Caveolae are involved in clathrin-independent endocytosis, transcytosis, signal transduction, and tumor suppression - all of which depend on their main constituent protein caveolin families. The periodontal Ruffini ending has been reported to develop a caveola-like structure on the cell membrane of both the axon terminals and Schwann sheaths, suggesting the existence of an axon-Schwann cell interaction in the periodontal Ruffini endings. However, little information is available concerning the functional significance of these caveolae. The present study was undertaken to examine the immunolocalization of caveolin-1, -3 (Cav-1, Cav-3) and Ca(2+)-ATPase in the periodontal Ruffini endings of the rat incisor. Decalcified sections of the upper jaws were processed for immunocytochemistry at the levels of light and electron microscopy. Some immunostained sections were treated with histochemistry for nonspecific cholinesterase (nChE) activity. Observations showed the periodontal Ruffini endings were immunopositive for Cav-1, but not Cav-3. Immunoreactive products for Cav-1 were confined to caveola-like structures in the cell membranes of the cytoplasmic extensions and cell bodies of the terminal Schwann cells associated with the periodontal Ruffini endings. However, the axonal membranes of the terminals did not express any Cav-1 immunoreaction. Double staining with Ca(2+)-ATPase and either protein gene product 9.5 (PGP 9.5) or S-100 protein disclosed the co-localization of immunoreactions in the axonal branches of the periodontal Ruffini endings, but not in the terminal Schwann cells. As Ca(2+) plays an important role in mechanotransduction, these characteristic immunolocalizations show Cav-1/Ca(2+)-ATPase might be involved in the quick elimination of intracellular Ca(2+) in mechanotransduction.

The lanceolate sensory endings that form palisades around the hair follicle associate with networks of branched Schwann cells. To define the properties of these glial networks as possible conduits of Ca2+ signals, lanceolate endings isolated from rat vibrissae were observed by confocal microscopy while the signaling was locally activated by mechanical stimulation. Intercellular coupling by gap junctions was also assessed by a technique employing fluorescence recovery after photobleaching (FRAP) and by transmission electron microscopy (TEM). Results showed that the glial Ca2+ signals can spread among the arrays of lanceolates in two forms: rapid signals that originate in individual Schwann processes covering the lanceolate axon terminals around the locus of mechanical stimulation, and delayed ones that travel from the stimulation locus through cytoplasmic arborization of the primarily activated cell to the adjacent cell processes. The former signaling was suppressed by the antipurinergic agents suramin and apyrase, whereas the latter was sensitive to the gap junction blocker carbenoxolon. FRAP experiments and TEM observations corroborated the presence of gap junction communications between the Schwann processes of different cell origins. These findings show that, in the Schwann networks, purinergically induced Ca2+ signals and those dependent on gap junctions are propagated in their own spatiotemporal patterns to constitute two distinct forms of communication among the mechanoreceptor palisades.

The present study utilized protein gene product 9.5 (PGP 9.5) and S-100 protein immunohistochemistry to examine if Ruffini endings, the primary mechanoreceptors in periodontal ligaments, can regenerate following nerve cross-anastomosis with an inappropriate nerve. Normally, axon terminals of periodontal Ruffini endings are extensively ramified, and terminal Schwann cells, identified by their S-100 immunoreactivity, are associated with axon terminals. Schwann cells are restricted to the alveolus-related part (ARP), but not tooth-related part (TRP) or the shear zone at the border between the ARP and the TRP of the lingual periodontal ligament of the lower incisor. When the central portion of the mental nerve (MN) was connected with the peripheral portion of the inferior alveolar nerve (IAN), regenerating MN fibers invaded the IAN around postoperative day 5 (PO 5). During the postoperative period, numerous S-100-immunoreactive (IR) cells, presumably terminal Schwann cells, began to migrate to the shear zone and the TRP. PGP 9.5-IR elements reappeared at PO 7 and gradually increased in number. Around PO 28, the terminal portion of the regenerating Ruffini endings appeared dendritic, but less expanded, and the rearrangement of terminal Schwann cells was noted. Regenerated periodontal Ruffini endings were slightly smaller in number. The number of trigeminal ganglion neurons sending peripheral processes beyond the site of injury was smaller compared to those of normal MN, but their cross-sectional areas were almost comparable. Expressions of calbindin D28k and calretinin, normally localized in axonal elements in Ruffini endings, were first detected around PO 56. The present results show that parts of periodontal Ruffini endings can regenerate following nerve cross-anastomosis with mental nerve.

