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1.
Summary Immunocytochemical techniques were used to investigate the distribution and co-localization of neuronspecific enolase (NSE) and serotonin (5-HT) in the skin of the conger eel, Conger conger. NSE and 5-HT immunoreactivity were found in Merkel cells; these cells were also identified at the electron-microscope level by the presence of characteristic granules and their association with an intraepithelial nerve ending. For the first time, it was demonstrated that Merkel-cell granules of vertebrate skin exhibit in immunoreaction with 5-HT. The production of amines may indicate that the Merkel cells of C. conger have both secretory capabilities and transduction functions.However, immunocytochemical investigation of the synaptic zones at the electron microscope level will be necessary to confirm this hypothesis.The present histochemical results suggest that NSE and 4-HT may be marker substances for Merkel cells, and that immunocytochemistry is a useful tool for the light-microscopic localization of these cells.  相似文献   

2.
Using an ultrastructural postembedding immunogold technique, we demonstrated vasoactive intestinal polypeptide (VIP)- and calcitonin gene-related peptide (CGRP)-like immunoreactivity in the Merkel cell dense-cored granules of skin and sinus hair follicles of adult cat and dog. The VIP-like substance was located in cat Merkel cells while both VIP- and CGRP-like substances were colocalised in dog Merkel cells. In cat Merkel cells, the magnitude of labelling of VIP was qualitatively higher than in dog Merkel cells. In the dog Merkel cell, CGRP appeared as the most abundant peptide. Dense-cored granules were labelled for these peptides. In addition, mast cells encountered in the dermal region of dog skin were also found to be immunolabelled by VIP antiserum. The immunoreaction was found to be confined to the secretory granules of the cells. Furthermore, all non-myelinated nerve plexuses encountered in the dermal region of the skin and the sinus hair follicles of the various mammalian species studied were immunolabelled by CGRP antiserum. The specific location was again restricted to the dense-cored granules present in these nerves. As VIP and CGRP have potent vasodilatory effects, our observations suggest that Merkel cells may play a separate or synergistic role in regulatory functions of the skin neuroendocrine cell, exerting their influence by paracrine, endocrine and neurocrine pathways, or a combination of these. Different methodologies of double labelling with different sizes of gold particles are also discussed.  相似文献   

3.
Summary The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve, bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.  相似文献   

4.
The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.  相似文献   

5.
Summary The comparative distribution and coexistence of chromogranin A (CGA)-, serotonin (5-hydroxytryptamine; 5-HT)- and pancreastatin (PST)-like immunoreactivity in endocrine-like cells of the human anal canal was investigated by light-microscopic immunocytochemistry. The largest population of colorectal endocrine-like cells consisted of CGA-immunoreactive (ir) cells, followed by the 5-HT-ir and PST-ir cell population. In the anal transitional zone (ATZ), CGA-and 5-HT-immunoreactivity was equally distributed; ir-PST was confined to a smaller endocrine-like cell population. In the squamous zone and the perianal skin, Merkel cells in the basal layer of the epidermis and hair follicles exhibited ir-CGA and ir-PST but no ir-5-HT. Double immunofluorescence on identical sections revealed distinct coexistence patterns. In the colorectal zone, about 2/3 of the CGA-ir endocrine-like cells also stained for 5-HT, whereas in the ATZ epithelium, CGA- and 5-HT-immunoreactivity completely overlapped. No 5-HT-immunoreactivity could be detected in CGA-ir Merkel cells of the squamous zone of the anal canal and the perianal skin. PST-immunoreactivity was present in about 1/3 of the CGA-ir colorectal and anal transitional endocrine-like cells and in about 1/4 of the Merkel-cell population staining for CGA. These chemically heterogeneous phenotypes of the anal endocrine-like and Merkel cells may reflect a specific regulatory role of these cells in the various epithelial linings of the human anal canal and the perianal skin.  相似文献   

6.
Uptake, distribution and turnover of 5-Hydroxytryptamine (5-HT) was studied by cytofluorometric analysis of whole mast cells and individual granules. Injection of 5-HT as well as 5-Hydroxytryptophan (5-HTP) intraperitoneally or subcutaneously resulted in a parallel uptake of 5-HT in cells and granules. Intraperitoneal injections of 5-HT in such small quantities that may be available under physiological conditions resulted in an increase in fluorescence intensity of the mast cells, indicating a very efficient uptake mechanism for 5-HT in vivo. Much larger doses of 5-HTP were required to obtain a corresponding uptake of 5-HT in the mast cells. The 5-HT was rather rapidly taken up in the granules and eliminated very slowly, at the same rate both from granules and mast cells. The low elimination rate confirms our previous findings that the turnover of 5-HT is much lower in mast cells than in other amine containing cell systems. The combination of an extremely efficient, rapid uptake of 5-HT with a slow elimination suggests a specific function for mast cells in the regulation of free amine concentrations in tissues.  相似文献   

