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바닐로이드 수용체 (TRPV1) 활성화에 의한 in vivo and in vitro 중뇌세포 사멸 및 그 기전 연구
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 진병관 | - |
| dc.contributor.author | 김상룡 | - |
| dc.date.accessioned | 2018-11-08T06:58:45Z | - |
| dc.date.available | 2018-11-08T06:58:45Z | - |
| dc.date.issued | 2006-02 | - |
| dc.identifier.other | 1144 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/4718 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의학계열,2006. 2 | - |
| dc.description.tableofcontents | ACKNOWLEGEMENTS i ABSTRACT ii TABLE OF CONTENT v PAGE of FIGURES x LIST of TABLE xi LIST of ABBREVIATION xii I. INTRODUCTION 1 1. Transient receptor potential vanilloid subtype 1 (TRPV1) 1 1.1. Character of TRPV1 1 1.1.1. Anandamide (AEA) 2 1.1.2. 12-hydroperoxyeicosatetraenoic acid (12-HPETE) 3 1.1.3. TRPV1 antagonists 5 1.2. Distribution of TRPV1 6 1.3. Activation of TRPV1 8 1.3.1. Skin 9 1.3.2. Inner ear 9 1.3.3. Urinary tract 10 1.3.4. Airways 11 1.3.5. Brain 12 2. Cannabinoid type 1 (CB1) receptor 13 2.1. Character and role of CB1 receptor 13 2.1.1. Neuro-toxicity by CB1 receptors 15 2.1.2. Neuro-protection by CB1 receptors 16 3. Microglia in the CNS 17 3.1. Characterization of microglia 17 3.2. The normal and protective effect of microglia 18 3.3. The deleterious role of microglia 20 4. TRPV1 and CB1 receptor 21 4.1. Functional cross-talk between TRPV1 and CB1 receptors 21 4.2. Significance of TRPV1 and CB1 receptors in the central nervous system 22 5. The release of cytochrome c and activation of caspase-3 are mportant regulators on cell death of dopaminergic neurons 23 6. Aims of study 25 II. MATERIALS AND METHODS 26 1. Chemicals 26 2. Neuron-enriched mesencephalic cell cultures 27 3. Cortical microglia or astrocyte cultures 27 4. Primary human microglial cell line culture 28 5. Reverse-transcription-polymerase chain reaction (RT-PCR) 28 6. Western immunoblot analysis 29 7. Live and dead assay 30 8. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay 31 9. LDH release assay 32 10. Immunohistochemistry 32 11. Stereological cell counts 33 12. Measurement of intracellular Ca2+ 35 13. Mitochondrial morphology assay 35 14. Stereotaxic injection of drugs 36 15. Tomato lectin histochemistry 36 16. Statistical analysis 37 III. RESULTS 38 Part A. Transient receptor potential vanilloid subtype 1 (TRPV1) mediates cell death of mesencephalic dopaminergic neurons in vivo and in vitro 38 1. Expression of TRPV1 and CB1 receptors in neuron-enriched mesencephalic culture 38 2. TRPV1-mediated neurotoxicity in mesencephalic cultures 40 3. Ca2+ influx through TRPV1 and subsequent mitochondrial disruption contribute to degeneration of DA neurons in vitro 44 4. Effects of caspase-3 inhibitor 49 5. TRPV1-mediated neurotoxicity in the SN in vivo 52 Part B. Cannabinoid type 1 (CB1) receptors contribute to neurodegeneration in response to functional cross-talk with TRPV1 in neuron-enriched mesencephalic cultures 56 1. Effects of HU210 in neuron-enriched mesencephalic cultures 56 1.1. HU210- or WIN 55,212-2-mediated toxicity in neuron-enriched mesencephalic cultures 60 2. Ca2+ influx through TRPV1 via CB1 receptors and subsequent mitochondrial disruption in neuron-enriched mesencephalic cultures 66 3. 12-HPETE mediates neurotoxicity in the SN in vivo 75 Part C. Microglia express TRPV1 and this receptor mediates toxicity against microglia in vivo and in vitro, including immortalized human microglial cell line, HMO6 78 1. Expression of TRPV1 in microglia 78 2. TRPV1 mediates cell death of microglia in cultures 81 3. Ca2+ influx through TRPV1, subsequent mitochondrial disruption and cleaved caspase-3 contribute to cell death of microglia 82 4. TRPV1-mediated toxicity in human microglial cell line cultures 87 5. TRPV1 mediates cell death of microglia in the SN in vivo 91 IV. DISCUSSION 99 Part A. Transient receptor potential vanilloid subtype 1 (TRPV1) mediates cell death of mesencephalic dopaminergic neurons in vivo and in vitro 99 Part B. Cannabinoid type 1 (CB1) receptors contribute to neurodegeneration in response to functional cross-talk with TRPV1 in neuron-enriched mesencephalic cultures 103 Part C. Microglia express TRPV1 and this receptor mediates toxicity against microglia in vivo and in vitro, including immortalized human microglial cell line, HMO6 108 V. SUMMARY AND CONCLUSION 114 VI. BIBLIOGRAPHY 115 국문요약 147 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 바닐로이드 수용체 (TRPV1) 활성화에 의한 in vivo and in vitro 중뇌세포 사멸 및 그 기전 연구 | - |