The localization of the calcium-binding proteins (CaBPs) calbindin-D28K (CB) and parvalbumin (PV) in avian rapidly-adapting Herbst and Grandry sensory corpuscles was studied with the use of immunocytochemistry and monoclonal antibodies. Strongest immunostaining was detected in cells of the capsule in both receptor types. Staining was more pronounced in the vicinity of endoplasmic reticulum and mitochondria in the perinuclear regions, whereas staining was distinct in pinocytotic vesicles in peripheral cytoplasmic lamellae. Fibroblasts and macrophages in the subcapsular space of Herbst receptors also showed strong immunostaining in organelles in perinuclear regions. Modified Schwann cells in both receptor types revealed moderately-expressed immunostaining, which was more pronounced in perinuclear regions. The various parts of the receptor nerve fibers showed weak to strong staining. The physiological roles of the investigated CaBPs may be associated with cytoplasmic calcium ion (Ca++) storage, which is necessary for either active metabolism in the immunostained structures and/or their transfer to sensory axonal regions where Ca++ channels are present.

We reviewed the regeneration of periodontal Ruffini endings, primary mechanoreceptors in the periodontal ligament, following injury to the inferior alveolar nerve (IAN) in adult and neonatal rats. Morphologically, mature Ruffini endings are characterized by an extensive arborization of axonal terminals and association with specialized Schwann cells, called lamellar or terminal Schwann cells. Following injury to IAN in the adult, the periodontal Ruffini endings of the rat lower incisor ligament regenerate more rapidly than Ruffini endings in other tissues. During regeneration, terminal Schwann cells migrate into regions where they are never found under normal conditions. The development of periodontal Ruffini endings of the rat incisor is closely associated with the eruption of the teeth; the morphology and distribution of the terminal Schwann cells became almost identical to those in adults during postnatal days 15-18 (PN 15-18d) when the first molars appear in the oral cavity, while the axonal elements showed extensive ramification around PN 28d when the functional occlusion commences. When the IAN was injured in neonates, the regeneration of periodontal Ruffini endings was delayed compared with the adults. The migration of terminal Schwann cells is also observed following IAN injury, after which the distribution of terminal Schwann cells became almost identical to that of the adults, i.e., PN 14d. Since the interaction between axon and Schwann cell is important during regeneration and development, further studies are required to elucidate its molecular mechanism during the regeneration as well as the development of the periodontal Ruffini endings.

Current knowledge on the Ruffini endings, primary mechanoreceptors in the periodontal ligament is reviewed with special reference to their cytochemical features and regeneration process. Morphologically, they are characterized by extensive ramifications of expanded axonal terminals and an association with specialized Schwann cells, called lamellar or terminal Schwann cells, which are categorized, based on their histochemical properties, as non-myelin-forming Schwann cells. Following nerve injury, the periodontal Ruffini endings of the rat incisor ligament can regenerate more rapidly than Ruffini endings in other tissues. During regeneration, terminal Schwann cells associated with the periodontal Ruffini endings migrate into regions where they are never found under normal conditions. Also during regeneration, alterations in the expression level of various bioactive substances occur in both axonal and Schwann cell elements in the periodontal Ruffini endings. Neuropeptide Y, which is not detected in intact periodontal Ruffini endings, is transiently expressed in their regenerating axons. Growth-associated protein-43 (GAP-43) is expressed transiently in both axonal and Schwann cell elements during regeneration, while this protein is localized in the Schwann sheath of periodontal Ruffini endings under normal conditions. The expression of calbindin D28k and calretinin, both belonging to the buffering type of calcium-binding proteins, was delayed in periodontal Ruffini endings, compared to their morphological regeneration. As the importance of axon-Schwann cell interactions has been proposed, further investigations are needed to elucidate their molecular mechanism particularly the contribution of growth factors during the regeneration as well as development of the periodontal Ruffini endings.

