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系統識別號 U0026-2407201312484200
論文名稱(中文) 鈣激活性氯離子通道在大白鼠背根神經節細胞中所扮演的生理角色
論文名稱(英文) Physiological Roles of Ca2+-activated Chloride Channels in Rat Dorsal Root Ganglion Neurons
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 101
學期 2
出版年 102
研究生(中文) 許雅涵
研究生(英文) Ya-Han Hsu
電子信箱 yahankayhsu@gmail.com
學號 s36994063
學位類別 碩士
語文別 英文
論文頁數 37頁
口試委員 指導教授-吳豐森
召集委員-黃阿敏
口試委員-游一龍
中文關鍵字 鈣激活性氯離子通道  辣椒素  辣椒素受體  背根神經節細胞  感覺神經細胞  急性痛  熱痛過敏現象 
英文關鍵字 CaCC  Capsaicin  Capsaicin receptors  DRG neurons  Sensory neurons  Nocifensive responses  Thermal hyperalgesia 
學科別分類
中文摘要 在感覺神經細胞中,鈣激活性氯離子通道(CaCC)在譯解感覺刺激所誘發的訊息上扮演著重要的角色。CaCC最重要的特徵為:其氯離子流動所產生的電流是由細胞內鈣離子濃度的上升來驅動。先前的研究已經顯示,CaCC確實存在於背根神經節細胞中,但其在背根神經節細胞中所扮演的功能並不清楚。例如,目前尚不清楚CaCC所誘發的電流是否會因辣椒素[一種能打開具鈣離子通透性的陽離子通道的化學物質]受體(TRPV1)的活化進而被激活。我們實驗室最近的研究進一步指出,大約有20%電生理記錄到的背根神經節細胞,同時具有CaCC及TRPV1活化所產生的電流。因此在本研究中我們提出一個假說:當TRPV1受到活化會增加細胞內的鈣離子濃度,進而活化CaCC,而CaCC所產生的電流會增大TRPV1所產生的電流反應。在本研究中,我們先選用四種CaCC的亞型蛋白的抗體(Bestrophin, TTYH3, TMEM16a, and TMEM16b),利用西方點墨法來偵測這四種CaCC亞型蛋白在大白鼠快速分離的背根神經節細胞中的表現量。我們的結果顯示,這四種CaCC在背根神經節細胞中皆有表現。我們更進一步利用免疫螢光染色法,發現有三種CaCC亞型蛋白(Bestrophin, TMEM16a, and TMEM16b)與TRPV1會表現在同一個背根神經節細胞中。約有35%表現TRPV1的細胞同時也表現CaCC,這些表現CaCC的細胞大多是屬於中型大小(直徑30-40μm)的神經細胞。此外,在大白鼠痛覺行為測試中,注射CaCC抑制劑到大白鼠後腳掌可降低辣椒素在雄鼠所誘發的急性痛及熱痛過敏現象。感覺神經細胞TRPV1的活化,在痛覺的傳遞及發炎所誘發的熱痛過敏現象上,擔任一個很重要的角色。我們的研究結果不但有助於瞭解CaCC與TRPV1間的互動關係,且能提供將來在臨床上一個新的止痛策略。
英文摘要 In sensory neurons, the Ca2+-activated chloride channel (CaCC) plays a critical role in decoding sensory stimuli. One important feature of CaCC is that its Cl- current is driven by the elevation of the cytosolic Ca2+concentration. Previous studies have reported that CaCCs are present in dorsal root ganglion (DRG) neurons, but the exact function in DRG neurons is unknown. For example, it is unclear whether the CaCC current is one of components in the current induced by capsaicin, a compound that opens the Ca2+-permeable cation channel [transient receptor potential vanilloid subtype 1 (TRPV1)]. Our recent studies have further indicated that ~20% of the recorded DRG neurons contain both the CaCC current and the capsaicin-induced current. We hypothesize that the opening of TRPV1 channels may cause an increase of intracellular Ca2+ concentration, leading to a further activation of the CaCC current that could amplify the capsaicin-induced current. In the present study, we initially selected four subtypes of CaCC protein antibodies (Bestrophin, TTYH3, TMEM16a, and TMEM16b) to detect protein levels of CaCCs in acutely dissociated rat DRG neurons by use of Western blotting. Our results revealed that all four subtypes of CaCC proteins were expressed in DRG neurons. Moreover, immunofluorescence of rat DRG neurons demonstrated that all three subtypes of CaCC proteins (TMEM16a, Bestrophin, and TMEM16b) were co-localized with TRPV1 in rat DRG neurons. About 35% of TRPV1-positive DRG neurons expressed CaCC proteins. Among them, the co-localization percentage of TMEM16b with TRPV1 was greater than that of TMEM16a or Bestrophin. In addition, the CaCCs-containing neurons were almost medium-size (30-40 μm) neurons. Furthermore, CaCC blockade reduces capsaicin-induced nocifensive responses and thermal hyperalgesia in rats in vivo. Capsaicin receptor activation has been implicated in nociception and inflammatory thermal hyperalgesia. Our results will provide a new understanding of how CaCCs interact with TRPV1 and this knowledge may offer new strategies to reduce the pain.
