Morphology and Cytochemistry of the Endocrine Epithelial System in the Lung

INTEKNATIONAL REVIEW OF CYTOLOGY, VOL. lob

Morphology and Cytochemistry of the Endocrine Epithelial System in the Lung D.W.SCHEUEKMANN Institrrtc>of Histology and Micwscwpic Anritomy, University of’Antwerp, 2020 Antwerp, Belgium

I. Introduction In his light microscopic study of 1938, Feyrter first described what he called “Helle Zellen” (clear cells), due to their almost transparent cytoplasm in hematoxylin- and eosin-stained sections, lying dispersed throughout the epithelial tissue of various organs. He assumed them to be endocrine-like cells belonging to a widespread endocrine epithelial system with paracrine, neurocrine, and hemocrine functions, located in various organs including the respiratory system. Later, in an extensive study, Frohlich (1949) provided a precise description of these clear cells in the epithelium of the tracheobronchial tract of several mammals (rabbit, cat, guinea pig, dog, wether, and monkey) including man (executed persons) by means of different conventional staining methods. Strikingly, in mammals, these clear cells-particularly those in man-were found to occur not only as solitary elements; indeed, Frohlich also outlined and illustrated the existence of distinctive groups of clear cells forming round or oval corpuscular structures. According to the same author, these clear cells are situated in the epithelial tissue, close to the basement membrane, the apical cytoplasm contacting the airway lumen only occasionally. He attributed to these cells a neurosensory function and realized a relationship with the dispersed endocrine epithelial system originally outlined by Feyrter ( 1938). Moreover, Frohlich provided the first demonstration of nerve endings in close contact with the basal cytoplasm of both solitary and groups of pulmonary clear cells, an observation later confirmed by several authors (e.g., Glorieux, 1963; Shul’ga, 1965; Lauweryns ef al., 1972, 1974; Hung et al., 1973; Lauweryns and Cokelaere, 1973b; Hung and Loosli, 1974; Lauweryns and Goddeeris, 1975; Wasano, 1977; Hung, 1980; Scheuermann et al., 1983a,b; Scheuermann, 1984; Stahlman and Gray, 1984). The early investigators demonstrated the reactivity of pulmonary clear cells to argentaffin and/or argyrophilic silver techniques and argued that they might possibly have a chemoreceptive and neurosecretory function, acting primarily at the pulmonary level (Frohlich, 1949; Feyrter, 1953, 35 Copyright l a 19x7 by Academic Pre% Inc. All rightr of reproduclion in any Iorm r r w v e d .

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1954, 1958). Since these first histological observations, the presence of endocrine-like clear cells among seemingly darker epithelial cells has been revealed by both light and electron microscopy in the extra- and intrapulmonary airways of several mammals, including man (Glorieux, 1963; Bensch et al., 1965; Lauweryns and Peuskens, 1969; Lauweryns et al., 1970, 1972; Cutz and Conen, 1972; Ericson et al., 1972; Hage, 1972, 1973a,b, 1974, 1980; Hung et a l . , 1973, 1979; Cutz et al., 1974, 1975, 1978a,b; Hung and Loosli, 1974; Jeffery and Reid, 1975; Lauweryns and Goddeeris, 1975; Hung, 1976, 1980; Hage et al., 1977; Hernandez-Vasquez et al., 1977, 1978a,b; Wasano, 1977; Sorokin and Hoyt, 1978; Edmondson and Lewis, 1980; Palisano and Kleinerman, 1980; Dey et al., 1981, 1983; Keith et al., 1981, 1982; Wasano and Yamamoto, 1981; Carabba et al., 1982; Sarikas et al., 1982; Pearsall et al., 1985), birds (Cook and King, 1969; Walsh and McLelland, 1974; Wasano and Yamamoto, 1979), amphibians (Rogers and Haller, 1978, 1980; Wasano and Yamamoto, 1978; Goniakowska-Witalinska, 1980a,b, 1981), and a reptile (Scheuermann et al., 1983a,b). These endocrine-like cells occur isolated or in distinctive groups of two or three cells, as well as in large clusters of more than 100 cells (Hoyt er al., 1982a,b) within the epithelium at every level of the bronchoalveolar tract. Combined histochemical, fluorescence microscopic, and ultrastructural investigations have shown the clear cells of the pulmonary epithelium to contain intracytoplasmic chemical mediators, such as 5-hydroxytryptamin (5-HT) (Lauweryns et al., 1974, 1982; Rogers and Haller, 1978; Keith et al., 1982; Scheuermann et al., 1983a) and neuropeptide hormones (bombesin, Wharton et al., 1978; Dayer et a l . , 1985; gastrin-releasing peptide; Iwanaga, 1983; Tsutsumi et al., 1983a,b; calcitonin, Becker er al., 1980; leu-enkephalin, Cutz et al., 1981), thereby displaying in many aspects a similarity to the elements of the APUD (amine precursor uptake and decarboxylation) endocrine system conceived by Pearse (1969, 1977). The structural resemblance of these cells to some known receptor cells (Cook and King, 1969; Lauweryns et al., 1972; Lauweryns and Peuskens, 1972; Hung et d., 1973; Wasano, 1977; Cutz et al., 1978a,b; Scheuermann et al., 1983a) has led to their classification in the paraneuronic system of Fujita (1977), indicating their functional relation to neurons. In the course of recent years, a plethora of names has been coined on the basis of morphological and presumed functional features to designate these cells. For instance, they have been referred to as Feyrter cells (Moosavi et al., 1973; Hernandez-Vasquez et al., 1977, 1978a,b; Taylor, 1982) after the pathologist who first described them. But they were also called enterochromaffin-like cells (Ericson et al., 1972) and Kultschitzkylike cells (or K cells) (Bensch et al., 1965; Cutz et al., 1974, 1975). since

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they display many features similar to those found in their counterparts in the gastrointestinal tract (Bensch et al., 1965; Cutz and Conen, 1970, 1972; Lauweryns et al.. 1970; Terzakis et al., 1972; Jeffery and Reid, 1975; Breeze and Wheeldon, 1977; Capella et al., 1978; Sorokin et ul., 1983). Another term suggested was argyrophil cells (Lauweryns and Peuskens, 1969; Taylor, 1977) or even AFG k e . , the initial letters of argyrophilic, fluorescent, and granulated) cells (Lauweryns et al., 1970). Other terms which have been proposed are chromafln-type cells (Basset et al., 1971), endocrine cells (Hage, 1972, 1973a,c, 1974), endocrine-like cells (Hage, 1976; Cutz and Conen, 1972; Ewen et al., 1972; Hage et al., 1977), neurosecretory cells (Becci et al., 1978) or neurosecretory-appearing cells (Terzakis et al., 1972). small granulated cells (McDowell et al., 1976), small granule endocrine cells (Sorokin and Hoyt, 1978), biogenic aminecontaining cells (Eaton and Fedde, 1978), or neuroendocrine cells (Keith et al., 1981). Their amino acid uptake characteristics have inspired some authors to call them APUD cells (Hage, 1973a; Sidhu, 1979), since they fulfill the principal criterium of this system (Pearse, 1969, 1977). However, except for the Feyrter and Kultschitzky terms, none of these names specify whether they are situated intraepithelially or in the pulmonary connective tissue. Since it was demonstrated that both solitary and groups of neuroendocrine cells containing biogenic amines may belong to intrapulmonary ganglia (McLean and Burnstock, 1967a,b; Bliimcke, 1968; Bock, 1970; Mann, 1971; Jacobowitz et al., 1973; Knight, 1980; Scheuermann and De Groodt-Lasseel, 1983; Scheuermann et al., 1983b, 1984a,b,c, 198% two populations of endocrine cells of the respiratory system should be included in the APUD series. Accordingly, it seems necessary to maintain a terminology which refers to the characteristic location of the cells in the epithelial tissue. This shortcoming applies equally to the term dense-core granulated cells introduced by Jeffery and Corrin (1984) in a recent study on the structural analysis of the respiratory tract. Indeed, the intraepithelial, granule-containing cells and the small intensely fluorescent cells occurring in the ganglia of the pulmonary interstitium are characterized by an abundance of dense-cored vesicles and are both assumed to have a chemoreceptor and endocrine or paracrine function (Bock, 1970; Knight, 1980; Scheuermann et al., 1983b, 1984a,b,c). Since the present work deals with neuroendocrine epithelial cells-and not with endocrine cells of the intrapulmonary ganglia-it seems justified to use the term neuroepithelial endocrine (NEE) cells. The well-demarcated epithelial organoid structures, composed of aggregated NEE cells-for the first time extensively described by Glorieux (1963), who called them “corpuscule kpithelial” (i.e., epithelial corpuscle)-will be referred to in the course of this review as neuroepithelial

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bodies (NEBs), a term proposed by Lauweryns et al., (1972) because of their prominent nerve supply. The endocrine clear cells grouped in NEBs have been compared to the solitary endocrine cells, lying dispersed in the epithelium of the respiratory system and described in human fetuses (Cutz and Conen, 1970, 1972; Hage, 1973a,b; Cutz et al., 1973, in children (Lauweryns and Peuskens, 1969; Cutz and Conen, 1970; Lauweryns et al., 1970; Rosan and Lauweryns, 1971; Cutz et al., 1975; Lauweryns and Goddeeris, 1975), in the adult human lung (Cutz and Conen, 1970; Terzakis et al., 1972; Lauweryns and Goddeeris, 1975; Hage et al., 1977) as well as in various mammalian species (Jeffery and Reid, 1973, 1975; Cutz et al., 1974; King et al., 1974; Hernandez-Vasquez et al., 1977; Sorokin and Hoyt, 1978; Edmondson and Lewis, 1980; Palisano and Kleinerman, 1980; Lauweryns et al., 1985), in birds (Cook and King, 1969), and in amphibians (Wasano and Yarnamoto, 1978; Goniakowska-Witalinska, 1980a). The corpuscular appearance of groups of NEE cells in NEBs and their conspicuous innervation are considered by some authors to be a separate neuroendocrine cell system, distinct from solitary NEE cells (Lauweryns and Cokelaere, 1973a; Lauweryns et d., 1974, 1978, 1985; Lauweryns and Goddeeris, 1975; Lauweryns and Liebens, 1977; Loosli and Hung, 1977; Hung et d., 1979; Sonstegard et al., 1979; Foliguet and Cordonnier, 1981). However, in developing rabbit lungs, it was demonstrated by electron microscopy (Sorokin et al., 1982) that scattered solitary clear cells appear very early during gestation as the first population in the pulmonary epithelium undergoing differentiation into NEE cells and that mature NEBs are derived from them. According to these authors, embryonal undifferentiated precursor cells, situated in the primary pulmonary epithelium, are observed in various stages of transformation to NEE cells, which subsequently appear in groups of two to three cells that will finally mature, at least partly, to NEBs. Similarly, Stahlman and Gray (l984), investigating electron microscopic preparations of the fetal human lung, describe putative neuroendocrine cells which, during development, differentiate into either singly occurring neuroendocrine cells or into NEBs. In a combined immunohistochemical and ultrastructural investigation of the development of NEE cells in the human lung from the early fetal to the perinatal period, Cutz et al. (1984) demonstrated the presence, in the canalicular stage, of NEE cells occurring either solitarily or packed in NEBs. As observed by these authors, the NEE cells grouped in NEBs share identical ultrastructural features with the isolated NEE cells. Both light and electron microscopic investigations seem to indicate that single NEE cells and NEBs represent stages of differentiation of one and the same embryonal precursor cell. In agreement with these findings, studies have shown that in the res-

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piratory system both the diffuse endocrine cells and the NEBs, which may be involved in the production of amines and/or polypeptide hormones, share similar histochemical properties (Cutz et al., 1984). Moreover, the innervation of NEE cells is not restricted to NEBs. Nerve terminals with synaptic contact have been described at the base of single NEE cells in the infant bronchial epithelium (Lauweryns et al., 1970). Unmyelinated axons were also found in close association with individual NEE cells of the pulmonary system in the rabbit (Hung, 1980), hamster (Edmondson and Lewis, 1980), rat (Jeffery and Reid, 1973), and guinea pig (DiAugustine et al., 1984). Since functions of these endocrine cells remain still unknown, the pulmonary single NEE cells and those arranged in NEBs will be treated as a single neuroepithelial endocrine system. 11. Light Microscopic Aspects

