Plasma membrane stretch activates transient receptor potential vanilloid and ankyrin channels in Merkel cells from hamster buccal mucosa

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons. Many transient receptor potential (TRP) channels have been identified in mammals; however, the activation properties of these channels in oral mucosal MCs remain to be clarified. We investigated the biophysical and pharmacological properties of TRP vanilloid (TRPV)-1, TRPV2, TRPV4, TRP ankyrin (TRPA)-1, and TRP melastatin (TRPM)-8 channels, which are sensitive to osmotic and mechanical stimuli by measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) using fura-2. We also analyzed their localization patterns through immunofluorescence. MCs showed immunoreaction for TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 channels. In the presence of extracellular Ca²⁺, the hypotonic test solution evoked Ca²⁺ influx. The [Ca²⁺]_i increases were inhibited by TRPV1, TRPV2, TRPV4, or TRPA1 channel antagonists, but not by the TRPM8 channel antagonist. Application of TRPV1, TRPV2, TRPV4, TRPA1, or TRPM8 channel selective agonists elicited transient increases in [Ca²⁺]_i only in the presence of extracellular Ca²⁺. The results indicate that membrane stretching in MCs activates TRPV1, TRPV2, TRPV4, and TRPA1 channels, that it may be involved in synaptic transmission to sensory neurons, and that MCs could contribute to the mechanosensory transduction sequence.     

Merkel cells and nerve endings in the labial epidermis of a lizard

Summary Examination of the labial epidermis of the lizard Lacerta sicula revealed cells displaying all features of Merkel cells. These cells are located in the stratum basale of epidermal pegs and are arranged in clusters.     

On the occurrence of merkel cells in the epidermis of teleost fishes

Summary The ultrastructure of a differentiated cell type in the epidermis of two species of teleost fish, Ictalurus melas and Phoxinus phoxinus, is described. This cell type has a synaptic association with nerve fibres, microvillus-like peripheral processes, and membrane-bounded inclusions, which together are the diagnostic features of the Merkel cells of tetrapod vertebrates. Other cytoplasmic features are shared with the epithelial cells. The appearance of the membrane-bounded granules depends on the fixative used; after fixation with glutaraldehyde the granules are of a size and electron-density comparable to that found in tetrapod Merkel cells, but after fixing in osmium tetroxide the granules are inconspicuous.Our thanks are due to Mr. A.C. Wheeler of the British Museum (Natural History) for help with the identification of the species of Ictalurus, and to Mr. E. Perry for technical assistance. One author (EBL) was supported by a SRC research studentship     

Species variability in the expression of met- and leu-enkephalin-like immunoreactivity in mammalian Merkel cell dense-core granules

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.     

Immunocytochemical evidence of a met-enkephalin-like substance in the dense-core granules of mouse Merkel cells

Summary The electron-microscopic immunogold method was applied to Merkel cells of adult mice to demonstrate the subcellular localization of met-enkephalin-like immunoreactivity. Post-embedding incubation with metenkephalin antisera showed that the gold particles were associated with the dense-core granules of the Merkel cells. The majority, but not all, of the dense-core granules were strongly labelled. Osmication caused a significant reduction in the number of gold particles on these granules. The nerve terminal associated with the Merkel cell did not show met-enkephalin-like immunoreactivity. To the best of our knowledge, this is the first report of the ultrastructural localization of a positive met-enkephalin immunoreactivity in the dense-core granules of Merkel cells in mice.     

Fine structure and innervation of the avian adrenal gland

Summary According to their ultrastructure and histochemistry three types of efferent nerve fibers can be distinguished in the bird's adrenal gland. The main part is made up of cholinergic fibers recognizable by a positive reaction for acetylcholinesterase and two specific populations of granules within the synaptic ending. Synaptic vesicles measuring 300 to 500 Å in diameter and dense-cored vesicles with a diameter of about 1 000 Å are discernible.In the periphery of the gland cholinergic axons for the innervation of adrenal cells form large bundles surrounded by a perineural sheath. The bundles cross the capsule and are situated within the adrenal chromaffin cords or at their periphery. Finally small groups of fibers enter a group of chromaffin cells which are surrounded by a basal lamina and which consist of about a dozen or more cells producing adrenaline and noradrenaline. Synaptic endings occur, above all in passeriform species, in the center of a chromaffin cell complex. They are either attached to the innervated cells or their dendrite-like processes, or embedded into the cells, or connected to short spines of the innervated cells. Synaptic and dense-cored vesicles leave the bouton by exocytosis. One synaptic terminal may innervate up to three A- or NA-cells. The existence of different types of synapses for A- and NA-cells cannot be excluded.Supported by a grant from the Deutsche Forschungsgemeinschaft (Un 34/1).     

