et al., 1993; Cutz et al., 1986; Gomi et al., 1994; Goniakowska-Witalińska et al., 1990, 1992). The results of the investigations are summarized in Table 2. 22 L. GONIAKOWSKA-WITALIŃSKA TABLE 1. Diameter ranges (in nm) of dense core vesicles (DCV) from solitary neuroendocrine cells (S) and cells grouped in NEB in the lungs of several species of Amphibia Species Bufo marinus Rana nigromaculata Hyla arborea Bombina variegata Bufo bufo Bufo viridis Bombina orientalis Triturus alpestris Salamandra salamandra Hynobius nebulosus Ambystoma mexicanum Ambystoma tigrinum Numerous, small DCV 60–100 60–110 54–110 52–130 30–140 and 170–420 60–100 120–200 69–189 I 90–150 and 150–270 II 150–450 I 46–69 II 80–110 I 58 II 73 I 70–140 II 140–260 and 320–700 Scarce, large DCV References — — 290–862 300–700 210–450 NEB NEB NEB 1 S NEB 1 S NEB 1 S 1 2 3 4 4 — NEB 1 S 5 — — 6 7 — S S S NEB 1 S 8 — NEB 1 S 9 300–600 NEB 1 S NEB 10 References: 1. Rogers and Haller, 1978; 2. Wasano and Yamamoto, 1978; 3. Goniakowska-Witalińska, 1981; 4. Goniakowska-Witalińska and Cutz, 1990; 5. Goniakowska-Witalińska et al., 1990; 6. Goniakowska-Witalińska, 1980a; 7. Goniakowska-Witalińska, 1982; 8. Matsumura, 1985; 9. Scheuermann et al., 1989; 10. Goniakowska-Witalińska et al., 1992. The biogenic amine serotonin, the principal amine present in NE cells of mammals and other vertebrates (Adriaensen and Scheuermann, 1993; Sorokin and Hoyt, 1989), was found in the NE cells of all investigated amphibian species with both techniques. Serotonin is suggested to be involved in the synthesis, storage and release of the coexisting regulatory peptides and to act as a broncho- and vasoconstrictor (Owman et al., 1973; Will et al., 1984). The immunocytochemical investigations of regulatory peptides showed great diversity in the NE cells of Amphibia. Bombesin, a predominant peptide in human lung (Cutz et al., 1984), stimulates the release of other regulatory peptides and acetylocholine and acts as bronchoconstrictor (Belvisi et al., 1991) as well as growth factor of epithelial cells in the respiratory tract (Sunday et al., 1990). Bombesin was found exclusively in the solitary cells of three species of tailed amphibians Triturus alpestris, Cynops pyrrhogaster and Hynobius nebulosus tokyoensis; however, in last species the NE cells were weakly stained and occurred in small number. Gastrin releasing peptide (GRP) is an airway constrictor (Palmer et al., 1987) and is closely related to bombesin. GRP was found in many mammals and reptiles (Andriaesen and Scheuermann, 1993). In Amphibia the GRP were found in solitary NE cells of Triturus and Hynobius. The co-existence of bombesin and GRP was demonstrated in the solitary NE cells of Triturus alpestris and in the submucosal nerves, close to smooth muscle of pulmonary septa in four species of anurans. The neuron specific enolase (NSE) is universal marker of the diffuse neuroendocrine system (Wharton et al., 1981) and was demonstrated in various mammals (Adriaensen and Scheuermann, 1993). In Amphibia NSE were found in solitary NE cells, NEB and associated nerves of five anuran species. The weak reaction of NSE was demonstrated in solitary NE cells of two urodele species. The co-existence of serotonin and NSE was found both in five anuran species and two urodele species. Calcitonin is often demonstrated peptide in NE cells of many mammals and lizards (Adriaensen and Scheuermann, 1993). Only in two species of urodele the solitary calcitonin positive NE cells were observed. Somatostatin was suggested to inhibit bronchoconstriction and secretion of regulatory peptides (Bloom, 1978) and was found in fetal and neonatal mammals. In amphibians somatostatin was detected in the solitary cells of Bombina variegata and in two species of Urodela. The met-enkephalin demonstrated in mammals (Cutz et al., 1981; Lauweryns and Van Ranst, 1987) was also found in Ambystoma tigrinum together with leuenkephalin and serotonin. Single leu-enkephalin positive solitary cells were also observed in Bombina variegata. Bodegas et al. (1993) with the immunocytochemical technique applied to the serial sections of the NEB of Rana temporaria revealed the co-localization of both serotonin and 7B2, a protein of 23 KDa isolated from human and porcine pituitary gland. Substance P (SP) was found only in the nerve fibres of five anuran species. SP containing nerves represent sensory type of innervation (Polak and Bloom, 1984). Cholecystokinin (CCK) was found