Epithelial cells with transparent cytoplasm are observed in routine light microscopic preparations of the entire respiratory tract, in particular, when using, after fixation with formaldehyde, hematoxylin-eosin, the trichrome method of Masson, or a modification of the Goldner-Masson staining method (Frohlich, 1949; Feyrter, 1958). However, since the translucent appearance is not a specific morphological feature, the solitary NEE cells remain in this way relatively inconspicuous. Conversely, NEBs can be readily detected by conventional staining techniques, forming clearly demarcated epithelial corpuscles. In some species, they protrude slightly into the airway lumen (Hung et al., 1973, 1979; Cutz et al., 1978b), but in others they are enveloped in the epithelium, indenting the underlying connective tissue (Pearsall et al., 1985). They can also be observed in a pitlike depression of the pulmonary epithelium. In most species, the more centrally located cells of the NEBs are characterized by a well-ordered appearance consisting of nonciliated cells, joined side-by-side, with their longitudinal axes more or less at right angles to the basal lamina, albeit slightly inclined to the center of the luminal surface. The shape of these corpuscular cells is almost columnar with a more or less oval nucleus. Sometimes, at the branching region of the airways, the NEE cells appear stratified or form ajumble of cells, often with a pyramidal form, the apex of which is directed to the airway lumen and the broad face situated against the basement membrane. Some profiles of NEBs do not contact the lumen of the airways, but seem isolated from the surface by dark nonciliated cells stretched over the luminal and lateral cytoplasm as it expands (Fig. 1). They are assumed to be modified Clara cells (Cutz et ul., 1978b; Hung et al., 1979; Pearsall e f al., 1985). From serially cut sections, it is apparent that, in most animal species,

FIG. I . In some sections, the NEB of the red-eared turtle appears elongated, with granulecontaining cells in a palissade-like row lying on the basal lamina. Most of the apical surfaces are covered with flattened Clara-like cells. The NEB is separated from capillaries by a narrow subendothelial space containing collagen fibers. To the left, Clara-like cells abut on ciliated epithelial cells. Silver method applied to semithin section of Epon-embedded material according to Lopez e/ d.(1983). Light microscopy. x 1100. FIG.2. NEB in the epithelial lining of the bronchiolus of a neonatal rabbit composed of yellow fluorescent, elongated cells, as revealed by formaldehyde-induced fluorescence. The contours at the base of the individual cells are hardly visible, because of their close apposition and very intense fluorescence. The emission and excitation spectra of the fluorophore of this NEB is rendered in Fig. 5 . Fluorescence microscopy. x 800. FIG.3. The same NEB of the neonatal rabbit as in Fig. 2, revealed by the argyrophilic method according to Grimelius. Light microscopy. x 800. FIG. 4. Whole-mount stretch preparation of the red-eared turtle lung treated for formaldehyde-induced fluorescence. An extensive group of intensely yellow-fluorescent neuroendocrine epithelial cells, with, in their neighborhood, a few solitary and grouped neuroendocrine epithelial cells. Green-fluorescent nerve fibers running to the yellow-fluorescent NEB. Fluorescence microscopy. x 90.

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a small portion of the apical cytoplasm often reaches the airway lumen (Fig. I). In most species, there is a striking similarity in NEB architecture and morphology; their size is highly variable (for review, see Foliguet and Cordonnier, 1981). However, in the toad lung, a NEB appears covered by a dark apical cell, provided with a single cilium protruding into the airway lumen (Rogers and Haller, 1978, 1980). The basement membrane rests on an usually thin lamina propria, which envelops one or more capillaries close to the NEE cells (e.g., Lauweryns and Goddeeris, 1975; Scheuermann er al., 1983a; Hung, 1984; Pearsall et ul., 1985). Fascicles of smooth muscle may closely approach the base of the NEE cells (Pearsall er al., 1985). In order to detect the NEE cells, besides conventional light microscopy, various convenient staining methods have been used, including the use of masked metachromasia (Solcia et ul., 1968) and such methods as lead hematoxylin (Solcia et ul., 1969) or periodic acid-Schiff and lead hematoxylin (Sorokin and Hoyt, 1978). Acid hydrolysis which precedes staining enhances the metachromasia to basic dyes of secretory granules in endocrine cells attributed to sidechain carboxyl or carboxamide groups of granule proteins (Pearse, 1969), whereas the diffuse basophilia, due to RNA, DNA, and acid polysaccharides, is not realized by extraction of these acid substances (Solcia et ul., 1968). A consecutive treatment with basic dyes after HCI hydrolysis stained the endocrine cells in pancreatic islets, in thyroid and parathyroid endocrine cells, in the gastroenteric endocrine cells, and in the adenohypophysis (Solcia et ul., 1968). It was believed that probably all cells from the APUD series contain metachromatic substances in their secretory granules in a “masked” form which can be unmasked by HCI hydrolysis (Bussolati et d.,1969; Fujita and Kobayashi, 1974). This technique was therefore applied to demonstrate the presence of endocrine cells in the lung. According to Hage ( 1972, 1976, 1980), HCI-toluidine blue-positive cells are distributed throughout the pulmonary epithelium of human fetuses, whereas in the human adult lung, these have not been found (Hage et d.,1977; Hage, 1980), nor, for that matter, in the rabbit, guinea pig, mouse (Hage, 19741, and rat (Cutz et al., 1974). The lead hematoxylin method is frequently used in light microscopy for staining secretory granules in endocrine cells known to produce polypeptide hormones (Solcia et al.. 1969). Some authors report a positive lead hematoxylin reaction in NEE cells of the lung of human fetuses (Hage, 1980) and in carcinoid lung tumors (Hage, 1976), but not in the normal adult human lung (Hage er al.. 1977). Lauweryns and Cokelaere (1973a) demonstrated weak lead hematoxylin-positive NEBS in the lung of neonatal animals (rabbit and mouse). However, Sorokin and Hoyt (1978) furnished

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evidence that, in lungs from young mice, rats, hamsters, kittens, as well as in late fetal and neonatal rabbits, lead hematoxylin alone produces little if any staining of the NEE cells. Conversely, a staining method which combines the periodic acid-Schiff (PAS) method to lead hematoxylin and which is applied to plastic-embedded sections seems particularly useful for granule-containing cell populations in the lung (Sorokin and Hoyt, 1978). The cells are recognizable by the magenta coloration of the cytoplasm, frequently heavier toward the cell base. In plastic-embedded material of control lungs and S h y droxytryptophan pretreated animals examined for the formaldehyde-induced fluorescence (FIF) technique and consecutively stained with the PAS-lead hematoxylin method, the NEE cells revealed a magenta staining corresponding precisely to sites of 5-HT fluorescence (Sorokin and Hoyt, 1978). In a systematic study on the infracardiac lobe of the hamster lung, five different types of NEE cells have been identified by the PAS-lead hematoxylin method (Hoyt et al., 1982a). Types I, 11, and V bear granules of about 0.2 pm in diameter, whereas types 111 and IV contain larger granules. Types I and I1 can be readily segregated, since only the granules of the first type stain reddish-pink without affinity for lead hematoxylin. Of the coarse-grained PAS-positive types 111 and IV, only the latter show affinity for lead hematoxylin. Type V cells appear reddish-purple with PAS and the small granules of these cells are stained by lead hematoxylin. Whereas types I, 11, 111, and 1V encompass columnar cells whose granules tend to accumulate at the basal pole of the cell, type V cells are apparent by their spheroidal shape. Types I, 11,111, and V may occur both solitarily and in organized clusters, whereas it was demonstrated that the PASpositive and lead hematoxylin-positive coarse-grained type IV cells, which never occur as single cells, are usually present in large NEBS. These structural differences of NEE cell subtypes are signifcant in view of the preferential relationship which each of them displays with nonendocrine cells and tissues, whether occurring solitarily or in organized clusters. For instance, NEE cell types I and I11 can be found in relation to the capillary network of the pulmonary artery; types 11, IV, and V may be linked with the capillaries of the pulmonary circuit; types I1 and IV can be related predominantly to smooth muscle cells. From these data, it might be concluded that, at least in the hamster lung, the NEE cell subtypes may have different functions, independent of their single or clustered appearance. The neuronal-like cytochemical features of NEE cells, which allow their detection in light microscopy, include silver techniques, cholinesteraseand neuron-specific enolase cytochemistry, FIF, and immunocytochemical detection of 5-HT and peptide hormones.

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111. Argentaffinity and Argyrophilia

The argentaffin method to reveal silver-reducing cells was first developed by Masson (l914), demonstrating that after formalin fixation the Kultschitzky cells contain an endogenous substance which reduces alkaline solutions of silver salts, resulting in these cells being impregnated by metallic silver; ever since, the term argentaffinity points to impregnation of the cells by silver after reduction of an alkaline silver solution without additional treatment with an extraneous reducing agent. After it was demonstrated that Kultschitzky cells contain 5-HT (Erspamer and Asero, 1952), Barter and Pearse (1953, 1955) showed that Kultschitzky cells in formalin-fixed tissue and synthetic 5-HT models reacted in a similar way with the usual argentaffin method, demonstrating that the silver-reducing power of these cells after formalin fixation is due to 5-HT. It was shown that, after subsequent treatment with alkaline or neutral solutions of silver salts and a weak extraneous reducing agent, not only were the argentaffin cells stained but also a wide range of other cells and tissue components (Hamperl, 1932). This other way to reveal tissue structures with silver salts is usually called the argyrophilic method, which is most frequently used for the demonstration of bronchopulmonary NEE cells; however, the NEE cells of the respiratory tract, being the pulmonary counterparts of the Kultschitzky cells, is the case in which it seems more obvious to apply the argentaffin reaction. Cytoplasmic argyrophilia was revealed in some clearly isolated NEE cells and in NEBS of paraffin-processed lung tissues from human fetuses and children (Hamperl, 1952; Feyrter, 1954, 1958; Hage, 1972, 1973b, 1976; Lauweryns and Peuskens, 1972; Cutz et a/., 1975; Lauweryns and Goddeeris, 1975), of adult human lungs (Tateishi, 1973; Lauweryns and Goddeeris, 1975; Hage et al., 1977; Hage, 1980), of rabbits, rats, sheep fetuses, and neonatal rats (Lauweryns et al., 1972, 1973, 1974; Moosavi et al., 1973; Hage. 1974, 1976; Cutz et al., 1975; Hung, 1980; Sorokin et al., 1982), of adult rabbits, dogs, and cats (Frohlich, 1949; Lauweryns et al., 1973; Taylor, 1977), and of a young armadillo (Cutz et al., 1975). This reaction yields fine, dark-brown argyrophilic granules either diffusely scattered throughout the cell or concentrated in its basal portion (Fig. 3). In this way, the solitary, pulmonary NEE cells of most species usually appear fusiform, pyramidal, or flask shaped, resting on the basement membrane, with a dark-stained, narrow, cytoplasmic, apical process pointed toward the airway lumen. In adult man, most NEE cells, observed only by Tateishi (1973) as extending to the airway lumen, lack luminal contacts. Some cells revealed dark, long, basolateral cytoplasmic processes extending along the basement membrane or between other epithelial

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cells. The NEBs exhibiting a strong and massive argyrophilia can be readily identified at the light microscopic level throughout the entire respiratory system. The argentaffin reaction on paraffin sections of formalin-fixed material in Bouin’s solution and immersed in Fontana’s ammoniacal silver nitrate solution (often with omission of gold toning and counterstaining) revealed, in fetal and neonatal rabbit NEE cells (Lauweryns et al., 1972, 1973; Sonstegard et al., 1982) and in NEBs of the turtle lung (Scheuerrnann et al., 1983a), dark-brown deposits in the cytoplasm, preferentially in the basal portion of the cell (Fig. 8). Consequently, these NEE cells possess silverreducing properties after formalin fixation. Nevertheless, several investigators observed little if any argentaffin reaction in NEE cells within the wall of the intrapulmonary airways of man and different animals (Lauweryns and Peuskens, 1969; Hage, 1972, 1974, 1976, 1980, 1984; Cutz et al., 1975). In the lung of Polypterus, NEE cells appeared to reduce the ammoniacal silver salt, some of them strongly, while in others this was only barely visible; hence, they may be assumed to be argentaffin. As shown by consecutive serial sections, the argyrophilic technique applied to the same cells impregnated them with silver as well (D. W. Scheuerrnann and M. H. A. De Groodt-Lasseel, unpublished work). Since the argyrophilic reaction seems more likely to occur than the argentaffin reaction, the former will also be positive whenever argentaffin cells are demonstrated. It therefore appears that argentaffin NEE cells are argyrophilic, whereas some argyrophilic cells do not seem to be argentah. This finding is consistent with reports by Tateishi (1973) and Hage (1980), who demonstrated several argyrophilic NEE cells in the human adult lung to be nonargentaffin. Hage (1980, 1984) attributed a negative argentaffin reaction to the very low concentration of the biogenic amine, as shown by the FIF technique. In this interpretation, it is not necessary to consider argentaffin and argyrophilic cells as distinct cell types. In fact, they have been thought of as representing different stages in the secretory cycle of a single cell (Hamperl, 1952), a hypothesis supported by electron microscopic investigations (Ratzenhofer and Leb, 1965; Ratzenhofer, 1966a.b) and by cytochemical studies on 5-HT fluorescence and argyrophilia (Penttila, 1966, 1967). It is known that the argentaffin silver technique requires a critical level of silver reduction, i.e., a minimal cytoplasm-reducing ability to effectively demonstrate amine-containing cells by light microscopy. As we have seen, the argentaffinity reflects the endogenous capability of the formalin-fixed cytoplasm to reduce silver salts; therefore, this reaction has a histochemical value. Although the argyrophilic reaction is more likely to occur than the argentah reaction, it will obviously become