論文目次 Abstract in Chinese…………………………………………………………..I
Abstract……………………………………………………………….........III
Table of Contents……………………………………………………………V
List of Figures..............................................................................................VII
Abbreviations……………………………………………………..………..VIII
Introduction…………………………………………………………...……..1
Physiological features and functions of CaCCs……………………………...........…..1
Signal transduction and regulation of CaCC activation………………………............2
Capsaicin, TRPV1, and nociception…………………………………………………...3
Rationale and the specific aim of this study……………………………....…………...6
Materials and Methods…………………………………………….......……7
Animals…………………………………………………………………..…………….7
Preparation of rat DRG neuron proteins……..……………………………………............7
Measurement of protein concentration…………………………..…………………............7
Western blotting analysis…………………………………………………...….………8
Antibodies for Western blotting……………………………...…………………...........9
Immunofluoresence of rat dorsal root ganglion neuron….............................................9
Antibodies for Immunofluoresence…..……………………………………………….10
Capsaicin test……………………………………...…………………………………10
Plantar test…………………………………………………...………………………11
Drugs and chemicals……………………………………………………………..…..11
Statistical analysis…....................................................................................................12
Experimental Design….……………………………………………..………………12
Results…………………...…………………………………………….…….14
Expression levels of CaCC protein in acute dissociated rat DRG neurons…………………………………………….…………………………………14
The co-expression pattern of CaCCs and TRPV1 in rat DRG neurons…………………………………………………………………………….…14
Intradermal pretreatment of the CaCC blocker reduces capsaicin-induced nocifensive responses in male rats in vivo……………………………………. …….…………...15
Intraplantar pretreatment of the CaCC blocker attenuates capsaicin-induced thermal hyperalgesia in male rats in vivo….………………...………………………………..15
Discussion……………………………..…………………………………….17
CaCC proteins are expressed in naïve rat peripheral niciceptive neurons…….………………………………………………………………….……...……17
Signal transductions of CaCC in DRG neurons…..…………….…………………………18
Mechanism of interaction between CaCCs and TRPV1 in thermal nociception………………………………………………………………………………...19
Conclusion………………………………………………………………......21
References…………………………………..………………………………28
About the Author……………………...……………………………………37

List of Figures
Figure 1. Protein levels of four subtypes of CaCCs in rat DRG neurons by use of Western blotting.…………...…………………………………………………………….22

Figure 2. Immunoreactivity of CaCCs and TRPV1 in rat DRG neurons…….............23

Figure 3. Quantitative analysis of the co-localization percentage of TRPV1 and CaCCs in rat DRG neurons………………………………………………...……………24

Figure 4. Pretreatment of the CaCC blocker niflumic acid reduces the capsiacin-induced nocifensive response in male rats….………………..………...........25

Figure 5. Pretreatment of the CaCC blocker niflumic acid decreases capsiacin-induced thermal hyperalgesia in male rats…...…………...………..……….26

Figure 6. Comparison of PWL differences between Veh, Cap and NFA, Cap groups at 15 min(A), 30 min (B), 60 min (C), and 120 min (D) after capsaicin injection ………………………..…………………………………….27
參考文獻 Acs, G., Palkovitts, M. and Blumberg, P. (1994) Comparison of [3H]resiniferatoxin binding by the vanilloid (capsaicin) receptor in dorsal root ganglia, spinal cord, dorsal vagal complex, sciatic and vagal nerve and urinary bladder of the rat. Life Sci 55:1017-1026.