The pronephros of the early ammocoete larva of lampreys (Cyclostomata,Petromyzontes): Fine structure of the renal tubules

Summary The renal tubules of the paired pronephros in early larvae (ammocoetes) of two lamprey species, Lampetra fluviatilis and Petromyzon marinus, were studied by use of light-, scanning- and transmission electron microscopy. They consist of (1) a variable number of pronephric tubules (3 to 6), and (2) an excretory duct. By fine-structural criteria, the renal tubules can be divided into 6 segments. Each pronephric tubule is divided into (1) the nephrostome and (2) the proximal tubule, the excretory duct consisting of (3) a common proximal tubule followed by (4) a short intermediate segment, and then by a pronephric duct composed of (5) a cranial and (6) a caudal section. The epithelium of the nephrostome displays bundles of cilia. The cells of the proximal tubule possess a brush border, many endocytotic organelles and a system of canaliculi (tubular invaginations of the basolateral plasmalemma). The same characteristics are encountered in the epithelium of the common proximal tubule; however, the number of these specific organelles decreases along the course of this segment in a posterior direction. In the intermediate segment, the epithelium appears structurally nonspecialized. The cells of the cranial pronephric duct lack a brush border; they have an extensive system of canaliculi and numerous mitochondria. The caudal pronephric duct is lined by an epithelium composed of light and dark cells; the latter are filled with mitochondria and the former contain mucus granules beneath the luminal plasmalemma. The tubular segments found in the pronephros are the same in structure and sequence as in the lamprey opisthonephroi. However, only the nephrostomes and proximal tubules occur serially in the pronephros, while the common proximal tubule, the intermediate segment and the cranial pronephric duct form portions of a single excretory duct.This paper is dedicated to the memory of Professor W. Bargmann, long-time editor of Cell and Tissue Research, the author of a splendid review on the structure of the vertebrate kidney and a master of German scientific writing.     

Fine structure and cytochemistry of specific granules in the lamprey atrium

Summary Fine structural and cytochemical studies were performed to examine the nature of three types of specific granules found in the atrium of lamprey; specific granules of the atrial muscle cell (ASG), interstitial cell granules (ICG) and endocardial endothelial granules (ESG).Ultrastructurally, ASG and ICG appeared quite similar in size, shape and electron opacity, while ESG were much larger and less dense in opacity than the other two.None of the granules showed positive DAB reaction or acid phosphatase reaction. Only ICG revealed positive chromaffin reaction, which agreed with formaldehyde induced green fluorescence along the atrial lumen. Phosphotungstic acid at low pH stained ICG and ASG strongly positive, and ESG weakly positive. Pronase treatment in Epon sections for 24h digested ASG alone, whereas in glycol-methacrylate embedded sections, ESG were digested first, ASG were digested thoroughly after 30 min, but ICG were not digested completely after 90 min.From these results it can be concluded that the three types of specific granules have different constituents. ESG consist of protein with some polysaccharides; ASG are composed of protein carbohydrate complexes and lack catecholamines; ICG contain catecholamine as well as protein carbohydrate complexes.This work was supported by a grant from the Ministry of Education, JapanThe authors would like to express their gratitude to K. Wasano, M.D. for his technical assistance in fluorescence microscopy     

Structural changes in the zonulae occludentes of the chloride cells of young adult lampreys following acclimation to seawater

Summary Thin sections and freeze-fracture replicas have been used to study the structure of the zonulae occludentes of the branchial chloride cells in young adults of the anadromous lamprey Geotria australis, caught during their downstream migration to the sea and after acclimation to full-strength seawater (35). The chloride cells in the epithelium of the gill filaments of both freshwater- and seawater-acclimated animals form extensive multicellular complexes. In freshwater animals, the majority of chloride cells (64%) are covered by pavement cells and are thus not exposed to the external environment. Most of the other chloride cells are separated from each other by pavement cells or their processes. The zonulae occludentes between chloride cells and pavement cells and between adjacent chloride cells are extensive and characterised by a network of 4 (range 3–7) superimposed strands. In seawater-acclimated animals, the pavement cells cover only 30% of the chloride cells and their processes no longer occur between chloride cells. Whereas the zonulae occludentes between chloride cells and pavement cells are still extensive, those between chloride cells are shallow and comprise only a single strand or two parallel strands. The zonulae occludentes between the chloride cells of lampreys acclimated to seawater are similar to those in the gills of teleosts in seawater, and are thus considered to be leaky and to provide a low-resistance paracellular pathway for the passive transepithelial movement of Na⁺.     