The periodontal ligament receives a rich sensory nerve supply and contains many nociceptors and mechanoreceptors. Although its various kinds of mechanoreceptors have been reported in the past, only recently have studies revealed that the Ruffini endings--categorized as low-threshold, slowly adapting, type II mechanoreceptors--are the primary mechanoreceptors in the periodontal ligament. The periodontal Ruffini endings display dendritic ramifications with expanded terminal buttons and, furthermore, are ultrastructurally characterized by expanded axon terminals filled with many mitochondria and by an association with terminal or lamellar Schwann cells. The axon terminals of the periodontal Ruffini endings have finger-like projections called axonal spines or microspikes, which extend into the surrounding tissue to detect the deformation of collagen fibers. The functional basis of the periodontal Ruffini endings has been analyzed by histochemical techniques. Histochemically, the axon terminals are reactive for cytochrome oxidase activity, and the terminal Schwann cells have both non-specific cholinesterase and acid phosphatase activity. On the other hand, many investigations have suggested that the Ruffini endings have a high potential for neuroplasticity. For example, immunoreactivity for p75-NGFR (low-affinity nerve growth factor receptor) and GAP-43 (growth-associated protein-43), both of which play important roles in nerve regeneration/development processes, have been reported in the periodontal Ruffini endings, even in adult animals (though these proteins are usually repressed or down-regulated in mature neurons). Furthermore, in experimental studies on nerve injury to the inferior alveolar nerve, the degeneration of Ruffini endings takes place immediately after nerve injury, with regeneration beginning from 3 to 5 days later, and the distribution and terminal morphology returning to almost normal at around 14 days. During regeneration, some regenerating Ruffini endings expressed neuropeptide Y, which is rarely observed in normal animals. On the other hand, the periodontal Ruffini endings show stage-specific configurations which are closely related to tooth eruption and the addition of occlusal forces to the tooth during postnatal development, suggesting that mechanical stimuli due to tooth eruption and occlusion are a prerequisite for the differentiation and maturation of the periodontal Ruffini endings. Further investigations are needed to clarify the involvement of growth factors in the molecular mechanisms of the development and regeneration processes of the Ruffini endings.

The postnatal expression of immunoreactivity for calretinin, one of the calcium binding proteins, and for protein gene product 9.5 (PGP 9.5), a general neuronal marker, was investigated in mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisor. Age-related changes in the expression of these two proteins in periodontal nerves were further quantified with a computerized image analysis. At 1 day after birth, a few PGP 9.5-immunoreactive nerve fibers and a still smaller number of calretinin-positive fibers were found in the periodontal ligament: they were thin and beaded in appearance and no specialized nerve terminals were recognized. Tree-like terminals, reminiscent of immature Ruffini endings, were recognizable in 4-day-old rats by PGP 9.5-immunohistochemistry, while calretinin-immunostaining failed to reveal these specialized endings. At postnatal 7-11 days when PGP 9.5-immunostaining could demonstrate typical Ruffini endings, calretinin-immunopositive nerve fibers merely tapered off without forming the Ruffini type endings. A small number of Ruffini endings showing calretinin-immunoreactivity began to occur in the periodontal ligament at 24-26 days after birth when the occlusion of the first molars had been established. At the functional occlusion stage (60-80 days after birth), the Ruffini endings showing calretinin-immunoreactivity drastically increased in number and density, but less so than those positive for PGP 9.5-immunoreaction. The delayed expression of calretinin suggests that the function of the periodontal Ruffini endings is established after the completion of terminal formation because Ca2+, which binds to calcium binding proteins including calretinin with high affinity, plays an important role in mechano-electric transduction.

