博碩士論文 962404003 詳細資訊




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姓名 黃雅涵(Ya Han Huang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 酸敏感G蛋白偶合受體調節的鈣離子訊息調控急性到慢性疼痛的轉換
(Proton-sensing GPCR-mediated calcium signals regulate the transition from acute to chronic pain.)
檔案 [Endnote RIS 格式]    [Bibtex 格式]    至系統瀏覽論文 (2021-8-31以後開放)
摘要(中) 組織受傷或發炎時,局部氫離子濃度會上升,此現象稱為組織酸化,組織酸化常伴隨著疼痛的感覺,且是造成疼痛的主要因子。辣椒素受體1以及酸敏感離子通道3已經被證實與酸引起的疼痛相關,而酸敏感G蛋白偶合受體的功能還未被確定。G蛋白偶合受體的家族含有四個基因:OGR1,GPR4,G2A與TDAG8。我們之前的研究發現酸敏感偶合受體的四個基因皆有表現在背根神經節的痛覺敏感神經內,並且與辣椒素受體1以及酸敏感離子通道3都會同時表現。而TDAG8的活化會使辣椒素受體1對辣椒素的反應敏感化。在CFA藥劑引起的發炎反應中,長時的痛覺敏感受到PKA與PKC兩條訊息路徑的調控,此兩條路徑調控的時間轉換點約在注射CFA藥劑的三到四小時之後。直接注射酸所引起的急性痛覺敏感也一樣受到PKA與PKC兩條訊息路徑的調控,而兩個路徑轉換的時間點約在酸注射二到四小時之後。因此,在發炎時,此兩條調控的訊息路徑轉換可能是由酸引起的。Gs-AC-PKA 訊息路徑可能負責調控四小時前的痛覺敏感,而Gi- PLC- PKC 訊息路徑則負責四小時之後的痛覺敏感。總括來說,酸敏感偶合受體可能參與在此兩條調控的訊息路徑。此篇研究發現,注射CFA藥劑兩小時後,TDAG8在酸引起的胞內鈣離子訊息變化上具有十分重要的調控作用,對於注射CFA藥劑二十四小時後的包內鈣離子濃度變化同樣具有重要的作用。而同時表現OGR1以及G2A使得細胞對酸的敏感度增加,並同時增加胞內的鈣離子濃度。進一步發現OGR1以及G2A同時表現時,下游涉及的訊息路徑是Gi- PLC- PKC,因此OGR1與G2A的異體偶合可能涉及發炎反應中觀察到的Gi- PLC- PKC調控路徑。
摘要(英) Tissue injury and inflammation raise local proton concentration (called tissue acidosis) and accompany with painful sensations. Tissue acidosis is a dominant factor that contributes to pain. Transient receptor potential vanilloid 1 (TRPV1) and acid-sensing ion channel 3 (ASIC3), one member of ASIC family, are proved to be related to acid-induced pain. Proton-sensing G-protein-coupled receptors (GPCRs) consists of ovarian cancer G-protein-coupled receptor 1 (OGR1), GPR4, G2A and T-cell death associated gene 8 (TDAG8). Our previous study indicates that the OGR1 family are expressed in nociceptors of DRG, and are co-localized with TRPV1 and ASIC3. TDAG8 activation sensitize TRPV1 response to capsaicin. In complete Freund’s adjuvant (CFA)-induced inflammation, prolonged hyperalgesia in mice is regulated by PKA and PKC. The switch time for PKA and PKC dependency is about 3 to 4 hours. Acute hyperalgesia induced by acidic solution (pH 5.5 or 5.0) depended on both PKA and PKC, as for prolonged hyperalgesia induced by CFA. The switch time for PKA and PKC dependency is about 2 to 4 hours. Therefore, the switch of PKA and PKC dependency in prolonged hyperalgesia induced by CFA can be due to acidosis signals. The Gs-AC-PKA pathway may be responsible for the early phase of hyperalgesia and Gi- PLC- PKC pathway for the late phase. Taken together, proton-sensing GPCRs might be the candidate to be involved in these two pathways. In this study, I have found the dominant role of TDAG8 to mediate proton-induced calcium signals after 2 hours of CFA injection, and TDAG8 is involved in the case after 24 hours of CFA injection as well. Co-expression of OGR1 and G2A increases the sensitivity to proton, and the magnitude of intracellular calcium signals. Co-expression of OGR1 and G2A is involved in a Gi- PLC- PKC pathway. OGR1 and G2A heteromer is the candidate responsible to the Gi- PLC- PKC pathway observed in inflammation.
