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Background: Lateral column lengthening (LCL) for flexible flatfoot is an effective surgery with powerful correction of deformity because it tightens only the lateral third of the long plantar ligament (LPL). However, LCL has been associated with joint damage at the osteotomy site and loss of foot flexibility owing to joint fixation. We focused on the cuboid and investigate a novel anatomical LCL osteotomy site that effectively tightens the LPL without damaging any joints. Methods: We studied 24 feet of 12 cadavers (mean age, 80.8 years). The lengths of the LPL and short plantar ligament, locations of the attachments, and shape and location of the cuneocuboid joint on the medial side of the cuboid were studied. ImageJ software was used to measure the osteotomy angle. Results: The lateral cuboid attachment of the LPL on average was located 4.6 mm from the calcaneocuboid joint, and the cuneocuboid joint on average was located 6.7 mm from the cuboid-metatarsal joint on the medial surface of the cuboid. The direct line connecting the anterior cuneocuboid joint and the oblique crest of the cuboid on average was at a 10.3-degree inclination posterior to the cuboid-metatarsal joint. Conclusion: A straight line must be selected between a point 4 mm from the calcaneocuboid joint laterally and 6 mm from the cuboid-metatarsal joint medially at a 10-degree posterior tilt to the cuboid-metatarsal joint to perform a cuboid osteotomy LCL without damaging the articular surface. Clinical Relevance: We investigated a potential novel cuboid osteotomy method for LCL.

Bradykinin (BK) and its receptors, B1 and B2, in trigeminal ganglion (TG) neurons are involved in the regulation of pain. Recent studies have revealed that B1 receptors are expressed in neonatal rat TG neurons; however, the intracellular signaling pathway following B1 receptor activation remains to be elucidated. To investigate the mechanism by which B1 receptor activation leads to intracellular Ca2+ mobilization, we measured the intracellular free Ca2+ concentration ([Ca2+]i) in primary-cultured TG neurons. The application of Lys-[Des-Arg9]BK (B1 receptor agonist) increased the [Ca2+]i in these TG neurons even in the absence of extracellular Ca2+. Pretreatment with inhibitors of ryanodine receptors or sarco/endoplasmic reticulum Ca2+-ATPase suppressed the increase in Lys-[Des-Arg9]BK-induced [Ca2+]i. The Lys-[Des-Arg9]BK-induced [Ca2+]i increase was unaffected by phospholipase-C inhibitor. B1 receptor activation-induced [Ca2+]i increase was suppressed by phosphodiesterase inhibitor and enhanced by adenylyl cyclase inhibitor. These results suggest that B1 receptor activation suppresses intracellular cAMP production via adenylyl cyclase inhibition and mobilizes intracellular Ca2+ via ryanodine receptors that access intracellular Ca2+ stores.

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons through synaptic contact. However, the detailed mechanisms for intercellular communication between MCs and neurons have yet to be clarified. The present study examined the increases in intracellular free Ca2+ concentration ([Ca2+]i) induced by direct mechanical stimulation of MCs. We also measured [Ca2+]i in the trigeminal ganglion neurons (TGs) following direct mechanical stimulation to the MCs in an MC-TGs coculture. The MCs were isolated from hamster buccal mucosa, while TGs were isolated from neonatal Wistar rats. Both cell populations showed depolarization-induced [Ca2+]i. Direct mechanical stimulation to MCs increased [Ca2+]i, showing stimulation strength dependence. In the MC-TGs coculture, the application of direct mechanical stimulation to MCs resulted in increased [Ca2+]i in the TGs. These changes were significantly suppressed by antagonists of glutamate-permeable anion channels (4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid; DIDS), and non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptors (MK801). Apyrase, an ATP-degrading enzyme, and suramin, a non-selective P2 purinergic receptor antagonist, did not exert inhibitory effects on these [Ca2+]i increases in the TGs following MC stimulation. These results indicated that MCs are capable of releasing glutamate, but not ATP, in response to cellular deformation by direct mechanical stimulation. The released glutamate activates the NMDA receptors on TGs. We suggest that MCs act as mechanoelectrical transducers and establish synaptic transmission with neurons, through the MC-neurite complex, to mediate mechanosensory transduction.

Various stimuli to the exposed surface of dentin induce changes in the hydrodynamic force inside the dentinal tubules resulting in dentinal pain. Recent evidences indicate that mechano-sensor channels, such as the transient receptor potential channels, in odontoblasts receive these hydrodynamic forces and trigger the release of ATP to the pulpal neurons, to generate dentinal pain. A recent study, however, has shown that odontoblasts also express glutamate receptors (GluRs). This implies that cells in the dental pulp tissue have the ability to release glutamate, which acts as a functional intercellular mediator to establish inter-odontoblast and odontoblast-trigeminal ganglion (TG) neuron signal communication. To investigate the intercellular signal communication, we applied mechanical stimulation to odontoblasts and measured the intracellular free Ca2+ concentration ([Ca2+]i). During mechanical stimulation in the presence of extracellular Ca2+, we observed a transient [Ca2+]i increase not only in single stimulated odontoblasts, but also in adjacent odontoblasts. We could not observe these responses in the absence of extracellular Ca2+. [Ca2+]i increases in the neighboring odontoblasts during mechanical stimulation of single odontoblasts were inhibited by antagonists of metabotropic glutamate receptors (mGluRs) as well as glutamate-permeable anion channels. In the odontoblast-TG neuron coculture, we observed an increase in [Ca2+]i in the stimulated odontoblasts and TG neurons, in response to direct mechanical stimulation of single odontoblasts. These [Ca2+]i increases in the neighboring TG neurons were inhibited by antagonists for mGluRs. The [Ca2+]i increases in the stimulated odontoblasts were also inhibited by mGluRs antagonists. We further confirmed that the odontoblasts express group I, II, and III mGluRs. However, we could not record any currents evoked from odontoblasts near the mechanically stimulated odontoblast, with or without extracellular Mg2+, indicating that N-methyl-d-aspartic acid receptor does not contribute to inter-odontoblast signal communication. The results suggest that a mechanically stimulated odontoblast is capable of releasing glutamate into the extracellular space via glutamate-permeable anion channels. The released glutamate activates mGluRs on the odontoblasts in an autocrine/paracrine manner, forming an inter-odontoblasts communication, which drives dentin formation via odontoblast-odontoblast signal communication. Glutamate and mGluRs also mediate neurotransmission between the odontoblasts and neurons in the dental pulp to modulate sensory signal transmission for dentinal sensitivity.

