Localization of serotonin-like immunoreactivity in the merkel cells of pig snout skin.код для вставкиСкачать
THE ANATOMICAL RECORD 225:267-271 (1989) Localization of Serotonin-Like lmmunoreactivity in the Merkel Cells of Pig Snout Skin T. GARCfA-CABALLERO, R. GALLEGO, E. ROSON, D. BASANTA, G. MOREL, AND A. BEIRAS Departamento de Ciencias Morfologicas (Catedra de Histologia Humana), Servicio de Microscopia Electronica, Facultad de Medicina, Hospital General de Galicia, Uniuersidad de Santiago de Compostela, Galicia, Spain (T.G.-C., R.G., E.R., D.B., A.B.); CNRS U R A 559, Laboratorie d‘Histologie-Embryologie, Facultd de Mkdecine Lyon Sud, France (G.M.) ABSTRACT The presence of serotonin in the Merkel cells of pig snout epidermis was investigated by the peroxidase-antiperoxidase immunohistochemical technique. Serotonin-like immunoreactive Merkel cells were found in groups located a t the base of epidermal rete pegs and in the external root sheath of sinus hair follicles (vibrissae). Immunoreactivity was stronger on the basal side of the Merkel cells, where dense-cored granules are most numerous. Neither the nerve terminal associated with the Merkel cell nor the neighbouring epidermal cells were immunostained. These results are the first evidence of serotonin-like immunoreactivity in mammalian Merkel cells. The fact that immunoreactivity is strongest in those parts of the Merkel cells with the highest granule density suggests that in these cells serotonin is probably localized in the dense-cored granules. Merkel cells were first described in 1875 by Friedrich Sigmund Merkel as “Tastzellen” (touch cells). Merkel suggested that these specialized epidermal cells, which appeared associated with nerve terminals, were receptors and transducers of mechanical stimuli (see Camisa and Weissmann, 1982). The mechanoreceptor function of Merkel corpuscles has been generally accepted. In mammals they act as slowly adapting type I units (Iggo and Muir, 1969; Tapper, 1970; Munger, 1971; Pubols et al., 1971; Andres and During, 1973; Burgess and Perl, 1973; Winkelmann and Breathnach, 1973; Horch et al., 1974; Smith, 1977). However, the function of the Merkel cells themselves is still unclear (Winkelmann and Breathnach, 1973). The roles suggested by morphological or physiological studies include their activity as: a) the actual mechanoelectric transducer as Merkel originally postulated (Andres, 1966; Iggo and Muir, 1969; Burgess and Perl, 1973; Chen et al., 1973; Mihara et al., 1979; Breathnach, 1980; Hartschuh and Weihe, 1980); b) neuromodulators of the response generated directly in the nerve ending by mechanical stimulus (Hartschuh et al., 1983; Diamond et al., 1986); c) tropic and/or trophic elements for the nerve terminals (Ram6n y Cajal, 1919; Smith and Creech, 1967; Munger et al., 1971; English, 1974, 1977; Diamond et al., 1976; Killackey, 1980; Scott et al., 1981; Gottschaldt and Vahle-Hinz, 1981, 1982); and d) passive abutments for the nerve endings (Gottschaldt and Vahle-Hinz, 1981). It has also been suggested that Merkel cells may affect neighbouring epidermal cells by paracrine action (Pearse, 1980; Hartschuh et al., 1984; Tachibana et al., 1984). Whatever the function of Merkel cells is, it involves the secretion of the contents of their dense-cored granules (Fox and Whitear, 1978; Tachibana et al., 1984; Salomon et al., 1987). Knowledge of the chemical nature 0 1989 ALAN R. LISS, INC. of these granules is essential for elucidation of Merkel cells functiods). Zaccone (1986) reported immunoreactivity for serotonin in Merkel cells in the skin of the conger-eel (Conger conger).The purpose of the work described here was to investigate immunohistochemically whether serotonin is present in Merkel cells of the pig. MATERIALS AND METHODS Samples were taken from the skin of pig snouts in which Merkel cells are abundant and exhibit morphologic and immunohistochemical similarities to human Merkel cells. The specimens were fixed by immersion in Bouin’s solution for 2 hrs and embedded in paraffin. Sections 5 pm thick were immunostained using the peroxidase-antiperoxidase (PAP) technique (Sternberger et al., 1970) by succesive incubation in: 1) hydrogen peroxide (3%) for 10 min at room temperature; 2) normal goat serum (1:lO) for 30 min at room temperature; 3) anti-serotonin raised in rabbits (Immuno Nuclear, Stillwater, MN, lot