TDAG8活化後經由PKA與PKCε增強辣椒素受體的敏感度

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張雁晴(Yen-ching Chang) 查詢紙本館藏

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論文名稱

TDAG8活化後經由PKA與PKCε增強辣椒素受體的敏感度
(TDAG8 activation sensitizes TRPV1 by PKA- and PKCε-dependent pathways)

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摘要(中)

在慢性發炎中，高濃度的氫離子在局部組織中累積（組織酸化）引起疼痛。疼痛的強度則與組織酸化的程度相關。組織酸化引起的疼痛主要是經由直接地激活傷害感覺性感覺神經元（nociceptive sensory neurons），或經由酸敏感的受體間接地調節。辣椒素受體（TRPV1）是一個酸敏感的離子通道，在發炎後的背根神經（dorsal root ganglion）節當中，TRPV1基因的表現量與功能增加因而參與發炎性疼痛的產生。近期的研究中發現，酸敏感的G蛋白偶合受體TDAG8，在發炎後的背根神經節中的基因表現量也增加；並且，活化後的TDAG8可以增強TRPV1的敏感度。然而，活化的TDAG8是如何增強TRPV1的敏感度目前仍不清楚。因此，本篇論文主要目的為釐清（1）TDAG8增強TRPV1敏感度的機制與（2）在背根神經節當中，經由酸化或慢性發炎增強TRPV1敏感度的機制。
在本篇論文中，我證實經由酸活化後的TDAG8可經由PKA與PKCε兩種激脢增強TRPV1的敏感度。在TDAG8表現的細胞中，TDAG8可經活化Gs蛋白，逕而藉由PKA與PKCε增強TRPV1的敏感度。特別的是，在小於pH 6.0的酸刺激下，TDAG8亦可經由能受到PTX與U73122所抑制的訊息傳導路徑增強TRPV1的敏感度。在培養的背根神經節細胞中，特別是IB4-positive細胞，pH 6.8的酸化可以經由PKA增加TRPV1的敏感度，也確實可以引起PKA與PKCε的活化並且轉移到細胞膜上。進一步研究結果指出，CFA所引起長時間性發炎也可經由PKCε增強TRPV1敏感度。因此，依據不同的情況下，PKA與PKCε皆可增強辣椒素TRPV1的敏感度。

摘要(英)

High local proton concentrations (tissue acidosis) found in some chronic inflammation complaints cause painfulness. The degree of associated pain is well correlated with magnitude of acidification. This is attributable to direct excitation of nociceptive sensory neurons or indirect modulation by proton-sensing receptors. Transient receptor potential vanilloid 1 (TRPV1), a proton-sensing ion channel, is involved in inflammatory pain because TRPV1 gene expression is increased and its function is enhanced in inflamed dorsal root ganglion (DRG) neurons. It is recently found that a proton-sensing G-protein-coupled receptors, TDAG8, has increased expression in inflamed DRG and its activation can enhance TRPV1 function. However, it remains unclear how TDAG8 activation sensitizes TRPV1 function. This thesis is to elucidate (1) the mechanism of TDAG8-mediated TRPV1 sensitization, and (2) the acidification- and chronic inflammation-induced TRPV1 sensitization in DRG neurons.
I have demonstrated that TDAG8 responds proton to sensitize TRPV1 through PKA- and PKCε-dependent pathways. In TDAG8-expressing cells, TDAG8 can activate Gs protein and then activate PKA or PKCε to sensitize TRPV1. Interestingly, at pH <6.0, TDAG8 can also sensitize TRPV1 through PTX- and U73122-sensitive pathways. In cultured DRG neurons, mild acidification (pH 6.8) induced TRPV1 sensitization through PKA-dependent pathways in IB4-positive neurons, although acidification induced PKA and PKCε translocation. Further studies revealed that CFA-induced long-term inflammation leading to TRPV1 sensitization through PKCε-dependent pathway. Accordingly, PKA and PKCε are involved in TRPV1 sensitization based on different situations.

關鍵字(中)

★ 辣椒素受體
★ T細胞死亡相關受體8
★ 蛋白質激脢A
★ 蛋白質激脢Cε
★ 發炎性疼痛
★ 組織酸化

關鍵字(英)