Al-Jumaily, M., Kozlenkov, A., Mechaly, I., Fichard, A., Matha, V., Scamps, F., Valmier, J. and Carroll, P. (2007) Expression of three distinct families of calcium-activated chloride channel genes in the mouse dorsal root ganglion. Neurosci Bull 23:293-299.
André, S., Boukhaddaoui, H., Campo, B., Al-Jumaily, M., Mayeux, V., Greuet, D., Valmier, J. and Scamps, F. (2003) Axotomy-induced expression of calcium-activated chloride current in subpopulations of mouse dorsal root ganglion neurons. J Neurophysiol 90:3764-3773.
Barish, M.E. (1983) A transient calcium-dependent chloride current in the immature Xenopus oocyte. J Physiol 342:309-325.
Bevan, S., Hothi, S., Hughes, G., James, I.F., Rang, H.P., Shah, K., Walpole, C.S. and Yeats, J.C. (1992) Capsazepine: a competitive antagonist of the sensory neurons excitant capsaicin. Br J Pharmacol 107:544-552.
Bevan, S. and Szolcsányi, J. (1990) Sensory neuron-specific actions of capsaicin: mechanisms and applications. Trends Pharmacol Sci 11:330-333.
Boudes, M., Sar, C., Menigoz, A., Hilaire, C., Pequignot, M.O., Kozlenkov, A., Marmorstein, A., Carroll, P., Valmier, J. and Scamps, F. (2009) Best1 is a gene regulated by nerve injury and required for Ca2+-activated Cl- current expression in axotomized sensory neurons. J Neurosci 29:10063-10071.
Caputo, A., Caci, E., Ferrera, L., Pedemonte, N., Barsanti, C., Sondo, E., Pfeffer, U., Ravazzolo, R., Zegarra-Moran, O. and Galietta, L.J. (2008) TMEM16A, a membrane protein associated with calcium-dependent chloride activity. Science 322:590-568.
Caterina, M.J. (2000) Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288:306-313.
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 389:816-824.
Chen, S.C., Chang, T.J. and Wu, F.S. (2004) Competitive inhibition of the capsaicin receptor-mediated current by dehydroepiandrosterone in rat dorsal root ganglion neurons. J Pharmacol Exp Ther 311:529-536.
Chen, T.Y. (2005) Structure and function of clc channels. Annu Rev Physiol 67:809-839.
Cho, H., Yang, Y.D., Lee, J., Lee, B., Kim, T., Jang, Y., Back, S.K., Na, H.S., Harfe, B.D., Wang, F., Raouf, R., Wood, J.N. and Oh, U. (2012) The calcium-activated chloride channel anoctamin 1 acts as a heat sensor in nociceptive neurons. Nat Neurosci 15:1015-1021.
Clapham, D.E. (2003) TRP channels as cellular sensors. Nature 426:517-524.
Cromer, B.A. and McIntyre, P. (2008) Painful toxins acting at TRPV1. Toxicon 51:163-173.
Davis, J.B., Gray, J., Gunthorpe, M.J., Hatcher, J.P., Davey, P.T., Overend, P., Harries, M.H., Latcham, J., Clapham, C., Atkinson, K., Hughes, S.A., Rance, K., Grau, E., Harper, A.J., Pugh, P.L., Rogers, D.C., Bingham, S., Randall, A. and Sheardown, S.A. (2000) Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 405:183-187.
Davis, K.D., Meyer, R.A. and Campbell, J.N. (1993) Chemosensitivity and sensitization of nociceptive afferents that innervate the hairy skin of monkey. J Neurophysiol 69:1071-1081.