no. 8616010) at a dilution of 1:2,000, overnight at 4°C;4) anti-rabbit IgG developed in goats (Sigma, St Louis, MO) at a dilution of 150, for 1 hr at room temperature; 5) PAP (rabbit) complex (Dakoppatts, Glostrup, Denmark) at 1:100, for 1 hr at room temperature; and 6) 3,3’diaminobenzidine-tetrahydrochloride (DAB) (Sigma, St Louis, MO) solution with 0.003% HzOZ,for 10 min. All the solutions were made up in PBS of pH 7.4. After steps Received December 5, 1988; accepted March 24, 1989. Address reprint requests to Prof. Andres Beiras, Servicio de Microscopia Electrbnica, Facultad de Medicina, CiSan Francisco s/n, 15705 Santiago de Compostela, La Coruna, Spain. 268 T. GARCIA-CABALLERO ET AL. 1,3,4, and 5 the sections were washed twice for 5 min with PBS of pH 7.4, and with distilled water after step 6. After step 2 the normal goat serum was drawn off before the primary antibody was applied. The serotonin-specificity of the antiserum was checked by preadsorbing diluted serotonin antiserum (1:2,000) with 5-HT creatinine sulphate (Sigma, St Louis, MO) at 100 pg/ml. The vials were placed in a 37°C oven for 1 hr and then stored overnight at 4°C. Additional vials of diluted antiserum were subjected to the same procedure and employed for staining controls. In addition, sections were incubated with normal (nonimmune) rabbit serum instead of anti-serotonin serum. For ultrastructural analysis, specimens were processed as previously described (Beiras et al., 1986). Briefly, samples were fixed by immersion in 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer of pH 7.3. Postfixation was performed in 2% osmium tetroxide in the same buffer. Tissue blocks were then dehydrated and embedded in Spurr’s epoxy resin (Spurr, 1969). RESULTS Serotonin-like immunoreactive Merkel cells were found parallel to the surface a t the tips of epidermal rete pegs of pig snout skin (Fig. 1). They appeared both isolated or, more frequently, forming groups (the latter generally at the tips of the widest rete pegs) (Fig. 1).No other epidermal cells were immunostained. The strongest immunoreactivity was seen on the basal side of the epidermal Merkel cells (Fig. 1).The area exhibiting the most intense immunostaining was the region of the Merkel cells that was closest to the nerve ending (Fig. l),where dense-cored granules are concentrated (Fig. 2). Neither the expanded nerve terminals associated with the Merkel cells nor the myelinated axons found in the superficial dermis were positive for serotonin (Fig. 1). Sinus hair follicles (vibrissae) exhibited immunostained Merkel cells in a row in the external root sheath (Fig. 3). Unlike epidermal Merkel cells, vibrissal Merkel cells were most intensely stained on the side facing into the follicle (Fig. 3). In vibrissae, the nerve endings were not situated on the dermal side of the Merkel cells (as in epidermis), but on their epithelial side, so that the Merkel cells lay between the nerve disc and the basal membrane (not shown). Control assays were performed in serial sections. Slides incubated with serotonin antiserum exhibited numerous immunostained Merkel cells (Fig. 4), but there was no immunoreaction in sections incubated with antiserum preadsorbed by serotonin (Fig. 5 ) , nor when the antiserum was replaced by normal rabbit serum. DISCUSSION ence of serotonin in the Merkel cells of conger-eel epidermis (Zaccone, 1986). Our results confirm that Merkel cells exhibit serotonin-like immunoreactivity and, to our knowledge, are the first observations of serotonin in mammalian Merkel cells. The specificity of the serotonin antiserum used in this study was verified by the absence of immunostaining after preadsortion of the antiserum by serotonin or replacement of the antiserum by normal rabbit serum. The finding of more intense immunostaining on the side of the Merkel cells with the highest granule density leads us to suppose that serotonin is stored in the Merkel cells’ dense-cored granules, as has been postulated by Zaccone (1986) for the Merkel cells of congereel (Conger conger). The different situations of the nerve disc described in the epidermal and vibrissal Merkel corpuscles (Andres and During, 1973; Hartschuh and Weihe, 1980) explain the difference in the localization of immunostaining in both Merkel corpuscles. Several peptides found in Merkel cell cytoplasm are