7.
Summary Immunogold staining failed to show met-enkephalin immunoreactivity in the Merkel cell dense-core granules of rats when examined by electron microscopy, but showed gold particle staining in the Merkel cell dense-core granules of mice and nude mice. Merkel cells of hamster, guinea pig, rabbit, cat and dog were also examined using a similar method, and different antisera dilutions. Immunogold particles were consistently found in the dense-core granules of mice and nude mice at all antisera dilutions, but not in the other species, except in the dog, where a very low labelling response was encountered. Merkel cells from skin touch domes or sinus hair follicles, did not exhibit any difference in peptide expression as far as met-enkephalin immunoreactivity was concerned. In addition, all species studied, including mice and nude mice, did not show leu-enkephalin immunoreactivity in their Merkel cell dense-core granules. It is concluded that species variability in peptide expression occurs in the Merkel cell dense-core granules, and may be closely related to the different methodologies used.  相似文献   

8.
Human Merkel cells were first described by Friedrich S. Merkel in 1875 and named "Tastzellen" (touch cells) assuming a sensory touch function within the skin. Only ultrastructural research revealed their characteristics such as dense-core granules, plasma membrane spines and dendrites as well as a loosely arranged cytoskeleton. Biochemical analysis identified the expression of very specific cytokeratins (most notably CK 20) allowing the immunohistochemical detection of Merkel cells. In humans, they occur within the basal epidermis, being concentrated in eccrine glandular ridges of glabrous skin and in Haarscheiben of hairy skin, within belt-like clusters of hair follicles, and in certain mucosal tissues. Within the human skin, the dense-core granules contain heterogeneously distributed neuropeptides, some of which might work as neurotransmitters through which Merkel cells and their associated nerves exert their classical function as slowly adapting mechanoreceptors type I. This is the case in the Haarscheiben, small sensory organs containing keratinocytes with a special program of differentiation that includes the expression of CK 17 and Ber-EP4. Other peptides may act as growth factors and thus might participate in growth, differentiation and homeostasis of cutaneous structures. It is not yet clear whether the Merkel cell carcinomas, aggressive skin carcinomas, indeed arise from Merkel cells. We summarize and discuss data on the distribution, function and heterogeneity of human Merkel cells in normal and diseased skin.  相似文献   

9.
Summary Merkel corpuscles in the lingual mucosa of the finch, Lonchura striata, were examined by means of the argyrophilic reaction and electron microscopy. These corpuscles are composed of 12 to 20 flattened Merkel cells and enclosed nerve terminals. The present study demonstrated for the first time argyrophilia in avian subepithelial Merkel cells with the use of Grimelius silver stain. Electron-microscopically, the Merkel cell was characterized by the presence of numerous densecore granules, approximately 80 to 140 nm in diameter, as well as specialized contacts with nerve terminals. The granules showed a tendency to accumulate in the cytoplasm in close association with both nerve terminals and basal lamina. This study also provided unequivocal evidence for exocytotic discharge of Merkel-cell granules at the plasma membrane facing not only the nerve terminals but also the basal lamina. The exocytotic figures toward the nerve terminals can be regarded as synaptic discharge of Merkel-cell granules, but the possibility also exists that the Merkel-cell granules may exert a trophic effect on the nerve terminals. The exocytotic release of Merkel-cell granules toward the basal lamina with no relation to nerve terminals may suggest an endocrine (paracrine) function for the Merkel cell. The avian subepithelial Merkel cells qualify as paraneurons, but their exact nature and function remain enigmatic as is the case of intraepithelial Merkel cells in other vertebrates.  相似文献   

