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TRPV 1的表达，香草酸受体1通道（瞬时受体电位（TRP）家族亚型，其基本上有助于检测痛觉），电压依赖性K+通道，（Kv通道;参与动作电位的产生）和Na^+-Ca^<2+>交换体采用免疫组化、细胞内Ca^<2 +>浓度测定和全细胞膜片钳技术，研究了NCX在大鼠成牙本质细胞内的作用. TRPV 1通道：免疫组化实验显示TRPV 1定位于成牙本质细胞膜的远端区域。RT-PCR分析显示成牙本质细胞表达TRPV亚家族1、2、3。在测定[Ca^<2+>]I的Fura-2荧光分析中，花生四烯酸酰胺（AEA; 10 pM;内源性TRPV ...更多信息 在细胞外Ca^<2+>存在的情况下，AEA引起[Ca^<2 +>]短暂升高，但短暂升高的激活稍有延迟。然而，在缺乏细胞外Ca^<2+>的情况下，我们不能观察到[Ca^<2+>]的任何瞬时升高，这表明AEA激活了Ca^<2+>内流。TRPV 1通道拮抗剂辣椒平可阻断Ca^2+内流。在我们以前的研究中，辣椒素的细胞外应用激活了大鼠成牙本质细胞的内向电流（Okumura，et.艾尔2006），但它们的电流幅度很小（约10 pA）。此外，在本实验中，AEA应用后[Ca^<2+>]的升高伴随激活延迟时间（约2-3分钟）。据报道，辣椒素以及AEA结合TRPV 1通道的胞内结构域。因此，我们研究是否细胞内应用辣椒素激活内向电流在成牙本质细胞。在保持电位为0 mV的连续电压钳条件下，经膜片钳向细胞内注入10 μM辣椒素，可引起内向电流，电流呈现短暂激活后衰减的现象。因此，这些结果表明成牙本质细胞表达功能性TRPV 1通道。2. KV通道：在急性分离的成牙本质细胞中，从-80mV的保持电位开始的去极化步骤诱发时间和电压依赖性外向电流。相对缓慢的活化动力学表现出强烈的依赖于膜电位。在膜电位为-50 mV时，灭活时间常数约为400 ms。这些结果表明，成牙本质细胞表达缓慢激活和失活的时间和电压依赖性K+电流。3. NCX：NCX抑制剂（KB-R7943和SEA 0400）以浓度依赖性方式阻断成牙本质细胞的反向交换Ca^2+内流。通过Na^+-Ca^<2+>正向交换的内向电流依赖于外部Na+。免疫组化结果显示，成牙本质细胞具有NCX。我们的研究结果表明，成牙本质细胞中TRPV 1、Kv通道和NCX的表达显著增加，提示成牙本质细胞可能对伤害性刺激产生直接反应，并通过与电压依赖性Na^+通道偶联产生动作电位。NCX在调节细胞内Ca^<2+>水平中起重要作用，其通过过量的内部Ca^<2+>调节细胞内Ca^<2+>水平，以及在Ca-<2 +>转运途径中起重要作用，其通过转运TRPV 1激活导致的细胞内Ca^<2+>而将其转运至牙本质矿化前沿。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