View : 234 Download: 0

Full metadata record

DC Field Value Language
dc.contributor.author노지현-
dc.creator노지현-
dc.date.accessioned2016-08-26T12:08:09Z-
dc.date.available2016-08-26T12:08:09Z-
dc.date.issued2005-
dc.identifier.otherOAK-000000012307-
dc.identifier.urihttp://dspace.ewha.ac.kr/handle/2015.oak/191434-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000012307-
dc.description.abstractThe neuroprotective actions of aspirin-derivatives, sulfasalazine (SULFA) and Neu2000 (NEU) have been recently reported. However, it is little known about the molecular mechanisms underlying the neuroprotective effect of SULFA and NEU. Therefore, in this Thesis, I will introduce the potential mechanisms for their roles as a neuroprotectant that, for the past several years, I have worked for. First, using a whole-cell voltage-clamp configuration, I have examined the effects of both drugs on either NMDA or non-NMDA responses of cultured single cortical neuron. SULFA acted as a typical noncompetitive inhibitor with neither agonist- nor use-dependency, and antagonized NMDA-evoked responses in a voltage-independent manner, suggesting that SULFA is not an open channel blocker. Meanwhile, NEU reduced NMDA responses uncompetitively. Its effect on NMDA receptor was dependent on an agonist concentration but not on a membrane potential. Single channel analyses showed that both drugs blocked NMDA responses by reducing the channel open probability without changing single channel conductance. Moreover, they accelerated the rate of NMDA desensitization without affecting the affinity of the receptor for NMDA, by decreasing the binding affinity for glycine in an allosteric manner. Interestingly, I found that NEU, unlike SULFA, potentiated the kainate (KA)-evoked responses in a noncompetitive manner. NEU partially augmented the apparent affinity of the receptor for KA and potentiated KA responses by increasing the channel opening probability without changing single channel conductance. Next, using either extracellular recording or thin slice patch recording, I have traced any characteristics of synaptic activities on rodent neuronal network by NEU that reduced the NMDA receptor activities and potentiated non-NMDA receptor activities. NEU attenuated the seizure-like synchronized spikes induced in entorhinal cortex by increasing excitatory activity and blocking inhibitory activity as well. NEU also lessened the paroxysmal activities in hippocampus CA3 induced by a hypoxic condition and increased the rate of recovery from the hypoxia-induced impairment. Interestingly and importantly, 100μM NEU which blocks NMDA response by 70% in a cultured cortical neuron displayed virtually little effect on NMDA receptor-dependent postsynaptic activity in slice circuit. It clearly says that NEU reduces NMDA response much more under pathological condition than physiological condition, suggesting that it may mitigate pathological condition in CNS. Therefore, I propose here that NEU should be an attractive pharmacological candidate for future drug development.;아스피린(aspirin) 유도체인 sulfasalazine(이하 SULFA)과 Neu2000(이하 NEU)은 NMDA 수용체를 통해 유도되는 신경세포사멸을 현저하게 막았다. 이는 SULFA 및 NEU가 glutamate 에 의한 신경세포사멸(glutamate toxicity) 를 저지할 수 있는 약물로써의 강력한 후보 물질의 가능성을 제안하는 것인데, 그러나 그들의 명확한 작용 기전에 대해서 보고된 바는 아직 없다. 본 논문은 설치류 뇌에서 SULFA 및 NEU 가 glutamate 수용체에 대한 강력한 조절자(modulator)로써 작용하는 약리학적 기전을 다양한 전기생리학 기법을 통해 조사하였다. 