臺灣博碩士論文加值系統

(RVLM)神經元在心血管功能的調控上扮演一個重要的角色，當我們在大白鼠的前腹外側延腦的位置微量注射NMDA（0.14 nmol, 0.2μl）可引起升壓反應。在我們實驗室先前的研究中發現，靜脈給予乙醇時會選擇性的抑制大白鼠的前腹外側延腦上NMDA所引發的升壓反應的現象，此抑制作用在持續輸注乙醇後會減弱，這表示乙醇對NMDA受體的抑制作用產生急性耐受性。
曝露乙醇會誘發蛋白激酶A（PKA）訊息路徑的活化，同時 NMDA受體的功能會受到蛋白激酶A（PKA）的調節。因此假設：PKA訊息路徑參與乙醇在大鼠前腹外側延腦上，所產生急性耐受性的形成。麻醉後大鼠（300-350克）由靜脈輸注乙醇0.16克（1毫升）後，接著以每小時0.16克的速率連續輸注乙醇。在連續輸注乙醇後的第十分鐘，乙醇會抑制NMDA所誘發的升壓反應，但是乙醇的抑制作用在連續輸注後的四十分鐘卻消失了。乙醇給予後，蛋白激酶活化劑或抑制劑與NMDA一同混合投與到前腹外側延腦位置時發現：乙醇抑制作用減弱的現象會被蛋白激酶A（PKA）的抑制劑KT5720（40, 400 and 4000 fmol）或環化腺甘酸單磷酸拮抗劑cAMP-Rp（20 and 200 fmol）所阻斷，並且都具有劑量依賴性的關係；但環化腺甘酸單磷酸的活化劑cAMPS-Sp（10 pmol），蛋白激酶C（PKC）的抑制劑 chelerythine（10 pmol）及蛋白激酶C（PKC）的活化劑phorbol 12-myristate 13-acetate（2 fmol）不影響乙醇抑制NMDA受體的急性耐受性的現象。此外在乙醇產生急性耐受性時，大鼠的前腹側延腦的NMDA受體NR1次單元上，受到PKA磷酸化位置絲胺酸897的磷酸化有明顯增加，而PKC磷酸化位置絲胺酸896的磷酸化沒有改變。以上結果證實，PKA訊息路徑參與乙醇對NMDA受體的抑制作用之急性耐受性之形成。

Rostral ventrolateral medulla (RVLM) neurons play an important role in regulation of the cardiovascular function. Microinjection of NMDA into the RVLM produces increases in blood pressure. Our recent study showed that intravenous ethanol selectively inhibited NMDA-induced responses in the RVLM. The inhibitory effects of ethanol were reduced over time during continuous infusion of ethanol, suggesting the development of acute tolerance.
Exposure of ethanol has been shown to activate cAMP dependent protein kinase (PKA) signaling pathways and NMDA receptor function is regulated by PKA. We hypothesize that PKA signal may participate in development of acute tolerance to ethanol inhibition of NMDA receptor activation in RVLM. Intravenous injection of ethanol (0.16g) followed by continuous infusion of ethanol (0.16g/hr) into anesthetized SD rats (300~350g) reduced NMDA-induced pressor effect at 10 min, but the inhibition disappeared at 40 min after the injection. The disappearance of ethanol inhibition was dose-dependently blocked by PKA inhibitor KT5720 (40, 400 and 4000 fmol) or a cAMP antagonist cAMPS-Rp (20 and 200 fmol) but not by a cAMP activator cAMPS-Sp (10 pmol), PKC inhibitor chelerythine (10 pmol),and PKC activator phorbol 12-myristate 13-acetate (2 fmol); these agents together with NMDA were microinjection into the RVLM after ethanol. A significant increase in the level of PKA-regulated phosphoserine 897 but not PKC-regulated phosphoserine 896 on NMDA NR1 subunit that was found in rostroventral medulla during acute ethanol tolerance. The results suggest that PKA signaling pathways participate in acute tolerance to ethanol inhibition of NMDA receptor function.

目錄
中文摘要……………………………………………………………………………01
英文摘要……………………………………………………………………………02
壹、背景介紹………………………………………………………………………03
一、何謂乙醇？乙醇對人類的影響如何？ ……………………………… 04
二、乙醇對神經系統的影響與相關研究……………………………………05
A、乙醇對GABA神經元的影響 ………………………………………05
B、乙醇對麩胺酸鹽受體的影響………………………………………06
A. NMDA受體…………………………………………………07
B. Non-NMDA受體……………………………………………10
三、乙醇的耐受性……………………………………………………………11
A、乙醇之急性耐受性…………………………………………………11
B、乙醇之慢性耐受性…………………………………………………11
四、前腹外側延腦（RVLM）與血壓的調控…………………………………12
五、在前腹外側延腦（RVLM）上酒精抑制NMDA誘發升壓反應之急性耐受性…………………………………………………………………………12
貳、研究目的與實驗設計…………………………………………………………14
一、實驗動機…………………………………………………………………14
二、實驗假設…………………………………………………………………14
三、實驗目的…………………………………………………………………15
四、實驗設計…………………………………………………………………15
A、在RVLM上以NMDA受體的升壓反應為指標來探討乙醇的急性耐受性 …………………………………………………………………15
B、Western blotting 來探討在乙醇產生急性耐受性時前腹側延腦上NMDA受體的磷酸化情形…………………………………………17
參、詳細實驗步驟…………………………………………………………………19
一、活體實驗…………………………………………………………………19
二、Western blotting 來觀察乙醇產生急性耐受性時，在前腹側延腦上NMDA受體的磷酸化情形…………………………………………20
三、實驗藥品及統計分析方法………………………………………………24
肆、結果……………………………………………………………………………25
一、投與NMDA至RVLM誘發升壓反應………………………………………25
二、乙醇抑制NMDA誘發升壓反應的急性耐受性 …………………………25
三、蛋白激酶活性對乙醇急性耐受性之影響 ……………………………26
A、直接抑制PKA………………………………………………………26
B、間接抑制PKA………………………………………………………27
C、間接活化PKA………………………………………………………27
D、抑制PKC……………………………………………………………28
E、活化PKC……………………………………………………………28
四、蛋白激酶活性對乙醇急性耐受性之影響以及酒精對NMDA受體磷酸化的影響………………………………………………………………29
A、前腹側延腦的NMDA受體上NR1次單元Serine897的磷酸化 …29
B、前腹側延腦的NMDA受體上NR1次單元Serine896的磷酸化 …29
伍、討論……………………………………………………………………………31
一、乙醇的急性耐受性的產生與cAMP-PKA訊息路徑 ……………………31
二、乙醇的急性耐受性的產生與PKC………………………………………33
陸、結論……………………………………………………………………………34
柒、參考文獻………………………………………………………………………35
捌、結果附圖………………………………………………………………………40
玖、附錄……………………………………………………………………………48

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