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positive whenever the argentaffin reaction is demonstrated. With the argyrophilic technique, however, the silver ions, which are mainly caused by addition of an extra reducer, deposit metallic silver on different tissue constituents. This staining characteristic mainly reflects the physical properties and not a chemical composition; it is therefore histochemically unspecific. As a result, the argyrophilic cells are by far more numerous than the argentaffin cells, but they are also much less precisely defined. In summary, a positive argentaffin reaction is indicative of the presence of reducing substances, whereas a negative reaction could imply that the reducing capacity is minimal. The situation is quite different for the argyrophilic reaction caused by treatment with an extraneous reducing agent. Although it is believed that here too the reducing substance of the cytoplasm first partly reduces the silver salt, whereafter additional silver is deposited on top, the latter deposit also occurs on a range of other tissue constituents that seem unrelated to the argentaffin components: it is not yet clear how and why this process occurs. It might be explained by the fact that 5-HT is closely linked to other nonamine components, which may interfere with its reactivity. Hence, as far as the NEE cells of the respiratory system are concerned, it is not surprising that different authors argue that the argyrophilic reaction is not entirely reliable (Cutz et af.. 1974; Sorokin et uf., 1982), probably due to species differences or to the age of the animals, as well as the development and maturation of the endocrine cells. Certainly these possibilities and restrictions should be taken into account when dealing with a comparative distribution of argentaffin and argyrophilic cells. Both silver methods can be applied to semithin sections of Aralditeembedded material according to the method described by Lopez ut af. (1983). Hence, the solitary NEE cells as well as the NEBs are readily identified in the lung of the adult monkey, pig, and red-eared turtle throughout the length of the airways by their low cytoplasmic density. The silver-impregnated granules are most numerous in the cytoplasm facing the basal lamina (Fig. 1). IV. Cholinesterase Activity

Cholinesterase activity was observed by some authors throughout the cytoplasm of NEE cells in the fetal lung of the rabbit (Lauweryns and Cokelaere, 1973a; Hung, 1980. 1984; Sonstegard et af., 1982)and rat (Morikawa rt d.,197th). Moreover, it has been demonstrated that NEBs of the embryonic rat lung, differentiating in virro and segregated from the central nervous system, revealed acetylcholinesterase-containing granules

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(Morikawa et al., 1978b). These observations are in agreement with the original view of Pearse (1969) that high levels of cholinesterase may characterize the cells of the APUD series. Indeed, monoamine-containing cells have been reported to contain cholinesterase activity, such as the chief cells of the carotid body (Koelle, 1950, 1951; Ballard and Jones, 1971; Korkala and Waris, I977), the cardiac (aorticopulmonary) glomus bodies (Papka, 1975, 1980), the catecholamine-containing cells in the pulmonary ganglia of the calf, goat, and fetal sheep (Mann, 1971), in the pelvic paraganglia (Thompson and Gosling, 1976), in adrenomedullary cells (Palkama, 1967), and in some adrenergic neurons (Jacobowitz and Koelle, 1965). As yet, the functional significance of this enzyme in monoaminecontaining cells is not clear. Since most of these cells receive a synaptic input from cholinergic nerve terminals, they are sensitive to acetylcholine. Acetylcholinesterase hydrolyzes and thereby inactivates the neurotransmitter acetylcholine. Hence, this cholinesterase, synthetized in NEE cells, transported on the outer surface of the cellular envelope, and released from this site, is likely to participate in modifying the microenvironment of the NEE cells, regulating the responsiveness to acetylcholine after its release from presynaptic axon terminals. It might also be that the presence of acetylcholinestemse is related to that of active acetylcholine metabolism in NEE cells, since, in some biogenic amine-containing cells, choline acetyltransferase (Ballard and Jones, 1972) as well as the uptake of [3H]choline was demonstrated (Fidone et al., 1976). Indeed, acetylcholine probably plays a role in modulating chemosensory discharge (Eyzaguirre and Zapata, 1968). Moreover, it was demonstrated that cultures of cells from pheochromocytoma may synthetize, store, and release acetylcholine (Greene and Rein, 1977). In adrenergic tissue (Burn and Rand 1959, 1965)and in parathyroid C cells (Welsch and Pearse, 1969), the release of catecholamines is mediated or facilitated by acetylcholine, so that cholinesterase activity may be correlated with physiological states of these cells. V. Neuron-Specific Enolase

Neuron-specific enolase is a protein which, in light microscopic immunocytochemistry, was found distributed in neuronal perikarya, dendrites, and axons (Pickel et al., 1976; Schmechel et ul., 1978a). It was shown to be a major neuronal protein correlated with neuronal development and differentiation; observations have apparently established it to be essential to the neuronal function (Marangos et al., 1978; Marangos

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and Schmechel, 1980). Since immunocytochemical investigations have revealed neuron-specific enolase to be present in endocrine cells of the APUD series (Schmechel et a/., 1978b) and in the larger cell group called paraneurons (Fujita et al., 1983), this enzyme seems to be a functional marker for the diffuse neuroendocrine system (Marangos et al., 1981; Tapia et a / . , 1981). Consistent with this finding, immunostaining using antibodies to neuron-specific enolase was found in both single NEE cells and in NEBS of the respiratory tract (Cole et a / . , 1980; Tapia et al., 1981; Wharton et al., 1981; Polak and Bloom, 1982, 1984; Sheppard et al., 1984). Although in the adult human lung distinctly immunostained NEE cells can be found, a considerably larger number is present in lungs from human neonates and perinates (Polak and Bloom, 1984). In serial sections of the human fetal lung, at least three different types of NEE cells on the basis of their immunoreactive content were identified containing ( 1) neuron-specific enolase, 5-HT, and bombesin; (2) neuronspecific enolase and 5-HT; and (3) neuron-specific enolase only (Wharton et a/., 1981). This enzyme was for the first time observed in lungs of 16week-old human fetuses; they assumed it to be a useful marker for the immunocytochemical detection of NEE cells in the lungs at any age and regarded its presence as indicative of the starting functional activity of these cells, since, in neuronal tissue, its appearance coincides with the initiation of synaptic contacts (Marangos et al., 1979). Some authors reported the first immunoreactive cells for neuron-specific enolase in the human fetal lung at about 8 weeks gestation, i.e., before neuropeptide could be detected (Sheppard et al., 1984), an observation strengthened by immunostaining for 5-HT and the electron microscopic findings of cytoplasmic secretory granules (Cutz et a / . , 1984). In contrast herewith, is a report by Takahashi and Yui (1983), who detected the first immunoreactive cells for 5-HT in the human fetal lung at 12 weeks. In rats chronically exposed to asbestos fibers, an increased number of large and irregular clusters of NEE cells may be demonstrated in the bronchopulmonary tree, using antibodies to neuron-specific enolase (Cole et a / . , 1982; Sheppard et a/., 1982; Polak and Bloom, 1984). According to these and other authors, using this antibody, neuroendocrine tumors can be identified in the lung (Tapia et a/., 1981; Polak, 1983). Although immunocytochemistry has detected neuron-specific enolase mostly in neurons and paraneurons (Marangos et a / . , 1981), it appeared that immunostaining for this neuronal protein in tissues and cells includes the reaction with a hybrid form of enolase (Marangos et al., 1980; Kato et al., 1982; Haimoto et al., 19851, warning that this way to detect NEE cells must be used with care.

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VI. Aspects of Induced Fluorescence

Recent microscopic studies with the FIF method (Falck and Owman, 1965) have made it clear that many NEE cells and NEBs in the wall of the respiratory system contain biogenic amines (Lauweryns and Peuskens, 1969, 1972; Lauweryns et al., 1970, 1973; Hage, 1972, 1976; Cutz et al., 1974; Cutz, 1982; Scheuermann et al., 1983a, 1984a; Scheuermann, 1984). After treatment with formaldehyde vapor, these cells exhibit a fluorescence of variable intensity (Fig. 4). The ring-closing condensation reaction, occumng between formaldehyde and indolylethylamines or catecholamines, yields heterocyclic compounds. The subsequent dehydrogenation, in the presence of proteins, produces strongly fluorescent dihydro-P-carbolines (Corrodi and Jonsson, 1965) and dihydroisoquinolines(Corrodi and Hillarp, 1964), respectively. There are authors who make use of a recently developed glyoxylic acidinduced fluorescent method for the demonstration of biogenic amines in NEE cells (Rogers and Haller, 1978; Hung, 1980). Because of the low endogenous amine content in APUD cells of some animals, it is difficult to reveal NEE cells directly by fluorescence microscopy without treatment with an amine precursor. Others, without treatment, are apparently devoid of a cellular store of biogenic amines. They can be revealed by in vivo or in vitro treatment of the specimens with an amine precursor, (3,4-dihydroxypheny1)-L-alanine or ~-5-hydroxytryptophan,which demonstrates their amine-handling properties (Ericson er al., 1972; Hage, 1973a, 1974, 1980,1984;Cutz etal., 1974,1975;Walsh and McLelland, 1974;Sonstegard et al., 1976; Hage et al., 1977; Lauweryns et al., 1977; Palisano and Kleinerman, 1980; Dey et al., 1981). The fluorescence appears predominantly in the basal, paranuclear cytoplasm; the nucleus is free of fluorescence (Figs. 2 and 7). After treatment with sodium borohydride solution, the fluorescence is nullified and then reestablished by further exposure to formaldehyde vapor, demonstrating the specificity of the histochemical reaction for monoamines (Corrodi et al., 1964). As determined in intrapulmonary NEBs of the rabbit (Lauweryns et al., 1973, 1974, 1977) and in single NEE cells of the same animal (Dey et al., 1981), as well as in human NEE cells (Keith er al., 1981), where the maximum intensity of the fluorescence emission is situated between 520 and 530 nm, it seems likely that the amine involved is 5-HT. Special caution, however, is required by the fact that the maximal emission intensity of catecholamines, usually about 480 nm, may under certain conditions be situated in the wavelength range from 500 to 540 nm, thus appearing yellowish. This spectral shift of catecholamines may occur when high concentrations are present, provided the catecholamine-protein ratio

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is very high (Caspersson et d . ,1966; Corrodi and Jonsson, 1967;Jonsson, 1967a. 1971a,b; Bjorklund et d . , 1968; Bjorklund and Falck, 1973; Laszlo, 1975). An exact differentiation between the fluorophores of indolylethylamine derivatives and catecholamines can only be achieved by microspectrofluorometric readings of excitation spectra. Indeed, at neutral pH, the maximal excitation intensity for catecholamine fluorophores is usually higher (i.e., 410 nm) as compared with that of fluorophores from indolylethylamine compounds (around 385 nm). Furthermore, upon acidification. the excitation maxima of catecholamine fluorophores change from 410 to 370 nm, with additional excitation peaks at 320 nm (Bjorklund c’t al.. 1972a.b) and, in an extended excitation range from 240 to 450 nm. a peak at about 260 nm (Reinhold and Hartwig, 1982; Scheuermann et d . , 1984b). On the other hand, it was found that, in some tissues, 5-HT and its metabolic precursor, 5-hydroxytryptophan, may be present simultaneously (Hartwig and Reinhold, 1981). In addition to the excitation peak at 385 nm, excitation recordings conducted in an extended wavelength range yield a distinct clear excitation peak at 310 nm. Moreover, the relative height of the peak at 385 nm, as compared to the excitation peak at 310 nm, appears much higher for 5-HT than for 5-hydroxytryptophan (Reinhold and Hartwig. 1982). Thus, microspectrofluorometrically in an extended excitation range, it is possible to distinguish clearly the fluorophores of 5-HT from those of 5-hydroxytryptophan. Additionally, the differentiation between fluorophores of indolylethylamine derivatives and catecholamines can be performed by studying the rate of fading of the fluorescence: the decrease in fluorescence intensity of the 5-HT fluorophore upon irradiation with the most effective wavelength is more rapid than that of the catecholamine fluorophores (Jonsson, 1967b). Moreover, the final fading rate of the 5-hydroxytryptophan fluorophore is much less pronounced than that of the 5-HT fluorophore (Reinhold and Hartwig, 1982). After formaldehyde condensation, the microspectrofluorometric measurements revealed, in NEBS of several vertebrates (rabbit, pig, and turtle), a maximal emission from 520 to 530 nm and an excitation maximum at 385 nm (Fig. 5). Furthermore, the characteristic spectral shift of catecholamine fluorophores after acid treatment do not materialize. These results allow a classification of the monoamine content in the NEE cells of the lung as an indolylethylamine derivative. Also, the excitation spectra revealed a much higher ratio of the 385-310 peak as compared to S h y droxytryptophan (Fig. 6). After irradiation at the most effective wavelength, the photodecomposition is very rapid, with a loss of 50% of the original fluorescence intensity during the first minute, followed by a much

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Qamar 1

0.5

c.

