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中文關鍵字 血壓;自主神經系統;交感神經;腦幹;下視丘

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• 系統編號 RG9811-0248

• 計畫中文名稱 中樞神經系統在肥胖型高血壓所扮演的角色:prolactin-releasing peptide 與 leptin

• 計畫英文名稱 --

• 主管機關 行政院衛生署 • 計畫編號 NHRI-EX98-9605NI

• 執行機構 台北醫學大學醫學系

• 本期期間 9801 ~ 9812

• 報告頁數 32 頁 • 使用語言 中文

• 研究人員 黃玲玲;戴妤妏;;;; Hwang, Ling-Ling;Dai, Yu-Wen;;;;

• 中文關鍵字 血壓;自主神經系統;交感神經;腦幹;下視丘;;;

• 英文關鍵字 orexin;RVLM;receptor;sympathetic;hypothalamus;brain stem;;

• 中文摘要

在台灣,高血壓及其相關疾病是主要死亡原因之一,肥胖是導致高血壓的主要危險因子之一,許多調控體內能量恆定的神 經信號分子亦會影響血壓恆定,因此,我們的主要假說為,調控體內能量恆定的神經信號分子藉由作用於自主神經系統,

與肥胖和高血壓的致病機轉有所連結。因此,本研究的主要目的在於評估這些神經信號分子在中樞自主神經上調節心血管 功能的作用,並試圖找出可能造成高血壓之關鍵因子。在本計畫的第三年,我們繼續前一年的研究探討一個能量恆定的神 經信號分子(稱為 orexin)對心血管與交感神經系統之影響。以下簡述我們所完成的事項。 (1) Orexin 對 RVLM 神經細胞之影 響 Orexin A 與 orexin B 是下視丘神經月生 肽 ,透過作用於 orexin 1 receptor (OX1R)與 orexin 2 receptor (OX2R)兩種受器,

它們參與多種生理功能之調節,當 orexin 被注入心血管中樞 rostral ventrolateral medulla (RVLM)核區後,動脈壓與心跳速率 上升,因此我們以神經電生理方法研究探討 orexin 對 RVLM 神經細胞之影響及其作用機轉,作用機轉之探討包括參與 orexin 作用之受器與神經細胞種類。 由去年的初步研究數據得知 orexin 使 RVLM 神經細胞產生去極化反應(即興奮反應),去年亦 初步評估 orexin 受器擷抗劑及致效劑之效用,現在我們大致完成相關實驗,並整理成果如下。Orexin A 與 B 使 RVLM 神經 細胞去極化之作用於 30~300 nM 濃度範圍內呈現濃度與效用之正相關現象,於 100 nM,orexin A or B (100 nM)各可興奮 42%RVLM 神經細胞,河豚毒素(tetrodotoxin)無法阻斷 orexin 在 RVLM 神經細胞誘發之去極化。OX1R 擷抗劑 SB-334867 存在下,orexin A (100 nM)依舊使 42% RVLM 神經細胞去極化,其去極化程度略微減小(4.9 0.8 vs. 7.2 1.1 mV)  ,但此減 小並未達統計差異。另外,OX2R 擷抗劑 Compound 29 存在下,受 orexin A (100 nM)興奮之 RVLM 神經細胞降低為 25%,

同時 orexin A 誘發之去極化有意義地大幅降低(1.7 0.5 mV) 。同時投與 SB-334867 與 Compound 29 完全抑制 orexin A 誘發

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之去極化。OX2R 致效劑[Ala11,D-Leu15]-orexin B 則具類似 orexin 的作用,亦能使 RVLM 神經細胞產生去極化反應,並具 有濃度與效用之正相關現象。我們進一步在 60 個 RVLM 神經細胞測試其對 100 nM orexin A 與 B 之反應,發現其中 27%RVLM 神經細胞可被 orexin A 與 B 興奮,但是約 10%與 8%RVLM 神經細胞只能被 orexin A 或 orexin B 所興奮。至於受 orexin 興 奮的 RVLM 神經細胞之種類,我們的研究發現約 88% RVLM 核區內 adrenergic 神經細胞及 43% non-adrenergic 神經細胞可 被 orexin 興奮。被 orexin 興奮之 RVLM 神經細胞亦包括該核區內之自發地節律性行奮型神經細胞。根據上述結果,我們的 結論是 orexin 可直接興奮 RVLM 神經細胞,這些細胞包括調節心血管功能之神經細胞,此作用主要透過 OX2R,OX1R 亦 有些許貢獻。這些結果曾以海報論文方式發表於 The 12th Society of Chinese Bioscientists in America International Symposium。