10.
Merkel cells are special neurosecretory cells which, in adult human skin, are usually very scarce. By immunofluorescence microscopy using antibodies to human cytokeratin polypeptide no. 18, we localized distinct non-keratinocyte cells in the glandular ridges of human fetal and adult plantar epidermis. Using electron and immunofluorescence microscopy, these cells were identified as Merkel cells containing typical neurosecretory granules as well as bundles of intermediate-sized filaments and desmosomes. Two-dimensional gel electrophoresis of the cytoskeletal fractions of microdissected epidermal preparations highly enriched in Merkel cells indicated the presence of cytokeratin polypeptides nos. 8, 18 and 19 which are typical of diverse simple epithelia of the human body. Double immunofluorescence microscopy showed that these human Merkel cells contain neither neurofilaments nor vimentin filaments. In human fetuses of 18-24 weeks of age, conspicuously high concentrations of Merkel cells, reaching a density of approximately 1,700 Merkel cells/mm2 skin, were found in the glandular ridges of plantar skin. The concentration decreased considerably at newborn and adult stages. Thin cell processes (up to 20 microns long) were observed in many fetal epidermal Merkel cells. In addition, we detected isolated Merkel cells deeper in the dermis (i.e. at distances of, at most, 100 microns from the epidermis) in fetal and newborn plantar skin. Our results show that Merkel cells are true epithelial cells which, however, differ profoundly from epidermal keratinocytes in their cytokeratin expression. The findings are discussed in relation to the much disputed question of the origin of Merkel cells. The present data speak against the immigration of Merkel cells from the neural crest, but rather suggest that they originate from epithelial cells of the skin, although most probably not from differentiated keratinocytes.  相似文献   

11.
Summary Uptake, distribution and turnover of 5-Hydroxytryptamine (5-HT) was studied by cytofluorometric analysis of whole mast cells and individual granules. Injection of 5-HT as well as 5-Hydroxytryptophan (5-HTP) intraperitoneally or subcutaneously resulted in a parallel uptake of 5-HT in cells and granules. Intraperitoneal injections of 5-HT in such small quantities that may be available under physiological conditions resulted in an increase in fluorescence intensity of the mast cells, indicating a very efficient uptake mechanism for 5-HT in vivo. Much larger doses of 5-HTP were required to obtain a corresponding uptake of 5-HT in the mast cells. The 5-HT was rather rapidly taken up in the granules and eliminated very slowly, at the same rate both from granules and mast cells. The low elimination rate confirms our previous findings that the turnover of 5-HT is much lower in mast cells than in other amine containing cell systems. The combination of an extremely efficient, rapid uptake of 5-HT with a slow elimination suggests a specific function for mast cells in the regulation of free amine concentrations in tissues.Supported by grants from the Swedish Medical Research Council, Project no 2235  相似文献   

12.
Cytofluorometric quantitation of 5-hydroxytryptamine (5-HT) and heparin in individual mast cell granules is described. The technique is based on micromanipulation of intact mast cells reacted with formaldehyde or stained with Berberine sulfate and the use of a cytofluorometer equipped with a sensitive peak detecting device. The quantities of 5-HT and heparin contained in mast cell granules which are of the order of 10(-16) and 10(-13) g, respectively were expressed as relative fluorescence guanta. The results of measurements on representative samples of mast cell granules indicate that all granules contain heparin as well as 5-HT, and that there are large variations in both 5-HT and heparin content within the granule populations of individual cells. A dose dependent increase in 5-HT content in both cells and individual mast cell granules occurred 24 hr after the injection of 10--50 mg L-5-hydroxytryptophan/kg intraperitoneally. There was no evidence for an increase in the heparin content of granules or cells, indicating that a new synthesis of granular macromolecules is not required for the 5-HT uptake. The results further suggest that 5-HT may be stored initially in a cytoplasmic extragranular pool and then taken up in the mast cell granules.  相似文献   

13.
The Merkel cell-neurite (MCN) complex generates slowly adapting type 1 (SA1) response when mechanically stimulated. Both serotonin (5-HT) and glutamate have been implicated in the generation of normal SA1 responses, but previous studies have been inconclusive as to what their roles are or how synaptic transmission occurs. In this study, excised dorsal skin patches from common water frogs (Rana ridibunda) were stimulated by von Frey hairs during perfusion in a tissue bath, and single-unit spike activity was recorded from SA1 fibres. Serotonin had no significant effect on the SA1 response at low (10?μM) concentration, significantly increased activity in a force-independent manner at 100?μM, but decreased activity with reduced responsiveness to force at 1?mM. Glutamate showed no effect on the responsiveness to force at 100?μM. MDL 72222 (100?μM), an ionotropic 5-HT3 receptor antagonist, completely abolished the responsiveness to force, suggesting that serotonin is released from Merkel cells as a result of mechanical stimulation, and activated 5-HT3 receptors on the neurite. The metabotropic 5-HT2 receptor antagonist, ketanserin, greatly reduced the SA1 fibre's responsiveness to force, as did the non-specific glutamate receptor antagonist, kynurenic acid. This supports a role for serotonin and glutamate as neuromodulators in the MCN complex, possibly by activation and/or inhibition of signalling cascades in the Merkel cell associated with vesicle release. Additionally, it was observed that SA1 responses contained a force-independent component, similar to a dynamic response observed during mechanical vibrations.  相似文献   