200

300

400

500 nm

FIG.5 . Excitation (left) and emission (right) spectra recorded by formaldehyde-induced fluorescent, neuroendocrine epithelial cells of the neonatal rabbit. FIG. 6. Microspectrofluorometric excitation spectrum from formaldehyde-induced fluorescent, neuroendocrine epithelial cells of the neonatal rabbit. The extended excitation range from 240 to 460 nm with the characteristic height of the peaks at 385 and 310 nm makes a differentiation possible between 5-hydroxytryptamine and 5-hydroxytryptophan.

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slower fading rate and ending in a final intensity decrease of about 70% after 5 minutes (Fig. 9). Hence, these microspectrofluorometric recordings point to the presence of 5-HT. Upon staining the same section with the argyrophilic reaction, the shape, size, location, and distribution of fluorescent cells correspond to that of the argyrophilic cells (Figs. 2 and 3) (Cutz et al., 1975; Dey et ul., 1981), although the argyrophilic reaction is assumed to be less sensitive than the FIF (Palisano and Kleinerman, 1980). When staining was camed out using the argentaffin reaction, those cells, which show a basal yellow fluorescence with spectral characteristics of 5-HT, revealed dark-brown deposits in the basal cytoplasm, indicating that the yellow fluorescent cells are identical to the argentaffin cells (Dey et al., 1981; Scheuermann et al., 19834. On the basis of their fluorescence color, following formaldehyde condensation, it has been suggested (Eaton and Fedde, 1977) that the NEBs of the mouse lung may occur in two different groups, one comprising yellow, quickly fading fluorescent cells, claimed to produce and store an indolylethylamine compound, whereas the other consists of blue-green fluorescent cells producing catecholamines. Consistent with this finding, in whole mounts of the lizard lung, yellow fluorescent cells and cells which fluoresce a faint green have been observed (McLean and Burnstock, 1967b). As mentioned before, the formaldehyde-induced, typically yellowish fluorescence, is in itself, i.e., without objective microspectrofluorometric measurements, inconclusive for the critical identification of a biogenic amine. As we have reported in the turtle lung, the yellow fluorescent cells show the microspectrofluorometric characteristics of 5-HT (Scheuermann et al., 1983a; Scheuermann, 1984). They are situated in the epithelial tissue and belong to the NEBs. The blue-green fluorescent cell groups, which appear as small intensely fluorescent cells, belong to pulmonary ganglia, located in the intraparenchymal connective tissue (Scheuermann et ul., 1984b,c). After formaldehyde condensation and microspectrofluorometry, the latter cells show excitation and emission maxima at about 415 and 480 nm, respectively, corresponding to the occurrence of catecholamines (Bjorklund et al., 1975). Moreover, the fading reaction follows the typical course for catecholamines. After acidification and analysis of the relative intensities of the excitation maxima, the identification of the catecholamine at the cellular level was performed (Scheuermann et al., 1984b). In the lungs of the monkey, pig, rat, and rabbit, these two types of intensely fluorescent cells, i.e., 5-HT-containing NEBs and intraganglionic, catecholamine-containing, small intensely fluorescent cells with the characteristic microspectrofluorometric recordings, can be demonstrated as

9

0

1

2

3

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5 min

FIGS.7 A N D 8. 5-HT fluorescence and argentaffinity demonstrated consecutively in the same tissue section of the basal portion of a NEB in the lung of the red-eared turtle. (Fig. 7) Cluster of yellow-fluorescent cells occurring in the trabecular epithelium. Note the nerve fibers. located in juxtaposition to the base of the yellow-fluorescent cells and emitting a blue-green fluorescence (arrow). x 1050. (Fig. 8) The same section as in Fig. 7 after subsequent exposure to the Masson-Hamper1 silver technique, showing argentaffinity of granular material in the basal portion of the cells (arrow). Cells displaying a basal formaldehyde-induced fluorescence are identical to those which reveal argentaffin granules in the basal portion of the cell. x 1050.

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well (D. W. Scheuermann. unpublished observations). Immunocytochemistry carried out at the light microscopic level, using a specific antiserum against 5-HT, confirmed the presence of 5-HT in both NEBS and solitary NEE cells (Cutz et a/., 1982; Lauweryns e l a / . , 1982; Sonstegard et al., 1982; Memoli el al., 1983). It should be made clear that the bluegreen fluorescent cells described in the mouse lung (Eaton and Fedde, 1977) contain catecholamines and do not belong to the NEBs, being intraganglionic, small intensely fluorescent cells located in the lung interstitium. It can be assumed that both populations of APUD cells, i.e., the NEE cells as well as the small intensely fluorescent cells of the intrapulmonary ganglia, might each serve as an origin for lung tumors belonging as they do to elements of the diffuse peripheral endocrine system (Scheuermann et a / . , 1983b). VII. Immunocytochemistry for Regulatory Peptides Since NEE cells and NEBs of the respiratory system have many features in common with cells from the APUD series initially described by Pearse (1966, 1968, 1969), they may be expected to produce polypeptide hormones. Indeed, as revealed by recent immunohistochemical studies in paraffin sections, pulmonary NEE cells may contain a variety of regulatory peptides. Bombesin-like immunoreactivity was detected in NEE cells of the bronchial and bronchiolar epithelium of the fetal and newborn human lung, both in single cells and in groups of cells (Wharton et a/., 1978; Johnson et a / . , 1982; Stahlman e l al., 1982, 1985); it was shown to be particularly abundant in the second trimester to term (Track and Cutz, 1982). Furthermore, bombesin-like immunoreactivity was detected in NEE cells at all levels of the tracheobronchiolar tract of the adult human lung (Cutz et d.,1981, 1984; Polak and Bloom, 1984) and in NEE cells of the airways of adult rats (Marchevsky and Kleinerman, 1982). Calcitonin-like immunoreactivity was revealed in single cells as well as in some NEBs of human fetuses and neonates (Becker et al., 1980; Stahlman et d.,1982, 1985), in fetal and adult human lungs (Cutz et a / . . 1981), FIG.9. Two fading curves from NEB cells and adjacent varicose nerve fibers. demonstrating the different photodecomposition rate of both fluorophores. The lower curve shows a rapid decomposition of the fluorescence in NEB cells after irradiation with the most effective wavelength. which is especially pronounced in the first minute and finally results in a total fluorescence decrease of about 70% after 5 minutes, typical of formaldehyde-induced 5-HT fluorophore. The upper curve renders the slow and gradual decrease (-20%) in fluorescence intensity of the nerve fibers under identical conditions. i.e., typical of formaldehyde-induced catecholamine fluorophores. [Fig. 7-9 modified from Scheuermann c’t ul. ( 1983a). Reprinted with permission of publisher.]

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and in NEBs of adult rats (Marchevsky and Kleinerman. 1982). However, calcitonin-positive NEE cells appeared most numerous in neonates compared with fetal and adult lungs. Leu-enkephalin antiserum revealed immunostaining in the peripheral airways of the fetal, neonatal, and adult human lung, but only in a few single NEE cells (Cutz et al., 1981). This was also true, to the same extent, during hyperplasia of pulmonary NEE cells in patients with bronchiectasis and bronchial epithelial neoplasms (Memoli et al., 1983). Immunoreactivity to gastrin-releasing peptide, the mammalian counterpart of amphibian bombesin (McDonald et al., 1979; Iwanaga, 1983; Tsutsumi et al., 1983b), was found in the fetal and adult human lung, both in single NEE cells and in NEBs (Takahashi and Yui, 1983; Tsutsumi et al., 1983a,b). However, bombesin immunoreactivity should be attributed to gastrin-releasing peptidelike molecules, which are present in the human lung (Price et al., 1983; Yoshizaki et al., 1984). Consecutive sections of the fetal human lung, alternatively immunostained for 5-HT and gastrinreleasing peptide, revealed the coexistence, within the same NEE cell, of a biogenic amine and a peptide (Takahashi and Yui, 1983). Chemical and immunocytochemical studies confirmed the simultaneous occurrence of neuropeptides and amines in NEE cells and lead to the concept that the coexistence of different bioactive substances in neuroendocrine cells is the rule rather than the exception (for reviews, see Owman et al., 1973; Fujita and Kobayashi, 1974; Pearse, 1976; Hokfelt et al., 1980; Sundler et al., 1980; Fujita, 1983): Using serial sections, immunoreactivity for 5-HT, bombesin, and somatostatin have been detected in the same NEBs of the fetal monkey lung (Dayer et al., 1985). According to their immunoreactivity, four groups of NEBS can be distinguished, i.e., NEBs containing ( I ) 5-HT, bombesin, as well as somatostatin; (2) 5-HT and somatostatin; (3) 5-HT and bombesin; (4) 5-HT only. Will et al. (1985) showed that, in the monkey, NEBs may also yield cholecystokinin immunoreactivity. Whether these peptides are present in different or in the same cells remains to be clarified. Although the simultaneous localization of more than one peptidergic antigen in a single cell has not frequently been reported, the coexistence of gastrin-releasing peptide and calcitonin was demonstrated within a subpopulation of NEE cells of the human bronchial tree using a serial section technique (Tsutsumi et al., 1983b). Whether these different peptides coexist in the same secretory granules of the NEE cells is unknown, although a coexistence in the same granules was recently described for met-enkephalin and oxytocin within nerve terminals of the posterior pituitary gland (Adachi et al., 1985). Species differences exist in relation to the immunoreactivity to regulatory peptides. For instance, in cats, bombesin

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is located in .scattered NEE cells of the upper respiratory tract and only occasionally in the bronchiolar epithelium, whereas immunoreactivity to bombesin could not be confirmed in rats and guinea pigs (Ghatei et al., 1982).Apparently, the NEE cell neoplasms of the respiratory system may express a larger spectrum of neuropeptides than has been found so far in normal NEE cells, e.g., they may demonstrate ectopic ACTH immunoreactivity (Gould et al., 1983a,b; Tsutsumi et al., 1983a; Polak and Bloom, 1984; Said, 1984). Since the above-mentioned peptides revealed in the NEE cells of the respiratory tract are known to be widely distributed in the central and peripheral nervous system (Becker et al., 1979; Yanaihara et al., 1981; Yui et al., 1981), it may be that they discharge peptides either into intercellular spaces, acting at least locally as neurotransmitters and/or neuromodulators, or as circulating hormones into adjacent blood capillaries. VIII. Electron Microscopic Aspects

