Yoshiyuki Shibukawa

Odontoblast TRP Channels and Thermo/Mechanical Transmission

Odontoblasts function as mechanosensory receptors because of the expression of mechanosensitive channels in these cells. However, it is unclear if odontoblasts direct the signal transmission evoked by heat/cold or osmotic changes. This study investigated the effects of heat/cold or osmotic changes on calcium signaling and the functional expression of the thermo/mechanosensitive transient receptor potential (TRP) channels in primary cultured mouse odontoblastic cells, with the use of RT-PCR, fluorometric calcium imaging, and electrophysiology. TRPV1, TRPV2, TRPV3, TRPV4, and TRPM3 mRNA was expressed, but TRPM8 and TRPA1 mRNA was not. The receptor-specific stimulation of TRPV1-3 (heat-sensing receptors) and TRPV4/ TRPM3 (mechanic receptors) caused increases in the intracellular calcium concentration. Moreover, the channel activities of TRPV1-4 and TRPM3 were confirmed by a whole-cell patch-clamp technique. These results suggest that primary cultured mouse odontoblasts express heat/mechanosensitive TRP channels and play a role in the underlying mechanisms of thermo/mechanosensitive sensory transmission.

Substitution of a Single Residue, Asp575, Renders the NCKX2 K+-dependent Na+/Ca2+ Exchanger Independent of K+

The Na+/Ca2+-K+ exchanger (NCKX) is a polytopic membrane protein that uses both the inward Na+ gradient and the outward K+ gradient to drive Ca2+ extrusion across the plasma membrane. NCKX1 is found in retinal rod photoreceptors, while NCKX2 is found in retinal cone photoreceptors and is also widely expressed in the brain. Here, we have identified a single residue (out of >100 tested) for which substitution removed the K+ dependence of NCKX-mediated Ca2+ transport. Charge-removing replacement of Asp575 by either asparagine or cysteine rendered the mutant NCKX2 proteins independent of K+, whereas the charge-conservative substitution of Asp575 to glutamate resulted in a nonfunctional mutant NCKX2 protein, accentuating the critical nature of this residue. Asp575 is conserved in the NCKX1–5 genes, while an asparagine is found in this position in the three NCX genes, coding for the K+-independent Na+/Ca2+ exchanger.

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.

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 ([Ca2+]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 [Ca2+]i were observed not only in mechanically stimulated odontoblasts, but also in neighboring odontoblasts and TG neurons. These increases in [Ca2+]i were abolished in the absence of extracellular Ca2+ and in the presence of mechanosensitive TRP channel antagonists. A pannexin-1 (ATP-permeable channel) inhibitor and ATP-degrading enzyme abolished the increases in [Ca2+]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 [Ca2+]i. An ionotropic ATP (P2X3) receptor antagonist inhibited the increase in [Ca2+]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 [Ca2+]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.

Ca2+ Signaling Mediated by IP3-Dependent Ca2+ Releasing and Store-Operated Ca2+ Channels in Rat Odontoblasts†

In the phospholipase‐C (PLC) signaling system, Ca2+ is mobilized from intracellular Ca2+ stores by an action of inositol 1,4,5‐trisphosphate (IP3). The depletion of IP3‐sensitive Ca2+ stores activates a store‐operated Ca2+ entry (SOCE). However, no direct evidence has been obtained about these signaling pathways in odontoblasts. In this study, we investigate the characteristics of the SOCE and IP3‐mediated Ca2+ mobilizations in rat odontoblasts using fura‐2 microfluorometry and a nystatin‐perforated patch‐clamp technique. In the absence of extracellular Ca2+ ([Ca2+]o), thapsigargin (TG) evoked a transient rise in intracellular Ca2+ concentration ([Ca2+]i). After TG treatment to deplete the store, the subsequent application of Ca2+ resulted in a rapid rise in [Ca2+]i caused by SOCE. In the absence of TG treatment, no SOCE was evoked. The Ca2+ influx was dependent on [Ca2+]o (KD = 1.29 mM) and was blocked by an IP3 receptor inhibitor, 2‐aminoethoxydiphenyl borate (2‐APB), as well as La3+ in a concentration‐dependent manner (IC50 = 26 μM). In TG‐treated cells, an elevation of [Ca2+]o from 0 to 2.5 mM elicited an inwardly rectifying current at hyperpolarizing potentials with a positive reversal potential. The currents were selective for Ca2+ over the other divalent cations (Ca2+ > Ba2+ > Sr2+ ≫ Mn2+). In the absence of [Ca2+]o, carbachol, bradykinin, and 2‐methylthioadenosine 5′triphosphate activated Ca2+ release from the store; these were inhibited by 2‐APB. These results indicate that odontoblasts possessed Ca2+ signaling pathways through the activation of store‐operated Ca2+ channels by the depletion of intracellular Ca2+ stores and through the IP3‐induced Ca2+ release activated by PLC‐coupled receptors.