Calcium hydroxide and mineral trioxide aggregate are widely used for indirect and direct pulp capping and root canal filling. Their dissociation into Ca2+ and OH- in dental pulp creates an alkaline environment, which activates reparative/reactionary dentinogenesis. However, the mechanisms by which odontoblasts detect the pH of the extracellular environment remain unclear. We examined the alkali-sensitive intracellular Ca2+ signaling pathway in rat odontoblasts. In the presence or absence of extracellular Ca2+, application of alkaline solution increased intracellular Ca2+ concentration, or [Ca2+]i. Alkaline solution–induced [Ca2+]i increases depended on extracellular pH (8.5 to 10.5) in both the absence and the presence of extracellular Ca2+. The amplitude was smaller in the absence than in the presence of extracellular Ca2+. Each increase in [Ca2+]i, activated by pH 7.5, 8.5, or 9.5, depended on extracellular Ca2+ concentration; the equilibrium binding constant for extracellular Ca2+ concentration decreased as extracellular pH increased (1.04 mM at pH 7.5 to 0.11 mM at pH 9.5). Repeated applications of alkaline solution did not have a desensitizing effect on alkali-induced [Ca2+]i increases and inward currents. In the presence of extracellular Ca2+, alkaline solution–induced [Ca2+]i increases were suppressed by application of an antagonist of transient receptor potential ankyrin subfamily member 1 (TRPA1) channels. Ca2+ exclusion efficiency during alkaline solution–induced [Ca2+]i increases was reduced by a Na+-Ca2+ exchanger antagonist. Alizarin red and von Kossa staining revealed increased mineralization levels under repeated high pH stimulation, whereas the TRPA1 antagonist strongly reduced this effect. These findings indicate that alkaline stimuli—such as the alkaline environment inside dental pulp treated with calcium hydroxide or mineral trioxide aggregate—activate Ca2+ mobilization via Ca2+ influx mediated by TRPA1 channels and intracellular Ca2+ release in odontoblasts. High pH–sensing mechanisms in odontoblasts are important for activating dentinogenesis induced by an alkaline environment.

The purpose of this study based on a cross-sectional internet survey was to investigate the relationship between risk of obstructive sleep apnea (OSA) and self-assessed oral health status. The participants, who comprised individuals registered with an online research company, were required to complete a self-reported questionnaire. Those answering in the affirmative to both of the following two questions were placed in the OSA-risk group, while those answering in the negative were assigned to the control group: 'Have other people noticed pauses in your breathing while you are sleeping?' and 'Do you feel excessively sleepy during the daytime?'. A total of 493 were included in the OSA-risk group and 2,560 in the control group. Among the total 3,053 respondents, the highest prevalence for OSA risk in men was in the 50-59-year age range, although this tended to level off after age 60 years. No such trend was observed in women, however. Multiple logistic regression analysis was performed to identify the relationship between risk of OSA and self-assessed oral health status. Significant correlations were observed with the following parameters: difficulty in opening mouth (odds ratio [OR]: 2.66; 95% confidence interval [CI]: 1.647-4.311), dry mouth (OR: 2.11; CI: 1.544-2.876), bad breath (OR: 1.69; CI: 1.309-2.186), gingival bleeding (OR: 1.48; CI: 1.134-1.932), and gingival swelling (OR: 1.44; CI: 1.046-1.981). These results suggest a relationship between risk of OSA and self-assessed oral health status, indicating that treating OSA might improve oral health status. Further study is needed to demonstrate a causal relationship between OSA and self-assessed oral health status, however.

Interleukin-1B (IL-1B) and -6 (IL-6) are inflammatory cytokines that are involved in bone resorption under pathological conditions. The cytokines are also involved in bone remodeling under physiological conditions. Voltage sensitive Ca 2+ channels (VSCCs) serve as crucial mediators of membrane excitability and many Ca 2+ -dependent functions such as growth of bone, regulate proliferation, differentiation, enzyme activity and gene expression. The effects of IL-1B and -6 on VSCCs in osteoblast cell line (MC3T3-E1) were investigated using patch-clamp recording. Our results showed that application of 50 pM-50 nM IL-1B facilitated VSCCs current (I Ca ) carried by Ba 2+ (I Ba ). Application of 50 pM-5 nM IL-6 facilitated I Ba . In contrast, 50 nM IL-6 inhibited I Ba in MC3T3-E1 cells. Treatment with MAPK inhibitor, PD98059, attenuated the 5 nM IL-6-induced facilitation of I Ba . Treatment with STAT3 inhibitor, staffic, attenuated the 50 nM IL-6-induced inhibition of I Ba . Treatment with PD98059 also attenuated the 50 nM IL-6-induced inhibition of I Ba . These results suggest that 5 nM IL-6 facilitates VDCCs involving MAPK pathways. In addition, 50 nM IL-6 inhibits VDCCs involving STAT3 and MAPK pathways in MC3T3-E1 cells.

Bradykinin (BK) and its receptors, B

Merkel cells (MCs), which form part of the MC-neurite complex, making contact with sensory afferents to drive mechanosensory transduction mechanisms, express transient receptor potential (TRP) cation channel subfamily vanilloid (V) members 1, 2, and 4, as well as ankyrin subfamily member 1. While these proteins are involved in sensing plasma membrane stretch, less is known about the functional properties of TRPV subfamily member 3 (TRPV3) during membrane stretch in MCs. The aim of this study was to determine whether TRPV3 channels were involved in mechanosensory activity by measuring intracellular free Ca(2+) concentrations ([Ca(2+)]i) in MCs isolated from hamster buccal mucosa. Application of a hypotonic extracellular solution to quinacrine-positive MCs elicited a transient increase in [Ca(2+)]i. When TRPV3 channel antagonist 2,2-diphenyltetrahydrofuran was added to the hypotonic extracellular solution, however, no effect was observed on hypotonic stimulation-induced increase in [Ca(2+)]i. These results suggest that TRPV3 channels are not involved in the mechanosensory mechanism during membrane stretch in MCs.