The NEE cells, whether occurring solitarily or clustered, may have various aspects and can be clearly recognized as intraepithelial elements (Fig. 10). The shape of single NEE cells is variable; but triangular, flask-shaped, or pear-shaped cells were regularly encountered, extending from the basement membrane to the airway surface, where they may terminate in a narrow tuft of microvilli (Fig. 18). Others, resting with a broad, basal pole on the basement membrane, prolong their narrow apical portion toward the luminal surface, without actually reaching the airway lumen; it is not unlikely that the latter transforms into the former. Grouped cells appear mostly in a palisade-like arrangement spanning the height of the epithelium between the airway lumen and the underlying connective tissue. Sometimes, the NEE cells revealed a stratification. The basal cells are oval or polygonal, resting on the basement membrane as well as interdigitating with the overlying rather pear-shaped cells with a major portion of the cytoplasm at their base. Some cells of the superficial layer contact the airway lumen by means of a narrow process (Fig. 11) (Ericson et d., 1972; Lauweryns et al., 1972; Terzakis ef al., 1972; Hung et al., 1973, 1979; Moosavi et al., 1973; Tateishi, 1973; Cutz et d., 1974; Hage, 1974; Walsh and McLelland, 1974; Lauweryns and Goddeeris, 1975; McDowell et ul., 1976; Taira and Shibasaki, 1978; Edmondson and Lewis, 1980; Johnson et al., 1980; Goniakowska-Witalinska, 1981; Scheuermann et al., 1983a,b, 1984a). As a result of the irregular course of the apical portion of these cells, it is not always possible in the same section to observe both the cell body and the apical

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process contacting the airway lumen. However, microvillous projections can be found arising from the apical surfaces. In some species, NEE cells of a NEB arising from a basal body may extend a modified or primary solitary cilium into the intercellular space or into the airway lumen (Rogers and Hailer, 1980; Scheuermann rt ul., 1983a). In transverse sections of the proximal portion of the cilium, the axonemal pattern appears as 9 + 0, representing only nine peripheral doublets without a central pair. Somewhat more distally, one of the peripheral paired tubules may be observed to be displaced gradually toward the center of the cilium, while eight pairs are arranged evenly around the periphery, resulting in an 8 + 1 axonemal configuration. An associated centriole connected to striated rootlets can be observed as a diplosomal basal structure. A similar kind of cilium has been reported in some neurons and sensory cells with a well-known receptor function (Barnes, 1961; Meyer and Bencosme, 1965; Dubois and Girod, 1970; Munger, 1971; Vigh and Vigh-Teichmann, 1973; Afzelius, 1975; Vigh-Teichmann et ul., 1976a,b. 1980; Kataoka, 1974). Although it should be borne in mind that the function of the single cilium remains unknown, the occurrence of such a modified cilium in some sensory receptor cells is suggestive of the detection of environmental conditions, such as chemical, osmotic, or local mechanical changes. Moreover, receptors on the microvillous apical cell membrane might recognize stimuli from the airway lumen and transduce them by an intracellular mechanism which triggers off or arrests the release of secretory granules. The morphological features of the apical portion of the NEE cells of the respiratory system are similar to those of the gastroenteric endocrine cells; hence, the hypothetical term of “taste cells,” proposed by Fujita and Kobayashi (1974) in their study on the gut, might be borrowed to designate similar cells in the lung. In lower vertebrates, as reported in the toad lung, when discussing light microscopic aspects, the apical surface of the NEE cells of a NEB can be observed in electron micrographs to be completely covered by an apical cell, the luminal pole of which is comparable with that of well-known receptor cells (Rogers and Hailer, 1980). This apical cell, provided with microvilli and a primary cilium, also contains microtubules, bundles of microfilaments, rough endoplasmic reticulum, and dense-cored granules. FIG. 10. NEB in the trabecular epithelium of the red-eared turtle. The neuroendocrine epithelial cells form an organoid structure between the basement membrane and the flattened Clara-like cells (arrow) covering most of the apical surface. Some neuroendocrine epithelial cells ( * I extend from the basement membrane to the airway lumen. The capillary lumen (C) is surrounded by a thin-walled endothelium and a narrow, subendothelial space. At lower left, the pulmonary ciliated epithelium. x 5600.

FIG.I I . Superficial neuroendocrine epithelial cell of a NEB of the red-eared turtle covered by cytoplasm from Clara-like cells. The remainder opens onto the air space and bears microvilli. Intercellular junctions (arrow). Note the concentration of dense-cored vesicles in the basal portion of the cell. x 8500.

FIG. 12. Putative neuroepithelial endocrine cell in the lung of a developing red-eared turtle, observed in mitotic division. The cytoplasm contains a moderate number of characteristic dense-cored vesicles. x 10,500.

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It was proposed that the apical cell functions as a receptor-transducer cell and that the underlying NEE cells serve as an additional source of peptides-biogenic amines to be released on stimulation of the apical cell. The combination of NEE cells buried in the respiratory epithelium with a specialized epithelial cell that could serve as a chemoreceptor stimulating the secretion by contacting NEE cells was also observed in the fetal human lung (Stahlman and Gray, 1984). In the adult human tracheobronchial tract, NEE cells occur singly (Bensch r t d . , 1965; Gmelich et a / . , 1967; Basset el ul., 1971; Terzakis c’t ul., 1972; Tateishi, 1973; Bensch et ul., 1968), adjacent to the basement membrane, rarely extending to the airway surface (McDowell et ul., 1976). Conversely, in the fetal and neonatal human lung, numerous NEE cells extend from the basement membrane to the luminal surface, terminating in a microvillous border (Hage, 1973b; Stahlman and Gray, 1984). In scanning electron microscopy, the luminal surface of NEBs of the fetal rabbit (Cutz et id., 1978b) and rat (Carabba et d . , 1985) appears partially covered with nonciliated cells showing dome-shaped protrusions, which make them similar in appearance to Clara cells (Fig. 14) (Kuhn et a / . , 1974; Kuhn, 1976). Correlated scanning and transmission electron microscopy revealed that, in neonatal mouse lungs, the Clara-like cells failed to display both the large amounts of endoplasmic reticulum and the secretory granules described as characteristic for mature Clara cells (Hung et r i l . , 1979). However, numerous large mitochondria and accumulations of particulate glycogen did occur (Sonstegard et ul., 1982). The differences in ultrastructural features between Clara cells of neonatal and adult mammals may reflect the immaturity of the former (Smith et al., 1974). Yet, in the red-eared turtle, the boundary of NEBs was outlined by flattened nonciliated cells containing numerous mitochondria as well as membranebound secretory granules and conspicuous cisterns of smooth and rough endoplasmic reticulum, which are all cytoplasmic features of Clara cells (Scheuermann et ul., 1983a). Interdigitating basolateral cytoplasmic processes of NEE cells may extend between other epithelial cells (Bensch et d . , 1965; Lauweryns and Peuskens, 1969; Tateishi, 1973; Dey et ul., 1981). Along the lateral interfaces next to the lumen of adjacent epithelial cells, tight junctions have been observed in the lung of the adult rat, the adult hamster (Edmondson and Lewis, 1980), and the adult mouse (Hung et ul., 1973; Hung and Loosli, 1974). In the adult human lung, the lateral cell membranes of NEE cells are linked to adjoining cells by desmosomal structures (Hage et ul., 1977). Junctional complexes, composed of zonulae occludens, zonulae. and maculae adherens, linking NEE cells to adjacent nonendocrine cells, are reported in the toad lung (Rogers and Haller, 1978). In the red-eared turtle,

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NEE cells are interconnected and linked to adjacent Clara-like cells by small desmosomes (Scheuermann er a / . , 1983a). As yet, there is no physiological evidence that would indicate a possible coupling between NEE cells. In large NEE cell clusters of the rat and hamster lung, narrow interstitial spaces reveal expansions, forming channels in which microvilli are seen to extend (Moosavi et al., 1973; Edmondson and Lewis, 1980). These intercellular canaliculi-like spaces resemble the channels between other endocrine cells, such as the hypophysis (Rennels, 1964) and the adrenomedullary cells (Wetzstein, 1957; Coupland, 1965; Elfvin, 1965; Coupland and Weakley, 1970; Grynszpan-Winograd, 1975). Hematogenous and interstitial substances may be transported through the channels in order to reach the receptor site of the basolaterdl cell membrane of the NEE cells, to which these may respond by triggering off or arresting a hormone release, in analogy to the D-glucose activation process described in the islets of Langerhans of the rat (Niki P r ul., 1974). The nucleus, usually spherical or ovoid with some small indentations, is located in the bottle-shaped cells at the entrance to the narrowed portion as well as in the polygonal cells almost at the center. This nucleus contains patches of dense chromatin, situated along the nuclear membrane and surrounding a prominent nucleolus. The nuclear envelope shows numerous pores. In the human fetal lung (Stahlman and Gray, 1984) and in the lung of the developing red-eared turtle, a putative NEE cell was observed in mitotic division (Fig. 12). A well-developed Golgi complex is often composed of greatly distended sacculi and different kinds of vesicles, partly filled with an electron-dense content (Fig. 16). It is usually present in a supranuclear position, although sometimes a lateral Golgi complex can be observed as well. This pattern, more evident in NEE cells abutting on the luminal endings, suggests some double functional polarity. There are investigations providing morphological indications for the involvement of the Golgi complex in the formation of dense granules (Gmelich et ul., 1967; Lauweryns and Cokelaere, 1973a; Hage, 1974; Taira and Shibasaki, 1978; Scheuermann et al., 1983a). FIG.13. Long strands of rough endoplasmic reticulum with attached and free polyribosomes visualized in the lateral region of a granule-containing cell in the red-eared turtle. x 38,000. FIG.14. NEB in the bronchiolus of a neonatal rabbit is largely covered by protruding nonciliated Clara-like cells. The uncovered part of neuroepithelial endocrine cells can be recognized in a craterlike depression between the Clara-like cells, where the stubby microvillous projections of the narrow tips of these cells reach the airway lumen. Arrow indicates the exposed surface of the NEE cells. Adjacent bronchiolar epithelium is composed of ciliated cells. Scanning electron microscopy. x 3200.

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In most species, the cytoplasm contains rather small mitochondria (Ericson et a/., 1972; Cutz et a/., 1974, 1975). Multivesicular bodies, lysosomes, pinocytotic vesicles, microtubules, and a few glycogen particles are present in variable numbers. The granular endoplasmic reticulum is usually dispersed in the cytoplasm, but sometimes a configuration with attached and free polyribosomes may resemble, to some degree, the Nissl substance of neurons (Fig. 13). Bundles of branching microfilaments, frequently packed in sheaves (Fig. 15) (Cook and King, 1969; Cutz and Conen, 1972; Ericson e f a / . , 1972; Lauweryns et a / . , 1972; Terzakis rf d . , 1972; Hung et ul., 1973; Hung and Loosli, 1974; Cutz et a / . , 1975; Taira and Shibasaki, 1978; Johnson et a / . , 1980; Scheuermann et a / . , 1983a; Hung, 1984; Pack and Widdicombe, 1984; Stahlman and Gray, 1984; Pearsall ef a / . , 1985), are considered by many authors as a major distinguishing feature of these cells. Although the role of microfilaments is as yet unknown, their presence is of special importance in the pathology-oriented classification of carcinoids of the bronchopulmonary tumors (Gould et a / ., 1983b). In electron micrographs, the most striking feature of the NEE cells is the presence of numerous vesicles with granular cores, referred to as dense-cored vesicles (DCV), which vary in number, sometimes scattered throughout the cytoplasm, but frequently tending to accumulate in the broad basal portion of the cell (Figs. 17 and 19). The shape of the cells, with their deep broad pole, the location of the nucleus, and the basal position of the secretory granules indicate that the pole of discharge is directed toward the connective tissue, in which capillaries are closely adjacent to the basis of the NEE cells. These features, in addition to the fact that the capillary endothelium sometimes appears fenestrated (Lauweryns ef d . , 1974; Scheuermann et d . , 1983a), strongly suggest that the NEE cells may function as endocrine glands. The DCV are usually spherical, but can also be irregular in shape. A limiting smooth-surfaced membrane encloses the osmiophilic content entirely. Usually there is a clear halo between the dense core and the membrane, ranging in width from a few nanometers to 20 nm. In larger vesicles, the dense core may be at the center of the vesicle or adhering eccentrically to the limiting membrane. The external diameter usually ranges from 60 to 200 nm in diameter (Bensch et a / . , 1965; Cook and King, 1969; Ericson et a / . , 1972; Lauweryns et a / . , 1972; Terzakis et d . , 1972; Hung ef a / . , 1973; Moosavi et a/., 1973; Cutz et a / . , 1974; Hung and Loosli. 1974; Walsh and McLelland, 1974; Cutz and Orange, 1977; Hage et u/., 1977; Becci et a / . , 1978; Taira and Shibasaki, 1978; Johnson et d . , 1980; Hung, 1984; DiAugustine and Sonstegard, 1984; Stahlman and Gray, 1984). The size of the DCV is sometimes described as specific for each animal species.