關鍵字(中) ★ 酸敏感G蛋白偶合受體 關鍵字(英) ★ proton-sensing GPCR
★ TDAG8
★ OGR1
★ G2A
論文目次 Abstracti
中文摘要ii
Contents iii
List of figuresvi
List of table vii
Abbreviationviii
Chapter 1 Decrease of TDAG8 receptor leads to declined proton-evoked
intracellular calcium signals in CFA-induced inflammation 1
1-1 Introduction 2
1-1-1 Tissue acidosis and inflammatory pain 3
1-1-2 Proton-sensing receptors 4
1-1-3 Proton-sensing ion channels 4
1-1-4 Proton-sensing ion GPCRs 6
1-1-5 The expression of proton-sensing GPCRs in nociceptors 7
1-1-6 Proton-sensing GPCRs and inflammation8
1-1-7 The roles of G2A and TDAG8 in long-term chronic inflammation 9
1-1-8 The objective of this chapter10
1-2 Materials and Methods 11
1-2-1 The agents 12
1-2-2 Animals12
1-2-3 Injection of mice 13
1-2-4 Injection of CG13
1-2-5 Primary DRG cultures 13

1-2-6 Treatment of coverslips with poly-L-lysine 14
1-2-7 Quantitation of intracellular calcium concentration 15
1-2-8 IB4 staining 16
1-3 Results17
1-3-1 Proton-induced calcium signals augment after 2 hours of CFA
injection. 18
1-3-2 Proton-induced calcium signals increase last to 24 hours after CFA
injection. 19
1-3-3 PKA inhibitor H89 has no effects on both magnitude and pattern of
calcium signals-induced by proton after CFA-injection. 21
1.3.4 PKCinhibitor KIE 1-1 significantly decreases the calcium signals
in both ipsilateral and contralateral side, and mainly in transient
pattern. 21
1-3-5 Knockdown of TD.AG8 gene blocks most proton-induced calcium
signals after 2 hours of CFA injection, and both sustained and
transient pattern are inhibited. 22
1-3-6 TDAG8 has effects on proton-induced calcium signals after 24 hours
of CFA injection. 22
1-3-7 Knockdown of TDAG8 gene switches the major pattern (transient) of
Calcium signals to sustained in acid-injection mice. 23
1-4 Discussion 25
Conclusion 28
Chapter 2 Co-expression of OGR1 and G2A leads to enhancement of
proton-induced intracellular calcium levels 30

2-1 Introduction31
2-1-1 Proton-sensing GPCRs 32

2-1-2 The objective of this chapter 33
2-2 Materials and Methods 34
2-2-1 Cell culture and transfection 35
2-2-2 Treatment of coverslips with poly-D-lysine 35
2-2-3 Quantitation of intracellular calcium concentration 36
2-2-4 Cyclic AMP assay 36
2-3 Results 37
2-3-1 OGR1 mediated-calcium levels is sensitive to U73122 and PTX, and G2A is
less sensitive to proton stimulation. 38
2-3-2 Co-expression of OGR1 and G2A was the only combination to increase
proton-induced intracellular calcium levels. 38
2-3-3 Co-expression of OGR1 and G2A increased the sensitivity to proton, and
augment the proton-induced intracellular calcium signals. 39
2-3-4 Co-expression of OGR1 and G2A was involved in a Gi- PLC- PKC
pathway. 40
2-4 Discussion41
Figures 43
Tables 72
Refferences 73
參考文獻 1. Julius, D. and Basbaum, A.I., 2001. Molecular mechanisms of nociception. Nature 413, pp. 203- 210.
