孤立束核(nucleus tractus solitarii, NTS)是感壓反射中第一個整合中樞，會將接收來自周邊感壓接受器的壓力訊號，同時和中樞神經系統中血壓調控中樞所下傳的訊號進行整合，因此孤立束核本身即扮演重要的調控角色。我們過去的研究顯示，在孤立束核中微量注射尼古丁(Nicotine)會導致血壓下降及心博舒緩的現象，由此項結果可知尼古丁在孤立束核中扮演著調控心血管系統的角色。本研究想進一步探討尼古丁在孤立束核中導致血壓下降及心跳下降的分子機制。我們使用WKY大鼠為動物模式，先微量注射一氧化氮合成酶(NOS)或ionotropic glutamate receptor抑制劑後，再觀察尼古丁所引起之心血管作用對抑制劑的反應。我們的結果發現，在孤立束核中微量注射尼古丁會使一氧化氮的產量增加。前處理calmodulin抑制劑(W-7)，非特異性一氧化氮合成酶抑制劑(L-NAME)和選擇性內皮細胞一氧化氮合成酶(eNOS)抑制劑(L-NIO)顯著地抑制了尼古丁所引起的心血管作用。同樣地，尼古丁的心血管作用會被選擇性NMDA接受器抑制劑所抑制，而不是被非NMDA接受器抑制劑所抑制。但是在西方墨點法及免疫組織染色的結果並沒有顯示出eNOS的磷酸化會因為尼古丁的作用而增加。我們進一步地使用MEK抑制劑(PD98059)及特異性神經細胞一氧化氮合成酶(nNOS)抑制劑(Vinyl-L-NIO和7-NI)發現尼古丁所引起的心血管作用不會被抑制，並且Akt的磷酸化也沒有因為尼古丁的作用而增加。我們的結果顯示尼古丁是透過calmodulin與eNOS結合，導致eNOS的活性增加，進而刺激麩胺酸的釋放以達到調控血壓的作用。

The nucleus tractus solitarii (NTS) is the primary integrative center for baroreflex. NTS not only integrates convergent information from peripheral baroreceptors and higher blood pressure (BP) control centers in CNS but itself is the site of substantial modulation. Our previous studies demonstrated that microinjection of nicotine into the NTS decrease BP and heart rate (HR), which indicates nicotine plays cardiovascular modulatory role in the NTS. However, the mechanisms how nicotine modulate cardiovascular functions in the NTS remained unclear. The aim of this study was to investigate the molecular mechanisms of nicotine-induced depressor and bradycardic effects in the NTS. Male anesthetized Wistar-Kyoto rats, with or without intra-NTS nitric oxide synthase (NOS) inhibitors or ionotropic glutamate receptor inhibitors pretreatment, received intra-NTS nicotine microinjection. BP and HR were monitored. Besides, NTS with/without nicotine microinjection were dissected and subjected to immunoblotting and nitric oxide (NO) analysis. Our results demonstrated that NO analysis study revealed intra-NTS NO production elevated after nicotine microinjection. The depressor and bradycardic effects of intra-NTS nicotine microinjection were diminished by pretreatment of calmodulin inhibitor (W7, 0.33 nmol), non-specific NOS inhibitor (L-NAME, 33 nmol) and eNOS specific inhibitor (L-NIO, 6 nmol). The cardiovascular effects of nicotine were also attenuated by NMDA receptor inhibitor (MK801, 1 nmol), not by non-NMDA receptor inhibitor (NBQX, 10 pmol). Immunoblotting and immunohistochemical studies did not revealed nicotine induced eNOSS1177 phosphorylation in NTS. Using MEK inhibitor, PD98059, and nNOS specific inhibitor,Vinyl-L-NIO and 7-NI, there were no effect on the depressor and bradycardic effects of intra-NTS nicotine microinjection, and the phosphorylation of AktT473 was not induced by nicotine. Therefore, our results indicate that nicotine-induced depressor and bradycardic responses maybe mediated through activating eNOS by calmodulin and stimulating glutamate release in the NTS.

Introduction+1
Specific Aims+10
Materials and Methods+11
Results+19
Discussion+27
Conclusion+33
Future Perspectives+34
References+36
Figures and Figure Lengend+43
Appendix+76

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