腎素-血管張力素系統 (renin-angiotensin system, RAS) 在哺乳動物調控血壓與鹽類平衡上面十分重要。過去文獻指出大鼠大腦中具有獨立的腎素-血管張力素系統，腎素為腎素-血管張力素系統中產生乙型血管張力素 (angiotensin II, Ang II) 的首要影響因子，而乙型血管張力素不管在周邊或是中樞神經皆有調控生理機能的作用。目前已有許多文獻報導腎素確實存在於大腦之中，而之前的文獻也指出腎素會透過以血管張力素為主和其他路徑來影響血壓。其中以血管張力素為主的路徑主要是透過與乙型血管張力素第一型接受器 (angiotensin II type-1 receptors, AT1R) 的結合來達成，而另一路徑則是透過與腎素接受器結合 [(pro)renin receptor, PRR] 來達成。文獻指出乙型血管張力素第一型接受器與腎素接受器都大量表現在對於中樞調控血壓上十分重要的孤立束核 (nucleus tractus solitarii, NTS)。更有文獻指出乙型血管張力素會影響一氧化氮在孤立束核的釋放，而一氧化氮對於孤立束核在中樞心臟血管系統之調控扮演極重要的角色。由此項結果可得知腎素在孤立束核中扮演著調控心血管系統的角色。但腎素如何調控血壓的分子機制目前還尚未明瞭。本研究想進一步探討腎素在孤立束核中心臟血管調控之分子機制。單側微量注射腎素到 WKY 大鼠的孤立束核中，產生明顯的血壓下降與心博舒緩現象。前處理非特異性一氧化氮合成酶抑制劑 (L-NAME) ，選擇性內皮性一氧化氮合成酶 (eNOS) 抑制劑 (L-NIO)，第四型Akt 抑制劑和PI3K 抑制劑 (LY294002) 顯著地抑制了腎素所引起的心血管作用。但是神經性一氧化氮合成酶 (nNOS) 專一性抑制劑 (Vinyl-L-NIO) 和 MEK抑制劑 (PD98059) 並不會引起顯著的變化。西方墨點分析結果顯示腎素會引起內皮性一氧化氮合成酶和Akt 磷酸化作用，而神經性一氧化氮合成酶與ERK1/2 則沒有此現象。而前處理LY294002 會顯著地降低內皮性一氧化氮合成酶和Akt 磷酸化作用。這部分的結果顯示腎素所引起的心血管作用是透過PI3K-Akt-eNOS傳遞路徑來活化內皮性一氧化氮合成酶，最後促使一氧化氮增加來影響血壓。進一步前處理腎素專一性抑制劑 (aliskiren)，血管張力素轉換酶抑制劑 (lisinopril)，第一型血管張力素接受器阻斷劑 (losartan) 和細胞內鈣離子抑制劑 (BAPTA-AM) 顯著地抑制了腎素所引起的心血管作用。但是Gβγ 蛋白抑制劑 (gallein)，PLC 抑制劑 (U73122) ，calmodulin抑制劑(W-7) 和 腎素接受器阻斷劑 (handle region peptide) 並不會引起顯著的變化。這部分的結果顯示腎素所引起的心血管作用主要是透過第一型血管張力素接受器。我們的結果顯示腎素是透過AT1R-PI3K-Akt 傳遞路徑，而後導致的內皮性一氧化氮合成酶活性增加來達到調控血壓的作用。