2. Shcolz, J. and Woolf, C.J., 2002. Can we conquer pain? Nature Neurosci 5 (Suppl), pp. 1062-1067.
3. Steen, K.H., Reeh, P.W., Anton, F., and Handwerker, H.O., 1992. Protons selectively induce lasting excitation and sensitization to mechanical stimulation of nociceptors in rat skin, in vitro. J. Neuroscience 12, pp. 86-95.
4. Steen, K.H. and Reeh, P.W., 1993. Sustained graded pain and hyperalgesia from harmless experimental tissue acidosis in human skin. Neuronsceince Letters 154, pp. 113-116.
5. Issberner, U., Reeh, P.W., Steen, K.H., 1996. Pain due to acidosis: a mechanism for inflammatory and ischemic myalgia? Neuronsceince Letters 208, pp. 191-194.
6. Reeh, P.W. and Steen, K.H., 1996. Tissue acidosis in nociception and pain. Prog. Brain res. 113, pp. 143-151.
7. Bevan, S. and Yeast, J., 1991. Protons activate a cation conductance in a sub-population of rat dorsal root ganglion neurons. J. Physiol. 433, pp. 145-161.
8. Steen, K.H., Steen, A. and Reeh, P.W., 1995. A dominant role of acid pH in inflammatory excitation and sensitization of nociceptors in rat skin, in vitro. J. neuroscience 15, pp. 3982-3968.
9. Steen, K.H., Steen, A., Kreysel, H. and Reeh, P.W., 1996. Inflammatory mediators potentiate pain induced by experimental tissue acidosis. Pain 66, pp. 163-170.
10. Waldmann, R., Champigny, G., Bassilana, F., Heurteaux, C. and Lazdunski, M., 1997. A proton-gated cation channel involved in acid-sensing. Nature 386, pp. 173–177.
11. Krishtal, O., 2003. The ASICs: signaling molecules? Modulators? Trends Neurosci. 9, pp 477-83.

12. Chen, C.C., England, S., Akopian, A.N. and WooD, J.N., 1998. A sensory neuron-specific, proton-gated ion channel. Proc. Natl. Acad. Sci. U. S. A. 95, pp. 10240-10245.
13. Alvarez-de-la Rosa, D., Zhang, P., Shao, D., White, F. and Canessa, C.M., 2002. Functional implications of the localization and activity of acid-sensitive channels in rat peripheral nervous system. Proc. Natl. Acad. Sci. U. S. A. 99, pp. 2326–2331.
14. Price, M.P., McIlwrath, S.L., Xie, J., Cheng, C., Qiao, J., Tarr, D.E., Sluka, K.A., Brennan, T.J., Lewin, G.R. and Welsh, M.J., 2001. The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice. Neuron 32, pp. 1071-1083.
15. Sluka, K.A., Price, M.P., Breese, N.M., Stucky, C.L., Wemmie, J.A., and Welsh, M.J., 2003. Chronic hyperalgesia induced by repeated acid injections in muscle is abolished by the loss of ASIC3, but not ASIC1. Pain 106, pp. 229-239.
16. Sluka, K.A., Radhakrishnan, R., Benson, C.J., Eshcol, J.O., Price, M.P., Babinski, K., Audette, K.M., Yeomans, D.C., and Wilsonf, S.P., 2007. ASIC3 in muscle mediates mechanical, but not heat, hyperalgesia associated with muscle inflammation. Pain 129, pp.102-112.