exclusively in the NE cells of Hyla arborea, while vasoactive intestinal peptide (VIP) was not detected in the NE cells of investigated amphibians. The results of immunohistochemistry presented above show great variability. The bioactive substances detected in NE cells of Amphibia are the same as were found in mammals. Thus, although the physiological generalisation of these results is difficult, the above data suggested the recepto-secretory function of NE cells in Amphibia. PHYLOGENETIC COMPARISONS OF THE ENDOCRINE SYSTEM IN DIPNOI, HOLOSTEI AND CHONDROSTEI In the lower vertebrates like fish and agnathans, the NE cells in the respiratory organs (gills, lungs or air Fig. 10. TEM. Ultrastructure of NE cells of Ambystoma tigrinum. From Goniakowska-Witalińska et al., 1992. Reprinted with permission from the publisher, Springer Verlag, Germany. a: Fragment of type I NE cell with two multivesicular bodies (arrows), elongated mitochondrium, several actin filaments (open arrow), ribosomes scattered over the cytoplasm, small DCV and a fragment of rough endoplasmic reticulum (arrow head). Note the desmosome between NE cell and pneumocyte in the lower left corner. 349,000. b: Type II NE cell with both types of DCV: larger with moderately dense interior (asterisk) and several smaller ones with electron dense core. G, goblet cell; L, lysosome. 332,000. c: Type I NE cell with Golgi complex (asterisk) and dense core vesicles in its vicinity. P, pneumocyte. 348,000. 24 L. GONIAKOWSKA-WITALIŃSKA Fig. 11. TEM. Ambystoma tigrinum. Fragments of type I (I) and type II (II) NE cells, with nerve endings (N), one between them. Nerve profile (N) is seen also in the connective tissue. The desmosome (arrow) is seen between NE cells in the upper left corner. Note the localization of DCV near basement lamina in type II cells and the difference in size of DCV in both types of NE cells. L, lysosome. 326,000. bladder) were also found (for review see Zaccone et al., 1994). The investigations on the evolutionarily ancient group of fish, which are interesting from a phylogenetic point of view, were performed only on a few species. The NE cells in the lungs and air bladder of fish, as in Amphibia, are associated with the ciliated epithelium. The NE cells reaching the air space are equipped with microvilli. No NEB in the fish respiratory organs were found. The dipnoan Protopterus possesses single innervated NE cells which are located in the pneumatic duct. Some of the NE cells reach the air space. In their cytoplasm DCV 75 to 150 nm in diameter are scattered and the cells are serotonin positive (Adriaensen et al., 1990; Zaccone et al., 1989b). In the two species of chondrostean Polypterus (P. ornatipinnis and P. delhezi) the single NE cells without innervation and with contact to the air space were found in small islets of ciliated epithelium. Numerous DCV (80–165 nm in diameter) at different stages of secretion to the basal lamina were observed (Zaccone et al., 1989a). The NE cells were serotonin positive (Scheuermann and De Groodt-Lasseel, 1982; Zaccone et al., 1989a). In the air bladder of holostean fish Amia calva, which estivate in solid mud during drought (Neill, 1950), three types of solitary non-innervated NE cells with differently sized DCV were observed (own unpublished data). The NE cells are located in the small patches of ciliated epithelium and between pneumocytes. Some NE cells from all three types were found to be in contact with the air space: type I, with numerous small DCV 70–190 nm in diameter, type II, rare cells equipped with a cilium on the free surface and with larger DCV—120–260 nm in diameter—and type III, with numerous DCV 447–800 nm in diameter with a characteristic wide halo between the dense core and the limiting membrane. THE ENDOCRINE SYSTEM IN AMPHIBIA AND OTHER VERTEBRATES AND ITS FUNCTION The NEB located in the airway epithelium of mammals is usually of the ‘‘open type,’’ i.e. it communicates with the air space via NE cells extending to the lumen and possessing numerous short microvilli on their NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS 25 Fig. 12. Schematic representation of the ‘‘open type’’ NEB in the lung of Ambystoma tigrinum. The NEB consist of three to five NE cells of type I (I) possessing small dense core vesicles, single NE cell of type II (II) with large DCV, and intracorpuscular nerve endings of afferent and efferent type (arrows). The type I NE cells are surrounded and separated