Photoswitchable molecular glue for RNA: reversible photocontrol of structure and function of the ribozyme.

Single-stranded RNA folds into a variety of secondary and higher-order structures. Distributions and dynamics of multiple RNA conformations are responsible for the biological function of RNA. We here developed a photoswitchable molecular glue for RNA, which could reversibly control the association of two unpaired RNA regions in response to light stimuli. The photoswitchable molecular glue, NCTA, is an RNA-binding ligand possessing a photoisomerizable azobenzene moiety. Z-NCTA is an active ligand for the target RNA containing 5'-WGG-3'/5'-WGG-3' (W = U or A) site and stabilizes its hybridized state, while its isomer E-NCTA is not. Photoreversible isomerization of NCTA enabled control of the secondary and tertiary structure of the target RNA. The RNA-cleaving activity of hammerhead ribozyme, where appropriate RNA folding is necessary, could be reversibly regulated by photoirradiation in cells treated with NCTA, demonstrating precise photocontrol of RNA structure and function by the photoswitchable molecular glue.

Functional Expression of Mechanosensitive Piezo1/TRPV4 Channels in Mouse Osteoblasts.

Mechanical stress is an important regulatory factor in bone homeostasis. Mechanical stimulation of osteoblasts has been shown to elicit an increase in the concentration of intracellular free Ca2+ ([Ca2+]i). The pattern of functional expression of mechanosensitive ion channels remains unclear, however. Therefore, the purpose of this study was to investigate the pharmacological characteristics of [Ca2+]i in response to direct mechanical stimulation in osteoblasts. The morphological expression of mechanosensitive ion channels was also examined. Mouse osteoblast-like cells (MC3T3-E1 cells) were loaded with fura-2-acetoxymethyl ester, after which [Ca2+]i was measured. Increased levels of [Ca2+]i were observed in MC3T3-E1 cells in response to direct mechanical stimulation by means of a glass micropipette, but no desensitization. Application of a hypotonic solution also induced an increase in [Ca2+]i but was accompanied by a desensitizing effect. Extracellular Gd3+, GsMTx4, or RN-1734 reversibly inhibited this mechanical stimulation-induced increase in [Ca2+]i, whereas no inhibitory effect was observed with HC030031 or clemizole. When osteoblasts were stimulated with Yoda1, an increase was observed in [Ca2+]i together with a significant desensitizing effect. Immunoreactivity against Piezo1 and TRPV4 channel antibodies was detected in MC3T3-E1 cells. These results suggest that osteoblasts express Piezo1 and TRPV4 channels, which are involved in mechanosensitive processes during mechanical stress.

Direct Mechanical Stimulation Mediates Cell-to-Cell Interactions in Cultured Trigeminal Ganglion Cells.

Trigeminal neuralgia occurs in the orofacial region, characteristically causing pain that feels like a transient electric shock. Some histopathological studies have reported that trigeminal neuralgia is caused by mechanical compression of the demyelinated trigeminal nerve; the pathophysiological mechanism behind this phenomenon remains to be clarified, however. Cell-cell interactions have also been reported to be involved in the development and modulation of some types of neuropathic pain. The purpose of this study was to investigate the potential contribution of cell-cell interactions to trigeminal neuralgia by measuring intracellular free Ca2+ concentrations ([Ca2+]i) in primary cultured trigeminal ganglion (TG) cells. Direct mechanical stimulation of TG cells induced an increase in [Ca2+]i in both neuronal and non-neuronal cells, such as glial cells. Moreover, this increase was stimulus intensity-dependent and non-desensitizing. Direct mechanical stimulation increased [Ca2+]i in neighboring cells as well, and this increase was inhibited by application of carbamazepine. These results indicate that direct mechanical stimulation affects Ca2+ signaling. Trigeminal ganglion cells establish intercellular networks between themselves, suggesting that this is involved in the development and generation of trigeminal neuralgia.

Functional Coupling between the P2X7 Receptor and Pannexin-1 Channel in Rat Trigeminal Ganglion Neurons.

