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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.
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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 (HOâ¢) and superoxide anions (O2 â¢-). 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 HOâ¢-scavenging activities, with IC50 values of 0.029%, 0.019%, and 0.014%, respectively. Lidocaine and prilocaine showed concentration-dependent O2 â¢--scavenging activity, with IC50 values of 0.033% and 0.057%, respectively. However, articaine did not scavenge O2 â¢-. 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.
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The oral cavity serves as the initial segment of the digestive system and is responsible for both nutritional supplementation and the mechanical breakdown of food. It comprises distinct hard and soft tissues; the oral mucosa is subject to mechanical stress and interaction with microbiota. In oral cancer, tumors exhibit abnormal cellular networks and aberrant cell-cell interactions arising from complex interplays between environmental and genetic factors. This presents a challenge for clinicians and researchers, impeding the understanding of mechanisms driving oral cancer development and treatment strategies. Lesions with dysplastic features are categorized under oral potentially malignant disorders, including oral leukoplakia, erythroplakia, oral submucous fibrosis, and proliferative verrucous leukoplakia, carrying a high malignancy risk. In this review, we discuss oral cancer cell characteristics and the stiffness of the surrounding matrix. We also discuss the significance of stiffness equilibrium in oral potentially malignant disorders, particularly oral submucous fibrosis, possibly triggered by mechanical stress such as betel quid chewing.
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Carcinoma de Células Escamosas , Neoplasias de la Boca , Fibrosis de la Submucosa Bucal , Lesiones Precancerosas , Humanos , Fibrosis de la Submucosa Bucal/complicaciones , Fibrosis de la Submucosa Bucal/patología , Lesiones Precancerosas/complicaciones , Lesiones Precancerosas/patología , Leucoplasia Bucal/complicaciones , Leucoplasia Bucal/patología , Neoplasias de la Boca/etiología , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/patologíaRESUMEN
Introduction This prospective, randomized, double-blind, crossover trial aimed to investigate the effect of different types of local anesthetics on regional tissue blood oxygenation on the stellate ganglion block (SGB). Methods Twenty eligible patients were recruited for this study; 16 of whom were allocated to the study protocol. Participants were randomized into one of the two crossover sequences: 1% lidocaine hydrochloride or 1% mepivacaine hydrochloride, and received SGBs with 6 mL of any one of the local anesthetics, followed by a washout period of more than 24 hours, and then received SGBs by substituting the two local anesthetics. The tissue oxygenation index (TOI) in the skin near the mental foramen on the blocked side was recorded using near-infrared spectroscopy at 15 minutes after the local anesthetic injection. Results One participant did not complete the study. As such, variation in regional tissue oxygenation was compared between the 15 participants. There was no difference in the increase in regional tissue blood flow or tissue oxygenation after SGB between the patients who were given lidocaine and the ones who were given mepivacaine; however, the kinetics of the increase in regional tissue oxygenation were significantly faster with mepivacaine than with lidocaine. Conclusions Different types of local anesthetics do not affect the intensity of the increase in regional tissue oxygenation after SGB, but they do affect the kinetics of the increase. These differences in local anesthetics may affect how patients feel after receiving SGB, the duration of SGB effects, and the frequency of adverse events associated with SGB.
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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.
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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.
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Receptores de Péptido Intestinal Vasoactivo , Serotonina , Humanos , Serotonina/farmacología , Serotonina/metabolismo , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Odontoblastos , Línea Celular , Transducción de Señal , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Cannabinoides/metabolismoRESUMEN
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.
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Osteoblastos , Canales Catiónicos TRPV , Animales , Ratones , Canales Catiónicos TRPV/metabolismo , Canales Iónicos/metabolismoRESUMEN
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.
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Receptores de Péptido Relacionado con el Gen de Calcitonina , Ganglio del Trigémino , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/farmacología , Odontoblastos , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina/metabolismo , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina/farmacología , Neuronas/metabolismo , Transducción de Señal , AMP Cíclico/metabolismo , DentinaRESUMEN
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.
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Ganglio del Trigémino , Neuralgia del Trigémino , Comunicación Celular , Células Cultivadas , Humanos , Ganglio del Trigémino/patología , Neuralgia del Trigémino/etiología , Neuralgia del Trigémino/patologíaRESUMEN
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.
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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.
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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.
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Calcio/metabolismo , Dentina/enzimología , Odontoblastos/enzimología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Calcificación de Dientes , Animales , Línea Celular , Humanos , Ratas , Ratas WistarRESUMEN
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.
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Conexinas/genética , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Receptores Purinérgicos P2X7/genética , Ganglio del Trigémino/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Cultivo Primario de Células , Antagonistas del Receptor Purinérgico P2X/farmacología , Ratas , Ganglio del Trigémino/efectos de los fármacosRESUMEN
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.