The renin-angiotensin system (RAS) is critical for the control of blood pressure (BP) and salt balance in mammals. Studies reveal that local RAS are present in the rat brain and renin is the first effector of the brain RAS for generating angiotensin II (Ang II) which exerts diverse physiological actions in both peripheral and central nervous system. The existence of renin within the brain has now been demonstrated by numerous studies. Previous studies suggest that renin may go through angiotensin-dependent and independent pathway to influence vascular tone, by Ang II type 1 receptor (AT1R) and renin specific (pro)renin receptor (PRR), respectively. Studies also indicate that AT1R and PRR are highly expressed in the nucleus tractus solitarii (NTS), which is important for central feedback regulation of BP. Further studies have shown that Ang II contributes to the release of NO, which plays an important role in cardiovascular regulation in the NTS. These results indicate that renin plays cardiovascular modulatory role in the NTS. However, the mechanisms how renin modulate cardiovascular functions in the NTS remained unclear. In the present study, I investigated the molecular mechanisms of renin-induced cardiovascular effects in the NTS. Unilateral microinjection of renin into the NTS of WKY rats produced prominent depressor and bradycardic effects. Pretreatment with a non-selective NOS inhibitor L-NAME, eNOS specific inhibitor L-NIO, Akt inhibitor IV, and PI3K inhibitor LY294002 significantly attenuated the cardiovascular response evoked by renin, whereas nNOS specific inhibitor Vinyl-L-NIO and MEK inhibitor PD98059 did not cause significant changes. Western blot studies showed renin increased eNOSS1177 and AktS473 phosphorylation instead of nNOSS1416 and ERK1/2T202/Y204 phosphorylation, and pretreatment with LY294002 blocked renin-induced eNOSS1177 and AktS473 phosphorylation. These results indicated that renin might go through PI3K-Akt-eNOS pathway to increase eNOS activity and ultimately result in NO release. The cardiovascular effects of renin were also attenuated by renin specific inhibitor aliskiren, angiotensin converting enzyme inhibitor lisinopril, AT1R antagonist losartan, and intracellular Ca2+ chelator, BAPTA-AM instead of G protein βγ subunit inhibitor gallein, PLC inhibitor U73122, calmodulin inhibitor (W-7) and (pro)renin receptor blocker, handle region peptide. These results indicated that renin mainly through AT1R to regulate BP. Therefore, my results indicated that the modulation of cardiovascular effects of renin in the NTS involves AT1R-PI3K-Akt pathway to activate eNOS activation.

1. Introduction………………………………………………………….....……….1
1.1 Renin and prorenin……………………………………………………………1
1.2 Angiotensin II and its receptors……………………………………………….3
1.3 Characteristics of the (pro)renin receptor……………………………………..5
1.4 Role of (pro)renin receptor in cardiovascular system and brain………...........6
1.5 Renin-angiotensin system in brainstem nuclei………………………………..7
1.6 Cardiovascular regulation by brainstem nuclei……………………………….9
1.7 Nitric oxide signaling in central cardiovascular regulation………………….11
2. Specific Aims………………………………………………………………….14
3. Materials and Methods…………………………………...………………….15
3.1 Reagents and chemicals…………………………………………….……….15
3.2 Animals……………………………………………………………………...15
3.3 Experimental Procedures……………………………………………………16
3.4 Microinjection of drugs……………………………………………………..18
3.5 Baroreflex measurement……………………………………………....…….18
3.6 Intracerebroventricular measurement……………………………………….19
3.7 Blood pressure measurement………………………………………………..20
3.8 Western blot analysis………………………………………………………...20
3.9 Determination of NO in NTS………………………...……………………....22
3.10 Immunohistochemistry analysis……………………………………….......22
3.11 Determination of renin in NTS………………………………………..…..24
3.12 Statistical analysis……………………………………………………...….26
4. Results……………………………………………………………...…….…..…27
4.1 Renin had cardiovascular effects in the NTS…………………………… …..27
4.2 Prior administration of NOS inhibitor, L-NAME, significantly attenuated the cardiovascular responses of renin in the NTS of WKY rats…...…………….......27
4.3 Renin increased NO production in the NTS of WKY rats………….…….…28
4.4 Prior administration of eNOS inhibitor, L-NIO, significantly attenuated the cardiovascular responses of renin in the NTS of WKY rats……………………..28
4.5 Renin induced eNOSS1177 phosphorylation in the NTS of WKY rats……….29
4.6 Prior administration of Vinyl-L-NIO did not attenuate the cardiovascular effects of renin in the NTS of WKY rats………………...………………………30
4.7 Renin did not increase nNOSS1416 phosphorylation and iNOS protein expression in the NTS of WKY rats……………………………………………..30
4.8 Prior administration of Akt inhibitor IV significantly attenuated the cardiovascular responses of renin in the NTS of WKY rats…………….……….31
4.9 Renin increased AktS473 phosphorylation in the NTS of WKY rats…...……. 31
4.10 Prior administration of PI3K inhibitor, LY294002, significantly attenuated the cardiovascular responses of renin in the NTS of WKY rats……………........32
4.11 Attenuation of renin induced eNOSS1177 phosphorylation by LY294002 in the NTS of WKY rats……………………………………………………………..….33
4.12 Attenuation of renin induced AktS473 phosphorylation by LY294002 in the NTS of WKY rats……...……………………………………………………...….33
4.13 Prior administration of PD98059 did not attenuate the cardiovascular responses of renin in the NTS of WKY rats……………………………..……….34
4.14 Renin did not induce ERKT202/Y204 phosphorylation in the NTS of WKY rats………………………………………………………………………………..34
4.15 Prior administration of W-7 did not attenuate the cardiovascular responses of renin in the NTS of WKY rats……………..…………….................................…35
4.16 Prior administration of renin inhibitor, aliskiren, significantly attenuated the cardiovascular responses of renin in the NTS of WKY rats………………...…...36
4.17 Prior administration of ACE inhibitor, lisinopril, significantly attenuated the cardiovascular responses of renin in the NTS of WKY rats……..….…………...36
4.18 Prior administration of AT1R antagonist, losartan, significantly attenuated the cardiovascular responses of renin in the NTS of WKY rats………………....37
4.19 Prior administration of G protein βγ subunit inhibitor, gallein, did not attenuate the cardiovascular responses of renin in the NTS of WKY rats……….38
4.20 Prior administration of PLC inhibitor, U73122, did not attenuate the cardiovascular responses of renin in the NTS of WKY rats…...………………...38
4.21 Prior administration of intracellular Ca2+ chelator, BAPTA-AM, significantly attenuated the cardiovascular responses of renin in the NTS of WKY rats……...39
4.22 The PRR existed in the NTS but may not participate the cardiovascular effects of renin in WKY rats……………………………………………………..39
5. Discussion………………………………………………………………...…......41
6. Conclusion…………………………………………………………....…………47
7. Future Perspectives…………………………………………………..……......48
8. References……………………………………………………………………....49
9. Figures and Figure Legends………………………………………………….60
F.1 Representative diagram of the set up for the stereotaxic microinjection studies on the rat brain stem nuclei……………………..……………...….……………..60

