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Abstract: 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 Ca2+ concentration ([Ca2+]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 Ca2+, the hypotonic test solution evoked Ca2+ influx. The [Ca2+]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 [Ca2+]i only in the presence of extracellular Ca2+. 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. [Copyright &y& Elsevier]

Abstract: Introduction: A number of transient receptor potential (TRP) channels have been identified as membrane-bound sensory proteins in odontoblasts. However, the activation properties of these channels remain to be clarified. The purpose of this study was to investigate hypotonic stimulation–induced Ca2+ entry via TRP vanilloid subfamily member (TRPV) 1, TRPV2, and TRPV4 channels, which are sensitive to osmotic and mechanical stimuli, and their functional coupling with Na+-Ca2+ exchangers (NCXs) in mouse odontoblast lineage cells. Methods: We examined TRP channel activity by measuring intracellular-free Ca2+ concentration by using fura-2 fluorescence and ionic current recordings with whole-cell patch-clamp methods. Protein localization and messenger RNA expression were characterized using immunofluorescence and reverse-transcription polymerase chain reaction analyses. Results: Extracellular hypotonic solution–induced stretching of plasma membrane resulted in the activation of Ca2+ influx and inward currents. TRPV1, TRPV2, and TRPV4 channel antagonists inhibited the hypotonic stimulation–induced Ca2+ entry and currents. Their respective agonists activated Ca2+ entry. Although the increase in the intracellular free Ca2+ concentration decayed rapidly after the applications of these TRPV channel agonists, NCX inhibitors significantly prolonged the decay time constant. The messenger RNA expression of TRPV1, TRPV2, and TRPV4 channels; NCX isoforms 2 and 3; and dentin sialophosphoprotein were up-regulated after 24 hours of exposure to the hypotonic culture medium. Conclusions: These results indicate that stretching of the odontoblast membrane activates TRPV1-, TRPV2-, and TRPV4-mediated Ca2+ entry, and increased intracellular-free Ca2+ concentration is extruded via NCXs. These results suggest that odontoblasts can act as sensors that detect stimuli applied to exposed dentin and drive a number of cellular functions including dentinogenesis and/or sensory transduction. [Copyright &y& Elsevier]

Abstract: 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 Ca2+ signaling and expression of Ca2+-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 Ca2+-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 Ca2+-activated K+ channels. Conclusions: Human odontoblasts expressed voltage-dependent Na+ channels, bradykinin receptors, and Ca2+-activated K+ channels, which play an important role in driving cellular functions by channel-receptor signal interaction and membrane potential regulation. [Copyright &y& Elsevier]

Abstract: Odontoblasts are involved in the transduction of stimuli applied to exposed dentin. Although expression of thermo/mechano/osmo-sensitive transient receptor potential (TRP) channels has been demonstrated, the properties of TRP vanilloid 1 (TRPV1)-mediated signaling remain to be clarified. We investigated physiological and pharmacological properties of TRPV1 and its functional coupling with cannabinoid (CB) receptors and Na+–Ca2+ exchangers (NCXs) in odontoblasts. Anandamide (AEA), capsaicin (CAP), resiniferatoxin (RF) or low-pH evoked Ca2+ influx. This influx was inhibited by capsazepine (CPZ). Delay in time-to-activation of TRPV1 channels was observed between application of AEA or CAP and increase in [Ca2+] i . In the absence of extracellular Ca2+, however, an immediate increase in [Ca2+] i was observed on administration of extracellular Ca2+, followed by activation of TRPV1 channels. Intracellular application of CAP elicited inward current via opening of TRPV1 channels faster than extracellular application. With extracellular RF application, no time delay was observed in either increase in [Ca2+] i or inward current, indicating that agonist binding sites are located on both extra- and intracellular domains. KB-R7943, an NCX inhibitor, yielded an increase in the decay time constant during TRPV1-mediated Ca2+ entry. Increase in [Ca2+] i by CB receptor agonist, 2-arachidonylglycerol, was inhibited by CB1 receptor antagonist or CPZ, as well as by adenylyl cyclase inhibitor. These results showed that TRPV1-mediated Ca2+ entry functionally couples with CB1 receptor activation via cAMP signaling. Increased [Ca2+] i by TRPV1 activation was extruded by NCXs. Taken together, this suggests that cAMP-mediated CB1-TRPV1 crosstalk and TRPV1-NCX coupling play an important role in driving cellular functions following transduction of external stimuli to odontoblasts. [Copyright &y& Elsevier]

Abstract: Bradykinin (BK) is involved in bone resorption in chronic inflammatory diseases. During bone formation, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays an important role in the regulation of Ca2+. In osteoblasts, 1,25(OH)2D3 stimulates transmembrane influx of Ca2+ through voltage-sensitive Ca2+ channels (VSCCs). Voltage sensitive Ca2+ channels serve as crucial mediators of membrane excitability and many Ca2+-dependent functions, including bone growth, regulation of proliferation, enzyme activity and gene expression. The purpose of this study was to investigate the effects of BK and 1,25(OH)2D3 on VSCC currents carried by Ba2+ (I Ba). Application of 1,25(OH)2D3 facilitated I Ba in a voltage-dependent manner. Pretreatment with SQ22536 (an adenylate cyclase inhibitor) attenuated 1,25(OH)2D3-induced facilitation of I Ba. Bradykinin and BK1 receptor agonist [Lys-des-Arg9]-BK also facilitated I Ba. After 24h or 7days exposure to BK, that is, under chronic inflammatory conditions, application of 1,25(OH)2D3 inhibited I Ba. In addition, pretreatment with PD98,059, a mitogen-activated protein kinase (MAPK) tyrosine kinase inhibitor, attenuated 1,25(OH)2D3-induced inhibition of I Ba. These results indicate that, under normal conditions, 1,25(OH)2D3 acts with adenylate cyclase to facilitate VSCCs, whereas under chronic inflammatory conditions it acts with MAPK to inhibit VSCCs in pre-osteoblasts. [Copyright &y& Elsevier]