Odontoblasts play a crucial role in dentin formation and sensory transduction following the application of stimuli to the dentin surface. Various exogenous and endogenous stimuli elicit an increase in the intracellular free calcium concentration ([Ca2+]i) in odontoblasts, which is mediated by Ca2+ release from intracellular Ca2+ stores and/or Ca2+ influx from the extracellular medium. In a previous study, we demonstrated that the depletion of Ca2+ stores in odontoblasts activated store-operated Ca2+ entry (SOCE), a Ca2+ influx pathway. However, the precise biophysical and pharmacological properties of SOCE in odontoblasts have remained unclear. In the present study, we examined the functional expression and pharmacological properties of Ca2+ release-activated Ca2+ (CRAC) channels that mediate SOCE and evaluated the alkali sensitivity of SOCE in rat odontoblasts. In the absence of extracellular Ca2+, treatment with thapsigargin (TG), a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, induced an increase in [Ca2+]i. After [Ca2+]i returned to near-resting levels, the subsequent application of 2.5 mM extracellular Ca2+ resulted in an increase in [Ca2+]i which is a typical of SOCE activation. Additionally, application of 2-methylthioadenosine diphosphate trisodium salt (2-MeSADP), a P2Y1,12,13 receptor agonist, or carbachol (CCh), a muscarinic cholinergic receptor agonist, in the absence of extracellular Ca2+, induced a transient increase in [Ca2+]i. The subsequent addition of extracellular Ca2+ resulted in significantly higher [Ca2+]i in 2-MeSADP- or CCh-treated odontoblasts than in untreated cells. SOCE, that is activated by addition of extracellular Ca2+ in the TG pretreated odontoblasts was then suppressed by Synta66, BTP2, or lanthanum, which are CRAC channel inhibitors. Treatment with an alkaline solution enhanced SOCE, while treatment with HC030031, a TRPA1 channel antagonist, inhibited it. The amplitude of SOCE at pH 9 in the presence of HC030031 was higher than that at pH 7.4 in the absence of HC030031. These findings indicate that CRAC channel-mediated alkali-sensitive SOCE occurs in odontoblasts. SOCE is mediated by P2Y and muscarinic-cholinergic receptors, which are activated by endogenous ligands in odontoblasts.

Various stimuli to the dentin surface elicit dentinal pain by inducing dentinal fluid movement causing cellular deformation in odontoblasts. Although odontoblasts detect deformation by the activation of mechanosensitive ionic channels, it is still unclear whether odontoblasts are capable of establishing neurotransmission with myelinated A delta (Aδ) neurons. Additionally, it is still unclear whether these neurons evoke action potentials by neurotransmitters from odontoblasts to mediate sensory transduction in dentin. Thus, we investigated evoked inward currents and evoked action potentials form trigeminal ganglion (TG) neurons after odontoblast mechanical stimulation.We used patch clamp recordings to identify electrophysiological properties and record evoked responses in TG neurons.We classified TG cells into small-sized and medium-sized neurons. In both types of neurons, we observed voltage-dependent inward currents. The currents from medium-sized neurons showed fast inactivation kinetics. When mechanical stimuli were applied to odontoblasts, evoked inward currents were recorded from medium-sized neurons. Antagonists for the ionotropic adenosine triphosphate receptor (P2XOdontoblasts established neurotransmission with myelinated Aδ neurons via P2X

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 Ca(2+) concentration ([Ca(2+)]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 Ca(2+), the hypotonic test solution evoked Ca(2+) influx. The [Ca(2+)]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 [Ca(2+)]i only in the presence of extracellular Ca(2+). 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.

Transient receptor potential (TRP) cation channels are unique cellular sensors involved in multiple cellular functions. Their role in salivary secretion remains to be elucidated. The expression and localization of temperature-sensitive TRP channels in salivary (submandibular, sublingual and parotid) glands were analyzed by immunohistochemistry and quantitative real-time reverse transcription plus the polymerase chain reaction (RT-PCR). The effects of various TRP channel agonists on carbachol (CCh)-induced salivary secretion in the submandibular gland and on the intracellular Ca(2+) concentration ([Ca(2+)]i) in a submandibular epithelial cell line were also investigated. Immunohistochemistry revealed the expression of TRP-melastatin subfamily member 8 (TRPM8) and TRP-ankyrin subfamily member 1 (TRPA1) in myoepithelial, acinar and ductal cells in the sublingual, submandibular and parotid glands. In addition, TRP-vanilloid subfamily member 1 (TRPV1), TRPV3 and TRPV4 were also expressed in myoepithelial, acinar and ductal cells in all three types of gland. Quantitative real-time RT-PCR results demonstrated the mRNA expression of TRPV1, TRPV3, TRPV4, TRPM8 and TRPA1 in acinar and ductal cells in these salivary glands. Perfusion of the entire submandibular gland with the TRPV1 agonist capsaicin (1 μM) via the submandibular artery significantly increased CCh-induced salivation, whereas perfusion with TRPM8 and TRPA1 agonists (0.5 μM WS12 and 100 μM allyl isothiocyanate) decreased it. Application of agonists for each of the thermosensitive TRP channels increased [Ca(2+)]i in a submandibular epithelial cell line. These results indicate that temperature-sensitive TRP channels are localized and distributed in acinar, ductal and myoepithelial cells in salivary glands and that they play a functional role in the regulation and/or modulation of salivary secretion.

