| Budget Amount *help |
¥3,680,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥180,000)
Fiscal Year 2007: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000)
Fiscal Year 2006: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Research Abstract |
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
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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
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