The ionotropic P2X receptor, P2X7, is believed to regulate and/or generate nociceptive pain, and pain in several neuropathological diseases. Although there is a known relationship between P2X7 receptor activity and pain sensing, its detailed functional properties in trigeminal ganglion (TG) neurons remains unclear. We examined the electrophysiological and pharmacological characteristics of the P2X7 receptor and its functional coupling with other P2X receptors and pannexin-1 (PANX1) channels in primary cultured rat TG neurons, using whole-cell patch-clamp recordings. Application of ATP and Bz-ATP induced long-lasting biphasic inward currents that were more sensitive to extracellular Bz-ATP than ATP, indicating that the current was carried by P2X7 receptors. While the biphasic current densities of the first and second components were increased by Bz-ATP in a concentration dependent manner; current duration was only affected in the second component. These currents were significantly inhibited by P2X7 receptor antagonists, while only the second component was inhibited by P2X1, 3, and 4 receptor antagonists, PANX1 channel inhibitors, and extracellular ATPase. Taken together, our data suggests that autocrine or paracrine signaling via the P2X7-PANX1-P2X receptor/channel complex may play important roles in several pain sensing pathways via long-lasting neuronal activity driven by extracellular high-concentration ATP following tissue damage in the orofacial area.

Paternal allelic mutation at the Kcnq1 locus reduces pancreatic ß-cell mass by epigenetic modification of Cdkn1c.

Genetic factors are important determinants of the onset and progression of diabetes mellitus. Numerous susceptibility genes for type 2 diabetes, including potassium voltage-gated channel, KQT-like subfamily Q, member1 (KCNQ1), have been identified in humans by genome-wide analyses and other studies. Experiments with genetically modified mice have also implicated various genes in the pathogenesis of diabetes. However, the possible effects of the parent of origin for diabetes susceptibility alleles on disease onset have remained unclear. Here, we show that a mutation at the Kcnq1 locus reduces pancreatic ß-cell mass in mice by epigenetic modulation only when it is inherited from the father. The noncoding RNA KCNQ1 overlapping transcript1 (Kcnq1ot1) is expressed from the Kcnq1 locus and regulates the expression of neighboring genes on the paternal allele. We found that disruption of Kcnq1 results in reduced Kcnq1ot1 expression as well as the increased expression of cyclin-dependent kinase inhibitor 1C (Cdkn1c), an imprinted gene that encodes a cell cycle inhibitor, only when the mutation is on the paternal allele. Furthermore, histone modification at the Cdkn1c promoter region in pancreatic islets was found to contribute to this phenomenon. Our observations suggest that the Kcnq1 genomic region directly regulates pancreatic ß-cell mass and that genomic imprinting may be a determinant of the onset of diabetes mellitus.

Restoration of Ribozyme Tertiary Contact and Function by Using a Molecular Glue for RNA.

Some RNA classes require folding into the proper higher-order structures to exert their functions. Hammerhead ribozyme (HHR) requires a folding conformation stabilized by tertiary interaction for full activity. A rationally engineered HHR was developed that was inactive, but could be activated by a synthetic RNA-binding ligand, naphthyridine carbamate tetramer with Z-stilbene linker (Z-NCTS). Binding of Z-NCTS could induce the formation of an active folding structure and thereby restore ribozyme activity, where Z-NCTS acts as a molecular glue to bring two isolated RNA loops into contact with each other. Next, we designed a Z-NCTS-responsive genetic switch using the HHR sequence inserted into the 3' untranslated region as a cis-acting element. We demonstrated that the rationally designed ribozyme switch enabled regulation of gene expression by Z-NCTS and was functional in mammalian cells.

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.

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.

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.

Upregulation of Amy1 in the salivary glands of mice exposed to a lunar gravity environment using the multiple artificial gravity research system.