| dc.title.alternative | Sang Ryong Kim | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Sang Ryong Kim | - |
| dc.contributor.department | 일반대학원 의학계열 | - |
| dc.date.awarded | 2006. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 565208 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000001144 | - |
| dc.description.alternativeAbstract | Transient receptor potential vanilloid subtype 1 (TRPV1, also known as VR1) has an oligomeric structure formed by subunits having six transmembrane segments with a pore domain formed by the fifth and sixth transmembrane regions. This receptor is a nonselective cation channel activated by the vanilloids or products of lipoxygenases, and protein kinase C and phospholipase C mediate the sensitization of TRPV1. Moreover, the widespread distribution of TRPV1 including substantia nigra (SN) in the brain has suggested that this receptor plays a significant role in the central nervous system (CNS). However, little is known about toxicity via TRPV1 in the SN. Intranigral injection of the TRPV1 agonist capsaicin (CAP) into the rat brain, or treatment of rat mesencephalic cultures with CAP, resulted in cell death of dopaminergic (DA) neurons, as visualized by immunocytochemistry. This in vivo and in vitro effect was ameliorated by the TRPV1 antagonist capsazepine (CZP) or iodo-resiniferatoxin (I-RTX), suggesting the direct involvement of TRPV1 in neurotoxicity. In cultures, both CAP and anandamide (AEA), an endogenous ligand for both TRPV1 and cannabinoid type 1 (CB1) receptors, induced degeneration of DA neurons, increases in intracellular Ca2+ ([Ca2+]i), and mitochondrial damage, which were inhibited by CZP, the CB1 antagonist AM251 or the intracellular Ca2+ chelator BAPTA/AM. We also found that CAP or AEA increased mitochondrial cytochrome c release as well as immunoreactivity to cleaved caspase-3, and that the caspase-3 inhibitor z-DEVD-fmk protected DA neurons from CAP- or AEA-induced neurotoxicity. Additional studies demonstrated that treatment of mesencephalic cultures with CB1 receptor agonists HU210 or WIN 55,212-2 also produced degeneration of DA neurons and increases in [Ca2+]i, which were inhibited by CZP, AM251 or BAPTA/AM. The CAP-, AEA-, HU210-, or WIN 55,212-2-induced increases in [Ca2+]i were dependent on extracellular Ca2+, with significantly different patterns of Ca2+ influx. Surprisingly, CZP and AM251 reversed HU210-, WIN 55,212-2 or CAP-induced neurotoxicity by inhibiting Ca2+ influx, respectively, suggesting the existence of functional cross-talk between TRPV1 and CB1 receptors. Moreover, 12-hydroperoxyeicosatetraenoic acid (12-HPETE, known as TRPV1 agonist) produced via activation of CB1 receptors by HU210 or WIN 55,212-2 induced neuronal toxicity via activation of TRPV1 in mesencephalic cultures, and intranigral injection of 12-HPETE into the rat brain also resulted in neuronal cell death. In addition, this study examined whether microglia express TRPV1 and activation of TRPV1 contributes to cell death of microglia. In cultures, RT-PCR, Western blot analysis and immunocytochemical staining showed that microglia, but not astrocytes, expressing TRPV1 underwent cell death following the treatment with TRPV1 agonist CAP or RTX. Moreover, treatment with CAP or RTX induced cell death of immortalized human microglial cell line HMO6 expressing this receptor. CAP- or RTX-induced cell death of microglia was accompanied by increases in cytosolic Ca2+ concentration in the presence of extracellular Ca2+ and mitochondrial damage as well as mesencephalic neurons. This toxicity was also ameliorated by TRPV1 antagonists or BAPTA/AM, suggesting involvement of increases in cytosolic Ca2+ via influx through the direct activation of TRPV1. Additional study demonstrated that intranigral injection of CAP or 12-HPETE into the rat brain produced cell death of microglia, but not astrocytes in the SN, visualized by immunocytochemistry, and this in vivo effect was ameliorated by CZP or I-RTX, suggesting involvement of TRPV1 in the toxicity. This study is the first to demonstrate that the activation of TRPV1 and/or CB1 receptors mediates cell death of DA neurons, microglia express TRPV1, and activation of TRPV1 also mediates cell death of microglia. The findings in this study suggest that these two types of receptors, TRPV1 and CB1 receptors, may contribute to neurodegeneration in response to endogenous ligands such as AEA or 12-HPETE. | - |
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