Anatomical Study of the Cuboid and Its Ligamentous Attachments and Its Implications for a Cuboid Osteotomy.

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.

Merkel Cells Release Glutamate Following Mechanical Stimulation: Implication of Glutamate in the Merkel Cell-Neurite Complex.

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.

Intracellular Ca2+ mobilization pathway via bradykinin B1 receptor activation in rat trigeminal ganglion neurons.

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.

High pH-Sensitive Store-Operated Ca2+ Entry Mediated by Ca2+ Release-Activated Ca2+ Channels in Rat Odontoblasts.

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.

Activation of Mechanosensitive Transient Receptor Potential/Piezo Channels in Odontoblasts Generates Action Potentials in Cocultured Isolectin B4-negative Medium-sized Trigeminal Ganglion Neurons.

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.

Temporal Expression Patterns of Clock Genes and Aquaporin 5/Anoctamin 1 in Rat Submandibular Gland Cells.

Circadian rhythms are essential for health and regulate various physiological functions. These rhythms are regulated by a negative-feedback loop involving clock genes in the suprachiasmatic nucleus (SCN) and peripheral tissues. The rate of secretion of salivary substances, ions, and water follows a circadian rhythm, however, the relationship between the molecular mechanism of salivary secretion and peripheral circadian rhythm is not yet clear. Anoctamin 1 (ANO1, also known as TMEM16A) and Aquaporin 5 (AQP5) play an important role in the transport of ions and water in the submandibular glands (SGs). We examined the interaction between the rhythmic expression pattern of the clock genes, Ano1 and Aqp5, in rat whole SGs as well as isolated acinar and ductal cells. Circadian rhythmic expression for Bmal1, Per1, Per2, Clock, Cry1, Cry2, Rorα, and Rev-erbα mRNAs, also called the clock genes, was observed in rat SGs by semi-quantitative RT-PCR analysis. We also observed rhythmic patterns in Ano1 and Aqp5 mRNA expression. The expression of ANO1 protein also showed circadian rhythm, as confirmed by western blot analysis. We could not observe any time delay between the peak expression of ANO1 protein and its mRNA. Expression levels of the clock gene mRNAs in the ductal cells was higher than that in acinar cells, however, rhythmic oscillations were observed in both. Our results suggest that SGs have peripheral clocks, and rhythmic expressions of Ano1 and Aqp5 along with the clock genes, may play an important role in the circadian regulation of salivary secretion.

Ionotropic P2X ATP Receptor Channels Mediate Purinergic Signaling in Mouse Odontoblasts.

ATP modulates various functions in the dental pulp cells, such as intercellular communication and neurotransmission between odontoblasts and neurons, proliferation of dental pulp cells, and odontoblast differentiation. However, functional expression patterns and their biophysical properties of ionotropic ATP (P2X) receptors (P2X1-P2X7) in odontoblasts were still unclear. We examined these properties of P2X receptors in mouse odontoblasts by patch-clamp recordings. K+-ATP, nonselective P2X receptor agonist, induced inward currents in odontoblasts in a concentration-dependent manner. K+-ATP-induced currents were inhibited by P2X4 and P2X7 selective inhibitors (5-BDBD and KN62, respectively), while P2X1 and P2X3 inhibitors had no effects. P2X7 selective agonist (BzATP) induced inward currents dose-dependently. We could not observe P2X1, 2/3, 3 selective agonist (αß-MeATP) induced currents. Amplitudes of K+-ATP-induced current were increased in solution without extracellular Ca2+, but decreased in Na+-free extracellular solution. In the absence of both of extracellular Na+ and Ca2+, K+-ATP-induced currents were completely abolished. K+-ATP-induced Na+ currents were inhibited by P2X7 inhibitor, while the Ca2+ currents were sensitive to P2X4 inhibitor. These results indicated that odontoblasts functionally expressed P2X4 and P2X7 receptors, which might play an important role in detecting extracellular ATP following local dental pulp injury.

Localization and expression patterns of TRP channels in submandibular gland development.

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.

Relationship between Obstructive Sleep Apnea and Self-assessed Oral Health Status: An Internet Survey.

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.

Transient Receptor Potential Cation Channel Subfamily Vanilloid Member 3 is not Involved in Plasma Membrane Stretch-induced Intracellular Calcium Signaling in Merkel Cells.

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.

Intercellular Odontoblast Communication via ATP Mediated by Pannexin-1 Channel and Phospholipase C-coupled Receptor Activation.

Extracellular ATP released via pannexin-1 channels, in response to the activation of mechanosensitive-TRP channels during odontoblast mechanical stimulation, mediates intercellular communication among odontoblasts in dental pulp slice preparation dissected from rat incisor. Recently, odontoblast cell lines, such as mouse odontoblast lineage cells, have been widely used to investigate physiological/pathological cellular functions. To clarify whether the odontoblast cell lines also communicate with each other by diffusible chemical substance(s), we investigated the chemical intercellular communication among cells from mouse odontoblast cell lines following mechanical stimulation. A single cell was stimulated using a glass pipette filled with standard extracellular solution. We measured intracellular free Ca(2+) concentration ([Ca(2+)]i) by fura-2 in stimulated cells, as well as in cells located nearby. Direct mechanical stimulation to a single odontoblast increased [Ca(2+)]i, which showed sensitivity to capsazepine. In addition, we observed increases in [Ca(2+)]i not only in the mechanically stimulated odontoblast, but also in nearby odontoblasts. We could observe mechanical stimulation-induced increase in [Ca(2+)]i in a stimulated human embryo kidney (HEK) 293 cell, but not in nearby HEK293 cells. The increase in [Ca(2+)]i in nearby odontoblasts, but not in the stimulated odontoblast, was inhibited by adenosine triphosphate (ATP) release channel (pannexin-1) inhibitor in a concentration- and spatial-dependent manner. Moreover, in the presence of phospholipase C (PLC) inhibitor, the increase in [Ca(2+)]i in nearby odontoblasts, following mechanical stimulation of a single odontoblast, was abolished. We could record some inward currents evoked from odontoblasts near the stimulated odontoblast, but the currents were observed in only 4.8% of the recorded odontoblasts. The results of this study showed that ATP is released via pannexin-1, from a mechanically stimulated odontoblast, which transmits a signal to nearby odontoblasts by predominant activation of PLC-coupled nucleotide receptors.

