Tamiko Tachibana

Serotonin (5-HT) has been a candidate for neurotransmitters in cutaneous type I mechanoreceptors (i.e., Merkel cell-nerve endings). Although recent electrophysiological studies have suggested the presence of the 5-HT2 and 3 receptors in the Merkel cell-nerve endings, the histological localization of these receptors are obscure. We thus immunohistochemically examined the presence of 5-HT1, 2, 3 receptors in Merkel cell-nerve endings in sinus hair follicles of the rat whisker pad. We also studied the immunohistochemical localization of the 5-HT transporter to confirm the site of 5-HT secretion. For this purpose, we used antibodies for the 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C and 5-HT3 receptors, and for the 5-HT transporter, as well as antibodies for cytokeratin 20 (as a marker of Merkel cells) and neurofilament H (a marker of type I sensory nerve terminals). The immuno-stained sections were analyzed under a laser-scanning microscope. It was found that the sensory nerve terminals in the Merkel cell-nerve endings showed strong positive immunoreactions of 5-HT1A and 1B receptors but not 5-HT2A, 2C, and 3 receptors. Furthermore, both the Merkel cells and related axon terminals showed strong immunoreactions of the 5-HT transporter. These findings support the idea that 5-HT molecules are released from the Merkel cells during mechanical reception and indirectly regulate neural actions of sensory neurons via 5-HT1 receptors. The localization of the 5-HT transporter found in this study also suggests a possibility that axon terminals in the Merkel cell-nerve endings also release 5-HT.

The role of Merkel cells in type I cutaneous mechanoreceptors remains enigmatic though mechanical transduction or neuromodulation function has been proposed. It has been shown that mammalian Merkel cells express immunohistochemical reactions of met-enkephalin, VIP, substance P, and CGRP, though the reactivity differs between species. If any one of these peptides acts as a transmitter or modulator for Merkel nerve terminals, these structures must have a specific receptor for the substance. We therefore studied the immunohistochemical localization of the above-mentioned neuropeptides and their receptors in Merkel cell-nerve endings in rat whisker pads. Specimens were doubly stained with polyclonal antibodies to neuropeptides and their receptors combined with a monoclonal antibody to cytokeratin 20, which was used for the labeling of Merkel cells. Merkel cells in the rat sinus hair follicles showed positive immunoreactions for all peptides studied, whereas the immunoreactions of receptors to these peptides were localized on Merkel cell membranes but not on the axon terminals. These results suggest that neuropeptides released from Merkel cells act on Merkel cells themselves by an autocrine mechanism.

We previously found that Merkel cells (MCs) of the rat and monkey show a strong immunoreaction of the α-subunit of Gq protein. The Gαq-subunit isoform activates isozymes of phospholipase C-β (PLC-β), which produces inositol-(1,4,5)-triphosphate (IP3) which mobilizes intracellular Ca⁺⁺ from calcium stores via IP3 receptors. Glutamate and adenosine triphosphate (ATP), which are candidates for neurotransmitters in Merkel endings, are known to couple to Gαq. Although MCs showed positive immunoreactions of metabotropic glutamate receptor 5 (mGluR5) in our preliminary study, these cells were not reactive to all antibodies to PLC-β isozymes. We, therefore, reinvestigated immunohistochemical affinities to MCs of antibodies to PLC-β isozymes and mGluRs using frozen sections of rat sinus hair follicles that were briefly postfixed in formaldehyde. We also studied the immunohistochemical expressions of P2Y receptors for ATP and IP3 receptor subtypes using similar sections. Merkel cells showed positive immunoreactions of PLC-β1 and mGluR5. It was also found that MCs show positive immunoreactions of P2Y2, IP3R-I, and IP3R-II receptors. These results suggest that the Gαq isoform in MCs couples to both the P2Y2 receptor and mGluR5 and regulates the intracellular Ca⁺⁺ concentration via the PLC-β–IP3 cascade.

Merkel cells ubiquitously distribute in the skin of vertebrates, from cyclostomes to mammals. It is well known that mammalian Merkel cells coupled with axon terminals of type I sensory nerve fibers form slowly adapting mechanoreceptors, Merkel endings, within the epidermis. However, there are still many unresolved problems in the biology of Merkel cells. We reviewed recently acquired knowledge about the histochemical nature of Merkel cell granules, the morphpological heterogeneity of Merkel cells and the roles of neurotrophins and their receptors for the development and survival of the cells. We discuss the functional significance of Merkel cell granules and the heterogeneity of Merkel cell populations.

The true function of Merkel cells (MCs) is still enigmatic, though the localization of various kinds of neurotransmitter-like substances in MCs has been revealed by immunohistochemistry. Most of the neurotransmitters act on target cells via seven-transmembrane receptors coupled to heterotrimeric G proteins. The heterotrimeric G proteins include various subfamilies that contribute to different signal transduction pathways. Therefore investigation of specific types of G proteins in MCs and related axon terminals (MC-axon terminals) should contribute to the elucidation of the function of MCs. In this study, we investigated the expression patterns of α-subunit isoforms of G proteins in MC-neurite complexes of the rat and monkey by enzymatic and fluorescence immunohistochemistry. MC-axon terminals of the rat and monkey showed positive immunoreactions of Gαo and Gαi1. Those of the monkey also showed a weak immunoreaction of Gαs. On the other hand, MCs of both animals showed positive immunoreactions of Gαo, Gαi1, Gαq, and Gαz. In addition, MCs of the monkey showed weak immunoreactions of Gαs. Gαo- and Gαi1-like immunoreactions in the MC-axon terminals suggest that MCs suppressively regulate receptive functions of type I mechanosensory nerve terminals. On the other hand, the localization of Gα-subunits in MCs suggests that these cells are regulated with hormones, neurotransmitter-like substances, or growth factors.

The origin and mechanism of the differentiation and proliferation of Merkel cells are enigmatic. A preliminary study in our laboratory showed that Merkel cells in the rat palatine mucosa emerge after birth. This is in contrast to the case of similar cells in the skin that differentiate during the embryonic period prior to the establishment of peripheral nerve innervation. We studied immunohistochemically the developmental timings of Merkel cells and peripheral nerves in the rat palatine mucosa using antibodies to cytokeratins 18 and 20, PGP 9.5, and CGRP using developing palates of prenatal and postnatal rats. We also studied the potency of mitosis in Merkel cells by immunohistochemistry using antibodies for a cell proliferation marker Ki67 and cyclin D-kinase inhibitors p16, p21 and p27. It was shown that Merkel cells in the rat palatine mucosa differentiate postnatally, after the development of peripheral nerve fiber terminals was almost established. The emergence and increase in number of Merkel cells progressed in an anterior-to-posterior wave. Newly appearing Merkel cells were usually negative for anti-cytokeratin 20 antibody but gained affinity for the antibody with progress of maturation. All Merkel cells in the palatine mucosa were negative for anti-Ki67 antibody but positive for anti-p27 antibody. These results indicate that Merkel cells in the rat palatine mucosa are not responsible for the development of peripheral nerve fiber terminals and that these cells differentiate in situ from intraepithelial stem cells.