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.

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.

Sodium-calcium exchangers in rat trigeminal ganglion neurons.

BACKGROUND: Noxious stimulation and nerve injury induce an increase in intracellular Ca(2+) concentration ([Ca(2+)]i) via various receptors or ionic channels. While an increase in [Ca(2+)]i excites neurons, [Ca(2+)]i overload elicits cytotoxicity, resulting in cell death. Intracellular Ca(2+) is essential for many signal transduction mechanisms, and its level is precisely regulated by the Ca(2+) extrusion system in the plasma membrane, which includes the Na(+)-Ca(2+) exchanger (NCX). It has been demonstrated that Ca(2+)-ATPase is the primary mechanism for removing [Ca(2+)]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. RESULTS: NCX 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 [Ca(2+)]i in TG neurons was elevated beyond physiological levels. CONCLUSIONS: Our results suggest that NCXs in TG neurons play an important role in regulating Ca(2+)-homeostasis and somatosensory information processing by functionally coupling with voltage-dependent Na+ channels.

Hypotonic-induced stretching of plasma membrane activates transient receptor potential vanilloid channels and sodium-calcium exchangers in mouse odontoblasts.

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 Ca(2+) 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(+)-Ca(2+) exchangers (NCXs) in mouse odontoblast lineage cells. METHODS: We examined TRP channel activity by measuring intracellular-free Ca(2+) 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 Ca(2+) influx and inward currents. TRPV1, TRPV2, and TRPV4 channel antagonists inhibited the hypotonic stimulation-induced Ca(2+) entry and currents. Their respective agonists activated Ca(2+) entry. Although the increase in the intracellular free Ca(2+) 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 Ca(2+) entry, and increased intracellular-free Ca(2+) 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.

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.

Distribution of macrophages, osteoclasts and the B-lymphocyte lineage in osteolytic metastasis of mouse mammary carcinoma.

The purpose of this study was to examine the localization of macrophages, B-lymphocytes and osteoclasts in tumoral lesions of mammary carcinoma metastasized to bone of non-immunocompromised mice. Mouse mammary carcinoma cells (BALB/c-MC) were injected through the left cardiac ventricle into 5-week-old female wild-type Balb/c mice. The femora and tibiae of mice with metastasized cancer were extracted, and thereafter processed for histochemical analyses. The foci of metastasized tumor cells occupied the metaphyseal area, and the cell death zones could be identified within the tumor mass. Abundant tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were found among the alkaline phosphatase (ALP)-reactive osteoblastic cell layer that covered the bone surface neighboring the metastatic lesion. In contrast, F4/80-positive macrophages/monocytes were localized adjacent to, or invading the metastatic tissue. In addition, some F4/80-positive cells were found in the aforementioned cell death zones. Unlike F4/80-positive cells, CD45R-positive B-lymphocytes did not accumulate at the surfaces of the tumor lesions, nor infiltrate into them, but were found scattered over bone marrow. Interestingly, some CD45R-positive cells were observed close to TRAP-positive osteoclasts in the stromal tissue surrounding the tumor lesion. Our findings suggest that, in the bone metastatic lesions of non-immunocompromised mice, F4/80-positive macrophages/monocytes accumulated on and/or infiltrated into the tumor nests, while CD45R-positive B-lymphocytes were associated with osteoclasts, rather than attacking metastatic tumor cells.