from the lumen by goblet cells (G), while the type II is surrounded by ciliated cells (CL) and communicate with the air space via the single modified cilium. The main innervation of NEB is localized in the basal part, where cytoplasmic continuity between afferent and efferent nerve endings is seen (star). From GoniakowskaWitalińska et al., 1992. surface (Cutz, 1982; Cutz and Conen, 1972; Cutz et al., 1975; Ericsson et al., 1972; Hung et al., 1973; Lauweryns and Van Lommel, 1987; Sorokin et al., 1982). The NEB and the solitary NE cells in amphibians are located in the strategic positions at the apical parts of septa. These structures are of ‘‘closed type’’ and are surrounded and covered with a layer of ciliated cells, goblet cells or pneumocytes. This layer varies in thickness and in some species is very thin. Only in two species, Bufo marinus and Ambystoma tigrinum, the NEB communicate with the air space. This communication is established through one cell equipped with short microvilli and a single modified cilium. The same arrangement was found only in the turtle, Pseudemis scripta elegans (Pastor et al., 1987; Scheuermann et al., 1983). The ultrastructure of these modified cilia (811 of axonemal configuration) suggests a sensory function of these cells (Afzelius, 1975; Sorokin, 1968) and the NEB of these species are suggested to act as chemoreceptors (Goniakowska-Witalińska et al., 1992; Rogers and Haller, 1980; Scheuermann et al., 1983). In Bufo marinus, Ambystoma tigrinum, Bombina variegata, B. orientalis and Hyla arborea the NEB show complex structure and innervation. This can be explained by relatively high metabolic rates of Ambys- toma (Full et al., 1988) and of these anuran amphibians. In these species a basally located plexus with numerous nerve endings is prominent. In Bombina and Ambystoma the nerve terminals are also situated apically and between the cells. Additionally, the afferent and efferent nerve endings in Ambystoma and Bombina form a cytoplasmic continuity. Such continuity and apically situated nerve endings were previously reported by Lauweryns and Van Lommel (1987) and Van Lommel and Lauweryns (1993) in the NEB of rabbits and newborn cats. The immunohistochemical investigations show the occurrence of monoamine serotonin in fish and cooccurrence of serotonin and several regulatory peptides in NE cells of amphibians, reptiles, birds, and mammals (see review Cutz et al., 1986; Adriaensen and Scheuermann, 1993; Sorokin and Hoyt, 1989; Zaccone et al., 1994). The physiological experiments performed on mammals suggest that the NEB are hypoxia sensitive (Lauweryns and Van Lommel, 1986) and oxygen sensing chemoreceptors (Youngson et al., 1993). On the other hand no physiological experiments on NEB were done in Amphibia. However, the endocrine system in the amphibian lungs shows great diversity in the structure, innervation and histochemical composition. 26 L. GONIAKOWSKA-WITALIŃSKA Witalińska et al., 1992) or mechanoreceptors in the case of deeper localization of NEB in the epithelium (Wasano and Yamamoto, 1978). Fig. 13. Fragments of two types of NE cells in the larval lungs of Salamandra salamandra, situated on basal membrane and surrounded by epithelial cells (EP). CT, connective tissue. From Goniakowska-Witalińska, 1982. a: Type I NE cell with small DCV. 325,000. b: Type II NE cell with large DCV. 324,600. The solitary NE cells as well as NEB in amphibians are similar in their location, ultrastructure and immunohistochemistry to the NEB observed in the higher vertebrates, including mammals (for review see Scheuermann, 1987; Adriaensen and Scheuermann, 1993; Fujita et al., 1988; Sorokin and Hoyt, 1989). For this reason NEB in Amphibia are supposed to be oxygen sensing chemoreceptors (Goniakowska-Witalińska, 1981, 1994; Goniakowska-Witalińska and Cutz, 1990; Goniakowska- CONCLUDING REMARKS In the course of ontogeny the air tract originates from the alimentary canal. Consequently, the neuroendocrine cells occurring in the lungs are kind of gastroenteric paraneurons sensitive to the stimuli that originate in the lumen of these organs (Fujita et al., 1988). In Amphibia the structure and innervation of NE cells as well as the occurrence of serotonin and neuropeptides suggested possible release of active substances both to the intracellular space and basement membrane (paracrine function) and to the afferent nerve terminals (receptor function) evolving local or systemic reflex changes. The bioactive