Angiotensin II (Ang II) plays a major role in the maintenance of extracellular fluid volume and blood pressure. In addition to its well-established role in circulatory homeostasis, it has been implicated in the process of bone formation. Osteoblasts play a major role in bone formation, employing intracellular Ca(2+) as a second messenger to modulate hormonal responses and as a cofactor for mineralization. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. The purpose of this study was to investigate the effects of Ang II on VDCC currents in osteoblasts using a patch-clamp recording method. To our knowledge, the data presented here demonstrate for the first time that Ang II facilitates VDCCs in osteoblasts.

Objective Expression of Transient receptor potential (TRP) channels: TRP canonical (TRPC)1, TRP vanilloid (TRPV)3, TRPV4 and TRP melastatin (TRPM)8 in adult rat salivary gland has recently been reported. The authors investigated expression of these TRP channels in the submandibular gland during early developmental stage in which the cell constitution is different, and discussed the function of TRP in the submandibular gland in early development. Design Using rat submandibular gland at embryonic days (E)18 and E20 and postnatal days (PN)0 and PN5 and PN28, expression of TRPV3, TRPV4, TRPC1 and TRPM8 was investigated using real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Results All TRP channels were expressed in cells constituting the submandibular gland in early developmental stage, but an increase in the expression level at PN5 on RT-PCR was significant compared with those at E18, PN0 and PN28 in TRPC1 and TRPV4 channels, whereas an increase was observed but not significant in the others. On immunohistochemical staining at PN5, whereas strong reactions of anti-TRPM8 antibody, anti-TRPV3 and anti-TRPV4 antibodies were observed in cells which proliferated from a terminal portion of cells arranged tubular structure which previously constituted mostly the submandibular gland. Conclusion It was clarified that TRP channels are expressed in the rat submandibular gland in early developmental stage although cells constituting the submandibular gland are different from those in adult animals, suggesting that these TRP channels are involved in cell differentiation in at PN5 into the adult submandibular gland during early development.

Introduction Transmembrane ionic signaling regulates many cellular processes in both physiological and pathologic settings. In this study, the biophysical properties of voltage-dependent Na + channels in odontoblasts derived from human dental pulp (HOB cells) were investigated together with the effect of bradykinin on intracellular Ca 2+ signaling and expression of Ca 2+ -activated K + channels. Methods Ionic channel activity was characterized by using whole-cell patch-clamp recording and fura-2 fluorescence. Results Mean resting membrane potential in the HOB cells was −38 mV. Depolarizing steps from a holding potential of −80 mV activated transient voltage-dependent inward currents with rapid activation/inactivation properties. At a holding potential of −50 mV, no inward current was recorded. Fast-activation kinetics exhibited dependence on membrane potential, whereas fast-inactivation kinetics did not. Steady-state inactivation was described by a Boltzmann function with a half-maximal inactivation potential of −70 mV, indicating that whereas the channels were completely inactivated at physiological resting membrane potential, they could be activated when the cells were hyperpolarized. Inward currents disappeared in Na + -free extracellular solution. Bradykinin activated intracellular Ca 2+ -releasing and influx pathways. When the HOB cells were clamped at a holding potential of −50 mV, outward currents were recorded at positive potentials, indicating sensitivity to inhibitors of intermediate-conductance Ca 2+ -activated K + channels. Conclusions Human odontoblasts expressed voltage-dependent Na + channels, bradykinin receptors, and Ca 2+ -activated K + channels, which play an important role in driving cellular functions by channel-receptor signal interaction and membrane potential regulation.

Extracellular ATP, an essential pain mediator, is received by cell-surface ionotropic P2X and/or metabotropic P2Y receptors. Although the contribution of P2X₃ and/or P2X(2/3) receptors toward the pain mechanism is well described in trigeminal ganglion neurons, the expression of other subtypes of P2X receptor remains to be clarified. We examined expression of P2X receptor mRNA and measured intracellular free Ca²⁺ concentration ([Ca²⁺]i) by the activation of these receptors by fura-2 fluorescence in primary cultured rat trigeminal ganglion neurons. Real-time reverse transcription-PCR analysis revealed mRNA expression of P2X receptor subtype P2X₁, P2X₃, and P2X₄ in trigeminal ganglion neurons. In the presence of extracellular Ca²⁺, the application of P2X receptors agonists, ATP, α,β-methylene ATP or β,γ-methylene ATP induced Ca²⁺ influx significantly. The ATP-induced increase in [Ca²⁺]i was inhibited by a series of selective antagonists for P2X₁, P2X₃, or P2X₄ receptors. These results indicate that trigeminal ganglion neurons functionally express P2X₁, P2X₃, and P2X₄ receptors and that these receptors are involved in the mediation of not only nociceptive but also neuropathic pain in the orofacial area.

It is established that neuropeptide Y (NPY) is a transmitter of parasympathetic secretory impulses in submandibular gland. The neuropeptides substance P, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) are likely mediators of secretory parasympathetic responses of the gland. Previously, we have shown that substance P, VIP and CGRP modulate voltage-dependent Ca(2+) channels (VDCCs) in hamster submandibular ganglion (SMG) neurons. In this study, we attempt to characterize the effect of NPY on VDCCs current using Ba(2+) (I(Ba)) in SMG neurons. Application of NPY caused both facilitation and inhibition of L-type and N/P/Q-type I(Ba), respectively. Intracellular dialysis of the Gα(s)-protein antibody attenuated the NPY-induced facilitation of I(Ba). The adenylate cyclase (AC) inhibitor, as well as protein kinase A (PKA) inhibitor attenuated the NPY-induced facilitation of I(Ba). Intracellular dialysis of the Gα(i)-protein antibody attenuated the NPY-induced inhibition of I(Ba). Application of a strong depolarizing voltage prepulse attenuated the NPY-induced inhibition of I(Ba). These results indicate that NPY facilitates L-type VDCCs via Gα(s)-protein involving AC and PKA. On the other hand, NPY also inhibits N/P/Q-type VDCCs via Gα(i)-protein βγ subunits in the SMG neurons.