Introduction: Space is a unique environment characterized by isolation from community life and exposure to circadian misalignment, microgravity, and space radiation. These multiple differences from those experienced on the earth may cause systemic and local tissue stress. Autonomic nerves, including sympathetic and parasympathetic nerves, regulate functions in multiple organs. Saliva is secreted from the salivary gland, which is regulated by autonomic nerves, and plays several important roles in the oral cavity and digestive processes. The balance of the autonomic nervous system in the seromucous glands, such as the submandibular glands, precisely controls serous and mucous saliva. Psychological stress, radiation damage, and other triggers can cause an imbalance in salivary secretion systems. A previous study reported that amylase is a stress marker in behavioral medicine and space flight crews; however, the detailed mechanisms underlying amylase regulation in the space environment are still unknown. Methods: In this study, we aimed to elucidate how lunar gravity (1/6 g) changes mRNA expression patterns in the salivary gland. Using a multiple artificial gravity research system during space flight in the International Space Station, we studied the effects of two different gravitational levels, lunar and Earth gravity, on the submandibular glands of mice. All mice survived, returned to Earth from space, and their submandibular glands were collected 2 days after landing. Results: We found that lunar gravity induced the expression of the salivary amylase gene Amy1; however, no increase in Aqp5 and Ano1, which regulate water secretion, was observed. In addition, genes involved in the exocrine system, such as vesicle-associated membrane protein 8 (Vamp8) and small G proteins, including Rap1 and Rab families, were upregulated under lunar gravity. Conclusion: These results imply that lunar gravity upregulates salivary amylase secretion via Rap/Rab signaling and exocytosis via Vamp8. Our study highlights Amy1 as a potential candidate marker for stress regulation in salivary glands in the lunar gravity environment.

Effect of Dental Local Anesthetics on Reactive Oxygen Species: An In Vitro Study.

Introduction Oxidative stress, an imbalance between reactive oxygen species (ROS) production and antioxidant defenses, plays an important role in various dental diseases. Local anesthetics are frequently used in dentistry. The potential antioxidant activity of dental local anesthetics can contribute to dental practice. Therefore, this study aimed to investigate the ROS-scavenging activities of three commonly used dental local anesthetics, lidocaine, prilocaine, and articaine, focusing on their effects on hydroxyl radicals (HO•) and superoxide anions (O2 •-). Materials and methods The electron spin resonance (ESR) spin-trapping technique was employed to specifically measure the ROS-scavenging activities of these local anesthetics at varying concentrations. Results Lidocaine, prilocaine, and articaine exhibited concentration-dependent HO•-scavenging activities, with IC50 values of 0.029%, 0.019%, and 0.014%, respectively. Lidocaine and prilocaine showed concentration-dependent O2 •--scavenging activity, with IC50 values of 0.033% and 0.057%, respectively. However, articaine did not scavenge O2 •-. Conclusions The proactive use of dental local anesthetics may mitigate oxidative injury and inflammatory damage through direct ROS scavenging. However, further research is needed to elucidate the specific mechanisms underlying the antioxidant effects of these dental local anesthetics and their potential impact on the dental diseases associated with oxidative stress.

A synthetic riboswitch that operates using a rationally designed ligand-RNA pair.

The construction of an artificial riboswitch is based on a ligand-RNA pair without any molecular biology-based selection processes. The ligand selectively and significantly stabilized an RNA duplex containing an r(XGG)/r(XGG) sequence (X=U, A, G). The integration of the ligand-binding sequences into the 5'-untranslated region of mRNA provided an artificial riboswitch that was responsive to Z-NCTS.

Functional Expression of IP, 5-HT4, D1, A2A, and VIP Receptors in Human Odontoblast Cell Line.

Odontoblasts are involved in sensory generation as sensory receptor cells and in dentin formation. We previously reported that an increase in intracellular cAMP levels by cannabinoid 1 receptor activation induces Ca2+ influx via transient receptor potential vanilloid subfamily member 1 channels in odontoblasts, indicating that intracellular cAMP/Ca2+ signal coupling is involved in dentinal pain generation and reactionary dentin formation. Here, intracellular cAMP dynamics in cultured human odontoblasts were investigated to understand the detailed expression patterns of the intracellular cAMP signaling pathway activated by the Gs protein-coupled receptor and to clarify its role in cellular functions. The presence of plasma membrane Gαs as well as prostaglandin I2 (IP), 5-hydroxytryptamine 5-HT4 (5-HT4), dopamine D1 (D1), adenosine A2A (A2A), and vasoactive intestinal polypeptide (VIP) receptor immunoreactivity was observed in human odontoblasts. In the presence of extracellular Ca2+, the application of agonists for the IP (beraprost), 5-HT4 (BIMU8), D1 (SKF83959), A2A (PSB0777), and VIP (VIP) receptors increased intracellular cAMP levels. This increase in cAMP levels was inhibited by the application of the adenylyl cyclase (AC) inhibitor SQ22536 and each receptor antagonist, dose-dependently. These results suggested that odontoblasts express Gs protein-coupled IP, 5-HT4, D1, A2A, and VIP receptors. In addition, activation of these receptors increased intracellular cAMP levels by activating AC in odontoblasts.