★ inflammatory pain
★ tissue acidosis
★ TDAG8
★ PKCε
★ TRPV1
★ PKA

論文目次

Contents
Abstract (English) i
Abstract (Chinese) ii
Acknowledgements iii
Contents iv
List of figures vii
List of tables ix
Abbreviations x
Chapter 1 Introduction 1
1.1 Pain 1
1.1.1 Nociception and nociceptors 1
1.1.2 Physiology of Aδ-fiber nociceptors 2
1.1.3 Physiology of C-fiber nociceptors 2
1.2 Inflammatory pain 3
1.2.1 Abnornal pain reseponses in inflammation 4
1.2.2 Inflammatory mediatros modulate nociceptors by PKA and PKCε 4
1.2.3 Tissue acidosis-linked pain 8
1.3 Proton receptors 8
1.3.1 Proton-gated ion channels 9
1.3.2 Proton-sensing G-protein-couple-receptors 11
1.4 The objective of the thesis 13
Chapter 2 Materials and Methods 14
2.1 Preparation of all solutions 14
2.2 Amplification and purification of plasmids 14
2.2.1 Transformation and bacteria cultures 14
2.2.2 Plasmid midi preparation 15
2.3 Measurement of intracellular cAMP 15
2.3.1 Cell treatment 16
2.3.2 Cell lysate collection 16
2.3.3 ELISA assay 17
2.4 Cell cultures and transfection 17
2.4.1 Subculture and sample preparation 17
2.4.2 Transfection 18
2.5 Measurement of intracellular calcium 18
2.5.1 Incubation of Fura-2 18
2.5.2 Single cell calcium imaging 18
2.6 Inflamed mice experiments 19
2.7 Primary DRG cultures 20
2.8 Immunohistochemistry and immunocytochemistry 21
2.8.1 Preparation of tissue section 21
2.8.2 Immunosatining 21
2.8.3 Fluorescence microscopy 22
2.8.4 Confocal microscopy 22
2.9 Statistical analysis 23
Chapter 3 Results 24
3.1 Accumulation of cAMP in TDAG8-exrpessing HEK293T cells 24
3.2 An increase of intracellular calcium levels by pH <6.0 extracellular acidification in TDAG8-expressing HEK293T cell 25
3.3 TDAG8-mediated calcium increase is through PTX- and U73122-sensitive pathway. 26
3.4 TRPV1 and ASIC1a are activated by pH <6.0 acidic stimulation. 26
3.5 TDAG8-mediated calcium increase is due to extracellular calcium influx through TRPV1 27
3.6 TDAG8 activation regulates TRPV1 function by PKA- and PKCε-dependent pathway at pH <6.0 28
3.7 TDAG8 activation sensitizes TRPV1 through PKA- and PKCε- dependent pathway at pH >6.0 29
3.8 Acidic stimulation sensitizes TRPV1 in IB4-positive neurons through a PKA- dependent pathway at pH >6.0 29
3.9 Acidic stimulation induces translocation of PKA and PKCε in DRG neurons 30
3.10 Small-to-medium diameter of DRG neurons express PKCε 31
3.11 CFA-induced inflammation cause paw edema and enhance capsaicin response 32
Chapter 4 Discussion 34
4.1 TDAG8 activation by proton 34
4.2 TDAG8 activation sensitizes TRPV1 36
4.3 Acidification-induced TRPV1 sensitization in DRG neurons 38
4.4 PKCε contributes to TRPV1 sensitization in CFA-induced long-term inflammation 39
Chapter 5 References 41
Appendix 77
List of figures
Figure 3-1 Changes of intracellular cAMP levels at different pH value in TDAG8-expressing HEK293T cells. 45
Figure 3-2 Changes of intracellular cAMP levels in TDAG8-expressing and G-protein-expressing HEK293T cells. 47
Figure 3-3 An increase of intracellular calcium levels in TDAG8-expressing cells exposed to pH <6.0 acidic buffers. 49
Figure 3-4 TDAG8-mediated calcium increase is through PTX- and U73122- sensitive pathway. 51
Figure 3-5 TRPV1 and ASIC1a were activated by pH <6.0 acidic stimulation. 52
Figure 3-6 TDAG8-mediated calcium increase is due to extracellular calcium influx through TRPV1 at pH <6.0. 54
Figure 3-7 TDAG8 activation regulates TRPV1 function by PKA- and PKCε- dependent pathway at pH <6.0. 56
Figure 3-8 TDAG8 activation sensitizes TRPV1 through a PKA- and PKCε- dependent pathway at pH >6.0. 58
Figure 3-9 Acidic stimulation induce TRPV1 sensitization through a PKA- dependent pathway at pH >6.0. 59
Figure 3-10 Acidic pH-stimulation induces redistribution of subcellular PKA in DRG neurons. 61
Figure 3-11 Acidic pH-stimulation induces redistribution of subcellular PKCε in DRG neurons. 63
Figure 3-12 Distribution of PKCε and non-peptidergic neurons in DRG tissue. 65
Figure 3-13 Distribution of PKCε in DRG tissue. 67
Figure 3-14 Paw thickness of mice was increased after CFA-injection 60 minutes and 25 hours. 69
Figure 3-15 Capsaicin response is not significant changed in acute inflamed DRG neurons.
70
Figure 3-16 KIE1-1 suppresses CFA-enhanced response to capsaicin in sub-acute -phase inflamed DRG neurons. 71

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指導教授

孫維欣(Wei-hsin Sun)

審核日期

2011-12-28

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