also thought t o be stored in the dense-cored granules, namely met-enkephalin in various rodents (Hartschuh et al., 1979b, 1980); vasoactive intestinal polypeptide in man, cat, dog, and pig (Hartschuh et al., 1983,19841, and calcitonin gene-related peptide in cats (Alvarez et al., 1988). The coexistence of serotonin and polypeptides in the same cell is not an exceptional circumstance and has been long recognized in neurons (Hokfelt et al., 1980, 1984; Pelletier et al., 1981) and in a variety of endocrine cells (Gylfe, 1978; Larsson 1980; Nunez et al., 1980). Merkel corpuscles may be considered analogous to the neuroepithelial bodies (NEBS) of the respiratory mucosa since both consist of granulated cells in close contact with nerve endings (Chen et al., 1973; Winkelmann, 1977; Stahlman et al., 1985). NEBS have been shown t o contain various peptides (bombesin, calcitonin, leu-enkephalin, calcitonin gene-related peptide, and somatostatin) and serotonin (Lauweryns et al., 1973; Wharton et al., 1978; Becker et al., 1980; Cutz et al., 1981; Dayer et al., 1985; Lauweryns and Van Ranst, 1987; Scheuermann et al., 1987). The presence of serotonin in Merkel cells thus increases the analogy. The role of serotonin in most paracrine cells is not well known. In the gastrointestinal tract it seems to act on both neural elements (Eklund et al., 1980; Johnson et al., 1980) and smooth muscle cells (Paton and Aboo Zar, 1968). For NEBS it has been suggested that serotonin has: 1) a role in the mechanism of hypoxia-induced pulmonary vasoconstriction (Lauweryns et al., 1982); 2) specific interaction with efferent vagal impulses (Lauweryns et al., 1982); and 3) a trophic role in the process of capillary invasion during fetal lung development (Stahlman et al., 1985).For the Merkel cells The morphological similarity between Merkel cell granules and monoamine storing granules led to the suggestion that the former contained monoamines too (Mustakallio and Kiistala, 1967; Winkelmann and Fig. 1. Isolated Merkel corpuscles located a t the tips of epidermal Breathnach, 1973). However, Smith (1967, 19701, rete pegs, showing immunoreactivity for serotonin on the side closest Hartschuh et al. (1979a1, and Diamond et al. (1986) to the associated nerve endings (arrowheads), which are not immu( x 1,100). failed to confirm this hypothesis by pharmacological nostained. Fig. 2. Ultrastructural appearance of a Merkel corpuscle. Most procedures or fluorescence techniques. Immunohisto- dense-cored granules are on the side of the Merkel cell apposed to the chemical techniques have recently revealed the pres- nerve terminal (N). ( x 31,200). MERKEL CELLS CONTAIN SEROTONIN Figs. 1-2. 269 T. GARCIA-CABALLERO ET AL. 270 Fig. 3.Sinus hair follicle (vibrissa), showing immunoreactivity on the epithelial side of Merkel cells ( X 500). Figs. 4 and 5. Antibody specificity controls. Serial sections were incubated with serotonin antiserum (Fig. 4) and with the antiserum preadsorbed by serotonin (Fig. 5). Observe the absence of immunostaining in the Merkel cells of the control (Fig. 5) ( X 270). of Conger conger, Zaccone (1986) postulated that serotonin may either stimulate the attached nerve ending, thus acting as a neurohormone or be a synaptic mediator that transduces received stimuli from the cell to the nerve ending. However, all these hypotheses are quite speculative; and it has also been suggested that serotonin coexisting in cells with polypeptides is involved in the synthesis, storage, andlor release of the latter (Owman et al., 1973). To sum up, serotonin is present in Merkel cells in pig snout skin, probably located in the dense cored granules of these cells. Further investigations are needed to confirm the subcellular localization of this amine and to discover the exact role that serotonin plays in the physiology of Merkel corpuscles. related peptide and vasoactive intestinal polypeptide in cat Merkel cells and cutaneous free nerve endings: Light and electron microscopic study. Cell Tissue Res., 254:429-437. Andres, K.H. 1966 Uber die Feinstruktur der Rezeptoren an Sinushaaren. Z. Zellforsch., 75t339-365. Andres, K.H., and M.V. During 1973 Morphology of cutaneous receptors. In: Handbook of Sensory Physiology. A. Iggo, ed. Springer Verlag, Berlin, Vol. 11, pp. 3-28. Becker, K.L., K.G. 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