substances can be also transported by the blood stream; in several species the capillaries or veins are situated in the direct vicinity or just under the NEB. The common localization of single noninnervated NE cells in the ciliated epithelium as well as between pneumocytes in amphibians suggests a paracrine role for NE cells. Such a role can be related to the increased activity of the ciliary and mucous cells as well as to the stimulation of smooth muscle cells. From the phylogenetic point of view it should be emphasized that in the history of vertebrates the endocrine system in the air-breathing organs developed in several evolutionary lineages which were independent from each other, e.g., in different systematic groups of Crossopterygii and Dipnoi, closely related to the terrestrial vertebrates, as well as ancient groups of fish as Holostei and Chondrostei. The investigations of morphology and ultrastructure of NE cell in the present (highly specialised) groups with ancient genealogy as Dipnoi, Holostei and Chondrostei do not necessarily reflect the past evolution of this system. However, considering the facts reviewed above, the following pattern of evolution of endocrine system could be proposed. The first step: the solitary non-innervated cells deeply embedded in the ciliated epithelium (as in the larval lungs of Salamandra) and located near the epithelial surface (as solitary NE cells in all investigated amphibians) or the non-innervated NE cells with contact with the air space (as in Polypterus and Amia). The second step: the innervated solitary cells of the ‘‘closed type’’ (as in Triturus) or innervated NE cells which communicate with the air space (as in dipnoan Protopterus). And the third step: NEB of the ‘‘closed type’’ (as in most amphibians and some other vertebrates), evolving from a simple to a complicated structure, innervation and histochemical composition, and NEB of the ‘‘open type’’ (as in some amphibians and the higher vertebrates). In conclusion, the role of NEB in vertebrate airways has not yet been fully elucidated. Earlier experiments (Lauweryns and Van Lommel, 1986) and recent investigations (Youngson et al., 1993) indicate that NEB can be viewed as the oxygen-sensing chemoreceptors. However, no physiological experiments were done on amphibian NEB. Their ultrastructure, localization, immunohis- NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS Fig. 14. TEM. Ambystoma tigrinum. Innervations of NEB. From Goniakowska-Witalińska et al., 1992. a: Morphologically afferent nerve ending with small lucent vesicles and numerous mitochondria containing long cristae. 342,000. b: Morphologically efferent type of nerve ending with numerous small lucent vesicles and some larger ones. 342,000. c: Basal part of the NEB with nerve plexus containing 27 afferent and efferent nerve endings. The cytoplasmic continuity between the upper efferent and lower afferent ending (asterisk) is evident. The synapse (arrow) between the cell membrane of type 1 cell and the afferent nerve ending reveals the presence of an electron dense grid on the NE cell side. 341,000. 28 L. GONIAKOWSKA-WITALIŃSKA TABLE 2. Results of immunohistochemical staining in the lungs of Amphibia Enkephalin Bufo marinus C Bombina bombina C N Bombina variegata C N Rana lessonae C N Bufo viridis C N Hyla arborea C N Triturus alpestris C N Bombina orientalis C Ambystoma mexicanum C N Ambystoma tigrinum C Rana temporaria C Hynobius nebulosus tokyoensis C Cynops pyrrhogaster C 7B2 Ser NSE Bom GRP Calc Leu Met Som CCK SP VIP Ref. x 1 x x x x x x x x x x 1 x x 1 2 1 1 2 1/2 2 1/2 2 2 2 2 x x 2 2 2 2 2 1 2 2 2 x x 1 2 1 1 2 1/2 2 1/2 2 2 1 2 x x 1 2 2 2 2 1 2 2 2 x x 1 2 1 1 2 1 2 1 2 2 2 2 x x 2 2 2 2 2 1 2 2 2 x x 1 2 1 1 2 1/2 2 1/2 2 2 2 2 x x 2 2 2 2 2 1 2 2 2 x x 1 2 1/2 1 2 1/2 2 1/2 2 2 2 2 x x 2 2 1 2 2 1 2 2 2 x x 1 2 1/2 x 1 x 1 x 2 2 2 2 x x 2 2 2 2 2 x 2 2 2 x 1 x x x x x x x x x x 3 x x 1 x x 1 x x x x x x x x x x x x x x x x x x 4 x 1 x x x x 1 1 x x x x 5 1 1 x x x x x x x x x x 6 x 1 1/2 1 1 1 x x 1 x x x 7 x 1 2 1 x 1 x x 1 x x x 8 C, NE cells; N, nerves; 1, positive; 1/2, weak positive; 2, negative; x, not observed. 7B2, protein from human and porcine pituitary gland; Ser, serotonin; NSE, neuron specific enolase; Bom, bombesin; GRP, gastrin releasing peptide; Calc, calcitonin; Som, somatostatin; CCK, cholecystokinin; SP, substance P; VIP, vasoactive intestinal peptide. 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