Receptors and transporter for serotonin in Merkel cell-nerve endings in the rat sinus hair follicle. An immunohistochemical study.

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.

Insulin-like growth factor-I stimulates cell proliferation in the outer layer of Hertwig's epithelial root sheath and elongation of the tooth root in mouse molars in vitro.

To elucidate the mechanism of root formation in tooth development, we examined the role of insulin-like growth factor I (IGF-I) on early root formation in mandibular first molar teeth from 5-day-old mice. Immunohistochemistry revealed the specific localization of the IGF-I receptor in Hertwig's epithelial root sheath (HERS) in the tooth root. The effect of IGF-I on root development, especially on HERS, was subsequently examined in vitro. The control culture showed normal development of HERS and the periodontium, resembling that in vivo. However, the presence of 100 ng/ml IGF-I resulted in elongation of HERS and increased cell proliferation in its outer layer. These effects were negated by the addition of antibodies specific for IGF-I. Thus, we propose that IGF-I is involved in early root formation by regulating the mitotic activity in the outer layer of HERS.

Immunohistochemical reactions of receptors to met-enkephalin, VIP, substance P, and CGRP located on Merkel cells in the rat sinus hair follicle.

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.

Induction of adipogenesis by the intrasplenic transplantation of chick serum clots.

Chick serum contains a factor that stimulates adipogenesis in Meckel's chondrocytes in vitro. The present study examined whether chick serum has a capacity for adipogenic induction in vivo, by transplanting serum clots (created by drying chick serum for up to 4 weeks) into mouse spleens. Specimens were harvested for histological analyses, which included light and electron microscopy and immunohistochemistry. The transplanted serum clots induced the appearance of lipid droplet-containing cells in splenic cords and sinus. Almost all the lipid droplet-containing cells were positive for sudan staining and consisted of multilocular lipid vacuoles. Immunostaining showed that the adipocytes induced by transplantation of the serum clots initially appeared as peroxisome proliferator-activated receptor-gamma (PPARgamma)-positive cells and developed into leptin and alpha-glycerophosphate dehydrogenase (GPDH)-producing cells, in addition to type III collagen synthesis. Furthermore, double immunofluorescence staining revealed that the immunoreactivity for GPDH was detected not only in stromal cells but also in macrophages. It was thus confirmed that stromal cells and macrophages in the spleen contain lipid droplets as seen in intact white adipose cells. The present results suggest that chick serum contains factors for adipocyte induction not only in vitro but also in vivo, and that the adipogenic potential does not depend on the supplements used during the cell culture.

Immunohistochemical expressions of mGluR5, P2Y2 receptor, PLC-beta1, and IP3R-I and -II in Merkel cells in rat sinus hair follicles.

We previously found that Merkel cells (MCs) of the rat and monkey show a strong immunoreaction of the alpha-subunit of Gq protein. The Galphaq-subunit isoform activates isozymes of phospholipase C-beta (PLC-beta), 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 Galphaq. 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-beta isozymes. We, therefore, reinvestigated immunohistochemical affinities to MCs of antibodies to PLC-beta 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-beta1 and mGluR5. It was also found that MCs show positive immunoreactions of P2Y2, IP3R-I, and IP3R-II receptors. These results suggest that the Galphaq isoform in MCs couples to both the P2Y2 receptor and mGluR5 and regulates the intracellular Ca(++) concentration via the PLC-beta-IP3 cascade.

Recent progress in studies on Merkel cell biology.

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 morphological 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.

Phenotypic characteristics of adipocytes generated from Meckel's chondrocytes in response to chick serum in vitro.

When present in the culture medium, chick serum (CKS) modulated the phenotypic change from chondrocytes of Meckel's cartilage to adipocytes in vitro, as revealed by light and electron microscopy, the incorporation of BrdU, and immunocytochemistry. CKS inhibited DNA synthesis in chondrocytes and the proliferation of these cells, while it facilitated the differentiation to adipocytes. CKS contributed to phenotypic changes in undifferentiated chondrocytes, but did not affect the characteristics of differentiated chondrocytes. Electron microscopy revealed that the lipid droplets in adipocytes were enclosed by limiting membranes that fused to yield larger lipid droplets. Immunocytochemical staining of adipocytes with stage-specific antibodies revealed the presence of immunoreactive uncoupling protein (UCP-1) and peroxisome proliferator-activated receptor (PPARgamma) in immature adipocytes, and leptin and glucose transporter (Glut-4) in mature adipocytes. The adipocytes that were formed in the present study were multilocular adipocytes that contained many small lipid droplets, but in many ways they resembled white adipocytes. CKS contains a high level of estrogen, compared with fetal bovine serum, and it is possible that estrogen might have induced the differentiation to adipocytes.

Phenotypic switching of in vitro mandibular condylar cartilage during matrix mineralization.

In order to analyze the phenotypic conversion of chondrocytes, mandibular condyles of mice and rabbits were cultured under cell and organ culture systems, and then examined by a combination of morphological and biochemical procedures. In organ culture, mandibular condylar cartilage (MCC) obtained from newborn mice began to mineralize from the central zone and then progressively widened towards the peripheral zone. Electron microscopic observations showed that with the increasing duration of the organ culture, chondrocytes at the central zone converted into spindle-shaped osteoblastic cells accompanying the formation of the bone type of thick-banded collagen fibrils. To obtain a better understanding of the chondrocytic conversion, immunolocalizations for type I and type X collagens and osteocalcin (OC) were examined in mouse MCC cells in cell culture. Type X collagen and OC were expressed almost simultaneously at the late stage of culture, and type I collagen was detected along the calcified nodules after the production of these proteins. Northern blot analysis in cell cultures of rabbit MCC indicated that type II collagen and alkaline phosphatase (ALPase) messenger ribonucleic acids (mRNAs) were highly expressed at day 7, but subsequently decreased. In contrast, mRNA for type I collagen was expressed at a low level on day 7 and peaked on day 12. The present results suggest that, morphologically and biochemically, cellular modification in MCC cells under culture conditions occurs at a cellular morphological level and also at marker-gene-expression level.