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F a . 15. Sheaves of microtilaments can be observed in the cytoplasm between the nucleus ( N ) and the Golgi complex ( G ) of a neuroepithelial endocrine cell of the red-eared turtle. x 40,000.

FIG. 16. Golgi complexes of a neuroendocrine epithelial cell of the red-eared turtle with electron-dense materials in some Golgi vesicles, indicated the involvement of this organelle in the formation of the specific granules. x 2 6 . 0 0 .

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FIG. 17. Base of a NEB, showing numerous secretory granules concentrated near the basement membrane. Some granules (arrow) contact the plasma membrane. In the connective tissue, numerous nonmyelinated nerve fibers occur immediately below the NEB. x 14,000.

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e.g., mouse, 107 nm, and rabbit, 142 nm (Hage, 1974). However, both the size and electron density of the DCV vary greatly not only from cell to cell, but even within the same cell (Lauweryns et ul., 1972, 1974; Taira and Shibasaki, 1978; Goniakowska-Witalinska, 1981). When the core is less dense, it may show a faintly granular substructure. In some DCV, a central constriction is seen, which appears to divide the granule into two portions, containing two dense cores as in a hourglass. They possess the same fine structure as the neurosecretory granules found as a normal component in the adrenomedullary cells. Different fixation and staining procedures may affect the ultrastructural appearance of the membrane-bound granules (Chen et a l . , 1969; Matthiessen et al., 1973; Schafer et ul., 1973). The dense core of the DCV in adult human bronchial NEE cells, stained intensely with phosphotungstic acid at low pH. suggests the presence of a glycoprotein (McDowell et d., 1976). Since glycosylation of proteins in order to form glycoproteins appears to be one of the functions of the Golgi complex (Dauwalder et ul., 1972), the close relationship between maturating DCV and the Golgi complex does not seem surprising. The material contained in the halo of the vesicle may be variously extracted by the fixation fluids or by other substances used for dehydration and embedding, thus accounting for the different electron density of the granule halos (Schafer et ul., 1973). but it is hard to accept that the procedures used for tissue processing can produce different effects in the same sample on identical cell components. Ultrastructural studies have revealed as many as three types of NEE cells in fetal human lungs (Cutz and Conen, 1972; Hage, 1972, 1973b, 1980; Capella et ul., 19781, mainly on the basis of the fine-structural morphology of their cytoplasmic secretory granules. The first and most frequent cell type, called P, cells, bears small secretory granules with a mean diameter of about I10 nm. Two varieties of granules may be present: ( I ) membrane-bound, spherical granules displaying a thin clear halo interposed between the central dense core and the membrane; (2) spherical to ovoid vesicles containing a small, eccentrically situated core of variable electron density. These P, cells are provided with an extensive rough endoplasmic reticulum, an expanded Golgi complex, scattered small vesicles, and microtubules. P, cells are located in all parts of the bronchial tree of the developing lung and give a positive argyrophilic reaction, whereas only FIG. 18. Enlargement of the luminal pole of a N E B , showing the apical portion of a neuroepithelial endocrine cell bearing a tuft of rnicrovilli. Junctional complexes (arrow).The Golgi complexes are supranuclear. The apical cytoplasm contains a moderate number of characteristic dense-cored vesicles and lysosomes. X 17,000.

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the cells with vesiculated granules are argentaffin. They can be observed forming clusters similar to the corpuscular arrangement of NEBS. The second type, called P2 cells, contains slightly larger, spherical granules with a mean diameter of about 140 nm and provided with a moderately electron-dense core surrounded by a thin transparent rim. Likewise, PI cells are found in all parts of the bronchial tree of the human fetus. The third type, P, endocrine cells, possess large, spherical, membranebound granules with a mean diameter of 190 nm, displaying a homogeneous electron-dense content. In the normal fetal human lung, these P, cells are restricted to the larger bronchial tubes, where they are present in small numbers. According to Hage (1973b), P, and P2 cells may be merely different functional stages of the same cell. In the normal adult human lung, she observed only a single type of endocrine cell, which she called Pa cells; they are provided with spherical, dense-cored. secretory granules characterized by their uniform size and homogeneous appearance. The diameter of the vesicle ranges from 110 to 140 nm (Hage et ul., 1977). There is a narrow clear rim between the core and the surrounding membrane, similar to the secretory granules of the PI cells in the fetal human lung. The P, cells of the fetal human lung and the P, cells from the adult human lung resemble certain endocrine cells in the stomach and pancreas (Solcia e t d.,1975; Capella et ul., 1978). A similar ultrastructural identification of NEE cells in the fetal human lung, which contain distinctive secretory granules, was described by Stahlman and Gray (1984). Some authors correlate these distinct types of NEE cells, defined by the features of vesicle appearance, with the three types of NEE cells distinguished according to their immunoreactivity to neuron-specific enolase, whether or not combined to 5-HT and bombesin (Polak and Bloom, 1982). However, it should be pointed out that biochemical data (Scrutton and Utter, 1968) as well as ultrastructural immunocytochemical observations on neuroendocrine cells (Zabel and Schafer, 1985) indicate that neuron-specific enolase apparently is not associated with secretory granules. Nevertheless, in electron micrographs of the fetal human lung, gastrin-releasing peptide immunoreactivity was found in cytoplasmic granules (Iwanaga, 1983) similar to the DCV of the cell type classified as P, (Hage, 1973b). FIG.19. Enlargement ofthe basal part o f a NEB. showing secretory granules of various shapes and electron density. At the upper right, a capillary separated from the neuroendocrine epithelial cell by a subendothelial space containing collagen fibers. x 28,000. FIG. 20. Part of ii NEB of the red-eared turtle stained with the Masson-Hamper1 argentaftin reaction on ii grid. Intense deposition of silver grains is shown in the granules. A slight background precipitation of silver is seen over the cytoplasm and nucleus. x 15.000.

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Some authors feel inclined to assume that every ultrastructural type of NEE cell produces one specific peptide, i.e., that each peptide is located in a different type of NEE cell (Cutz et al., 1981). However, this finding is difficult to reconcile with the observation, in serial sections of immunostained material, that the same NEE cell may contain different peptides (Tsutsurni et al., 1983b; Zabel, 1984). These peptides may even be present in the same secretory granule, as was demonstrated for met-enkephalin and oxytocin within nerve terminals of the neurohypophysis (Adachi et al., 1985). Conclusive correlation of the ultrastructurally defined types of NEE cells with the presence of specific peptides requires the application of a double immunocytochemical staining technique for the simultaneous demonstration of coexistent neuropeptides at the electron microscopic level. In fetal and adult human trachea as well as in the trachea of adult rabbits, Cutz et al. (1975) found NEE cells with only one type of spherical granules, measuring about 100 nm in diameter and displaying a homogeneous dense core surrounded by a clear space of 16-18 nm. DiAugustine et al. (1984) also described one kind of NEE cells in the trachea of the guinea pig. Conversely, in the tracheal mucosa of lamb and armadillo, Cutz et ul. (1975) demonstrated two distinct types of NEE cells whose DCV differ not only in their mean diameter, i.e., 168 versus 112 nm for the lamb and 175 versus 125 nm for the armadillo, but also in their configuration and electron density. It seems likely that NEE cells in the lung of man and various animals have ultrastructural differences particularly with respect to their DCV. This diversity could reflect species variation (Hage, 1974). but the differences might just as well be due to variations in the physiological and/or pathological state. Ultrastructural studies have revealed that a number of human bronchial carcinoid tumors and oat cell carcinomas of the lung (e.g., Bensch et ul., 1965, 1968; Toker, 1966; Gmelich et al., 1967; Hachmeister and Okorie. 1971; Hattori et al., 1972; Gould et a l , , 1983a,b, 1984) is composed of DCV-containing cells. Compared to the normal adult human bronchial epithelium, the secretory granules in these cells may display a wider range in size and shape, as well as a greater variation in both electron density and ultrastructural configuration (for reviews, see Hage et al., 1977; Taira and Shibasaki, 1978; Gould et ul., 1983a,b, 1984; Hage, 1984). This heterogeneity of the granules points to a similarity with some enterochrornafin cells of the human gastric mucosa (Pearse et al., 1970; Hage, 1973d; Solcia at ul., 1975),which reflects the common entodermal origin of the gut wall and the bronchoalveolar tree. It has been reported that morphometrical analysis, after glutaraldehyde fixation, of fetal rabbit NEBS might provide evidence for the existence

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of two types of DCV within the same cell (Lauweryns et al., 1972, 1974; Sonstegard et al., 1979). Type I DCV are wedge or ovoid, with a diameter of about I34 nm, containing an electron-dense amorphous core, usually with a narrow clear halo subjacent to the limiting membrane. In this halo, acetylcholinesterase was detected (Lauweryns and Cokelaere, 1973a). Near the center, the electron-opaque core may contain a compact deposit surrounded by a more grayish periphery extending up to the limiting membrane. Type I1 DCV have a more spherical shape with a diameter of about 112 nm and a less electron-dense core surrounded by a distinct, large, perigranular, clear halo of about 15-20 nm. Formalin pretreatment blocks the active sites of catecholamines but does not prevent the indolamines from reacting with glutaraldehyde in order to form a SchifT monobase. Since this is necessary for the subsequent reaction with potassium dichromate in order to obtain electron-opaque deposits, the formalin-glutaraldehyde-dichromate method is considered to be specific for indolamines (Wood, 1967; Jaim Etcheverry and Zieher, 1968). The latter method was applied to NEE cells, demonstrating that only type 1 DCV yield dense reactive granules, in contrast to type 11 DCV, which can be seen after uranyl acetate staining only (Lauweryns et d., 1972, 1977). This observation therefore suggests that only type I DCV contain 5-HT. In addition. the latter authors consider the possibility of a direct conversion of DCV I1 into DCV I, which may represent their mature form (Lauweryns et a / . , 1977). Thus, the differences in size, shape, and characteristics of the DCV within the same NEE cell are assumed to represent stages of granular genesis (Lauweryns et a / . , 1977; Sonstegard et al., 1979). These results might parallel the different maturation stages during the development of some granular vesicles in the adrenergic neuron (Machado, 1971). Their formation in adrenergic fibers seems to be initiated by an agranular vesicle in which the development takes the place of an eccentric small core attached to the vesicle membrane. The size of the core increases after further accumulation of dense or semidense material, finally resulting in a vesicle with a large dense core, apparently forming a mature DCV. This comparison is supported by the fact that secretory granules of NEE cells display transitional stages with regard to the density of their content. Thus, besides vesicles which are almost empty, there are granular vesicles whose dense core area is extremely small, i.e., with a large clear halo up to the limiting membrane, as well as others with a large dense core filling the vesicle nearly completely, i.e., up to the membrane. In controlled studies of hypoxia on neonatal rats, DCV reveal ultrastructural changes indicating almost the reverse phenomenon of what was suggested in connection with the development (Moosavi et al., 1973). A