De Petrocellis, L., Chu, C.J., Moriello, A.S., Kellner, J.C., Walker, J.M. and Di Marzo, V. (2004) Actions of two naturally occurring saturated N-acyldopamines on transient receptor potential vanilloid 1 (TRPV1) channels. Br J Pharmacol 143:251-256.
Docherty, R.J., Yeats, J.C., Bevan, S. and Boddeke, H.W. (1996) Inhibition of calcineurin inhibits the desensitization of capsaicin-evoked currents in cultured dorsal root ganglion neurones from adult rats. Eur J Physiol 431:828-837.
Dray, A. (1992) Mechanism of action of capsaicin-like molecules on sensory neurons. Life Sci 51:1759-1765.
Frings, S., Reuter, D. and Kleene, S.J. (2000) Neuronal Ca2+-activated Cl- channels — homing in on an elusive channel species. Prog Neurobiol 60:247-289.
Gilchrist, H.D., Allard, B.L. and Simone, D.A. (1996) Enhanced withdrawal responses to heat and mechanical stimuli following intraplantar injection of capsaicin in rats. Pain 67:179-188.
Green, B. and Shaffer, G. (1993) The sensory response to capsaicin during repeated topical exposures: differential effects on sensations of itching and pungency. Pain 53:323-334.
Gunthorpe, M.J., Benham, C.D., Randall, A. and Davis, J.B. (2002) The diversity in the vanilloid (TRPV) receptor family of ion channels. Trends Pharmacol Sci 23:183-191.
Hartzell, C., Putzier, I. and Arreola, J. (2005) Calcium-activated chloride channels. Annu Rev Physiol 67:719-758.
Hartzell, H.C. (2008) CaCl-ing channels get the last laugh. Science 322:534-545.
Holzer, P. (1988) Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 24:739-768.
Holzer, P. (1991) Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol Rev 43:144-201.
Huang, F., Rock, J.R., Harfe, B.D., Cheng, T., Huang, X., Jan, Y.N. and Jan, L.Y. (2009) Studies on expression and function of the TMEM16A calcium-activated chloride channel. Proc Natl Acad Sci USA 106:21413-21418.
Hwang, S.W., Cho, H., Kwak, J., Lee, S.Y., Kang, C.J., Jung, J., Cho, S., Min, K.H., Suh, Y.G., Kim, D. and Oh, U. (2000) Direct activation of capsaicin receptors by products of lipoxygenases: Endogenous capsaicin-like substances. Proc Natl Acad Sci USA 97:6155-6160.
Kurahashi, T. and Yau, K.W. (1993) Co-existence if cationic and chloride components in odorant-induced current of vertate olfactory receptor cells. Nature 363:71-74.
Labrakakis, C., Tong, C.K., Weissman, T., Torsney, C. and MacDermott, A.B. (2003) Localization and function of ATP and GABAA receptors expressed by nociceptors and other postnatal sensory neurons in rat. J Physiol 549:131-142.
Lee, M.G., Ohana, E., Park, H.W., Yang, D. and Muallem, S. (2012) Molecular mechanism of pancreatic and salivary gland fluid and HCO3 secretion. Physiol Rev 92:39-74.
Liu, B., Linley, J.E., Du, X., Zhang, X., Ooi, L., Zhang, H. and Gamper, N. (2010) The acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl– channels. J Clin Invest 120:1240-1252.
Lopshire, J.C. and Nicol, G.D. (1998) The cAMP transduction cascade mediates the prostaglandin E2 enhancement of the capsaicin-elicited current in rat sensory neurons: whole-cell and single-channel studies. J Neurosci 18:6081-6092.
Lowe, G. and Gold, H. (1993) Nonlinear amplification by calcium-dependent chloride channels in olfactory receptor cells. Nature 366:283-286.
Lu, Y.C., Chen, C.W., Wang, S.Y. and Wu, F.S. (2009) 17Beta-estradiol mediates the sex difference in capsaicin-induced nociception in rats. J Pharmacol Exp Ther 331:1104-1110.
