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

A significant increase of lysophosphatidylinositol 4-phosphate with insulin in isolated rat fat cells

AbstractWe studied the effects of insulin on the incorporation of 32Pi into phospholipids in rat fat cells. When the cells were treated with insulin, a new radioactive phospholipid was detected on thin layer chromatography. The substance migrated slower than phosphatidylinositol 4,5-bisphosphate and was hardly detectable in the absence of insulin. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 120 μU/ml. Pretreatment of insulin with anti-insulin antibody or the cells with anti-insulin receptor anti-body inhibited the effect of insulin. The product of phosphatidylinositol 4-phosphate hydrolysed by phospholipase A2 was coincided with the substance on thin layer chromatography. Quinacrine inhibited the formation of the substance in a dose-dependent manner. These results suggested that insulin stimulates the generation of lysophosphatidylinositol 4-phosphate through the insulin-receptor interaction

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

Anti‐inflammatory potential of remimazolam: A laboratory and clinical investigation

Abstract Background Anesthetic agents, particularly intravenous anesthetics, may affect immune function and tumorigenic factors. We herein investigated whether the anti‐inflammatory effects of anesthetic agents are attributed to their antioxidant properties. The antioxidant and anti‐inflammatory effects of remimazolam, a new anesthetic, remain unclear. We hypothesized that remimazolam exerts anti‐inflammatory effects due to its antioxidant properties, which may affect the postoperative inflammatory response. This retrospective clinical study examined this hypothesis using laboratory and clinical approaches. Methods The antioxidant effects of remimazolam and dexmedetomidine were assessed by electron spin resonance (ESR) spectroscopy, and postoperative inflammatory responses were compared in 143 patients who underwent transcatheter aortic valve replacement at Kindai University Hospital between April 2021 and December 2022. The primary endpoint was the presence or absence of the antioxidant effects of the anesthetics themselves using ESR. Results Remimazolam at clinical concentrations exerted antioxidant effects, whereas dexmedetomidine did not. Increases in C‐reactive protein (CRP) levels on POD3 from preoperative values were significantly smaller in the remimazolam group than in the dexmedetomidine group (1.33 ± 1.29 vs. 2.17 ± 1.84, p = .014). Conclusions Remimazolam exerted stronger anti‐inflammatory effects than dexmedetomidine, and these effects were enhanced by its antioxidant properties, which may have affected postoperative CRP production

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

Oxidative Synthesis of Acid Blue 7 Dye Catalyzed by CuO/Silicotungstic Acid in Water-Phase

A catalytic oxidation reaction for Acid Blue 7 dye synthesis was evaluated in water. Without lead oxide or manganese oxide derivatives as oxidants, polyoxometalate catalysts were investigated to reduce the usage of harmful heavy metal. A catalyst was prepared by mixing silicotungstic acid with copper oxide, and aqueous hydrogen peroxide (30%) was used as an oxidizing agent. This reaction proceeded to produce Acid Blue 7 from the corresponding leuco acid after 45 min at 95 °C and was viable for a 10 g-scale synthesis

Thermal and membrane stretch sensitivity of TRPM8 and TRPA1 channels.

<p>(<b>A and C</b>) Representative traces of [Ca²⁺]_i increase in response to cool-stimulation from 35°C to 22±1°C or cold-stimulation from 26°C to 13±1°C, with or without 10 µM 5-BOT (<b>A</b>) or 100 µM HC030031 (<b>C</b>), in presence of extracellular Ca²⁺ (white boxes at bottom). Black bars indicate when cool- or cold-stimulation was applied, and white bars show application of 5-BOT (<b>A</b>) or HC030031 (<b>C</b>) to external solution. (<b>B</b>) Summary bar graphs of increase in [Ca²⁺]_i upon cool-stimulation with (gray column) or without (open columns) 10 µM 5-BOT. (<b>D</b>) Summary bar graphs of increase in [Ca²⁺]_i upon cold-stimulation with (gray column) or without (open columns) 100 µM HC030031. Each bar indicates mean ± SE of 5 (<b>B</b>) and 8 (<b>D</b>) experiments. Statistically significant differences between columns (shown by solid lines) are denoted by asterisks: *<i>P</i><0.05. (<b>E and G</b>) Example traces of transient [Ca²⁺]_i increase in response to 200 mOsm/L hypotonic solution with or without 10 µM 5-BOT (<b>E</b>) or 100 µM HC030031 (<b>G</b>) in presence of extracellular Ca²⁺ (white boxes at bottom). Bars denote when hypotonic solution (black) and 5-BOT (<b>E</b>) or HC030031 (<b>G</b>) (white) were added to external solution. (<b>F and H</b>) Summary bar graphs of increase in [Ca²⁺]_i upon addition of 200 mOsm/L hypotonic solution without (open columns in <b>F</b> and <b>H</b>) or with (gray column in <b>F </b><b>and </b><b>H</b>) 5-BOT or HC030031. Each bar indicates mean ± SE of several experiments (see text). Statistically significant differences between columns (shown by solid lines) are denoted by asterisks: *<i>P</i><0.05.</p

Localization and distribution of TRPM8 channels in odontoblasts.

<p>(<b>A–D</b>) Tall, columnar, and polarized secretory odontoblasts positive for TRPM8 channel immunoreactivity (brown and green) were observed at dental pulp section in the mandibular incisor (<b>A and B</b>) and molar (<b>C and D</b>) by immunohistochemical (<b>A and </b><b>C</b>) and immunofluorescence (<b>B </b><b>and D</b>) analysis. Immunoreactivity was also observed in mature odontoblasts and those obtained from incisors and molars of 16-week-old rats (not shown). TRPM8 channels were localized across entire cell membrane. Arrowheads indicate distal portion of membrane, and asterisks show cellular processes inside dentinal tubules of odontoblasts. Nuclei are shown in blue. Arrows: dentopulpal junction. Scale bars: 20 µm. No fluorescence was detected in negative controls (insets of <b>A–D</b>).</p