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widening of the clear halo between the osmiophilic core and the limiting membrane goes hand-in-hand with a decrease in size and electron density of the core, which often becomes not only minute but eccentric, lying attached to the limiting membrane. Moreover, many empty vesicles are formed. Hence, one gains the impression that type 1 and type 11 DCV are the extremes of a continuous series of different degrees of amine and/or polypeptide filling. Thus, the type 11 DCV would contain little if any biogenic amines, which are hardly detectable by the poorly sensitive aldehyde-osmium tetroxide method. The morphological variations of DCV observed in chronic hypoxia might be considered as an enhancement of variations observed in DCV of normal controls. Quantitative fluctuations between these different types of granular vesicles occurring under normal conditions may reflect physiologic changes in the oxygen content of the inhaled air. Using Fontana’s ammoniacal silver technique at the fine-structural level (Hgkanson et al., 1971), the membrane-bound granules of the bronchopulmonary NEE cells, at least those of the frog (Rogers and Haller, 1978) and the turtle (Scheuermann et ul., 1983a, 1984a), stained selectively by silver deposits over the dense core of the vesicles, demonstrating the argentaffin reaction of the DCV (Fig. 20). This is in agreement with electron microscopic studies of Ericson and co-workers (l972), who have shown that tritiated 3,4-dihydroxyphenyl-~-alanine and 5-hydroxytryptophan are taken up and incorporated into DCV of NEE cells of the mouse lung (trachea). This indicates the presence in the granules of a synthetized amine from exogenous precursors. Moreover, electron microscopic immunocytochemical studies on NEE cells of the respiratory system of human fetuses revealed that gastrin-releasing peptide immunoreactivity is located in DCV (Iwanaga, 1983). Thus, the results obtained so far by light and electron microscopy seem to indicate that, as has also been assumed for endocrine cells in other organs, the DCV of the NEE cells belonging to the respiratory system may be considered as a storage site of biogenic amines and polypeptide hormones (Owman et ul., 1973). There is evidence suggesting that a biogenic amine, whether in combination with coexisting substances, is liberated from the NEE cells by vesicular exocytosis, i.e., direct extrusion of the entire vesicular content to the extracellular space after fusion of the vesicular limiting membrane and the basolateral plasma membrane. Invaginations of the plasma membrane, sometimes containing an amorphous material, similar in size to the DCV content, are indicative of this phenomenon (Lauweryns and Cokelaere, 1973a; Taira and Shibasaki, 1978; Scheuermann et al., 1983a). This suggests that the secretion from the NEE cells could be directed to struc-

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tures below the basement membrane, such as capillaries, smooth muscle cells, and mucosal glands. Granular release at the luminal surface has never been observed. In bronchopulmonary NEE cells of the rabbit, during hypoxia or after intake of certain drugs, such as nicotin or reserpin, the DCV are clearly shifted to the basal pole of the cell. Eventually their bounding membranes are in contact with the basal cytoplasmic membrane, a feature which is rather exceptional in normal animals (Lauweryns et d . , 1977). The content of the granular material appears to empty into the intercellular space at an increased rate by emiocytosis. This phenomenon is correlated with a decrease of 5-HT, as shown by FIF (Lauweryns et d . , 1977). IX. Location

The presence of NEE cells in the epithelium of the respiratory system of man and every vertebrate species examined is well established, although they are not evenly distributed. As early as 1949, Frohlich reported argyrophilic cells to occur mainly at the bifurcations of large and small bronchi as well as at the sites of transition from the bronchioli terminalis to the bronchioli respiratorii. This was later confirmed by several investigators (e.g., Lauweryns et d.,1972; Lauweryns and Goddeeris, 1975; Hage, 1976; Hung et al., 1979; Foliguet and Cordonnier, 1981; Cutz et (11.. 1984; Sarikas et ul., 1985a.b). Single NEE cells were found distributed over almost the entire respiratory system [e.g., larynx (Ewen et a / . , 1972; Kirkeby and Rgmert, 19771, trachea (Ericson et al., 1972; Cutz et al., 1975; Dey et al., 19x1, 19831, and bronchi and bronchioli terminalis (Lauweryns and Peuskens, 1969; Terzakis et d.,1972; Moosavi et d.,1973; Hernandez-Vasquez et al., 1977; Stahlman and Gray, 1984; Stahlman et d . , 1985)l. whereas NEBS seemed to be restricted to the intrapulmonary airways (Cutz et d.,1975). Frohlich (1949) and various later investigators reported that the number of these NEE cells increases in a distal direction up to the smallest bronchi and that the distance between NEE cells increases from the bifurcations of the bronchioli respiratorii onward. As for the upper airways, the ventral mucosa of the trachea revealed more NEE cells than the dorsal mucosa. predominantly in the cranial segment (Dey et d . , 1981),a finding confirmed in the guinea pig (Kirkeby and Rgmert. 1977; DiAugustine et a/., 1984) and rat (Kleinerman et al., 1981). In the lungs of fetal and newborn rabbits (Lauweryns e t al., 1972) and mice (Hung et al., 1979), the number of NEE cells appears high. Hemandez-Vasquez and co-workers ( 1977, 1978a) have shown the number of identifiable NEE cells in fetal rabbit to decrease from 26 to 29 days,

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followed by an increase between 29 days of gestation and I day of extrauterine life, and finally by an initial decrease after birth. The decrease observed in the rat pulmonary NEE cells after the fourth postnatal day is consistent with this pattern (Moosavi et al., 1973). In the adult rat, these cells occur predominantly in the trachea, gradually decreasing in the smaller airway branchings (Kleinerman et al., 1981). Postnatally, the NEB density and average diameter in rabbits were found to decrease in conjunction with the increase in lung volume (Redick and Hung, 1984). Systematic studies have revealed large numbers of NEE cells in fetal human lungs in the early canalicular period (Cutz and Orange, 1977). These were more numerous in proximally differentiated bronchial tubes than in terminal buds. Thereafter, a gradual decrease in the number of NEE cells was observed, although the number per airway remained unchanged (Cutz and Orange, 1977). In the lungs of infants and adult man, NEE cells were found to be more numerous in small bronchi and proximal bronchioli, as compared with major bronchi and bronchioli terminalis (Lauweryns and Peuskens, 1969; Tateishi, 1973). The number of NEE cells is generally reported to decrease with age (for review, see Cutz, 1982; Keith and Will, 1982; DiAugustine and Sonstegard, 1984; Hung, 1984; Pack and Widdicombe, 1984). In some mammalian species, after an initial increase in NEE cells demonstrable in the fetal lung, an apparent decrease close to term was reported, followed by a considerable increase at birth. Since the apparent decrease close to term may be the result of a depletion of cytoplasmic secretory material, some authors ascribed an important role in the respiratory adaptation at birth to the activity of these cells (Lauweryns et al., 1982; Hernandez-Vasquez et al., 1978a; Cutz et al., 1984; Redick and Hung, 1984). However, in contrast to other mammalian species studied, maturation of NEE cells in hamsters does not appear to have been completed at birth (Sarikas et al., 1985a). The significance of these findings is strengthened by quantitative studies in this animal, demonstrating that, I day before birth, most peripheral bronchioles were devoid of NEE cells (Sarikas et al., 1985b). Obviously, it is not inconceivable that the apparently higher frequency of NEE cells in developing lungs of some animal species might be due to their early differentiation, or possibly, also to the smaller dimensions of the fetal lung, i.e., it could be argued that, with peri- and postnatal development of alveoli and growth of the airways, the NEE cells are distributed over an enlarged surface, as a result of which they cannot readily be detected. It appears that the number, as well as the presence, of NEE cells in the different parts of the airways varies not only according to the age of an animal, but also with respect to the species involved. Therefore, in the

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author's opinion, it is not yet possible to draw a final conclusion with regard to the actual significance of NEE cells at birth.

X. Innervation Using a modified silver impregnation technique for the staining of nervous tissue, Frohlich (1949) revealed, in the bronchial epithelium of rabbits and cats, fine nerve terminals to the very surface of the NEE cells and entering NEBs. He therefore suggested that these cells constitute an afferent' chemosensitive system comparable to the specific cells in the carotid and aortic bodies. Following Frohlich, a number of investigators has observed both light and electron microscopically a distinct innervation of the single and grouped NEE cells in the pulmonary tree of various animal species and in man (Cook and King, 1969; Lauweryns et al., 1970; Hung et al., 1973; Jeffery and Reid, 1975; Hung, 1976, 1980, 1984; Taira and Shibasaki, 1978; Goniakowska-Witalinska, 1980a, I98 I ; Al-Ugaily et a / . , 1983; Stahlman and Gray, 1984). Some authors described nerve endings on NEBs only (Lauweryns et al., 1972, 1974, 1985; Lauweryns and Cokelaere. 1973a; Cutz et al., 1974; Hung and Loosli, 1974; Rogers and Haller, 1978, 1980; Scheuermann et al., 1983a); others have not observed them on solitary NEE cells (Bensch et al., 1965; Terzakis et al., 1972; Cutz et al., 1974, 1975; Hage, 1974; Hage et al., 1977; Hage, 1980), which argues in favor of a subclassification in multicellular NEBs and solitary NEE cells. Different methods have been used to differentiate the nerves associated with NEE cells in the respiratory system. In silver-impregnated sections, unmyelinated nerve endings are described near the basement membrane and surrounding the epithelial cells of NEBs in newborn infants (Lauweryns and Peuskens, 1972) and various vertebrate species (Lauweryns et ul., 1972. 1973, 1974; Hage, 1976; Hung, 1980; Scheuermann et a / . , 1983a). They apparently originate from bundles of nerve processes running in the subepithelial connective tissue, where they are ensheathed by Schwann cells. Although some nerve endings may be traced in the immediate vicinity of argyrophil NEE cells, the pronounced argyrophilia of the latter often impedes a clear recognition of nerve terminals. Some authors reported a dense network of acetylcholinesterase-positive fibers in apposition to NEE cells [lamb (Cutz and Orange, 19771, neonatal rabbit and mouse (Lauweryns and Cokelaere, 1973a), fetal rabbit (Hung, 'The author uses the terms afferent and efferent for terminals on NEE cells of nerve fibers that conduct to and from the central nervous system, respectively.

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1980), fetal rat (Morikawa et al., 1978a,b), and fetal human lungs (Taylor and Smith, 1971)l. In some species, a blue-green fluorescent nerve plexus with microspectrofluorometric characteristics of catecholamines was demonstrated by the FIF method in the subepithelial connective tissue, with presumed nerve terminals contacting the yellow fluorescent NEBS (Lauweryns et al., 1972; Hung, 1980; Scheuermann et al., 1983a; Redick and Hung, 1984). The recorded peak of maximum emission is situated at 480 nm, with an excitation maximum at 4 10 nm, characteristic for catecholamines (Bjorklund r t al., 1975). After irradiation at the most effective wavelength, the photodecomposition showed a slow, almost linear decrease in fluorescence intensity, with a loss of less than 20% of the original intensity (Fig. 9). This fading characteristic is in accordance with the results of excitation and emission maxima, arguing for a catecholamine-dependent FIF (Ritzen, 1966; Bjorklund rt al., 1972a,b), and is in contrast with those results obtained for 5-HT-containing cells. In electron microscopic studies, isolated or small groups of nerve fibers and presumed nerve terminals are reported invaginating between NEE cells of a NEB (Hung et al., 1973; Lauweryns and Cokelaere, 1973a; Hung and Loosli, 1974; Lauweryns et al., 1974; Rogers and Haller, 1978, 1980; Goniakowska-Witalinska, 1981; Scheuermann et al., 1983a; Hung, 1984; Stahlman and Gray, 1984) or in close apposition to the basolateral plasma membrane of a single NEE cell (Lauweryns ef al., 1970; Hung et ul., 1973; Jeffery and Reid, 1973; Hung, 1976; Goniakowska-Witalinska, 1980a; Stahlman and Gray, 1984). From the examination of serial sections, it appears that the same nerve fiber may innervate, after branching, several NEE cells by means of bulbous and basket endings or fusiform “en passant” dilatations (Bensch et al., 1965). As a result, the course of one nerve terminal may display a range of appearances. The axoplasm is characterized by the presence of neurotubules. neurofilaments, and small mitochondria (e.g., Hung, 1984). Sometimes, glycogen particles are condensed in larger amounts. Many nerve endings feature an accumulation of various types of vesicles. Clusters of densely packed agranular vesicles of about 60 nm in diameter are almost always present in the approximately oval nerve endings (Figs. 21 and 23) (Cutz et al., 1974; Rogers and Haller, 1978; GoniakowskaWitalinska, 1980a; Scheuermann et al., 1983a). Between the clear vesicles, a few large granular vesicles can usually be observed, ranging in diameter from 90 to 110 nm. These nerve terminals and NEE cells are separated by an extracellular space, about 20 nm wide. Some authors reported nerve processes forming junctions with NEE cells, quite specific for synapses. Features characteristic of these synapses are the presence of cytoplasmic