Abstract: Introduction: Intracellular Ca2+ is essential to many signal transduction pathways, and its level is tightly regulated by the Ca2+ extrusion system in the plasma membrane, which includes the Na+-Ca2+ 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. Ca2+ influx by reverse NCX activity was measured by fura-2 fluorescence. Ca2+ 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 Ca2+ influx by reverse NCX activity depended on extracellular Ca2+ concentration, and that this influx was blocked by NCX inhibitor KB-R7943 in a concentration-dependent manner. However, Ca2+ 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 Ca2+ extrusion system as well as in the directional Ca2+ transport pathway from the circulation to the dentin-mineralizing front. [Copyright &y& Elsevier]

Abstract: Dental pulp has various characteristics in the pulp chamber, but only a few biological evaluations about the effect of age on dental pulp tissue have been reported. The purpose of this study was to compare dental pulp from young and adult rats to characterize the homeostatic mechanism. Dental pulp cells (DPCs) were obtained from the first molar of rats, weighing 150 g each for the young group and 350 g each for the adult group. The expression of core-binding factor alpha-1 (Cbfa-1), vascular endothelial growth factor (VEGF), or heat shock protein (HSP) 27 messenger RNAs (mRNAs) by cultured pulp cells was determined by using a quantitative real-time PCR system after 3, 7, or 14 days. The expression of Cbfa-1 mRNA in the young group was higher than in the adult group. Expression of VEGF and HSP27 mRNAs in the adult group was higher than in the young group. The self-defense system in young DPCs is undertaken by calcification, but in adult DPCs it is carried out by the expression of self-defense proteins and the regeneration of vessels. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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 (P2X 3 ), transient receptor potential channel subfamilies, and Piezo1 channel significantly inhibited these inward currents. Mechanical stimulation to odontoblasts also generated action potentials in the isolectin B 4 –negative medium-sized neurons. Action potentials in these isolectin B 4 –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 P2X 3 receptor activation. The results also indicated that mechanosensitive TRP/Piezo1 channels were functionally expressed in odontoblasts. The activation of P2X 3 receptors induced an action potential in the Aδ neurons, underlying a sensory generation mechanism of dentinal pain. [ABSTRACT FROM AUTHOR]

Abstract: 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 Ca2+ channels (VDCCs) in hamster submandibular ganglion (SMG) neurons. In this study, we attempt to characterize the effect of NPY on VDCCs current using Ba2+ (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 Gαs-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 Gαi-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 Gαs-protein involving AC and PKA. On the other hand, NPY also inhibits N/P/Q-type VDCCs via Gαi-protein βγ subunits in the SMG neurons. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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 P2Y 12 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 P2Y 12 receptors in rat TG neurons. P2Y 12 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 P2Y 1, 12, 13 receptors) transiently increased intracellular free Ca 2+ concentrations ([Ca 2+ ] i ), an effect that was abolished by P2Y 12 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 P2Y 12 receptors are expressed in TG neurons, and act via a cAMP-dependent pathway to release intracellular Ca 2+ from ryanodine-sensitive Ca 2+ stores. [ABSTRACT FROM AUTHOR]

Abstract: 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-Gαs-protein and pretreatment of SQ22536 (an adenylate cyclase inhibitor). Conclusions: ADM and CGRP facilitates VDCCs mediated by Gαs-protein and adenylate cyclase in SMG. [ABSTRACT FROM AUTHOR]

Abstract: Calcitonin gene-related peptides (CGRP) and adrenomedullin (ADM) belong to the calcitonin family of peptides and are structurally related. Both peptides are found in the neurons of the CNS and play a role in many neuronal functions, including the control of blood pressure. The nucleus tractus solitarius (NTS) is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Recently, hypotension and bradycardia were observed after CGRP and ADM injection in the NTS. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+-dependent functions, such as neurotransmitter release, enzyme activity, and gene expression. The purpose of this study is to investigate the effects of CGRP and ADM on VDCC currents (I Ca) carried by Ba2+ (I Ba) in the NTS, using patch-clamp recording methods. Application of CGRP and ADM caused facilitation of I Ba in a concentration-dependent manner. Intracellular dialysis of the anti-Gαs-protein antibody attenuated CGRP-induced facilitation of I Ba. Intracellular dialysis of the anti-Gαi-protein antibody attenuated ADM-induced facilitation of I Ba. Pretreatment with SQ22536 (an adenylate cyclase inhibitor) and intracellular dialysis of PKI(5–24) (a protein kinase A inhibitor) attenuated CGRP-induced facilitation of I Ba. In contrast, pretreatment with PD98,059 (a mitogen-activated protein kinas inhibitor) attenuated ADM-induced facilitation of I Ba. Mainly L-type VDCCs were facilitated by both CGRP and ADM. These results indicate that CGRP facilitates L-type VDCCs via Gαs-protein involving adenylate cyclase and protein kinase A. In contrast, ADM facilitates L-type VDCCs via Gαi-protein involving mitogen-activated protein kinase in the NTS. [Copyright &y& Elsevier]

Abstract: 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 Ca2+ 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. [Copyright &y& Elsevier]

Abstract: 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 Ca2+-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 IBa. M617, GAL, and AR-M961 caused inhibition of IBa 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 IBa. Intracellular dialysis of the Gαi-protein antibody also attenuated the Gal-induced inhibition of IBa. These results indicate that GAL inhibits N- and P/Q-types VDCCs via Gαi-protein βγ subunits mediated by GalR1 in NTS. [Copyright &y& Elsevier]