(2) 投射至 RVLM 之 orexin 神經細胞表現 M3 muscarinic 受器 此部分研究乃延續去年的研究。orexin 神經細胞位於下視丘 之 lateral 與 perifornical 區域,曾有研究顯示 orexin 神經細胞是連結心血管交感系統活性與精神壓力及 somatomotor 相關神 經結構的其中一個主要整合中心,RVLM 核區內有一群神經細胞直接投射至並興奮脊髓內之心血管交感節前神經細胞,將 orexin 注射入 RVLM 可引發升血壓作用,因此,由 orexin 神經細胞至 RVLM 的神經投射可能參與 orexin 對心血管及交感 神經之興奮作用。研究報告指出 orexin 神經細胞中只有一小部分受到 acetylcholine 調節,此調節是透過 M3 muscarinic 受器,

本研究透過檢視投射至 RVLM 之 orexin 神經細胞(RVLM-projecting orexin 神經細胞)是否表現 M3 muscarinic 受器,以了解 acetylcholine 是否透過 orexin 神經細胞參與調節心血管及交感神經系統。使用神經細胞軌跡追蹤(track-tracing)與免疫組織化 學染色等技術,我們發現 RVLM-projecting orexin 神經細胞密集地聚集在與追蹤染劑注射側同側的腦室旁核,並且分散在同 側的下視丘外側區域,該區亦為 orexin 神經元主要所在區域。大約 15% RVLM-projecting 下視丘外側區域神經細胞為 orexin 神經細胞,M3 muscarinic 受器的確存在於一小群 RVLM-projecting orexin 神經細胞上。曾有報告(Sakurai, et al., 2005)指出,

orexin 神經細胞上之 cholinergic 神經投射來自 basal forebrain cholinergic neurons,這些 cholinergic 神經細胞於清醒與快速動 眼期睡眠狀況下活性增加,因此 RVLM-projecting orexin 神經細胞受 M3 muscarinic 受器調節一事,可能與清醒與快速動眼 期睡眠狀況下之心血管功能調節有關。這些研究結果曾以海報論文方式發表於 the XXXVIth International Congress of

Physiological Sciences (IUPS) in July, 2009 in Japan。 (3) Orexin 對大鼠血壓之調節 如上述所提,orexin 系統在調控心血管功 能上扮演著重要的角色。orexin 系統可能在維持基礎血壓上扮演要角,因為研究報告指出 orexin 系統功能異常之人類與動 物之動脈壓低於正常值,除此之外, orexin 系統參與壓力引發之心血管反應(Kayaba et al., 2003)。Orexin 參與這些功能調控 之作用位置究竟在哪裡?我們的研究結果顯示 RVLM 可能是其中一個重要作用位置,除此之外,位於下視丘之 paraventricular nucleus (PVN)參與心血管系統功能之調節是早已確定的,orexin 在此核區亦有很顯著的作用。 我們目前正正在測試 orexin 系統透過 RVLM 與 PVN 在正常大鼠之血壓調控功能。這系列的實驗還在持續進行,並且已有初步成果。

• 英文摘要

Hypertension and its related diseases have been one of the leading causes of death in Taiwan. Obesity is one of the major risk factors of hypertension. Many neuronal signaling molecules of energy homeostasis (NSMEH) were found to elevate arterial pressure and promote sympathetic activity. Our main hypothesis is that the NSMEH link the pathogenic mechanisms of obesity and hypertension through their actions on the autonomic nervous system. The aim of our study is to evaluate the effects of these molecules in central

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autonomic regulation of cardiovascular functions and to identify potential molecules of the development of hypertension. In the third year of the study, we have continued to explore the effects and mechanisms of an NSMEH, orexin, in the central regulation of

cardiovascular and sympathetic system and have accomplished the following works in the third year. (1) Effects of orexin on the RVLM neurons Hypothalamic neuropeptides, orexin A and B, are involved in various physiological functions by acting on orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R). Microinjections of orexins into the rostral ventrolateral medulla (RVLM) elevated arterial pressure and heart rate. This study aimed to determine the effects of orexins on the activity of RVLM neurons and

characterize the receptor subtype(s) and neuronal phenotype(s) involved in the effects of orexins. In last year, we had observed preliminary data of orexin-induced depolarization in the RVLM neurons recorded from rat brainstem slices. The effects of orexin receptor antagonist and agonist had also been started last year. We now have collected significant data to show that orexin A and B (30~300 nM) concentration-dependently depolarized RVLM neurons. Orexin A or B (100 nM) excited 42% of RVLM neurons.