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densities attached asymmetrically to apposed plasma membranes of both the nerve fiber and the NEE cell, as well as numerous small clear vesicles forming clusters close to the junctional material (Lauweryns ef u / . , 1970, 1972. 1974; Lauweryns and Cokelaere. 1973a; Rogers and Haller, 1978; Scheuermann et ( I / . , 1983a; Hung, 1984; Stahlman and Gray, 1984).These structures display the characteristics of efferent cholinergic nerve endings. Furthermore, electron microscopic observations have shown that some adrenergic nerve fibers appear related not only to blood vessels, but also to NEE cells, forming distinct synaptic contacts (Rogers and Haller, 1978). In addition to clear vesicles, these are characterized by small granular vesicles of about 60 nm in diameter, typical of adrenergic nerve varicosities (Rogers and Haller. 1978; Scheuermann et uI., 1983a; Stahlman and Gray, 1984).They can be correlated with the nerve endings observed using the FIF method, since this kind of vesicle is shown to store noradrenaline (Bisby and Fillenz. 1971). Rogers and Haller (1978) argue that the function of the adrenergic nerve fibers might be efferent, since there is no indication of transmission from the NEE cells to the nerve varicosities. In certain vertebrates. two kinds of synaptic regions may be recognized on the same afferent nerve terminal, arranged side-by-side. One region is postsynaptic' and the other presynaptic' to the NEE cell. In the former, an accumulation of small, dense-cored, and clear vesicles occurs along the surface of the presynaptic membrane thickenings in the cytoplasm of the NEE cell. In the latter synaptic component, clusters of small, agranular vesicles (25-50 nm in diameter) are aggregated near dense presynaptic projections on the surface membrane of the NEE cell, without accumulation of dense-cored vesicles in the NEE cell (Rogers and Haller, 1978). These complex paired synaptic contacts were described for the first time as reciproccd synupses in the central nervous system (Reese and Shepherd, 1972). In the peripheral nervous system, they were encountered in the carotid body (King et a / . , 1975; McDonald and Mitchell, 1975; Osborne and Butler, 1975) and cardiac ganglia (Yamauchi el d.,I975a,b). It has been suggested that the regions of reciprocal synaptic junctions, where the nerve terminal is postsynaptic to the NEE cell, are involved in a synaptic mechanism from NEE cell to nerve terminal, while the adjacent synaptic component interacts in the reverse direction. Nerve profiles containing another type of filled vesicle with a wider range in size (80-225 nm in diameter) and a moderately electron-dense content appear in apposition to single NEE cells and to NEBS (Stahlman and Gray, 1984), without yielding synaptic structures. These large granular 'The author uses the tcrms presynaptic and postsynaptic to designate the direction of synaptic transmission.

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vesicles may be compared with similar granules observed in peptidergic neurosecretory systems (Rinne, 1966; Bargmann et af., 1967; Krisch, 1974; Gibbins and Haller, 1979; Helen and Hervonen, 1981), which are presumed to belong to nonadrenergic, noncholinergic pathways (Baumgarten et a l . , 1970; Burnstock, 1982). Other enlarged nerve endings on NEE cells are crowded with slender mitochondria (Fig. 22). These terminals have been observed close (6-20 nm) to the plasma membrane of NEE cells of the respiratory system in different mammalian species, e.g., in the mouse (Hung et af., 1973) and rabbit (Lauweryns and Cokelaere, 1973a), and in nonmammalian species, e.g., in birds (Cook and King, 1969; King et al., 1974) and reptiles (Scheuermann et ul., 1983a). Some authors considered these nerve endings as characteristic for sensory (afferent) nerve fibers (Cook and King, 1969; Lauweryns and Cokelaere, 1973a; King et al., 1974; Rogers and Haller, 1978; Hung, 1980; Stahlman and Gray, 1984), with mitochondria serving as a source of energy for the transformation of the stimulus into a nerve impulse. As revealed by serial sections of the turtle lung, the number of mitochondria appeared to vary greatly from one region of a given nerve ending to another (Scheuermann et al., 1983a), confirming observations on nerve terminals in the carotid body (Verna, 1973). In comparison with the innervation of other tissues, the nerve terminals, tightly packed with mitochondria, are mostly considered as sensory (Rees, 1967; Bock et al., 1970; Chiba and Yamauchi, 1970; Kobayashi, 1971; Kondo, 1971; Munger. 1971; Chiba, 1972; Verna, 1973; King et al., 1974). Other authors attributed the accumulation of mitochondria to a degenerative change associated with a process of aging (Seitelberger, 1971; Leonhardt, 1976). However, in the latter case, mitochondria display various stages of disintegration and transitional forms between mitochondria

FIG.21. Cross section through a bundle of subendothelial nerve fibers in the lung of a red-eared turtle near neuroendocrine epithelial cells. Some nerve fibers, partially encased by Schwann cell cytoplasm, contain ( I ) peptidergic granules (PI,(2) cholinergic granules (arrow), (3) adrenergic nerve varicosities (arrowhead). x 19,000. FIG.22. A nerve ending associated with a neuroendocrine epithelial cell in the red-eared turtle lung. No synapse is present. but the nerve ending is densely packed with numerous slender mitochondria. x 47,000. FIG.23. Nerve terminal on the perikaryonal region of a neuroendocrine epithelial cell in the red-eared turtle lung filled with small clear vesicles. Part of a synapse is visible between the arrows. The basal lamina of the neuroendocrine epithelial cell runs at the outer side of the nerve terminal. x 56.000.

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and dense bodies. Nevertheless, in the lateral vestibular nucleus of the rat, dendritic growth cones packed with slender mitochondria are suggestive of regeneration (Sotelo and Palay, 1968). Alternating degenerative and regenerative processes might be considered to occur in nerve fibers abutting the NEE cells as suggested for nerve terminals in the carotid sinus (Knoche and Addicks, 1976). Although no strictly morphological criteria exist for the establishment of the afferent or efferent nature of the NEE cell innervation, selective nerve degeneration experiments are of considerable importance. It was shown that, after unilateral cervical infranodose vagotomy, degenerating intraepithelial axons appeared in the trachea of both the rat (Hoyes and Barber, 1981) and the cat, as well as in the bronchi (Das et a / . , 1979). This is in agreement with the concept that there exist intraepithelial afferent nerve fibers in the tracheobronchial tree. Recently it was shown that most axon endings in the NEBS rapidly degenerate after unilateral sectioning of the homolateral vagus nerve below the nodose ganglion, a process which does not take place after homolateral supranodose vagotomy. Hence, it appears that the cell bodies of these nerve endings are located in the nodose ganglion (Lauweryns and Van Lommel, 1983; Lauweryns et a / . , 1985). Selective labeling with tritiated amino acids of the nodose ganglia proved that the wall of the respiratory system possesses an afferent innervation, at least in the adult hen (Bower et al., 1978), where labeled fibers are observed to enter groups of NEE cells, suggesting a role as receptor. Although great differences in the innervation of the lung may exist among animal species (Richardson, 1979), it appears that the NEE cells are provided with a cholinergic, adrenergic, and nonadrenergic, noncholinergic innervation. The cholinergic and adrenergic nerve terminals are generally considered to be stimulatory, the nonadrenergic, noncholinergic nerve terminals playing an inhibitory role. Although the unequivocal identification of afferent nerve terminals remains difficult, the presumed function of NEE cells, i.e., subserving as chemoreceptors of the airways, leads to assume the existence of an afferent innervation. Finally, it should be mentioned that the morphology and histochemistry of the pulmonary NEE cells were compared to the structure of type 1 cells of the carotid body. The dual innervation with afferent and efferent pathways as well as the morphological features that are produced after hypoxic conditions have both cell types in common (e.g., Keith and Will, 1982; Gould et d.,1983b; Becker, 1984). Nonetheless, the NEE cells differ from principal cells of the carotid body in several respects. For instance, the former contain 5-HT, while type I cells of the carotid body store catecholamines (for review, see Verna, 1979). Moreover, the latter are situated

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in the carotid body among ganglionic cells, which were never observed between NEE cells. In addition, NEE cells seem to react directly to hypoxia of inhaled air, whereas, in contrast to cells of the carotid body, these do not respond to hypoxemic conditions (Lauweryns et al., 1977; Cutz et a / . , 1982). In consequence, NEE cells do not appear to be vascular chemoreceptors. Conversely, granule-containing cells of the intrapulmonary ganglia have many structural features in common with both type I cells of the carotid body and glomus cells of the aorticopulmonary bodies (Verna, 1979; Papka, 1980; Bock, 1982; Scheuermann et a / . , 1984b). The latter three types of cells contain catecholamines in their secretory granules and, being associated with ganglionic cells, are thought to have a receptor-secretory function. It should be given thought whether the reaction of the lung to hypoxemic conditions may be effectuated through stimulation by vascular chemoreceptors, including the granule-containing cells of the pulmonary ganglia and their release of catecholamines, since the latter substances may produce vasoconstriction in the lung (Bergofsky, 1980; Becker, 1984). The functional relations between the NEE cells, sensing oxygen levels in the pulmonary airways, and the vascular chemoreceptors to oxygen levels in the blood, e.g., the carotid body, the aortic bodies, and perhaps the granule-containing cells of the pulmonary ganglia, are not yet fully understood. A further investigation of the efferent and afferent innervation pattern of the receptors located near gas and blood in the lung is thus required. XI. Concluding Remarks

To date, the typical histological, histochemical, and ultrastructural characteristics as well as the bioactive substances of NEE cells of the lung are fairly well-known. The function of these cells, however, is at present far from elucidation and therefore remains subject to speculative thought. The shape of the NEE cells, whether solitary or grouped into clusters, with a narrow apical portion bearing villous projections into the airway lumen, is indicative of a receptor function. The basal portion is found adjacent to capillaries and may be synaptically connected with varicosities of subepithelial nerve fibers. Ultrastructurally, the nervous connections are suggestive of both afferent and efferent innervation. Furthermore, most of these cells are located in strategic positions at the bifurcations of the bronchial tree. It seems likely that these structures perceive changes in

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the intraluminal environment of the lung, upon which they respond by releasing their secretory products. This assumption is supported by the fact that the NEE cells are degranulated by hypoxia releasing 5-HT. Possibly 5-HT, presumably released in association with polypeptides, could then influence their specific target cells via synaptic structures, local action, or in a vascular way. The central nervous system may modulate, by means of efferent cholinergic nerve fibers, the release from NEE cells of 5-HT and/or polypeptides in response to intraluminal stimuli, e.g., changes in the airway gases. This assumption is supported by the fact that synaptic contact between nerve terminals and NEE cells has been demonstrated with certainty. Thus, the secretory products released from NEE cells may activate afferent nerve terminals, evoking local or systemic reflex changes. The release and diffusion of neurally active substances into nonsynaptic intercellular spaces may provide a morphological basis for a paracrine function, e.g., a local response of the bronchial and vascular smooth muscle and perhaps of the intrapulmonary neuronal plexuses. Moreover, the release of 5-HT and/or polypeptides may be transported by the blood stream, either systemic or specific, from the NEE cells to remote targets. The regularity with which capillaries, at times fenestrated, are observed in proximity to the enlarged basal foot of NEE cells provides a strong indication that bioactive substances secreted by the NEE cells diffuse into the blood circulation, making them widely accessible. Much work remains to be done in the field of lung endocrinology. An improved knowledge of the secretory activities of the NEE cells and a clarification of the functional relationships between these cells and the nervous system represent challenges demanding further research. REFERENCES Adachi. T., Hisano, S.. and Daikoku, S. (1985). J . Hisrochem. Cytochem. 33, 891-899. Afzelius, B. A. (1975). In "Handbook of Molecular Cytology" (A. Lima-de-Faria. ed.), pp. 1219-1242. North-Holland Publ., Amsterdam. Al-Ugaily, L. H., Pack, R. J . . and Widdicombe, J . G . (1983). J . Physiol. (London) 340,54P. Ballard, K. J . , and Jones. J . V. (1971). J . Physiol. (London) 219, 747-753. Ballard, K . J . . and Jones, J . V. (1972). J . Physiol. (London) 227, 87-94. Bargmann, W., Lindner, E., and Andres, K. H . (1967). Z . ZelUorsch. 77, 282-298. Barnes, B. G . (1961). J . Ulrrustruct. Res. 5, 453-467. Barter, R . , and Pearse. A. G. E. (1953). Nutitre (London) 172, 810. Barter, R . , and Pearse, A. G . E. (1955). J . Purhol. Bucreriol. 69, 25-31. Basset, F., Poirier, J . , Le Crom, M., and Turiaf, J . (1971). Z . Zellforsch. 116, 425-442. Baumgarten, H. G . , Holstein, A.-F., and Owman, C. (1970). Z . Zellforsch. 106, 376-397. Becci, P. J . , McDowell, E. M., and Trump, B. F. (1978). J . Null. Cuncer Inst. U.S.61, 551-561.

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