Cellular mechanisms of dentin sensation by odontoblast as senory receptor cell(expression of TRP and voltage-dependent K^+ channels, and analysis by odontoblast-neuron co-culture system)

成牙本质细胞作为感觉受体细胞的牙本质感觉的细胞机制（TRP和电压依赖性K^通道的表达，以及成牙本质细胞-神经元共培养系统的分析）

• 批准号: 18592050
• 负责人: SHIBUKAWA Yoshiyuki
• 金额: $ 2.36万
• 依托单位: Tokyo Dental College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18592050/
• 关键词: Dentistry; Signal Transduction; Neuroscience; Oral Physiology; Dental Pulp; Odontoblast; TRP(transient receptor notential)Channels; Na^+; Ca^<2+> exchanger(NCX); TRP(transient recetor potential; Ca^<2+> exchanger (NCX); transient receptor poten; Dentistry; Signal Transduction; Neuroscience; Oral Physiology; Dental Pulp; Odontoblast; TRP(transient receptor notential)Channels; Na^+; Ca^<2+> exchanger(NCX); TRP(transient recetor potential; Ca^<2+> exchanger (NCX); transient receptor poten

This study aimed to clarify the role of odontoblasts in cellular mechanisms of dentin sensation. During the research period from April 2006 to March 2008, we investigated expression of TRPV1 (vanilloid receptor 1; VR1) channel (a transient receptor potential (TRP) family subtype, which contributes essentially for the detection of pain sensation), voltage-dependent K+ channels (Kv channles; which involve in the generation of action potential), and Na^+-Ca^<2+> exchangers (NCX; which regulate ntracellular Ca^<2+> concentartion ([Ca^<2+>]_i) by extrusion of [Ca^<2+>]_i)in rat odontoblasts by immunohistochemical, intracellular Ca^<2+> concentartion measurement and whole-cell patch-clamp technique.1. TRPV1 channels: Immunohistochemical experiments showed localization of TRPV1 on the distal regions of odontoblast membranes. RT-PCR analysis showed that odontoblasts express TRPV subfamily 1, 2, 3. In the fura-2 fluorescence analysis to measure[Ca^<2+>]I, anandamide (AEA; 10 pM; endogenous TRPV … More 1 activator) evoked a transient rise in [Ca^<2+>], in the presence of extracellular Ca^<2+> with slight delay in activation of transient rise after AEA application. However, in the absence of extracellular Ca^<2+>, we could not observe any transient rise in [Ca^<2+>], indicating that AEA activates Ca^<2+> influx. The Ca^<2+> influx was blocked by a TRPV1 channel antagonist, capsazepine. In our previous study, that extracellular application of capsaicin activated inward currents in rat odontoblast (Okumura, et. Al., 2006), but their current amplitude was small (ca, 10 pA). In addition, in the present experiments, rise in [Ca^<2+>], after AEA application accompanied with delay time (ca, 2-3 min)in activation. It has been reported that capsaicin as well as AEA binds intracellular domain of TRPV1 channels. Therefore, we examine whether or not intracellular application of capsaicin activate inward currents in odontoblasts. Under the continuous voltage-clamp condition with holding potential of 0 mV, an intracellular application of 10 μM capsaicin via patch-pipette elicited inward currents, showing transient activation followed by current decaying. Therefore, these results indicate that odontoblasts express functional TRPV1 channels. 2. Kv channels: In acutely isolated odontoblasts, depolarizing steps from a holding potential of -80mV evoked time- and voltage-dependent outward currents. The relatively slow activation kinetics exhibited strong dependence on the membrane potential. Time constant of inactivation was approximately 400 ms at membrane potential of -50 mV. These results indicate that odontoblasts express slowly-activating and -inactivating time- and voltage-dependent K+ currents. 3. NCX: The reverse exchange Ca^<2+>influx in odontoblasts was blocked by an NCX inhibitor (KB-R7943 and SEA0400) in a concentration-dependent manner. The inward currents via forward Na^+-Ca^<2+>exchange had a dependence on external Na+. Immunohistochemical localization of NCX was detected at distal membrane of odontoblasts.These results indicate that odontblasts possess NCX. Our research results indicate significant expression of TRPV1, Kvchannels and NCX in odontoblasts, suggesting that odontoblasts may sirectly respond to the noxious stimuli, and generate action potential by coupling with voltage-dependent Na^+ channels. NCX play an important role in the regulation of intracellular Ca^<2+> levels by excessive internal Ca^<2+>, as well as in Ca-<2+> transport pathway to the dentin mineralizing front by transporting increased intracellular Ca^<2+> as a result of activation of TRPV1. Less