Functional expression of bradykinin B1 and B2 receptors in neonatal rat trigeminal ganglion neurons.

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.

Depolarization-induced Intracellular Free Calcium Concentration Increases Show No Desensitizing Effect in Rat Odontoblasts.

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.

Expression and function of purinergic P2Y12 receptors in rat trigeminal ganglion neurons.

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.

Odontoblasts as sensory receptors: transient receptor potential channels, pannexin-1, and ionotropic ATP receptors mediate intercellular odontoblast-neuron signal transduction.

Various stimuli induce pain when applied to the surface of exposed dentin. However, the mechanisms underlying dentinal pain remain unclear. We investigated intercellular signal transduction between odontoblasts and trigeminal ganglion (TG) neurons following direct mechanical stimulation of odontoblasts. Mechanical stimulation of single odontoblasts increased the intracellular free calcium concentration ([Ca(2+)]i) by activating the mechanosensitive-transient receptor potential (TRP) channels TRPV1, TRPV2, TRPV4, and TRPA1, but not TRPM8 channels. In cocultures of odontoblasts and TG neurons, increases in [Ca(2+)]i were observed not only in mechanically stimulated odontoblasts, but also in neighboring odontoblasts and TG neurons. These increases in [Ca(2+)]i were abolished in the absence of extracellular Ca(2+) and in the presence of mechanosensitive TRP channel antagonists. A pannexin-1 (ATP-permeable channel) inhibitor and ATP-degrading enzyme abolished the increases in [Ca(2+)]i in neighboring odontoblasts and TG neurons, but not in the stimulated odontoblasts. G-protein-coupled P2Y nucleotide receptor antagonists also inhibited the increases in [Ca(2+)]i. An ionotropic ATP (P2X3) receptor antagonist inhibited the increase in [Ca(2+)]i in neighboring TG neurons, but not in stimulated or neighboring odontoblasts. During mechanical stimulation of single odontoblasts, a connexin-43 blocker did not have any effects on the [Ca(2+)]i responses observed in any of the cells. These results indicate that ATP, released from mechanically stimulated odontoblasts via pannexin-1 in response to TRP channel activation, transmits a signal to P2X3 receptors on TG neurons. We suggest that odontoblasts are sensory receptor cells and that ATP released from odontoblasts functions as a neurotransmitter in the sensory transduction sequence for dentinal pain.

Arg-vasopressin facilitates calcium channel currents in osteoblasts.

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 facilitates calcium channel currents 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.

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

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons. Many transient receptor potential (TRP) channels have been identified in mammals; however, the activation properties of these channels in oral mucosal MCs remain to be clarified. We investigated the biophysical and pharmacological properties of TRP vanilloid (TRPV)-1, TRPV2, TRPV4, TRP ankyrin (TRPA)-1, and TRP melastatin (TRPM)-8 channels, which are sensitive to osmotic and mechanical stimuli by measurement of intracellular free 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.

Functional expression of TRPM8 and TRPA1 channels in rat odontoblasts.

Odontoblasts produce dentin during development, throughout life, and in response to pathological conditions by sensing stimulation of exposed dentin. The functional properties and localization patterns of transient receptor potential (TRP) melastatin subfamily member 8 (TRPM8) and ankyrin subfamily member 1 (TRPA1) channels in odontoblasts remain to be clarified. We investigated the localization and the pharmacological, biophysical, and mechano-sensitive properties of TRPM8 and TRPA1 channels in rat odontoblasts. Menthol and icilin increased the intracellular free Ca(2+) concentration ([Ca(2+)]i). Icilin-, WS3-, or WS12-induced [Ca(2+)]i increases were inhibited by capsazepine or 5-benzyloxytriptamine. The increase in [Ca(2+)]i elicited by allyl isothiocyanate (AITC) was inhibited by HC030031. WS12 and AITC exerted a desensitizing effect on [Ca(2+)]i increase. Low-temperature stimuli elicited [Ca(2+)]i increases that are sensitive to both 5-benzyloxytriptamine and HC030031. Hypotonic stimulation-induced membrane stretch increased [Ca(2+)]i; HC030031 but not 5-benzyloxytriptamine inhibited the effect. The results suggest that TRPM8 channels in rat odontoblasts play a role in detecting low-temperature stimulation of the dentin surface and that TRPA1 channels are involved in sensing membrane stretching and low-temperature stimulation. The results also indicate that odontoblasts act as mechanical and thermal receptor cells, detecting the stimulation of exposed dentin to drive multiple cellular functions, such as sensory transduction.

Immunolocalization and distribution of functional temperature-sensitive TRP channels in salivary glands.

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.