Maki Tsumura

TRPV1-mediated calcium signal couples with cannabinoid receptors and sodium–calcium exchangers in rat odontoblasts

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(+)-Ca(2+) exchangers (NCXs) in odontoblasts. Anandamide (AEA), capsaicin (CAP), resiniferatoxin (RF) or low-pH evoked Ca(2+) 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 [Ca(2+)](i). In the absence of extracellular Ca(2+), however, an immediate increase in [Ca(2+)](i) was observed on administration of extracellular Ca(2+), 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 [Ca(2+)](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 Ca(2+) entry. Increase in [Ca(2+)](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 Ca(2+) entry functionally couples with CB1 receptor activation via cAMP signaling. Increased [Ca(2+)](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.

Ca2+ extrusion via Na+-Ca2+ exchangers in rat odontoblasts.

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.

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 Ca2+ concentration ([Ca2+]i). Icilin-, WS3-, or WS12-induced [Ca2+]i increases were inhibited by capsazepine or 5-benzyloxytriptamine. The increase in [Ca2+]i elicited by allyl isothiocyanate (AITC) was inhibited by HC030031. WS12 and AITC exerted a desensitizing effect on [Ca2+]i increase. Low-temperature stimuli elicited [Ca2+]i increases that are sensitive to both 5-benzyloxytriptamine and HC030031. Hypotonic stimulation-induced membrane stretch increased [Ca2+]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.

Voltage-dependent Sodium Channels and Calcium-activated Potassium Channels in Human Odontoblasts In Vitro

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.

Sodium-Calcium Exchangers in Rat Trigeminal Ganglion Neurons

BackgroundNoxious stimulation and nerve injury induce an increase in intracellular Ca2+ concentration ([Ca2+]i) via various receptors or ionic channels. While an increase in [Ca2+]i excites neurons, [Ca2+]i overload elicits cytotoxicity, resulting in cell death. Intracellular Ca2+ is essential for many signal transduction mechanisms, and its level is precisely regulated by the Ca2+ extrusion system in the plasma membrane, which includes the Na+-Ca2+ exchanger (NCX). It has been demonstrated that Ca2+-ATPase is the primary mechanism for removing [Ca2+]i following excitatory activity in trigeminal ganglion (TG) neurons; however, the role of NCXs in this process has yet to be clarified. The goal of this study was to examine the expression/localization of NCXs in TG neurons and to evaluate their functional properties.ResultsNCX isoforms (NCX1, NCX2, and NCX3) were expressed in primary cultured rat TG neurons. All the NCX isoforms were also expressed in A-, peptidergic C-, and non-peptidergic C-neurons, and located not only in the somata, dendrites, axons and perinuclear region, but also in axons innervating the dental pulp. Reverse NCX activity was clearly observed in TG neurons. The inactivation kinetics of voltage-dependent Na+ channels were prolonged by NCX inhibitors when [Ca2+]i in TG neurons was elevated beyond physiological levels.ConclusionsOur results suggest that NCXs in TG neurons play an important role in regulating Ca2+-homeostasis and somatosensory information processing by functionally coupling with voltage-dependent Na+ channels.

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.

Calcitonin gene-related peptide- and adrenomedullin-induced facilitation of calcium current by different signal pathways in nucleus tractus solitarius

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 Ca(2+) 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 is to investigate the effects of CGRP and ADM on VDCC currents (I(Ca)) carried by Ba(2+) (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-Galpha(s)-protein antibody attenuated CGRP-induced facilitation of I(Ba). Intracellular dialysis of the anti-Galpha(i)-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 Galpha(s)-protein involving adenylate cyclase and protein kinase A. In contrast, ADM facilitates L-type VDCCs via Galpha(i)-protein involving mitogen-activated protein kinase in the NTS.

Calcitonin gene-related peptide- and adrenomedullin-induced facilitation of calcium current in submandibular ganglion

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.

Ca2+ Channels in Odontoblasts

Abstract 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.