Migration of Merkel cells in the labial mucous epithelium of adult rabbits following mental nerve resection

Summary Merkel cells in the lower labial mucosa of adult rabbits were studied electron microscopically, 9, 21, 28, and 50 days after resection of the mental nerves. By day 9, nerve fibers were completely retracted from the epithelial layer of the mucosa. On and after day 21, Merkel cells were located not only in the basal layer but also in the prickle or more superficial cell layers. The ultrastructure of the migrating Merkel cells was unchanged, both as to the amount and location of the specific cored granules in the cytoplasm, until the cells reached the granular cell layer. The position of the migrating Merkel cells differed from cell to cell, and migration continued for at least 50 days. A remarkably large number of immature Merkel cells was observed in the basal and suprabasal cell layers of the denervated epithelium even by day 50. Therefore, the possibility of the reproduction of Merkel cells exists. The migrating Merkel cells, as well as the keratinocytes in the same cell layer, had degenerated drastically in the parakeratinized cell layer. This seems to indicate that the Merkel cells belong to the line of keratinocytes.     

Sensory innervation of the tentacles of the polychaete,Sabella pavonina

Summary Following observation of conical groups of stiff, but motile cilia on the tentacles of the branchial crown of Sabella pavonina, these were examined with the electron microscope. The bundles consist of about 40 unenclosed standard cilia supported by one or two primary sense cells with centrally directed axons of 0.1–0.2 diameter. Axons in the distal portions of the branchial crown occur in small bundles surrounded by a basement membrane. More centrally, glial elements appear and the nerves are surrounded by a collagenous sheath. The branchial nerve trunk shows similarities in organisation to other previously investigated annelid central nervous tissue in that the whole nerve is surrounded by a fibrous sheath central to which there is a layer of glial cells with processes penetrating a central neuropile. The 0.1–0.2 axons commonly occur in glial-enveloped groups of < 40 whilst other axons of larger and mixed diameter are found together.Each tentacle has two branchial nerves on the oral side, and each nerve gives rise to two small 75-axon branches running to each pinnule. The branchial nerves fuse to form the branchial nerve trunk running to the supra-oesophageal ganglia.Sections of the branchial nerves of the branchial crown at progressively more central levels show that the branchial nerve trunk contains enough axons of 0.1–0.2 diameter to account for all the sensory cells on the tentacles. This is taken as evidence for the sensory cells having axons terminating within the central nervous system and that there is no peripheral confluence or fusion of these afferent axons.     

Neural crest origin of mammalian Merkel cells

Here, we provide evidence for the neural crest origin of mammalian Merkel cells. Together with nerve terminals, Merkel cells form slowly adapting cutaneous mechanoreceptors that transduce steady indentation in hairy and glabrous skin. We have determined the ontogenetic origin of Merkel cells in Wnt1-cre/R26R compound transgenic mice, in which neural crest cells are marked indelibly. Merkel cells in whiskers and interfollicular locations express the transgene, beta-galactosidase, identifying them as neural crest descendants. We thus conclude that murine Merkel cells originate from the neural crest.     

Mammalian Merkel cells are descended from the epidermal lineage

Merkel cells are specialized cells in the skin that are important for proper neural encoding of light touch stimuli. Conflicting evidence suggests that these cells are lineally descended from either the skin or the neural crest. To address this question, we used epidermal (Krt14^Cre) and neural crest (Wnt1^Cre) Cre-driver lines to conditionally delete Atoh1 specifically from the skin or neural crest lineages, respectively, of mice. Deletion of Atoh1 from the skin lineage resulted in loss of Merkel cells from all regions of the skin, while deletion from the neural crest lineage had no effect on this cell population. Thus, mammalian Merkel cells are derived from the skin lineage.     

