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)

本研究旨在阐明成牙本质细胞在牙本质感觉细胞机制中的作用。在2006年4月至2008年3月的研究期间，我们研究了TRPV1 （vanilloid receptor 1, VR1）通道（瞬时受体电位（TRP）家族亚型，主要参与痛觉的检测）、电压依赖性K+通道（Kv通道，参与动作电位的产生）和Na^+-Ca^<2+>交换器(NCX；通过免疫组化、细胞内Ca^<2+>浓度测定和全细胞膜片钳技术，通过挤压[Ca^<2+>] i)调节大鼠成牙细胞内Ca^<2+>浓度（[Ca^<2+>] i）。TRPV1通道：免疫组化实验显示TRPV1定位于成牙细胞膜远端区域。RT-PCR分析显示成牙细胞表达TRPV亚家族1,2,3。在测量[Ca^<2+>]I的fura-2荧光分析中，anandamide （AEA; 10 pM；内源性TRPV…More 1激活剂）在细胞外Ca^<2+>存在的情况下引起了[Ca^<2+>]的短暂上升，AEA应用后短暂上升的激活略有延迟。然而，在没有细胞外Ca^<2+>的情况下，我们没有观察到任何短暂的[Ca^<2+>]升高，这表明AEA激活了Ca^<2+>内流。Ca^<2+>内流被TRPV1通道拮抗剂capsazepine阻断。在我们之前的研究中，细胞外应用辣椒素激活了大鼠成牙细胞的内向电流（Okumura等，2006），但其电流振幅很小（约10 pA）。此外，在本实验中，应用AEA后，[Ca^<2+>]升高，并伴有激活延迟时间（Ca, 2-3 min）。据报道，辣椒素和AEA结合胞内TRPV1通道结构域。因此，我们研究了细胞内应用辣椒素是否会激活成牙细胞的内向电流。在保持电位为0 mV的连续电压箝位条件下，通过膜片移液器向细胞内注入10 μM辣椒素，细胞内产生向内电流，呈现瞬时激活后电流衰减的过程。因此，这些结果表明成牙细胞表达功能性TRPV1通道。2. Kv通道：在急性分离的成牙细胞中，从- 80mv保持电位开始的去极化步骤诱发了与时间和电压相关的外向电流。相对缓慢的活化动力学表现出对膜电位的强烈依赖性。在-50 mV的膜电位下，失活时间常数约为400 ms。这些结果表明，成牙细胞表达慢激活和慢失活的时间和电压依赖的K+电流。3. NCX：一种NCX抑制剂（KB-R7943和SEA0400）以浓度依赖的方式阻断成牙细胞中反向交换Ca^<2+>内流。通过正向Na^+-Ca^<2+>交换的向内电流依赖于外部Na+。在成牙细胞远端膜检测NCX的免疫组织化学定位。这些结果表明成牙细胞具有NCX。我们的研究结果表明，TRPV1、Kvchannels和NCX在成牙细胞中显著表达，表明成牙细胞可能直接响应有害刺激，并通过与电压依赖性Na^+通道偶联产生动作电位。NCX通过过量的Ca^<2+>调节细胞内Ca^<2+>水平，并通过TRPV1激活细胞内Ca^<2+>的增加，在Ca-<2+>向牙本质矿化前沿的转运途径中发挥重要作用。少

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