우선 배양된 대뇌피질 단일 신경세포(cultured single cortical neuron)에서 SULFA 및 NEU 가 NMDA/non-NMDA 수용체에 미치는 영향을 whole-cell voltage clamp recording 기법을 통해 살펴보았다. SULFA는 NMDA 수용체에 대하여 전형적인 noncompetitive antagonist로써 작용한 반면 NEU는 agonist의 농도에 따라 그 작용효과가 달리 나타나는 uncompetitive antagonist 로써 작용하였다. SULFA와 NEU 모두 막전위에서는 무관하게 작용하였고, open channel blocker 로써의 가능성은 관찰되지 않았다. 두 약물이 NMDA 수용체를 저지하는 방법은 주로 channel open probability 의 감소와 NMDA 수용체의 desensitization 속도의 증가에 의한 것이었고, 또한 NMDA 수용체에 co-against로 작용하는 glycine 에 대한 affinity 를 alloteric 하게 감소시키는 효과에 의한 것이었다. 흥미롭게도 NEU는 SULFA와 달리 kainate 에 의해 유도되는 non-NMDA 수용체의 역할을 noncompetitive 방식으로 증가시켰다. Non-NMDA 수용체에 대한 NEU의 증가 기전은 수용체에 대한 kainate의 affinity 의 증가에 의하며 또한 single channel conductance의 변화없이 channel opening probability 의 증가에 의하 것으로 판명되었다. 다음으로 단일 신경세포에서의 NEU에 의한 NMDA 및 non-NMDA 수용체의 변화를 근거로 쥐의 뇌 신경망에서의 NEU에 의한 변화를 extracellular recording과 thin slice patch recording 기법을 통해 살펴보았다. NEU는 간질(seizure)을 유도한 조건에서 발생된 synchronized spike 를 흩트리는 방식으로 좀더 정상적인 spike 모습으로 변화시켰으며, 이러한 현상은 흥분성 신경전달(excitatory neurotransmission)을 증가시키고 억제성신경전달(inhibitory neurotransmission)을 감소시키는 조건을 동시에 주었을 때 관찰되었다. 한편 좀더 명확한 NEU의 병리학적 역할을 살펴보기 위하여 간질과는 좀더 다른 방식으로 병리학적 조건을 유도하여 보았다. NEU는 신경망 내에서 산소결핍(hypoxia) 및 허혈(ischemia)과 같은 병리학적 조건에서 관찰된느 순간적으로 유발된 폭발적 spike의 수의 증가를 막아주었고, 병리학적 조건이 유도된 후 정상 상태로 회복되는 속도 또한 빠르게 진행 할 수 있도록 도와주었다. 흥미롭게도 단일세포에서 관칠된 바와 유사하게 신경망에서도 NEU가 NMDA 수용체에 대해서 저지하는 기전을 보일지를 알아보기 위하여, 신경망에서 NMDA component 관련 EPSC 에 대한 NEU의 역할을 살펴본 결과, 단일세포에서 직접적인 NMDA의 처리에 의해 유도되는 NMDA current를 매우 현저하게 감소시켰던 NEU의 농도에 대해서 신경망 내 NMDA 수용체를 통해 유발되는 EPSC 변화를 관찰할 수 없었다. 이는 NEU의 역할이 정상적인 상태의 신경망 패턴에는 크게 영향을 주지 않지만, 매우 비정상적으로 NMDA receptor의 활성이 발생하였을 때에는 그 현상을 감쇠키는 것으로 병리학적 상황을 완화시켜 줄 수 있는 가능성을 지니고 있음을 시사하여, 따라서 NEU는 미래의 신경세포사멸과 관련된 질병을 위한 약무로써의 후보로써 뿐만 아니라 좀더 효과적인 약물 개발을 위한 발판으로써 제안된다.-
dc.description.tableofcontentsAbstract I (English) = xi Abstract II (Korean) = xiii Chapter I. General Introduction = 1 Glutamate receptors = 2 Glutamate and neurodegeneration = 5 Pharmacological inhibition of glutamate toxicity-NMDA receptor antagonist = 6 Role of aspirin-derivatives as NMDA receptor antagonist = 10 Aims of this study = 11 References = 12 Chapter II. Underlying mechanism for NMDA receptor antagonism by the anti-inflammatory drug, sulfasalazine, in mouse cortical neurons = 21 Introduction = 22 Materials and Methods = 25 Preparation of mouse cortical neurons = 25 Electrophysiological recordings = 26 Stationary noise analysis of membrane current fluctuation = 26 Single-channel recordings = 28 Solutions and chemicals = 29 Results = 30 SULFA acts as an antagonist for NMDA receptors = 30 Agonist- and use-independent antagonism of SULFA = 34 Noncompetitive antagonism of SULFA = 37 Analysis of SULFA kinetics = 39 Acceleration of NMDA desensitization by SULFA = 40 SULFA-induced reduction of the apparent open channels of the NMDA receptor: noise analysis = 44 Reduction of the channel opening frequency: single channel recording = 46 Voltage-independent NMDA current reduction by SULFA treatment = 49 Potential linkage of SULFA sites to NMDA receptor glycine site = 53 Potential linkage of SULFA sites to NMDA receptor polyamine site = 56 Discussion = 60 Significance of the SULFA-induced blockade of NMDA receptors = 60 Pharmacological profiles of SULFA = 62 Molecular mechanisms underlying the SULFA blockade of