Intracellular cAMP Signaling Pathway via Gs Protein-Coupled Receptor Activation in Rat Primary Cultured Trigeminal Ganglion Cells.

G protein-coupled receptors in trigeminal ganglion (TG) neurons are often associated with sensory mechanisms, including nociception. We have previously reported the expression of P2Y12 receptors, which are Gi protein-coupled receptors, in TG cells. Activating P2Y12 receptors decreased the intracellular free Ca2+ concentration ([Ca2+]i). This indicated that intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels can mediate Ca2+ signaling in TG cells. Here, we report more extensive-expression patterns of Gs protein-coupled receptors in primary cultured TG neurons isolated from 7-day-old newborn Wistar rats and further examine the roles of these receptors in cAMP signaling using the BacMam sensor in these neurons. To identify TG neurons, we also measured [Ca2+]i using fura-2 in TG cells and measured intracellular cAMP levels. TG neurons were positive for Gαs protein-coupled receptors, beta-2 adrenergic (ß2), calcitonin gene-related peptide (CGRP), adenosine A2A (A2A), dopamine 1 (D1), prostaglandin I2 (IP), and 5-hydroxytriptamine 4 (5-HT4) receptor. Application of forskolin (FSK), an activator of adenylyl cyclase, transiently increased intracellular cAMP levels in TG neurons. The application of a phosphodiesterase inhibitor augmented the FSK-elicited intracellular cAMP level increase. These increases were significantly suppressed by the application of SQ22536, an adenylyl cyclase inhibitor, in TG neurons. Application of agonists for ß2, CGRP, A2A, D1-like, IP, and 5-HT4 receptors increased intracellular cAMP levels. These increases were SQ22536-sensitive. These results suggested that TG neurons express ß2, CGRP, A2A, D1, IP, and 5-HT4 receptors, and the activations of these Gαs protein-coupled receptors increase intracellular cAMP levels by activating adenylyl cyclase.

A small molecule regulates hairpin structures in d(CGG) trinucleotide repeats.

Unusual expansion of trinucleotide repeats has been identified as a common mechanism of hereditary neurodegenerative diseases. Although the actual mechanism of repeat expansion remains uncertain, trinucleotide repeat instability may be related to the increased stability of an alternative DNA hairpin structure formed in the repeat sequences. Here we report that a synthetic ligand naphthyridine carbamate dimer (NCD) selectively bound to and stabilized an intra-stranded hairpin structure in CGG repeat sequences. The NCD-CGG hairpin complex was a stable structure that efficiently interfered with DNA replication by Taq DNA polymerase. Considering the sequence preference of NCD, the use of NCD would be valuable to investigate the genetic instabilities of CGG/CCG repeat sequences in human genomes.

Gαs-Coupled CGRP Receptor Signaling Axis from the Trigeminal Ganglion Neuron to Odontoblast Negatively Regulates Dentin Mineralization.