Osteoblasts play a major role in bone formation. Osteoblasts employ intracellular Ca(2+) as a second messenger to modulate hormonal responses and a cofactor for bone mineralization. Adrenomedullin (ADM) promotes osteoblast growth and proliferation, inducing an increase in bone mass. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. Voltage-dependent Ca(2+) channels serve as crucial mediators of many Ca(2+)-dependent functions, including growth of bone and regulation of proliferation. The purpose of this study was to investigate the effects of ADM on VDCC currents in osteoblasts using a patch-clamp recording method. To our knowledge, the data presented here demonstrate for the first time that ADM facilitates VDCCs in osteoblasts.

Objective The control of saliva secretion is mainly under parasympathetic control. The submandibular ganglion (SMG) is a parasympathetic ganglion which receives inputs from preganglionic cholinergic neurons, and innervates the submandibular salivary gland to control saliva secretion. The aim of this study was to investigate if adrenomedullin (ADM) and/or calcitonin gene-related peptide (CGRP) modulate voltage-dependent calcium channel (VDCCs) current ( I Ca ) in SMG. Design The profile of CGRP and ADM actions in SMG was studied using the whole-cell configuration of the patch-clamp technique. Results Both ADM and CGRP facilitated I Ca . These facilitations were attenuated by intracellular dialysis of the anti-Gα s -protein and pretreatment of SQ22536 (an adenylate cyclase inhibitor). Conclusions ADM and CGRP facilitates VDCCs mediated by Gα s -protein and adenylate cyclase in SMG.

During palatine rugae development in mice, a cell cluster appeared transiently in the palatine rugae epithelium. The characteristics of these cells were examined histologically and immunohistochemically for anti-cytokeratin (CK) and anti-protein gene product 9.5 (PGP9.5) antibodies using mice from embryonic day 15 (E15) to postnatal day 1 (PN1). At E15, the epithelium of both palatine rugae located in the anterior portion and inter-rugae area consisted of 2-3 cell layers, and palatine rugae have papillae under the epithelium. However, palatine rugae located in the posterior portion have cell clusters in the epithelium. These cells expressed immunopositive reactions for anti-CK18, although they did not react with anti-CK14. Under the anti-CK18 immunopositive cell cluster in palatine rugae epithelium, anti-PGP9.5 immunoreactivity was observed in papillae. Moreover, the basal cells of the cell cluster showed anti-CK7 immunoreactivity. The cell cluster in palatine rugae epithelium disappeared gradually and anti-CK18 immunopositive cells also disappeared. However, anti-PGP9.5 immunoreactivity in the papilla was maintained after the disappearance of anti-CK18 immunoactivity in the palatine rugae epithelium. At PN1, the epithelium of the palate, including the palatine rugae, was stratified and keratinized. These findings suggested that the cell cluster in the palatine rugae epithelium may be considered as a taste bud-like structure according to the expression pattern for anti-CK and may play an important role in nerve innervation to the papilla under the palatine rugae epithelium.

Introduction Various stimuli to the dentin surface elicit dentinal pain by inducing dentinal fluid movement causing cellular deformation in odontoblasts. Although odontoblasts detect deformation by the activation of mechanosensitive ionic channels, it is still unclear whether odontoblasts are capable of establishing neurotransmission with myelinated A delta (Aδ) neurons. Additionally, it is still unclear whether these neurons evoke action potentials by neurotransmitters from odontoblasts to mediate sensory transduction in dentin. Thus, we investigated evoked inward currents and evoked action potentials form trigeminal ganglion (TG) neurons after odontoblast mechanical stimulation. Methods We used patch clamp recordings to identify electrophysiological properties and record evoked responses in TG neurons. Results We classified TG cells into small-sized and medium-sized neurons. In both types of neurons, we observed voltage-dependent inward currents. The currents from medium-sized neurons showed fast inactivation kinetics. When mechanical stimuli were applied to odontoblasts, evoked inward currents were recorded from medium-sized neurons. Antagonists for the ionotropic adenosine triphosphate receptor (P2X3), transient receptor potential channel subfamilies, and Piezo1 channel significantly inhibited these inward currents. Mechanical stimulation to odontoblasts also generated action potentials in the isolectin B4–negative medium-sized neurons. Action potentials in these isolectin B4–negative medium-sized neurons showed a short duration. Overall, electrophysiological properties of neurons indicate that the TG neurons with recorded evoked responses after odontoblast mechanical stimulation were myelinated Aδ neurons. Conclusions Odontoblasts established neurotransmission with myelinated Aδ neurons via P2X3 receptor activation. The results also indicated that mechanosensitive TRP/Piezo1 channels were functionally expressed in odontoblasts. The activation of P2X3 receptors induced an action potential in the Aδ neurons, underlying a sensory generation mechanism of dentinal pain.

Introduction Intracellular Ca 2+ is essential to many signal transduction pathways, and its level is tightly regulated by the Ca 2+ extrusion system in the plasma membrane, which includes the Na + -Ca 2+ exchanger (NCX). Although expression of NCX1 isoforms has been demonstrated in odontoblasts, the detailed properties of NCX remain to be clarified. In this study, we investigated localization and ion-transporting/pharmacologic properties of NCX isoforms in rat odontoblasts. Methods We characterized both the reverse and forward modes of NCX activity in odontoblasts in a dental pulp slice preparation. Ca 2+ influx by reverse NCX activity was measured by fura-2 fluorescence. Ca 2+ efflux by forward NCX activity elicited inward Na + current as measured by perforated-patch clamp recording. For immunohistochemical analysis, cryostat sections of incisors were incubated with antibodies against NCX. Results Immunohistochemical observation revealed localization of NCX1 and NCX3 in the distal membrane of odontoblasts. Inward currents by forward NCX activity showed dependence on external Na + . Fura-2 fluorescence measurement revealed that Ca 2+ influx by reverse NCX activity depended on extracellular Ca 2+ concentration, and that this influx was blocked by NCX inhibitor KB-R7943 in a concentration-dependent manner. However, Ca 2+ influx by NCX showed a slight sensitivity to SEA0400 (a potent NCX1 inhibitor), indicating that expression potencies in odontoblasts were NCX3 > NCX1. Conclusions These results suggest that odontoblasts express NCX1 and NCX3 at the distal membrane, and that these isoforms play an important role in the Ca 2+ extrusion system as well as in the directional Ca 2+ transport pathway from the circulation to the dentin-mineralizing front.