Immunocytochemical analysis of calcitonin gene-related peptide and vasoactive intestinal polypeptide in Merkel cells and cutaneous free nerve endings of cats

Summary Calcitonin gene-related peptide (CGRP)-and vasoactive intestinal polypeptide (VIP)-immunoreactivity were observed to coexist in Merkel cells of cats. No differences in peptide content were found between Merkel cells located in epithelia of the hard palate, in hairy and glabrous skin of the upper lip, and in vibrissae follicles. CGRP-and VIP-immunoreactive nerve fibres were also found near CGRP/VIP-immunoreactive Merkel cells. In the vibrissae follicles some CGRP-and VIP-immunoreactive nerve terminals end abutting on the glassy membrane. Other CGRP immunoreactive nerve fibres penetrate the epithelium of the skin and end within it. Electron microscopy of vibrissae follicles revealed that Merkel cell neuntes are not immunostained and that immunostained nerve fibres form unmyelinated bundles before ending freely. Thus, CGRP-and VIP immunoreactive nerve fibres in cat skin do not end as Merkel cell neuntes but as different kinds of free nerve endings.     

Merkel cell distribution in human hair follicles of the fetal and adult scalp

The distribution of Merkel cells in fetal and adult terminal hair follicles of human scalp was studied immunohistochemically using cytokeratin (CK) 20 as a specific Merkel cell marker. In hair follicles of adult scalp, abundant Merkel cells were found enriched in two belt-like clusters, one in the deep infundibulum and one in the isthmus region. No Merkel cells were found in the deep follicular portions including the bulb, or in the dermis. In early fetal hair follicles (bulbous peg stage), Merkel cells were only detected in the basal layer of the developing infundibulum but not in deeper follicular areas. In later stages, Merkel cells were also present in the isthmus and bulge. No Merkel cells were seen in the dermis around developing hair follicles. Nerve growth factor receptor was not only present in nerves but was found to be widely distributed within fetal skin. In adult skin, this receptor was localized to the basal cell layers of the outer root sheath of the bulb and the suprabulbar area, but was not detectable in the areas containing Merkel cells. The present study localizing Merkel cells within the permanent hair follicle structures close to their possible stem cells suggests that they have paracrine functions.     

Lamprey (<Emphasis Type="Italic">Lethenteron japonicum</Emphasis>) IL-17 upregulated by LPS-stimulation in the skin cells

We report here the first evidence for interleukin-17, a pro-inflammatory cytokine, in cyclostomes. To detect the novel molecules involved in the immune response in the skin of the lamprey Lethenteron japonicum, subtractive hybridization was performed with 6-h-cultured skin cells with or without lipopolysaccharide (LPS). In approximately 100 partially sequenced clones analyzed, we identified an interesting sequence similar to that of the IL-17 genes in teleosts and mammals. Subsequent rapid amplification of cDNA ends was used to obtain the cDNA of lamprey IL-17 (LampIL-17) that contains a 519-bp open reading frame encoding a mature protein of 154 amino acids and a 19-residue NH₂-terminal signal peptide. The phylogenetic tree indicated that LampIL-17 is clustered into IL-17D, which is a subgroup of the IL-17 family. Southern blot analysis showed that the lamprey harbors a single copy of the LampIL-17 gene in its genome. The LampIL-17 gene was constitutively expressed in most tissues examined as well as in the skin, where the basal layer epithelial cells expressed LampIL-17 mRNA. Real-time-polymerase chain reaction (RT-PCR) demonstrated that the LampIL-17 gene expression in LPS-stimulated skin cells tended to be greater than that in non-stimulated cells. These results suggest that LampIL-17 is responsible for defense against bacterial infections in the lamprey skin. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB303391.     

Ultrastructural evidence for a possible secretory function of Merkel cells in the barbels of a teleost fish,Cyprinus carpio

Summary Examination of barbels of the carp (Cyprinus carpio) revealed cells showing the characteristics of Merkel cells. Some ultrastructural features of these cells suggest a secretory function.     

Fine structure of complex ocelli of a cubomedusan,Tamoya bursaria Haeckel

Summary The ultrastructure of adult male rat pinealocytes was studied after orchidectomy and orchidectomy followed by LH-RH administration. Castration causes an increased development of the rough endoplasmic reticulum and the Golgi apparatus as well as an increase in the number of lipid droplets and lysosomes. The changes after orchidectomy followed by LH-RH administration were more marked than after castration alone.The ultrastructural features observed after castration can be interpreted as a morphological equivalent of increased protein synthesis. The observations reported appear to indicate mutual interaction between the pineal organ and the hypothalamo-hypophyseo-gonadal system. A working hypothesis is put forward implying the presence of a negative feedback mechanism between the pineal and the adenohypophysis.Dedicated to Professor W. Bargmann in honour of his 70th birthdayIBRO/UNESCO fellow Acknowledgements. We thank Dr. P. Pevet for fruitful discussions and Miss M.T. Mud and Mr. P.S. Wolters for skillful technical assistance