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.

Cortical Representation Area of Human Dental Pulp

To elucidate the dental pulp-representing area in the human primary somatosensory cortex and the presence of A-beta fibers in dental pulp, we recorded somatosensory-evoked magnetic fields from the cortex in seven healthy persons using magnetoencephalography. Following non-painful electrical stimulation of the right maxillary first premolar dental pulp, short latency (27 ms) cortical responses on the magnetic waveforms were observed. However, no response was seen when stimulation was applied to pulpless teeth, such as devitalized teeth. The current source generating the early component of the magnetic fields was located anterior-inferiorly compared with the locations for the hand area in the primary somatosensory cortex. These results demonstrate the dental pulp representation area in the primary somatosensory cortex, and that it receives input from intradental A-beta neurons, providing a detailed organizational map of the orofacial area, by adding dental pulp to the classic “sensory homunculus”.

Magnetoencephalography study of right parietal lobe dysfunction of the evoked mirror neuron system in antipsychotic-free schizophrenia.

Introduction Patients with schizophrenia commonly exhibit deficits of non-verbal communication in social contexts, which may be related to cognitive dysfunction that impairs recognition of biological motion. Although perception of biological motion is known to be mediated by the mirror neuron system, there have been few empirical studies of this system in patients with schizophrenia. Methods Using magnetoencephalography, we examined whether antipsychotic-free schizophrenia patients displayed mirror neuron system dysfunction during observation of biological motion (jaw movement of another individual). Results Compared with normal controls, the patients with schizophrenia had fewer components of both the waveform and equivalent current dipole, suggesting aberrant brain activity resulting from dysfunction of the right inferior parietal cortex. They also lacked the changes of alpha band and gamma band oscillation seen in normal controls, and had weaker phase-locking factors and gamma-synchronization predominantly in right parietal cortex. Conclusions Our findings demonstrate that untreated patients with schizophrenia exhibit aberrant mirror neuron system function based on the right inferior parietal cortex, which is characterized by dysfunction of gamma-synchronization in the right parietal lobe during observation of biological motion.

Cerebral cortical dysfunction in patients with temporomandibular disorders in association with jaw movement observation

Abstract Temporomandibular disorders (TMD) represent a group of chronic painful conditions in the masticatory musculature and temporomandibular joint. To examine possible changes in cortical machinery in TMD patients, we compared neuromagnetic signals evoked by cortical neurons between healthy subjects and TMD patients while they were carefully observing the video frames of jaw‐opening movements performed by another person. During the movement observation task in the healthy subjects, we found cortical activation in the following sequence with left hemisphere dominance: (1) the occipitotemporal region near the inferior temporal sulcus (human homologue of MT/V5 in monkeys), (2) the inferior parietal cortex (IPC), and (3) the anterior part of the inferior‐lateral precentral gyrus (PrCG). In the TMD patients, however, we found deficit or marked attenuation of the neuromagnetic responses in the PrCG and IPC, while the activity of the MT/V5 showed no differences from that in the healthy subjects. In addition, we could not find any differences in cortical magnetic responses between healthy subjects and TMD patients when they were observing palm‐opening movements, indicating that cortical dysfunction associated with jaw‐movement observation is specific phenomena in the patients of TMD. Thus the present study provides new neuropathological evidence that TMD patients exhibit dysfunction of recognition mechanisms in cerebral cortex during motor observation, and suggests that disturbance of cortical functions regulating visuomotor integration would play a crucial role in development as well as aggravation of TMD.