An inflammatory response following dental pulp injury and/or infection often leads to neurogenic inflammation via the axon reflex. However, the detailed mechanism underlying the occurrence of the axon reflex in the dental pulp remains unclear. We sought to examine the intracellular cyclic adenosine monophosphate (cAMP) signaling pathway in odontoblasts via the activation of Gs protein-coupled receptors and intercellular trigeminal ganglion (TG) neuron-odontoblast communication following direct mechanical stimulation of TG neurons. Odontoblasts express heterotrimeric G-protein α-subunit Gαs and calcitonin receptor-like receptors. The application of an adenylyl cyclase (AC) activator and a calcitonin gene-related peptide (CGRP) receptor agonist increased the intracellular cAMP levels ([cAMP]i) in odontoblasts, which were significantly inhibited by the selective CGRP receptor antagonist and AC inhibitor. Mechanical stimulation of the small-sized CGRP-positive but neurofilament heavy chain-negative TG neurons increased [cAMP]i in odontoblasts localized near the stimulated neuron. This increase was inhibited by the CGRP receptor antagonist. In the mineralization assay, CGRP impaired the mineralization ability of the odontoblasts, which was reversed by treatment with a CGRP receptor antagonist and AC inhibitor. CGRP establishes an axon reflex in the dental pulp via intercellular communication between TG neurons and odontoblasts. Overall, CGRP and cAMP signaling negatively regulate dentinogenesis as defensive mechanisms.

Piezo1-pannexin-1-P2X3 axis in odontoblasts and neurons mediates sensory transduction in dentinal sensitivity.

According to the "hydrodynamic theory," dentinal pain or sensitivity is caused by dentinal fluid movement following the application of various stimuli to the dentin surface. Recent convergent evidence in Vitro has shown that plasma membrane deformation, mimicking dentinal fluid movement, activates mechanosensitive transient receptor potential (TRP)/Piezo channels in odontoblasts, with the Ca2+ signal eliciting the release of ATP from pannexin-1 (PANX-1). The released ATP activates the P2X3 receptor, which generates and propagates action potentials in the intradental Aδ afferent neurons. Thus, odontoblasts act as sensory receptor cells, and odontoblast-neuron signal communication established by the TRP/Piezo channel-PANX-1-P2X3 receptor complex may describe the mechanism of the sensory transduction sequence for dentinal sensitivity. To determine whether odontoblast-neuron communication and odontoblasts acting as sensory receptors are essential for generating dentinal pain, we evaluated nociceptive scores by analyzing behaviors evoked by dentinal sensitivity in conscious Wistar rats and Cre-mediated transgenic mouse models. In the dentin-exposed group, treatment with a bonding agent on the dentin surface, as well as systemic administration of A-317491 (P2X3 receptor antagonist), mefloquine and 10PANX (non-selective and selective PANX-1 antagonists), GsMTx-4 (selective Piezo1 channel antagonist), and HC-030031 (selective TRPA1 channel antagonist), but not HC-070 (selective TRPC5 channel antagonist), significantly reduced nociceptive scores following cold water (0.1 ml) stimulation of the exposed dentin surface of the incisors compared to the scores of rats without local or systemic treatment. When we applied cold water stimulation to the exposed dentin surface of the lower first molar, nociceptive scores in the rats with systemic administration of A-317491, 10PANX, and GsMTx-4 were significantly reduced compared to those in the rats without systemic treatment. Dentin-exposed mice, with somatic odontoblast-specific depletion, also showed significant reduction in the nociceptive scores compared to those of Cre-mediated transgenic mice, which did not show any type of cell deletion, including odontoblasts. In the odontoblast-eliminated mice, P2X3 receptor-positive A-neurons were morphologically intact. These results indicate that neurotransmission between odontoblasts and neurons mediated by the Piezo1/TRPA1-pannexin-1-P2X3 receptor axis is necessary for the development of dentinal pain. In addition, odontoblasts are necessary for sensory transduction to generate dentinal sensitivity as mechanosensory receptor cells.

Large-Conductance Calcium-Activated Potassium Channels and Voltage-Dependent Sodium Channels in Human Cementoblasts.