The biologically active form of vitamin D, 1,25-dihydroxy vitamin D3 (VD), regulates the synthesis of the bone Ca-binding proteins osteocalcin and osteopontin. The actions of VD are mediated through the vitamin D receptor (VDR). Liganded VDR heterodimerizes with the retinoid X receptor and interacts with a vitamin D response element (VDRE). Recently, it has been demonstrated that vitamin D responses elicited in osteoblasts can be rapid as well as long-term. The purpose of this study was to elucidate the mechanism of Ca 2 signaling of VD in osteoblasts using intracellular Ca 2 ([Ca

Odontoblasts, well-polarized columnar cells at the periphery of the dental pulp, originate from neural crest cells. They are primarily involved in dentin formation (dentinogenesis) as sites of the synthesis and secretion of collagenous and non-collagenous matrix proteins, and also participate in the directional transport of Ca 2+ from the circulation to the dentin/mineralizing front. Dentinogenesis is activated by : 1) pulpal patho-physiological events, such as intra-pulpal inflammatory responses, physiological or developmental processes, and 2) events at the dentin surface, such as variousimechanical/heat/coldjstimuli applied to the tooth surface. Therefore, the aim of this communication is to give an overview of the Ca 2+ -signaling system, which may have a major role in dentin formation in both physiological and pathological settings. Special attention will be given to discussion of the following : 1) sequential Ca 2+ signaling pathways from internal inositol 1,4,5-trisphosphate (IP 3 ) production via the activation of phospholipase C-coupled receptors to the release of Ca 2+ from IP 3 -sensitive Ca 2+ stores, 2) subsequent Ca 2+ influx via store-operated Ca 2+ channels, and 3) the expression of transient receptor potential channels coupled with Na + -Ca 2+ exchangers in odontoblasts.

Mechanical stress such as occlusal and orthodontic loading has been suggested to induce a homeostatic and regenerative response in periodontal ligament (PDL), but the underlying mechanism remains to be clarified. The purpose of this study was to investigate expression of mRNAs encoding proteins involved in osteogenesis and homeostasis by PDL cells following application of tensile stress and characterize the relationship between such expression and the regenerative and homeostatic functions of the PDL. PDL cells were obtained from rats and stretched by 9% or 18% at a frequency of 6 cycles/min for 12 hr to 5 days in a FX-4000Ttrade; culture system. After stretching, expression of mRNAs encoding collagen type I (Col-I), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-4 (BMP-4), heat shock protein 70 (HSP70) and basic fibroblast growth factor (bFGF) was investigated. The highest levels of Col-I, ALP and BMP-2 mRNA expression occurred at 12 hr, while those of BMP-4 and HSP70 occurred at 1 day and 5 days, respectively. Expression levels of Col-I, ALP, BMP-2, BMP-4 and HSP70 increased magnitude-dependently with stretching force in the stretching groups. In contrast, expression of bFGF mRNA showed statistically significant reduction in both stretching groups, with the largest reduction seen in the 9% stretching group (p0.01). These results suggest that stretching of PDL cells provokes significant increases in expression of factors promoting osteogenic differentiation and HSP70, which protects PDL cells undergoing mechanical stress and contributes to maintenance of PDL homeostasis. However, expression of bFGF was restrained. Reduced expression of bFGF mRNA suggested that there was an optimum magnitude of stretching force for increasing expression.

Temporomandibular disorders are a group of chronic and painful conditions in the masticatory musculature and temporomandibular joint. The pathological mechanisms underlying temporomandibular disorders remain to be clarified; however, it is known that disturbances in neurophysiological, cognitive, behavioral and neuromuscular functions are involved in the development and persistence of such disorders. In addition, it has been suggested that neuropathological changes in the central nervous system are involved in the development of temporomandibular disorders. This review describes changes in the cortical machinery in patients with temporomandibular disorders by comparing neuromagnetic signals between healthy subjects and patients elicited by observing jaw opening movements made by another person.

Objective Neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), promote neuronal development and neuronal survival, but their mechanisms remain controversial. This study aimed to investigate the hypothesis that NGF and BDNF interfere with angiotensin-II- and glutamate-induced facilitation of voltage-dependent Ca 2+ channels (VDCCs) in nucleus tractus solitarius (NTS) neurons. Design The profile of NGF and BDNF actions in acutely dissociated rat NTS was studied using the whole-cell configuration of the patch-clamp technique. Results Pretreatment with NGF and BDNF attenuated angiotensin-II-induced facilitation of VDCCs, but did not attenuate glutamate-induced facilitation of the L-type VDCC current in NTS neurons. NGF-induced attenuation was antagonised by pretreatment with a tyrosine kinase A (TrkA) receptor antagonist K-252a. Conclusions NGF attenuated angiotensin-II-induced facilitation of L-type VDCCs mediated by TrkA receptors in NTS neurons.

Galanin (GAL), a 29-amino-acid neuropeptide, is involved in various neuronal functions, including the regulation of food intake, hormone secretion and central cardiovascular regulation. The nucleus tractus solitarius (NTS) is known to plays a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca(2+)-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of GAL on VDCCs currents (ICa) carried by Ba2+ (IBa) in the NTS using patch-clamp recording methods. An application of M617 (GalR1 specific agonist), AR-M961 (GAL receptor GalR 1/2 agonist) and GAL caused inhibition of N- and P/Q-types I(Ba). M617, GAL, and AR-M961 caused inhibition of I(Ba) in a concentration-dependent manner, with IC50s of 678 nM, 325 nM and 573 nM, respectively. This inhibition was relieved, albeit incompletely, by a depolarizing prepulse. Pretreatment with M35 (GalR non-specific antagonist) attenuated the M617-induced inhibition of I(Ba). Intracellular dialysis of the Galpha(i)-protein antibody also attenuated the Gal-induced inhibition of IBa. These results indicate that GAL inhibits N- and P/Q-types VDCCs via Galpha(i)-protein betagamma subunits mediated by GalR1 in NTS.