Subcellular localization of S-100 protein, a kind of calcium binding proteins, was examined immunohistochemically in the Ruffini ending, a primary mechanoreceptor, in the periodontal ligament of the rat incisor. The periodontal ligament of the rat incisor was found to contain many S-100beta-immunoreactive (-IR) structures but no S-100alpha-IR elements. The S-100beta-IR structures ramified extensively to form Ruffini endings and were frequently associated with round cells, the terminal Schwann cells, which also showed S-100beta-like immunoreactivity. In many periodontal Ruffini endings, S-100beta-IR products were recognized in the cytoplasm of Schwann cells, but not in the axoplasm. However, some axon terminals which had fewer or shorter axonal fingers, were filled with S-100beta-IR products. The present findings indicated the existence of S-100beta, not S-100alpha, in axon terminals of the periodontal mechanoreceptive endings which were identified as type II Ruffini endings.

Caveolae are specialized plasmalemmal microdomains originally studied in numerous cell types for their involvement in the transcytosis of macromolecules. They are enriched in glycosphingolipids, cholesterol, sphingomyelin, and lipid-anchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4 degreesC. Once the identification of the marker protein caveolin made it possible to purify this specialized membrane domain, it was discovered that caveolae also contain a variety of signal transduction molecules. This includes G protein-coupled receptors, G proteins and adenylyl cyclase, molecules involved in the regulation of intracellular calcium homeostasis, and their effectors including the endothelial isoform of nitric oxide synthase, multiple components of the tyrosine kinase-mitogen-activated protein kinase pathway, and numerous lipid signaling molecules. More recent work has indicated that caveolae further serve to compartmentalize, modulate, and integrate signaling events at the cell surface. This specialized plasmalemmal domain warrants direct consideration in future investigations of both normal and pathological signal transduction in pulmonary cell types.

The cell biology of caveolae is a rapidly growing area of biomedical research. Caveolae are known primarily for their ability to transport molecules across endothelial cells, but modern cellular techniques have dramatically extended our view of caveolae. They form a unique endocytic and exocytic compartment at the surface of most cells and are capable of importing molecules and delivering them to specific locations within the cell, exporting molecules to extracellular space, and compartmentalizing a variety of signaling activities. They are not simply an endocytic device with a peculiar membrane shape but constitute an entire membrane system with multiple functions essential for the cell. Specific diseases attack this system: Pathogens have been identified that use it as a means of gaining entrance to the cell. Trying to understand the full range of functions of caveolae challenges our basic instincts about the cell.

Regeneration of calretinin (CR)-like immunoreactive (IR) nerve fibers was investigated in the periodontal ligament of the rat lower incisor following resection of the inferior alveolar nerve (IAN). In addition, the degeneration and regeneration processes of periodontal nerve fibers were examined by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker. In normal animals, the periodontal nerve fibers showing PGP 9.5-like immunoreactivity (LI) formed either periodontal Ruffini endings with expanded arborization and thin free nerve endings in the alveolar half of the ligament. Thick CR-IR nerve fibers also appeared in a dendritic fashion in the same region, but thin CR-IR nerve fibers were rarely observed. Five days following resection of the IAN, a major population of PGP 9.5-IR and all CR-IR nerve fibers disappeared except for some thin PGP 9.5-IR nerves in the periodontal ligament. Regenerated PGP 9.5-IR nerve fibers appeared around 7 days following resection, in contrast to a very small number of regenerated CR-IR nerve fibers. Around 14-21 days following resection, the number and terminal morphology of regenerated PGP 9.5-IR nerve fibers were comparable to those observed in normal animals, but the number of regenerated CR-IR nerve fibers was still smaller than that of normal animals. The number of regenerated CR-IR nerve fibers increased to return to normal by 56 days following injury. The delay of expression of CR-LI in the regenerated periodontal Ruffini endings suggests that functional recovery of periodontal Ruffini endings occurred after the completion of the regeneration of periodontal nerve fibers.