这项研究旨在阐明牙本质细胞在牙本质感觉的细胞机制中的作用。 During the research period from April 2006 to March 2008, we investigated expression of TRPV1 (vanilloid receptor 1; VR1) channel (a transient receptor potential (TRP) family subtype, which contributes essentially for the detection of pain sensation), voltage-dependent K+ channels (Kv channels; which involve in the generation of action potential), and Na^+-Ca^<2+> exchangers (NCX; which regulate通过免疫组织化学，细胞内CA^<2+>浓度伸展[Ca^<2+>] _ I）ntrocellular Ca^<2+>通过延伸[Ca^<2+>] _ I）通过免疫组织化学，细胞内Ca^<2+>浓度>全细胞贴剂技术。1。 TRPV1通道：免疫组织化学实验，显示了TRPV1在Odontoblast膜远端区域的定位。 RT-PCR analysis shown that odontoblasts express TRPV subfamily 1, 2, 3. In the fura-2 fluorescence analysis to measure[Ca^<2+>]I, anandamide (AEA; 10 pM; endogenous TRPV … More 1 activator) evoked a transient rise in [Ca^<2+>], in the presence of extracellular Ca^<2+> with slight delay in activation of transient rise after AEA application.但是，在没有细胞外Ca^<2+>的情况下，我们无法观察到[Ca^<2+>]的任何短暂上升，表明AEA激活了Ca^<2+>影响。 Ca^<2+>影响被TRPV1通道拮抗剂辣椒粉阻断。在我们先前的研究中，辣椒素的细胞外应用激活了大鼠odontoblast的内向电流（Okumura等，等，2006），但它们的当前放大器很小（CA，10 PA）。另外，在目前的实验中，[Ca^<2+>]的上升幅度在AEA应用后，据报道辣椒素和AEA结合TRPV1通道的细胞内结构域。因此，我们检查了辣椒素的细胞内应用是否在牙糖细胞中激活内向电流。在连续的电压钳状态下，保持电位为0 mV，细胞内施用10μm辣椒素通过斑块夹板引起了向内电流，显示了瞬时激活，然后是当前衰减。因此，这些结果表明Odontoblasts表达功能性TRPV1通道。 2。KV通道：在急性隔离的Odontoblasts中，目前从保持-80MV的持有电势向外依赖于-80MV的势力和电压依赖性的步骤。相对缓慢的激活动力学暴露了对膜电位的强依赖性。在-50 mV的膜电位下，灭活的时间常数约为400毫秒。这些结果表明，牙本质细胞表达缓慢激活和激活的时间和电压依赖性K+电流。 3。NCX：ODONTOBLASTS中的反向交换Ca^<2+>以浓度依赖性的方式被NCX抑制剂（KB-R7943和SEA0400）阻止。通过正向Na^+-ca^<2+>交换的内向电流依赖于外部Na+。在Odontblast的离散膜上检测到NCX的免疫组织化学定位。这些结果表明Odontblasts具有NCX。我们的研究结果表明，在Odontblasts中，TRPV1，KVChannels和NCX的显着表达，表明Odontblasts可能直接响应有害的刺激，并通过与电压依赖性Na^+通道耦合来产生动作潜力。 NCX通过过量的内部Ca^<2+>以及Ca- <2+>在Ca- <2+>中的转运途径在向牙本质矿化前矿化的转运途径中提高了细胞内Ca^<2+>的运输途径在调节细胞内Ca^<2+>水平中起重要作用，这是TRPV1激活的结果。较少的