The purpose of this study was to evaluate age-related differences in expression of vascular endothelial growth factor (VEGF) by periodontal ligament (PDL) cells. PDL cells were obtained from Wistar male rats weighing approximately 150g each in the young group and 350g each in the old group. PDL cells derived from upper and lower incisors were seeded in 35-mm culture dishes after primary culture. For cell proliferation assays, cells were detached and counted at 1, 3, 5, 7, 11 and 14 days after culture. VEGF mRNA expression was analyzed with TaqMan ® . The number of cells in both groups increased day by day, but the rate of increase in the young group was higher than that in the old group. VEGF mRNA expression in the young group increased from 3 to 14 days, but in the old group increased only slightly over the same time period. Expression ratios in the young group were higher than those in the old group, and there were significant differences between the young and old groups at 7 and 14 days of culture. In conclusion, the data revealed that PDL cells varied with age, and suggest that in view of such changes in cell proliferation and VEGF mRNA expression, age should be taken into consideration in periodontal treatment.

Odontoblasts play an important role in the transduction of the sensory signals underlying dentinal pain. Transmembrane voltage-independent Ca(2+) influx in odontoblasts has been well described. Voltage-dependent Ca(2+) influx has also been reported, but its biophysical properties remain unclear. The aim of the present study was to investigate the desensitizing effect of voltage-dependent Ca(2+) influx in rat odontoblasts by measuring depolarization-induced intracellular free Ca(2+) concentrations ([Ca(2+) ]i ). Odontoblasts on dental pulp slices from newborn rats were acutely isolated and [Ca(2+) ]i measured by using fura-2 fluorescence. Repeated application of extracellular high-K(+) solution (50 mM), which induces membrane depolarization-elicited repeated and transient increases in [Ca(2+) ]i in the presence of extracellular Ca(2+). Increases in depolarization-induced [Ca(2+) ]i showed no significant desensitizing effect (p >0.05; Friedman test). These results suggest that odontoblasts express a voltage-dependent Ca(2+) influx pathway with no desensitizing properties.

Bradykinin (BK) and its receptors (B1 and B2 receptors) play important roles in inflammatory nociception. However, the patterns of expression and physiological/pathological functions of B1 and B2 receptors in trigeminal ganglion (TG) neurons remain to be fully elucidated. We investigated the functional expression of BK receptors in rat TG neurons. We observed intense immunoreactivity of B2 receptors in TG neurons, while B1 receptors showed weak immunoreactivity. Expression of the B2 receptor colocalized with immunoreactivities against the pan-neuronal marker, neurofilament H, substance P, isolectin B4, and tropomyosin receptor kinase A antibodies. Both in the presence and absence of extracellular Ca(2+) ([Ca(2+)]o), BK application increased the concentration of intracellular free Ca(2+) ([Ca(2+)]i). The amplitudes of BK-induced [Ca(2+)]i increase in the absence of [Ca(2+)]o were significantly smaller than those in the presence of Ca(2+). In the absence of [Ca(2+)]o, BK-induced [Ca(2+)]i increases were sensitive to B2 receptor antagonists, but not to a B1 receptor antagonist. However, B1 receptor agonist, Lys-[Des-Arg(9)]BK, transiently increased [Ca(2+)]i in primary cultured TG neurons, and these increases were sensitive to a B1 receptor antagonist in the presence of [Ca(2+)]o. These results indicated that B2 receptors were constitutively expressed and their activation induced the mobilization of [Ca(2+)]i from intracellular stores with partial Ca(2+) influx by BK. Although constitutive B1 receptor expression could not be clearly observed immunohistochemically in the TG cryosection, cultured TG neurons functionally expressed B1 receptors, suggesting that both B1 and B2 receptors involve pathological and physiological nociceptive functions.

The aim of the present study was to investigate the responses of Merkel cells that are numerous in the palatine rugae, due to the continuous mechanical stimulation exerted by the palatal plate. Forty golden hamsters were used in this experiment. The palatal plate was made of adhesive resin and it was set on the palate of the animal. To exert a continuous pressure, a 0.8 mm elevation on the internal surface of the palatal plate was created at the middle portion of the fourth palatine ruga. Thereafter, the number of Merkel cells in the mucosa was calculated by immunohistochemical observation. Morphological changes of Merkel cells were examined by electron microscopy. There was significant difference among the control and any of the treated groups on the number of CK20 positive Merkel cells (p < 0.05) and that numbers were decreased at the sites where continuous mechanical stimulation was exerted. Degeneration of the cytoplasm mitochondria and nerve endings, and a decrease in both the number of neurosecretory granules and cytoplasmic processes were observed. Furthermore, the presence of nuclear chromatin aggregation and fragmentation was recognized. The continuous mechanical stimulation by the palatal plate affected the responses of Merkel cells and nerve endings, thus inducing a decrease in the number of Merkel cells. A portion of these changes was also associated with the expression of apoptosis.

The purpose of this study was to investigate the relationship between Merkel cells and nerve elements during tissue regeneration after receiving dental implants. Golden hamsters were divided into 3 groups and titanium alloy implants were fixed in their left-side maxilla through the third palatine rug. Animals were sacrificed at 1, 2, 3, 4, 5, 6, and 7 days after the implantation and tissues were characterized at the immunohistochemical and morphological levels. CK 20 and PGP 9.5 antibodies which react with Merkel cells and nerve fibers were used. Immunohistochemically, no CK 20-positive Merkel cells were seen in the peri-implant epithelium throughout the 7 days. However, starting at day 4, PGP 9.5-positive nerve fibers appeared in the connective tissue, and by day 7, nerve fibers had invaded the more superficial layer of the peri-implant epithelium compared to the mucosa removal control group. At the electron microscopic level, the intercellular spaces of the regenerating epithelium in the mucosa removal control group were small. In contrast, intercellular spaces of the peri-implant epithelium tended to be wide and regenerating nerve fibers invaded those intercellular spaces. In both the mucosa removal control group and the implantation group, the basal lamina and connective tissues regenerated completely. However, clear Merkel cells containing neurosecretory granules were not observed. Taken together, our results indicate that Merkel cells in the hamster palatine mucosa do not regenerate in the peri-implant epithelium. However, regenerative nerve fibers seem to play essential roles as part of the defense and sensory systems around the peri-implant epithelium to compensate for the weakened defense mechanism.