F.2 Cardiovascular effects of unilateral microinjection of renin into the NTS in the anesthetized WKY rats………………………..……………………….…..…….61

F.3 The cardiovascular effects of renin in the NTS after administration of the NOS inhibitor, L-NAME………………………………….…………….......…………63

F.4 Effects of renin on NO3- levels on the NTS of WKY rats…………..…..…...65

F.5 The cardiovascular effects of renin the NTS after administration of the eNOS inhibitor, L-NIO………………………………………….…………….………...66

F.6 Effects of renin on eNOS phosphorylation……………………...…………...68

F.7 Immunohistochemical detection of renin-dependent activation of eNOS phosphorylation in the NTS of WKY rats…………………………..……….…...70

F.8 The cardiovascular effects of renin the NTS after administration of the selective nNOS inhibitor, Vinyl-L-NIO……………………………..….………..72

F.9 Effects of renin on nNOS phosphorylation……………………..……….…...74

F.10 Effects of renin on iNOS protein expression……………………….…..…..76

F.11 The cardiovascular effects of renin the NTS after administration of the Akt inhibitor IV…………………………………….……………………………..…..78

F.12 Effects of renin on Akt phosphorylation…………………………...……….80

F.13 The cardiovascular effects of renin the NTS after administration of the PI3K inhibitor, LY294002……………………………………….………………….….82

F.14 Effects of LY294002 on renin stimulated eNOS phosphorylation………....84

F.15 The effect of PI3K inhibitor LY294002 on renin stimulated eNOS phosphorylation in the NTS of WKY rats………………………..………..……..86

F.16 Effects of LY294002 on renin stimulated Akt phosphorylation……….…...88

F.17 The cardiovascular effects of renin the NTS after administration of the MAPK inhibitor, PD98059………………………………………………..……..90

F.18 Effects of renin on ERK1/2 phosphorylation…………………………….....92

F.19 The cardiovascular effects of renin the NTS after administration of the calmodulin inhibitor, W-7……………………………………....…..……………94

F.20 The cardiovascular effects of renin the NTS after orally administration of the specific renin inhibitor, aliskiren…………………………………..….…………96

F.21 The cardiovascular effects of renin the NTS after administration of the ACE inhibitor, lisinopril…………………………………….……………..…………..98

F.22 The cardiovascular effects of renin the NTS after administration of the AT1R antagonist, losartan……………………………………….………………….…100

F.23 The cardiovascular effects of renin the NTS after administration of the G protein βγ subunit inhibitor, gallein………………….………………….….…..102

F.24 The cardiovascular effects of renin the NTS after administration of the PLC inhibitor, U73122…………………………………….………………………....104

F.25 The cardiovascular effects of renin the NTS after administration of the intracellular Ca2+ chelator, BAPTA-AM………………………………..………106

F.26 The expression of PRR in the NTS of WKY rats………………….….…..108

F.27 The cardiovascular effects of renin the NTS after administration of the PRR blocker, HRP………………………….………………………………..……….109

F.28 Proposed renin signaling pathway in the regulation of blood pressure and heart rate in the NTS……………………………………………….…………...111

10. Appendix………………………………………………………………….…..112

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