14.
Serotonin immunoreactive material was localized to rat enterochromaffin cells (EC cells) at the subcellular level using antibodies to serotonin (5-HT) raised in rabbits. Ultrathin sections from paraformaldehyde fixed plastic embedded tissues were directly labelled with the 5-HT antiserum, using the protein A-gold technique to visualize the immunoreaction. The 5-HT immunoreactivity (5-HT-IR) in the rat gastrointestinal mucosa was exclusively localized to epithelial EC cells with a low background over other epithelial non-enterochromaffin cells. Quantitative evaluation of the immunoreaction revealed that most of the 5-HT-IR in the cytoplasm of EC cells (60%) was located over the dense cores of the secretory granules. However, a significant part of the cytoplasmic 5-HT-IR (40%) was located outside the dense cores of the secretory granules which suggests that different forms of 5-HT storage may exist.  相似文献   

15.
Intracellular localization of serotonin (5-HT) in the mast cells of two phenotypes in normal rat colon and dextran sodium sulphate-induced colitis was studied by immunoelectron microscopy with a quantitative analysis of the distribution of immunogold labelling. Mucosal mast cells in normal rats contained round shape secretory granules with varying electron density. Immunogold labelling for 5-HT was concentrated over the secretory granules. In mucosal mast cells from colitis rats, vacuolated granules without 5-HT labelling were frequently observed and immunogold labelling over the secretory granules was significantly increased compared to controls. On the other hand, connective tissue mast cells in normal rats contained oval shape secretory granules with homogeneous electron density. Their immunogold labelling was diffusely scattered over the secretory granules as well as over the cytoplasm. In connective tissue mast cells from colitis rats, secretory granules with high electron density were increased and the immunogold labelling over the secretory granules was much higher than that in controls. The present results suggest that intracellular localization of 5-HT is different in two phenotypes of mast cells and they may release 5-HT in a different manner. Mucosal mast cells may release 5-HT by a degranulation or exocytosis, while connective tissue mast cells may release 5-HT by a diacrine manner of secretion.  相似文献   

16.
Serotonin-like immunoreactivity (5-HT-LI) has been localized at the ultrastructural level in enterochromaffin (EC) cells of rat gastrointestinal tract. Ultra-thin sections of tissues embedded in epoxy resin were incubated with 5-HT antisera and antibody binding sites were visualized with protein A-gold. Three different antisera were compared and were shown to require different fixation regimens for optimal preservation of 5-HT-LI. For one antiserum, tissues fixed in glutaraldehyde and osmium tetroxide could be used to demonstrate 5-HT-LI in EC cells. Immunocytochemical localization of 5-HT can thus be performed with good ultrastructural preservation of tissues. Quantitative evaluation of the intracellular distribution of 5-HT-LI was performed on EC cells from antrum, duodenum, and proximal colon, fixed in glutaraldehyde only. In all three locations, the majority of the gold particles (90%) in EC cells were localized over the dense core of the secretory granules, while a minor fraction (10%) were localized in parts of the cytoplasm devoid of granules. In EC cells fixed in glutaraldehyde and post-fixed in osmium tetroxide, 5-HT-LI was reduced by about 85%, although intracellular distribution was essentially the same as in cells fixed in glutaraldehyde alone. The results indicate that 5-HT in EC cells is stored mainly in secretory granules, with a small fraction of 5-HT being localized outside the granules.  相似文献   