The topography of the insula and operculum responses to electrical stimulation, applied to the first and third transverse palatine ridge and mucosa of foramen palatinum majus of normal volunteers, was analyzed using a 306-channel, whole-head DC-superconducting quantum interference device (SQUID) magnetoencephalography (MEG). The following order of a location of equivalent current dipoles (ECD) was found: third transverse palatine ridge—Insula, Parietal operculum, SI-Frontal operculum, Parietal operculum-SI or SI. It has been reported that the primary gustatory area is the transition area between the operculum and insula in macaque monkeys and humans, and the neurons in the insular cortex of rats are mechanoreceptive. Stimulus to the oral mucosa, therefore, has the possibility to influence the sense of taste.

Purpose : The purpose of this study was to investigate Merkel cell behavior under the condition of continuous pressure by a denture base.Materials and Methods : A denture base made of 4-META/MMA-TBB adhesive resin was applied on the palatal rugae of golden hamsters, and the animals were sacrificed 2 and 3 weeks later.Results : Immunohistochemically, an aggregation of CK 20-positive cells was seen at the tip of the rugae in the control groups. However, a significant decrease in CK 20-positive cells was observed in the experimental groups at each of the time periods.Conclusion : These results suggest that the Merkel cells became apoptotic under continuous pressure and that the sensory system might have changed.

Clear findings relative to where in the brain the starting point of voluntary swallowing is controlled were obtained in the present magnetoencephalographic study. Namely, the cerebral activity was observed in the cingulate gyrus and supplementary motor area for about 80 ms between 1,000 and 1,500 ms before swallowing in all test subjects. Thus, it is clear that this type of central control mechanism also plays an important role in complicated swallowing movements.

Purinergic receptors play key signaling roles in neuropathic pain in the orofacial region, which is innervated by trigeminal ganglion (TG) neurons. The neuropathology of purinergic P2Y12 receptors is well characterized in glia; however, their physiological role in TG neurons remains to be fully elucidated. The present study investigated the expression and function of P2Y12 receptors in rat TG neurons. P2Y12 receptor immunoreactivity was intense in the soma, dendrites, and axons, and colocalized with a pan-neuronal marker, neurofilament H, isolectin B4, and substance P. In the presence of extracellular Ca(2+), 2-methylthio-ADP (an agonist of P2Y1, 12, 13 receptors) transiently increased intracellular free Ca(2+) concentrations ([Ca(2+)]i), an effect that was abolished by P2Y12 receptor antagonists. In the absence of extracellular Ca(2+), ryanodine receptor/channel inhibitors diminished the 2-methylthio-ADP-induced increases in [Ca(2+)]i. A sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor gradually increased [Ca(2+)]i, and after a plateau, application of 2-MeS-ADP induced a rapid and transient, but additive increase in [Ca(2+)]i. An adenylate cyclase inhibitor transiently increased [Ca(2+)]i, while a phosphodiesterase inhibitor prevented the 2-methylthio-ADP-induced increase in [Ca(2+)]i. Our study shows that P2Y12 receptors are expressed in TG neurons, and act via a cAMP-dependent pathway to release intracellular Ca(2+) from ryanodine-sensitive Ca(2+) stores.

The hypothalamic nonapeptide and neurohypophyseal hormone arg-vasopressin (AVP), also known as antidiuretic hormone, is best known for its effects on water reabsorption in kidney. Osteoblasts play a major role in bone formation, employing intracellular Ca(2+) as a second messenger to modulate hormonal responses and as a cofactor for mineralization. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. The purpose of this study was to investigate the effects of AVP on VDCC currents in osteoblasts using a patch-clamp recording method. An application of 1μM AVP facilitated VDCC currents in osteoblasts. To our knowledge, the data presented here demonstrate for the first time that AVP facilitates VDCCs in osteoblasts.

Adrenaline (Adr) is known to directly or indirectly modulate bone cell activity under physiological and pathological conditions. Osteoblasts play a major role in bone formation, employing intracellular Ca(2+) as a second messenger to modulate hormonal responses and as a cofactor for mineralization. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. The purpose of this study was to investigate the effects of Adr on VDCC currents in osteoblasts using a patch-clamp recording method. Application of 1 mM Adr facilitated VDCC currents in a concentration-dependent manner. Pre-treatment with b receptor antagonist propranolol blocked Adr-induced facilitation of VDCC currents carried by Ba(2+) (IBa). These results indicate that Adr-induced facilitation of IBa was mediated by b receptors in MC3T3-E1 osteoblast-like cells. To our knowledge, the data presented here demonstrate for the first time that Adr facilitates VDCCs in MC3T3-E1 osteoblast-like cells.

This paper revealed the changes occurred on the cylindrical cytoplasmic processes in Merkel cells after single cell isolation at hourly intervals observed under scanning electron microscopy (SEM). After 1 h of single Merkel cell isolation, the cylindrical cytoplasmic processes on the cell surface were bent and other numerous vestiges were observed parallely in contact with the cell surface. After 2 h, a decrease on the cylindrical cytoplasmic processes as well as agglutination to the cell membrane was observed. After 5 h, almost no cylindrical cytoplasmic processes were observed, thus the cell surface became smooth similar to a balloon's surface. The cylindrical cytoplasmic processes, which are considered to be receptors of mechanical stimulation, disappeared after isolation with the passing of time, leading to a Merkel cell with a spherical appearance. When mechanical stimulation by mean of Krebs solution perfusion was carried out, an absence of activity of the mechanically-gated channels, which are present on the cell membrane, was observed. It was thought that this might be due to the disappearance of the above mentioned cylindrical cytoplasmic processes. Then, we considered that the perfusion fluid was inadequate for showing the reception of mechanical stimulation of isolated single Merkel cells.