17. Mogil, J.S., Breese, N.M., Witty, M.F., Ritchie, J., Rainville, M.L., Ase, A., Abbadi, N., Stucky, C.L., and Sèguèla, P., 2005. Transgenic Expression of a Dominant-Negative ASIC3 subunit leads to increased sensitivity to mechanical and inflammatory stimuli. J. Neurosci. 25, pp. 9893-9901.
18. Deval, E., Noël, J., Lay, N., Alloui, A., Diochot, S., Friend, V., Jodar, M., Lazdunski, M., and Lingueglia, E., 2008. ASIC3, a sensor of acidic and primary inflammatory pain. EMBO J. 27, pp. 3047-3055.
19. Voilley, N., Weille, J.D., Mamet, J., Lazdunski, M., 2001. Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J. Neurosci. 21, pp 8026-8033.
20. Mament, J., Baron, A., Lazdunski, M., Voilley, N., 2002. Proinflammatory mediators, stimulators of sensory neuron excitability via the expression of acid-sensing ion channels. J. Neurosci. 22, pp 10662-10670.
21. Mament, J., Lazdunski, M., Voilley, N., 2003. How nerve growth factor drives physiological and inflammatory expression of acid-sensing ion channel 3 in sensory neurons. J. Bio. Chem. 278, pp 48907-48913.
22. Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D. and Julius, D., 1997. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, pp. 816-824.
23. Jordt, S.E., Tominaga, M., Julius, D., 2000. Acid potentiation of the capsaicin receptor determined by a key extracellular site. Proc. Natl. Acad. Sci. 97, pp. 8134-8139.
24. Caterina, M.J., Leffler, A., Malmberg, A.B., Martin, W.J., Trafton, J., Petersen-Zeitz, K.R., Koltzenburg, M., Basbaum, A.I. and Julius, D., 2000. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288, pp. 306-313.
25. Bhave G, Zhu, W., Wang, H., Brasier, D.J., Oxford, G.S., Gereau, R.W., 2002. cAMPdependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct phosphorylation. Neurons 35, pp.721-731.
26. Bhave, G., Gereau, R.W., 2004. Psottranslational mechanisms of peripheral sensitization. J Neurobiol 61, pp.88-106.
27. Bhave, G., Hu, H.J., Glauner, K.S., Zhu, W., Wang, H., Brasier, D.J., Oxford, G.S., Gereau, R.W., 2003. Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). Proc. Natl. Acad. Sci. 100. pp. 12480-12485.
28. Carlton, S.M. and Coggeshall, R.E., 2001. Peripheral capsaicin receptors increase in the inflamed rat hindpaw: a possible mechanism for peripheral sensitization. Neurosci. Lett. 310, pp. 53-6.
29. Amaya, F., Oh-hashi, K., Naruse, Y., Iijima, N., Ueda, M., Shimosato, G., Tominaga, M., Tanaka, Y., Tanaka, M., 2003. Local inflammation increases vanilloid receptor 1 expression within distinct subgroups of DRG neurons. Brain Res. 963, pp. 190-6.
30. Breese, N.M., George, A.C., Pauers, L.E., Stucky, C.L., 2005. Peripheral inflammation selectively increases TRPV1 function in IB4-positive sensory neurons from adult mouse. Pain 115, pp. 37-49.
31 Hucho, T.B., Dina, O.A., Levine, J.D.,.2005. Epac mediates a cAMP-to-PKC signaling in inflammatory pain: an isolectin B4 (+) neuron-specific mechanism. J. Neurosci. 25, pp. 6119-6126.
32. Aley, K.O., Messing, R.O., Mochly-Rosen, D., Levine, J.D., 2000. Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the ε isozyme of protein kinase C. J Neurosci 20, pp. 4680-4685.
33. Parada, C.A., Yeh, J.J., Reichling, D.B., Levine, J.D., 2003. Transient attenuation of protein kinase Cε can terminate a chronic hyperalgesic state in the rat. Neurosci. 120, pp. 219-226.