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OBJECTIVES: The exact timings associated with swallowing are difficult to measure with the currently available methods. In this study, we aimed to elucidate the timings of action of the swallowing organs in the oral and pharyngeal stages of swallowing by recording the barometric pressure (BP) and tongue muscle activities. METHODS: Dry and water swallows were studied in 10 adults using electromyography and small barometers. BPs were recorded during swallowing in the oral cavity (BP-o) and pharynx (BP-p), associated with muscle activities of the anterior (TA) and posterior (TP) parts of the tongue and of the suprahyoid (SHy) muscle. To analyze the temporal pattern of each activity, times of onset, cessation, and peak were measured. RESULTS: Two characteristic waveforms were obtained. BP-o peaked immediately after onset and decreased to atmospheric pressure following a short plateau. However, BP-p gradually increased, reached a peak, and returned to the atmospheric pressure immediately before the end of BP-o. Since pressure increments indicated that the sensor was compressed in a closed space, onset and cessation of BP-p could correspond to the duration of nasopharyngeal closure. The onset of BP-p and the peak time of BP-o occurred in close succession. Thus, nasopharyngeal closure could be evaluated from BP-o. The sensor and EMG measured durations of oral and pharyngeal stages as 0.4 and 0.6 sec, respectively. TA activation began earlier than the TP. TA and TP peaks appeared before the BP-o peak, suggesting that the tongue begins the activity for swallowing before nasopharyngeal closure. CONCLUSIONS: This study revealed movements of swallowing organs in the two stages with high temporal resolution. BP-o detected the duration of nasopharyngeal closure.
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Deglución , Orofaringe , Presión Atmosférica , Electromiografía , Humanos , Boca , LenguaRESUMEN
Motor neuron diseases are a group of progressive neurological disorders that degenerate motor neurons. The neuroblastoma × spinal cord hybrid cell line NSC-34 is widely used as an experimental model in studies of motor neuron diseases. However, the differentiation efficiency of NSC-34 cells to neurons is not always sufficient. We have found that prostaglandin E2 (PGE2) induces morphological differentiation in NSC-34 cells. The present study investigated the functional properties of PGE2-differentiated NSC-34 cells. Retinoic acid (RA), a widely-used agent inducing cell differentiation, facilitated neuritogenesis, which peaked on day 7, whereas PGE2-induced neuritogenesis took only 2 days to reach the same level. Whole-cell patch-clamp recordings showed that the current threshold of PGE2-treated cell action potentials was lower than that of RA-treated cells. PGE2 and RA increased the protein expression levels of neuronal differentiation markers, microtubule-associated protein 2c and synaptophysin, and to the same extent, motor neuron-specific markers HB9 and Islet-1. On the other hand, protein levels of choline acetyltransferase and basal release of acetylcholine in PGE2-treated cells were higher than in RA-treated cells. These results suggest that PGE2 is a rapid and efficient differentiation-inducing factor for the preparation of functionally mature motor neurons from NSC-34 cells.
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Diferenciación Celular/efectos de los fármacos , Dinoprostona/farmacología , Neuronas Motoras/citología , Acetilcolina/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Biomarcadores/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Ratones , Neuronas Motoras/efectos de los fármacos , Proyección Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Tetrodotoxina/farmacología , Tretinoina/farmacologíaRESUMEN
The specification of cell identity depends on the exposure of cells to sequences of bioactive ligands. All-trans retinoic acid (ATRA) affects neuronal development in the early stage, and it is involved in neuronal lineage reprogramming. We previously established a fibroblast-like dedifferentiated fat cells (DFATs) derived from highly homogeneous mature adipocytes, which are more suitable for the study of cellular reprogramming. Canine cognitive dysfunction is similar to human cognitive dysfunction, suggesting that dogs could be a pathological and pharmacological model for human neuronal diseases. However, the effect of ATRA on neuronal reprogramming in dogs has remained unclear. Therefore, in this study, we investigated the effect of ATRA on the neuronal reprogramming of canine DFATs. ATRA induced the expression of neuronal marker mRNA/protein. The neuron-like cells showed Ca2+ influx with depolarization (50 mM KCl; 84.75 ± 4.05%) and Na+ channel activation (50 µM veratridine; 96.02 ± 2.02%). Optical imaging of presynaptic terminal activity and detection of neurotransmitter release showed that the neuron-like cells exhibited the GABAergic neuronal property. Genome-wide RNA-sequencing analysis shows that the transcriptome profile of canine DFATs is effectively reprogrammed towards that of cortical interneuron lineage. Collectively, ATRA can produce functional GABAergic cortical interneuron-like cells from canine DFATs, exhibiting neuronal function with > 80% efficiency. We further demonstrated the contribution of JNK3 to ATRA-induced neuronal reprogramming in canine DFATs. In conclusion, the neuron-like cells from canine DFATs could be a powerful tool for translational research in cell transplantation therapy, in vitro disease modeling, and drug screening for neuronal diseases.