NMDA signals = 64 A new model for drug development? = 66 References = 69 Chapter III. Underlying mechanism for NMDA receptor antagonism by Neu2000 in mouse cortical neurons = 77 Introduction = 78 Materials and Methods = 80 Preparation of mouse cortical neurons & Electrophysiological recordings = 80 Single-channel recordings = 80 Solutions and chemicals = 80 Results = 81 NEU acts as an antagonist for NMDA receptors = 81 Agonist-dependent antagonism of NEU = 84 Uncompetitive antagonism of NEU = 85 Voltage- and use-independent NMDA current reduction by NEU treatment = 90 Acceleration of NMDA desensitization by NEU = 91 Reduction of the channel opening frequency: single channel recording = 96 Potential linkage of NEU to glycine and polyamine sites of NMDA receptor = 98 Discussion = 105 Molecular mechanisms underlying the NEU blockade of NMDA signals = 105 Pharmacological significance = 107 References = 111 Chapter IV. Underlying mechanism for non-NMDA response potentiation by Neu2000 in mouse cortical neurons = 115 Introduction = 116 Materials and Methods = 118 Preparation of mouse cortical neurons & Electrophysiological recordings = 118 Stationary noise analysis of membrane current fluctuation = 118 Solutions and chemicals = 118 Results = 119 NEU as a potentiator for non-NMDA receptors = 119 Effect of NEU on KA binding kinetics = 122 NEU augmentation of the channel opening probability by NEU = 125 NEU action on glutamate responses = 127 Discussion = 132 Molecular mechanisms underlying the augmentation of non-NMDA signals by NEU = 132 Significance of the NEU-induced augmentation of KA responses = 133 References = 135 Chapter V. General Discussion = 139 References = 145 Appendix I. Effects of Neu2000 on synaptic responses in cortical network = 149 Introduction = 150 Materials and Methods = 152 Entorhinal cortex slice preparation = 152 Spontaneous extracellular recordings = 152 Results = 154 Mg2+ free-induced epileptiform activity in rat entorhinal cortex = 154 Effects of NEU on Mg2+ free-induced epileptiform activity in rat entorhinal cortex = 155 Effects of NEU on Mg2+ free-induced epileptiform activity with inhibitory transmission blockade in rat entorhinal cortex = 156 Discussion = 158 References = 161 Appendix II. Effects of Neu2000 on synaptic activities in rat hippocampus = 171 Introduction = 172 Materials and Methods = 174 Hippocampus Slice preparation = 174 Induction of hypoxia conditions = 174 Spontaneous extracellular recordings = 175 Electrophysiological slice patch recordings = 175 Results = 177 Effects of NEU on hypoxia-induced abnormal activity in rat hippocampus = 177 Effects of NEU on physiological activity in rat hippocampus = 178 Discussion = 180 References = 184 Curriculum Vitae = 193-
dc.formatapplication/pdf-
dc.format.extent3297049 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.titleMechanisms for the role of sulfasalazine and Neu2000 as glutamate receptor modulators in rodent central nervous system-
dc.typeDoctoral Thesis-
dc.creator.othernameNOH, JI-HYUN-
dc.format.pagexv, 197 p.-
dc.identifier.thesisdegreeDoctor-
dc.identifier.major대학원 생명과학과-
dc.date.awarded2006. 2-
Appears in Collections:
일반대학원 > 생명·약학부 > Theses_Ph.D
Files in This Item:
There are no files associated with this item.
Export
RIS (EndNote)
XLS (Excel)
XML


qrcode

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

BROWSE