The distribution and ultrastructural localization of calretinin (CR)-like immunoreactivity (-LI) were investigated in the lingual periodontal ligament of rat incisors. Some thick nerve fibers within the nerve bundle displayed CR-LI; these CR-like immunoreactive (-IR) nerve fibers entered the alveolar half of the lingual periodontal ligament of the incisor where dendritic terminal arborization was exhibited. Thin and beaded CR-IR nerve fibers were rarely observed in the periodontal ligament. Observations of adjacent sections immunostained with protein gene-product 9.5 (PGP 9.5) revealed that most, if not all, PGP 9.5-IR nerve terminals showing a dendritic arborization expressed CR-LI. Immunoelectron microscopic observations showed that electron-opaque immunoreaction products were localized in the axoplasm of the axon terminals, except for the mitochondria, which were surrounded by Schwann sheaths and multiple-layered basal lamina. Neither cell bodies, the cytoplasmic extension of terminal Schwann cells, nor other cellular elements such as periodontal fibroblasts exhibited CR-LI. The present findings suggest that Ruffini endings, an essential mechanoreceptor in the periodontal ligament and categorized as a slowly adapting mechanoreceptor, express CR-LI, and that CR may participate in the Ca2+ homeostasis against external stimuli in the periodontal Ruffini endings.

It was examined whether calbindin D28k (CB) might be located in the rat incisor periodontal Ruffini ending, an essential mechanoreceptor in periodontal ligament, by light- and electron-microscopic immunohistochemistry. Some thick nerve fibers showing CB-like immunoreactivity (LI) entered the lingual half of the periodontal ligament of the incisor and showed the dendritic terminal arborization. Electron-dense immunoreaction products indicating CB-LI were distributed diffusely in axoplasm of the axon terminals, no mitochondria, however, were not labeled. Neither cell bodies nor cytoplasmic extensions of the terminal Schwann cells exhibited CB-LI. CB was presumed to be involved in the maintenance of Ca2+ homeostasis in the mechano-electric transduction in mechanoreceptors in the periodontal ligament.

This paper reviews the immunohistochemical characteristics of two kinds of human cutaneous sensory nerve formations (SNFs), the Meissner and Pacinian corpuscles. In both kinds of SNF the central axon might be easily identifiable because it displays immunoreactivity (IR) for the neuroendocrine markers neuron-specific enolase and protein gene product 9.5, as well as for neuron-specific intermediate filament proteins, i.e., neurofilaments. Other intermediate filament proteins such as vimentin are localized in the lamellar cells of Meissner corpuscles, and in the inner core, outer core and capsule of Pacinian corpuscles. However, they lack cytokeratins or glial fibrillary acidic protein IR. On the other hand, and in agreement with ultrastructural data, IR for basement membrane constituents laminin and type IV collagen is found underlying all SNF constituents, with the exception of the axon. One of the mechanisms involved in the maintenance of intracellular calcium ions (Ca2+) homeostasis is the calcium binding proteins. Ca2+ play a key role in the mechanoelectric transduction and have been localized in SNFs. In this way IR for the Ca(2+)-binding proteins calbindin D28K, parvalbumin and calretinin, is present and colocalized in both Meissner and Pacinian corpuscles; furthermore, S-100 protein is exclusively localized in the lamellar cells and the inner core. On the other hand, the skin is a main source of neurotrophins for a subset of neural crest sensory neurons, some of which end forming SNF. These factors are conveyed via retrograde axonal transport from the skin to the cell body of the responsive neurons. Interestingly, Meissner and Pacinian corpuscles also display IR for the pan-neurotrophin low-affinity receptor (p75), and for the trkA receptor protein, a basic constituent of the high-affinity receptor for some neurotrophins. Moreover, they express IR for the epidermal growth factor receptor. Finally, other antigens not proper to the cells forming human cutaneous SNF, such as the epithelial membrane antigen and the leucocytary antigen-7, have also been detected.