17.
Merkel cells, the neurosecretory cells of skin, are essential for light-touch responses and may probably fulfill additional functions. Whether these cells derive from an epidermal or a neural lineage has been a matter of dispute for a long time. In mice, recent studies have clearly demonstrated an epidermal origin of Merkel cells. Given the differences in Merkel cell distribution between human and murine skin, it is, however, unclear whether the same holds true for human Merkel cells. We therefore attempted to gain insight into the human Merkel cell lineage by co-immunodetection of the Merkel cell marker protein cytokeratin 20 (CK20) with various proteins known to be expressed either in epidermal or in neural stem cells of the skin. Neither Sox10 nor Pax3, both established markers of the neural crest lineage, exhibited any cell co-labeling with CK20. By contrast, β1 integrin, known to be enriched in epidermal stem cells, was found in nearly 70 % of interfollicular epidermal and 25 % of follicular Merkel cells. Moreover, LRIG1, also enriched in epidermal stem cells, displayed significant co-immunolabeling with CK20 as well (approximately 20 % in the interfollicular epidermis and 7 % in the hair follicle, respectively). Further epidermal markers were detected in sporadic Merkel cells. Cells co-expressing CK20 with epidermal markers may represent a transitory state between stem cells and differentiated cells. β1 integrin is probably also synthesized by a large subset of mature Merkel cells. Summarizing, our data suggest that human Merkel cells may originate from epidermal rather than neural progenitors.  相似文献   

18.
Merkel cells have been assumed to guide nerve fibers to the skin. However, there has been little in vitro evidence that supports this hypothesis, because there is no suitable established culture system of Merkel cells. Here we show that Merkel cells isolated from rat footpad skin were successfully cultured in a monolayer with keratinocytes. Keratinocytes did not affect any structural changes in Merkel cells. When nerve cells (NG108-15 or PC12) were added to the culture system, both nerve fibers and cytoplasmic processes of Merkel cells outgrew and cooperatively organized synapse-like structures at their contact points. Nerve cells promoted Merkel cell survival, compared with keratinocytes only. Merkel cell proliferation was not detected in all conditions, even with nerve growth factor, neurotrophin-3, interleukin-6 and tumor necrosis factor-alpha. The data suggest, firstly, that Merkel cells may guide nerve fibers to the skin by interacting with nerve cells; and, secondly, that nerve cells, but not keratinocytes, may produce some survival factors other than the cytokines above for Merkel cells, although Merkel cells may be a terminally differentiated cell type. Our method could open a way to study Merkel cell biology.  相似文献   

19.
Summary Serotonin immunoreactive material was localized to rat enterochromaffin cells (EC cells) at the subcellular level using antibodies to serotonin (5-HT) raised in rabbits. Ultrathin sections from paraformaldehyde fixed plastic embedded tissues were directly labelled with the 5-HT antiserum, using the protein A-gold technique to visualize the immunoreaction. The 5-HT immunoreactivity (5-HT-IR) in the rat gastrointestinal mucosa was exclusively localized to epithelial EC cells with a low background over other epithelial non-enterochromaffin cells. Quantitative evaluation of the immunoreaction revealed that most of the 5-HT-IR in the cytoplasm of EC cells (60%) was located over the dense cores of the secretory granules. However, a significant part of the cytoplasmic 5-HT-IR (40%) was located outside the dense cores of the secretory granules which suggests that different forms of 5-HT storage may exist.Supported by grants from the Swedish Medical Research Council (537, 2207, 5220). Göteborgs Läkaresällskap, and The Medical Faculty of Göteborg  相似文献   

20.
Electron microscopic observations of an originally established mouse mastocytoma cell line (BSP-MST-2) revealed that the cytoplasm of many of the MST-2 cells contained small and low osmiophilic granules and a few mature electron-dense granules. Fluorescent- and immuno-histochemical examinations also suggested the immaturity of granules as the cytoplasmic reaction for serotonin (5-HT) was weak. Induction of further maturation of granules was investigated by administration of various chemical agents. Among the chemicals examined, sodium butyrate and hydrocortisone were effective. In the presence of 1 mM sodium butyrate for 24 h, the cytoplasmic granules contained an abundant dense matrix. MST-2 cells incubated with hydrocortisone at 5 micrograms/ml for 24 h showed a somewhat different granulopoietic pattern from those incubated with sodium butyrate, including numerous electron-dense progranules. Fluorescent- and immuno-histochemical studies showed increased reactions of cytoplasmic 5-HT of both butyrate- and hydrocortisone-treated MST-2 cells. The specificity of these morphological and cytochemical changes was confirmed by treatment with reserpine, a drug which depletes cellular 5-HT; electron-dense materials were virtually diminished and cytochemical reactions were significantly decreased. The mode of induced production of 5-HT in mastocytoma granules is discussed, in relation to mastocyte differentiation.  相似文献   

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