Cementum, which is excreted by cementoblasts, provides an attachment site for collagen fibers that connect to the alveolar bone and fix the teeth into the alveolar sockets. Transmembrane ionic signaling, associated with ionic transporters, regulate various physiological processes in a wide variety of cells. However, the properties of the signals generated by plasma membrane ionic channels in cementoblasts have not yet been described in detail. We investigated the biophysical and pharmacological properties of ion channels expressed in human cementoblast (HCEM) cell lines by measuring ionic currents using conventional whole-cell patch-clamp recording. The application of depolarizing voltage steps in 10 mV increments from a holding potential (Vh) of -70 mV evoked outwardly rectifying currents at positive potentials. When intracellular K+ was substituted with an equimolar concentration of Cs+, the outward currents almost disappeared. Using tail current analysis, the contributions of both K+ and background Na+ permeabilities were estimated for the outward currents. Extracellular application of tetraethylammonium chloride (TEA) and iberiotoxin (IbTX) reduced the densities of the outward currents significantly and reversibly, whereas apamin and TRAM-34 had no effect. When the Vh was changed to -100 mV, we observed voltage-dependent inward currents in 30% of the recorded cells. These results suggest that HCEM express TEA- and IbTX-sensitive large-conductance Ca2+-activated K+ channels and voltage-dependent Na+ channels.

Plasma Membrane Ca2+-ATPase in Rat and Human Odontoblasts Mediates Dentin Mineralization.

Intracellular Ca2+ signaling engendered by Ca2+ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca2+ is exported by the Na+-Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA) to maintain Ca2+ homeostasis. We previously demonstrated a functional coupling between Ca2+ extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca2+ concentration ([Ca2+]i). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca2+]i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1-4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca2+]i in odontoblasts in both rat and human odontoblasts. The Ca2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca2+]i increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4-8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca2+]i homeostasis in odontoblasts by Ca2+ extrusion following [Ca2+]i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca2+ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.

Mechanical Stimulation-Induced Calcium Signaling by Piezo1 Channel Activation in Human Odontoblast Reduces Dentin Mineralization.

Odontoblasts play critical roles in dentin formation and sensory transduction following stimuli on the dentin surface. Exogenous stimuli to the dentin surface elicit dentinal sensitivity through the movement of fluids in dentinal tubules, resulting in cellular deformation. Recently, Piezo1 channels have been implicated in mechanosensitive processes, as well as Ca2+ signals in odontoblasts. However, in human odontoblasts, the cellular responses induced by mechanical stimulation, Piezo1 channel expression, and its pharmacological properties remain unclear. In the present study, we examined functional expression of the Piezo1 channel by recording direct mechanical stimulation-induced Ca2+ signaling in dentin matrix protein 1 (DMP-1)-, nestin-, and dentin sialophosphoprotein (DSPP)-immunopositive human odontoblasts. Mechanical stimulation of human odontoblasts transiently increased intracellular free calcium concentration ([Ca2+]i). Application of repeated mechanical stimulation to human odontoblasts resulted in repeated transient [Ca2+]i increases, but did not show any desensitizing effects on [Ca2+]i increases. We also observed a transient [Ca2+]i increase in the neighboring odontoblasts to the stimulated cells during mechanical stimulation, showing a decrease in [Ca2+]i with an increasing distance from the mechanically stimulated cells. Application of Yoda1 transiently increased [Ca2+]i. This increase was inhibited by application of Gd3+ and Dooku1, respectively. Mechanical stimulation-induced [Ca2+]i increase was also inhibited by application of Gd3+ or Dooku1. When Piezo1 channels in human odontoblasts were knocked down by gene silencing with short hairpin RNA (shRNA), mechanical stimulation-induced [Ca2+]i responses were almost completely abolished. Piezo1 channel knockdown attenuated the number of Piezo1-immunopositive cells in the immunofluorescence analysis, while no effects were observed in Piezo2-immunopositive cells. Alizarin red staining distinctly showed that pharmacological activation of Piezo1 channels by Yoda1 significantly suppressed mineralization, and shRNA-mediated knockdown of Piezo1 also significantly enhanced mineralization. These results suggest that mechanical stimulation predominantly activates intracellular Ca2+ signaling via Piezo1 channel opening, rather than Piezo2 channels, and the Ca2+ signal establishes intercellular odontoblast-odontoblast communication. In addition, Piezo1 channel activation participates in the reduction of dentinogenesis. Thus, the intracellular Ca2+ signaling pathway mediated by Piezo1 channels could contribute to cellular function in human odontoblasts in two ways: (1) generating dentinal sensitivity and (2) suppressing physiological/reactional dentinogenesis, following cellular deformation induced by hydrodynamic forces inside dentinal tubules.