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Desdiferenciación Celular/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Tretinoina/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Reprogramación Celular/efectos de los fármacos , Perros , Neurogénesis/genética , ARN Mensajero/genética , Sinapsis/efectos de los fármacos , Sinapsis/genéticaRESUMEN
Odontoblasts act as dentin formation and sensory receptors. Recently, it was reported that transient receptor potential ankyrin (TRPA) 1, TRP vanilloid (TRPV) 4 and pannexin 1 (PANX-1) play important roles in odontoblast sensory reception. However, it is not known when odontoblasts begin to possess a sense reception function. The aim of this study was to clarify the development of odontoblasts as sense receptors. Sections of mandibular first molars from postnatal day (PN) 0 to PN12 Wistar rats were prepared for hematoxylin-eosin staining. Immunohistochemically, we used anti-dentin sialoprotein (DSP), anti-TRPA1, anti-TRPV4, anti-PANX-1, and anti-neurofilament (NF) antibodies. In addition, we investigated TRPA1 and TRPV4 expression by reverse transcriptional quantitative polymerase chain reaction (RT-qPCR). At PN0, undifferentiated odontoblasts showed no immunoreaction to anti-DSP, anti-TRPA1, anti-TRPV4, or anti-PANX-1 antibodies. However, immunopositive reactions of these antibodies increased during odontoblast differentiation at PN3 and PN6. An immunopositive reaction of the anti-NF antibody appeared in the odontoblast neighborhood at PN12, when the odontoblasts began to form root dentin, and this appeared later than that of the other antibodies. By RT-qPCR, expression of TRPA1 at PN6 was significantly lower than that at PN0 (p < 0.05) and PN3 (p < 0.01). Expression of TRPV4 at PN6 was significantly lower than that at PN0 (p < 0.01) and PN3 (p < 0.01). The results of this study suggest that odontoblasts may acquire sensory receptor function after beginning to form root dentin, when TRPA1, TRPV4, PANX-1 channels, and nerve fibers are completely formed.
Asunto(s)
Diente Molar/crecimiento & desarrollo , Diente Molar/fisiología , Odontoblastos/metabolismo , Odontoblastos/fisiología , Células Receptoras Sensoriales , Canales de Potencial de Receptor Transitorio/fisiología , Animales , Inmunohistoquímica , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
Circadian rhythms regulate various physiological functions and are, therefore, essential for health. Light helps regulate the master and peripheral clocks. The secretion rates of saliva and electrolytes follow a circadian rhythm as well. However, the relationship between the molecular mechanism of saliva water secretion and the peripheral circadian rhythm in salivary glands is not yet clear. The transmembrane proteins aquaporin5 (Aqp5) and anoctamin1 (Ano1) are essential for water transport in the submandibular glands (SGs). The purpose of this study was to reveal the effect of light conditioning on the peripheral clock in SGs. We examined temporal expression patterns among clock genes, Aqp5 and Ano1, in rat SGs under light/dark (LD) and dark/dark (DD) conditions. We observed circadian rhythmic expression of Bmal1, Per2, Cry1, Aqp5, and Ano1 mRNAs under both LD and DD conditions. The expression levels of Aqp5 and Ano1 peaked 6 h earlier under the DD condition than under the LD condition. Maintenance of the circadian rhythm of Aqp5 and Ano1 expression even under the DD condition indicates that Aqp5 and Ano1 may be controlled by clock genes; such genes are called clock-controlled genes (CCGs). Western blot analysis revealed the circadian oscillation and peak shift of AQP5 and ANO1expression under DD conditions. Clock genes may regulate the rhythmic expression of Ano1 and Aqp5 and may control osmic gradients in SGs.
RESUMEN
Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons through synaptic contact. However, the detailed mechanisms for intercellular communication between MCs and neurons have yet to be clarified. The present study examined the increases in intracellular free Ca2+ concentration ([Ca2+]i) induced by direct mechanical stimulation of MCs. We also measured [Ca2+]i in the trigeminal ganglion neurons (TGs) following direct mechanical stimulation to the MCs in an MC-TGs coculture. The MCs were isolated from hamster buccal mucosa, while TGs were isolated from neonatal Wistar rats. Both cell populations showed depolarization-induced [Ca2+]i. Direct mechanical stimulation to MCs increased [Ca2+]i, showing stimulation strength dependence. In the MC-TGs coculture, the application of direct mechanical stimulation to MCs resulted in increased [Ca2+]i in the TGs. These changes were significantly suppressed by antagonists of glutamate-permeable anion channels (4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid; DIDS), and non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptors (MK801). Apyrase, an ATP-degrading enzyme, and suramin, a non-selective P2 purinergic receptor antagonist, did not exert inhibitory effects on these [Ca2+]i increases in the TGs following MC stimulation. These results indicated that MCs are capable of releasing glutamate, but not ATP, in response to cellular deformation by direct mechanical stimulation. The released glutamate activates the NMDA receptors on TGs. We suggest that MCs act as mechanoelectrical transducers and establish synaptic transmission with neurons, through the MC-neurite complex, to mediate mechanosensory transduction.