Tetrodotoxin failed to block orexin-induced depolarizations. In the presence of SB-334867, an orexin 1 receptor (OX1R) antagonist, orexin A excited 42% of RVLM neurons with a smaller, but not significantly different, amplitude of membrane depolarizations (4.90.8 vs. 7.21.1 mV). In the presence of Compound 29, an orexin 2 receptor (OX2R) antagonist, the percentage of orexin A-excited neurons decreased to 25% and orexin A-induced depolarizations became significantly smaller (1.70.5 mV).

Co-application of SB-334867 and Compound 29 completely eliminated orexin A-induced depolarization in all neurons tested. An OX2R agonist, [Ala11,D-Leu15]-orexin B, concentration-dependently depolarized RVLM neurons. In 60 RVLM neurons tested for their responsiveness to orexin A and B (100 nM), 27% were excited by either peptides, while 10% and 8% were excited only by orexin A or B. Regarding neuron phenotypes, 88% of adrenergic neurons and 43% of non-adrenergic neurons were depolarized by orexins. The orexin-excited RVLM neurons also included rhythmically firing neurons. We conclude that orexins may directly excite RVLM neurons, which include bulbospinal vasomotor neurons, mainly via OX2R, with a smaller contribution from OX1R. These results were published as a poster presentation entitled “Orexin A and orexin B depolarize C1 and non-C1 neurons in rostral

ventrolateral medulla via OX1R and OX2R.” in the 12th Society of Chinese Bioscientists in America International Symposium. (2) Expression of M3 muscarinic receptors on RVLM-projecting orexin neurons This part of study was continued from the study of last year. Orexin neurons are located in the lateral and perifornical regions of the hypothalamus. It has been demonstrated that

orexin/hypocretin neurons are one of the major groups of central neurons that link the cardiosympathetic system to motor or mental stress-related cortex. The RVLM contains neurons that directly project to cardiosympathetic preganglionic neurons. Microinjection of orexins into RVLM elicited a pressor effect. Therefore, the projections from orexin/hypocretin neurons to RVLM may be one of the orexin/hypocretin pathways responsible for the regulation of cardiosympathetic activity. A small population of orexin/hypocretin neurons is reported to be regulated by cholinergic inputs. The purpose of the present study is to determine whether the muscarinic regulation happen in the RVLM-projecting orexin/hypocretin neurons. With neuronal track-tracing and immunohistochemisty

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techniques, we found that the RVLM-projecting neurons are densely located in ipsilateral paraventricular nucleus and scattered in ipsilateral lateral hypothalamic area. About 15% of RVLM-projecting lateral hypothalamic neurons are orexin/hypocretin neurons.

We also found the expression of M3 muscarinic receptor on a population of RVLM-projecting orexin/hypocretin neurons. It had been reported that the cholinergic innervation to the orexin neurons are from basal forebrain cholinergic neurons (Sakurai, et al., 2005), which exhibit increased activity during wakefulness and rapid eye movement (REM) sleep. Therefore, the muscarinic

regulation on the RVLM-projecting orexin/hypocretin neurons might be involved in the regulation of cardiovascular function during wakefulness and REM sleep. These findings will be published as a poster presentation in the XXXVIth International Congress of Physiological Sciences (IUPS) in July, 2009 in Japan. (3)The roles of orexins in the regulation of arterial pressure in rats As

mentioned above, the orexin system is crucial in the regulation of cardiovascular functions. Orexin system may play important roles in the maintenance of basal arterial pressure since lower levels of arterial pressure were detected in both animals and human

possessing dysfunctional orexinergic system. In addition, orexin system has been reported to involve in stress-induced cardiovascular responses (Kayaba et al., 2003). Where does orexins exert their effects in cardiovascular regulation. Our data also support that the RVLM might be one of the sites of orexin’s actions. In addition to the RVLM, the importance of the paraventricular nucleus (PVN) of the hypothalamus in the regulation of cardiovascular functions is well recognized. We currently are examining the roles of

endogenous orexins in the RVLM and PVN in normal rats and in a high fat-diet induced obese rats, which we have established in our laboratory and possess an elevated arterial pressure, by pharmacological blockade approaches.

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