Manning, D.C., Raja, S.N., Meyer, R.A. and Campbell, J.N. (1991) Pain and hyperalgesia after intradermal injection of bradykinin in humans. Clin Pharmacol Ther 50:721-729.
Melvin, J.E., Yule, D., Shuttleworth, T. and Begenisich, T. (2005) Regulation of fluid and electrolyte secretion in salivary gland acinar cells. Annu Rev Physiol 67:445-469.
Oh, U., Hwang, S.W. and Kim, D. (1996) Capsaicin activates a nonselective cation channel in cultured neonatal rat dorsal root ganglion neurons. J Neurosci 16:1659-1667.
Reisert, J., Bauer, P.J., Yau, K.W. and Frings, S. (2003) The Ca-activated Cl channel and its control in rat olfactory receptor neurons. J Gen Physiol 122:349-363.
Rocha-Gonzalez, H.I., Mao, S. and Alvarez-Leefmans, F.J. (2008) Na+,K+,2Cl- cotransport and intracellular chloride regulation in rat primary sensory neurons: thermodynamic and kinetic aspects. J Neurophysiol 100:169-184.
Sanders, K.M., Koh, S.D. and Ward, S.M. (2006) Interstitial cells of cajal as pacemakers in the gastrointestinal tract. Annu Rev Physiol 68:307-343.
Sanders, K.M. and Ward, S.M. (2006) Interstitial cells of Cajal: a new perspective on smooth muscle function. J Physiol 576:721-726.
Schmelz, M., Schmid, R., Handwerker, H.O. and Torebjork, H.E. (2000) Encoding of burning pain from capsaicin-treated human skin in two categories of unmyelinated nerve fibres. Brain 123:560-571.
Schroeder, B.C., Cheng, T., Jan, Y.N. and Jan, L.Y. (2008) Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell 134:1019-1029.
Scott, R.H., Sutton, K.G., Griffin, A., Stapleton, S.R. and Currie, K.P. (1995) Aspects of calcium-activated chloride currents: a neuronal perspective. Pharmacol Ther 66:535-565.
Smart, D., Gunthorpe, M.J., Jerman, J.C., Nasir, S., Gray, J., Muir, A.I., Chambers, J.K., Randall, A.D. and Davis, J.B. (2000) The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol 129:227-230.
Stephan, A.B., Shum, E.Y., Hirsh, S., Cygnar, K.D., Reisert, J. and Zhao, H. (2009) ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification. Proc Natl Acad Sci USA 106:11776-11781.
Stucky, C.L., Koltzenburg, M., Schneider, M., Engle, G., Albers, K.M. and Davis, B.M. (1999) Overexpression of nerve growth factor in skin selectively affects the survival and functional properties of nociceptors. J Neurosci 19:8509-8516.
Szallasi, A. (1994) The vanilloid (capsaicin) receptor: receptor types and species differences. Gen Pharmacol 25:223-243.
Szallasi, A. and Blumberg, P.M. (1999) Vanilloid (Capsaicin) receptors and mechanisms. Pharmacol Rev 51:159-211.
Tominaga, M., Caterina, M.J., Malmberg, A.B., Rosen, T.A., Gilbert, H., Skinner, K., Raumann, B.E., Basbaum, A.I. and Julius, D. (1998) The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 21:531-543.
Tominaga, M. and Tominaga, T. (2005) Structure and function of TRPV1. Eur J Physiol 451:143-150.
Ward, S.M., McLaren, G.J. and Sanders, K.M. (2006) Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine. J Physiol 573:147-159.
Willis, W.D. (2006) John Eccles' studies of spinal cord presynaptic inhibition. Prog Neurobiol 78:189-214.
Wood, J.N., Winter, J., James, I.F., Rang, H.P., Yeats, J. and Bevan, S. (1988) Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture. J Neurosci 8:3208-3220.
Yang, Y.D., Cho, H., Koo, J.Y., Tak, M.H., Cho, Y., Shim, W.S., Park, S.P., Lee, J., Lee, B., Kim, B.M., Raouf, R., Shin, Y.K. and Oh, U. (2008) TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 455:1210-1215.
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