The localization of the calcium-binding proteins (CaBP) calbindin-D28k (CB), parvalbumin (PV) and S-100 protein (S100P) in the human cutaneous sensory corpuscles was studied by immunohistochemical procedure using monoclonal antibodies. Occurrence of CB, PV and S100P immunoreactivity (IR) was observed in the lamellar cells of Meissner's corpuscles. In the pacinian corpuscles, S100P IR was restricted to the inner-core cells whereas CB and PV IR were found labelling the axon, inner core, outer core and capsule. At the light-microscope level of resolution, the presence of IR in the axon of Meissner's corpuscles cannot be ensured. Since calcium ions (Ca2+) seem to participate in the mechanoreceptor electrogenesis, present results suggest that CaBP could be involved in buffering and/or transport of Ca2+ within the specialized cells surrounding the axon tips of sensory corpuscles, thus, maintaining the periaxonal microenvironment.

Influences of Ca++ transport inhibitors on the extrusion of cytosolic Ca++ from axon terminals of oral mucosal mechanoreceptors were cytochemically studied through the use of trifluoperazine (TFP), a Ca++ pump inhibitor, and amiloride, a Na+/Ca++ exchanger inhibitor. Palates of Mongolian gerbils were isolated after perfusion with normal, TFP-containing, or amiloride-containing HEPES-buffered saline, and then mechanically stimulated in the same buffer. Cytosolic Ca++ in axon terminals at 2 min after the stimulation was visualized by means of an oxalate-antimonate method using microwave fixation, and then evaluated electron-microscopically. TFP was found to inhibit the Ca++ extrusion from axon terminals of Meissner corpuscles, but not from those of Merkel cell-axon complexes. Conversely, amiloride severely reduced the Ca++ extrusion from axon terminals of Merkel cell-axon complexes, but not from those of Meissner corpuscles. These results suggest that cytosolic Ca++ extrusion from axon terminals of rapidly adapting and slowly adapting type I mechanoreceptors is regulated by a Ca++ pump and an Na+/Ca++ exchanger, respectively.

Cytochemical localization of Ca2+ in Meissner corpuscles and Merkel cell-neurite complexes in the palatine mucosa of the Mongolian gerbil was studied by a combined oxalate antimonate-microwave irradiation procedure. The reaction products obtained were identified as calcium antimonate by EGTA solubility and X-ray microanalysis. Meissner corpuscles in the normal palatine rugae could be roughly classified into three types by amount and localization of Ca2+. Type I corpuscles were characterized by a high Ca2+ content in both the terminal axoplasm and caveolae of the lamellar plates, type II, by a low Ca2+ content in the terminal axoplasm and a high Ca2+ content in the lamellar cytoplasm. Type III corpuscles showed intermediate characteristics. Palatine rugae stimulated mechanically during fixation contained an increased number of type I corpuscles. On the other hand, two patterns were distinguished in the distribution of Ca2+ in Merkel cells in palatine rugae fixed under normal conditions. One showed abundant Ca2+ dispersed throughout the cell, while in the other, Ca2+ was specifically localized in the Golgi apparatus and mitochondria. Similar distribution patterns also were observed in palatine rugae that had received mechanical stimulus during fixation. Axon terminals of most Merkel cell-neurite complexes in normal palatine rugae were poor in axoplasmic Ca2+, whereas those in most Merkel cell-neurite complexes in mechanically stimulated palatine rugae contained abundant Ca2+ in their axoplasm.