34. Parada, C.A., Reichling, D.B., Levine, J.D., 2005. Chronic hyperalgesic priming in the rat involves a novel interaction between cAMP and PKCε second messenger pathways. Pain 113, pp. 185-190.
35. Ludwig, M., Vanek, M., Guerini, D., Gasser, J.A., Jones, C.E., Junker, U., Hofstetter, H., Wolf, R.M. and Seuwen, K., 2003. Proton-sensing G-protein-coupled receptors. Nature 425, pp. 93-98.
36. Tomura, H., Wang, J., Komachi, M., Damirin, A., Mogi, C., Tobo, M., Kon, J., Misawa, N., Sato, K., Okajima, F., 2005, Prostaglandin I2 production and cAMP accumulation in response to acidic extracellular pH through OGR1 in human aortic smooth muscle cells. J. Biol. Chem. 280, pp. 34458-34464.
37. Tobo, M., Tomura, H., Mogi, C., Wang, J., Liu, J., Komachi, M., Damirin, A., Kimura, T., Murata, N., Kurose, H., Sato, K., Okajima, F., 2007, Previously postulated “ligandindependent“ signaling of GPR4 is mediated through proton-sensing mechanisms. Cellular Signaling 19, pp.1745-1753.
38. Murakami, N., Yokomizo, T., Okuno, T. and Shimizu, T., 2004. G2A is a proton-sensing G-protein-coupled receptor antagonized by lysophosphatidylcholine. J. Biol. Chem. 279, pp. 42484-42491.
39. Radu, C.G.., Nijagal, A., McLaughlin, J., Wang, L. and Witte, O.N., 2005. Differential proton sensitivity of related G protein-coupled receptors T cell death-associated gene 8 and G2A expressed in immune cells. Proc. Natl. Acad. Sci. 102, pp. 1632-1637.
40. Wang, J., Kon, J., Mogi, C., Tobo, M., Damirin, A., Sato, K., Komachi, K., Malchinkhuu, E., Murata, N., Kimura, T., Kuwabara, A., Wakamatsu, K., Koizumi, H., Uede, T., Tsujimoto, G., Kurose, H., Sato, T., Harada, A., Misawa, N., Tomura, H. and Okajima, F., 2004. TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor. J. Biol. Chem. 279, pp. 45626-45633.
41. Choi, J.W., Lee, S.Y. and Choi, Y., 1996. Identification of a putative G rotein-coupled receptor induced during activation-induced apoptosis of T cells. Cell Immunol. 168, pp. 78-84.
42. Essayan, D.M., 2001. Cyclic nucleotide phosphodiesterases. J Allergy Clin Immunol. 108, pp. 671-80.
43. Huang, C.W., Tzeng, J.N., Chen, Y.J., Tsai, W.F., Chen, C.C., Sun, W.H., 2007. Nociceptors of dorsal root ganglion express proton-sensing G-protein-coupled receptors. Mol. Cell. Neurosci. 36, pp. 195-210.
44. Zaslavsky, A., Singh, L.S., Tan, H., Ding, H., Liang, Z., Xu, Y., 2006. Homo- and hetero-dimerization of LPA/S1P receptors, OGR1 and GPR4. Biochim Biophys Acta. 1761, pp. 1200-12.
45. Chen, Y.J, Huang, C.W., Lin, C.S., Chang, W.H., Sun, W.H., 2009. Expression and function of proton-sensing G-proteincoupled receptors in inflammatory pain. Mol. Pain 5, pp. 39-57.
46. Huang, W.Y., Dai, S.P., Chang, Y.C., Sun, W.H., 2015. Acidosis Mediates the Switching of Gs-PKA and Gi-PKCε Dependence in Prolonged Hyperalgesia Induced by Inflammation. PLoS One 10(5), e0125022
47. Chang, Y.C., 2011. TDAG8 activation sensitizes TRPV1 by PKA- and PKCε-dependent pathways.
指導教授 孫維欣(Wei Hsin Sun) 審核日期 2016-8-31
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