| 摘要: | 高血壓常見於國人十大死因之一。而高血壓之致病因子相當多所以其致病機轉目前仍然不明確。食慾素(orexin)是由下視丘神經細胞所合成的神經胜肽會興奮交感神經中樞，也被證實了會引發升血壓作用與增加交感神經興奮，並與經常性及波動性的血壓調節作用的恆定有關。 Orexin亦稱為下丘腦泌素是以orexin-A及B兩種形式存在並且主要是透過第一型orexin受器(簡稱OX1R)與第二型orexin受器(簡稱OX2R)產生生物功能。它是否於原發性高血壓的病變扮演任何角色仍然不清楚，目前沒有中樞orexin系統跟高血壓之致病機轉相關的研究。我們首先探討在成熟雄性自發性高血壓大鼠(以下簡稱SHR;一種常用來研究人類原發性高血壓的實驗動物)的高血壓病變是否跟它本身中樞的orexin系統活性增加有關。我們分別利用OX1R和OX2R專一性的拮抗劑或特異性的抗體壓制SHR的中樞orexin系統活性，相同地，我們也使用相同的方法與條件測試另一種正常血壓的大鼠(以下簡稱WKY)當作SHR的實驗對照組。我們發現以測腦室注射方式給與30 和100 nmole兩種不同濃度的第I型orexin受器的拮抗劑SB334867，除了發現在100 nmole 濃度的SB334867會降低WKY的心跳； 30 或 100 nmole這兩種濃度的SB334867，都不影響SHR與WKY的血壓與心跳。然而，以測腦室注射方式給與0.3, 3, 10 和30 nmole四種不同濃度的第II型orexin受器的拮抗劑TCS-OX2-29，發現3, 10 和30 nmole這三種濃度的TCS-OX2-29會造成SHR的血壓與心跳產生具持久性地降低的現象，例如TCS-OX2-29在30 nmole濃度下，血壓會降低21±3毫米汞柱與心跳每分鐘降低22±2次。在SHR以測腦室注射方式給與OX2R特異性的抗體所引起的降血壓與降心跳的程度，顯著的大於WKY。但是，給與OX1R特異性的抗體或是一般非特異性免疫球蛋白時，並不影響SHR的血壓與心跳。而且，不管是OX1R或OX2R的特異性抗體或是一般非特異性免疫球蛋白，這三者皆不影響自WKY的血壓與心跳。在西方墨點法的實驗中，我們發現在SHR與WKY的下丘腦背內側、室旁核或孤立束核，這些與心血管調控有關的腦部核區的OX2R的蛋白表現量無統計差異。但是，跟WKY相同條件比較下，SHR的RVLM的OX2R的蛋白表現量低於WKY。而這四個腦部核區的OX1R的蛋白表現量在SHR與WKY相比下並無統計差異性。在RVLM微量注射方式給與OX2R的拮抗劑TCS-OX2-29的實驗，發現跟WKY相比，在SHR所引起的降血壓的程度較大。綜合以上研究發現，我們推論RVLM的OX2R所媒介的活性增加可能與SHR高血壓病變有關。
但是，非預期的結果發現SHR的RVLM的OX2R的蛋白表現量低於WKY。因此，我們進一步探討在SHR中RVLM的OX2R所媒介活性增加的機制。實驗結果我們發現: 1. 位在下視丘外側區orexin的神經元細胞數目，在4週大或16週大的SHR比相同週數大的WKY的神經元數目多。而且利用神經束追蹤技術(一種藉由神經元末端將傳送物質逆行傳輸回細胞體的方式)所被螢光標示會投射到RVLM的orexin的神經元數目，發現在16週大的SHR比相同週數大的WKY的神經元數目多，表示SHR位在下視丘外側區orexin的神經元細胞投射到RVLM比WKY多。2. 進一步分析發現，這些16週大的SHR的下視丘外側區orexin的神經元細胞數目或是它本身投射到RVLM的神經元細胞數目增加，主要是增加在下視丘腹背核和下視丘穹窿週區這兩個核區。3. 跟WKY相比，SHR在下視丘組織所測得orexin-A及B濃度比較高；而在RVLM的組織所測得orexin-A濃度比較高。4. 於RVLM區內微注射給與OX2R的致效劑(orexin-A或ALOXB)，在SHR所引起升血壓反應大於WKY。5. 於WKY或SHR的RVLM區內先預處裡微量注射給與具選擇性第一型NOS(簡稱nNOS)的抑制劑7-nitroindazole (2.5 pmol)，或是鳥核酸環化酶抑制劑甲基藍 (250 pmol)，再於同一位置給與OX2R的致效劑，結果會使ALOXB升血壓反應消失。相反地，給與具選擇性第二型一氧化氮合成酶(簡稱iNOS)的抑制劑氨基胍無法阻斷ALOXB的升血壓反應。6. 於WKY或SHR的RVLM核區內微量注射給與ALOXB造成這兩種品系大鼠RVLM核區內的nNOS的蛋白表現量與一氧化氮濃度皆增加，跟WKY相比，SHR增加比較多。7. 藉由免疫螢光染色實驗，發現RVLM有一部份的神經細胞會共同表達OX1R與nNOS或OX2R與nNOS。我們的結論是在SHR的RVLM中orexin的神經突觸釋放增加和OX2R所引發nNOS之一氧化氮生成訊息傳遞作用增強，可能導致OX2R所媒介活性增加並進而造成SHR高血壓的病變。
Hypertension is one of the leading cause of death in Taiwan. The pathological mechanisms of hypertension is not yet fully understood. Orexins, novel hypothalamic peptides, exert an excitatory effect on sympathetic activity and cardiovascular functions and are involved in tonic and phasic control of cardiovascular homeostasis. Orexin occurs in two forms, orexin-A and orexin-B and exert their actions through their interaction with two G protein-coupled receptors, orexin-1 receptor and orexin-2 receptor (OX1R and OX2R). However, it is still unclear whether orexin contributes to the pathogenesis of primary hypertension. We examined the contribution of central orexinergic activity in hypertension in spontaneously hypertensive rats (SHR), an animal model for primary hypertension, by suppressing central orexinergic activity in SHR and its normotensive control, Wistar-Kyoto rats (WKY), with specific antagonists or antibodies against OX1R and OX2R. Intracerebroventricular (ICV) administration of an OX1R antagonist, SB-334867 (30 and 100 nmole), induced no significant change in mean arterial pressure (MAP) or heart rate (HR) in SHR and WKY except that SB-334867 reduced HR in WKY at 100 nmole. In contrast, ICV administration of an OX2R antagonist, TCS-OX2-29 (3-30 nmole), induced long-lasting reductions (21±3 mmHg and 22±2 bpm at 30 nmole) of MAP and HR in SHR, but not in WKY. ICV administration of anti-OX2R IgG, but not anti-OX1R IgG or non-immune goat IgG, significantly lowered MAP and HR in SHR. None of the three IgGs affected MAP or HR in WKY. The OX2R protein level in the RVLM was lower in SHR than WKY, whereas no differences were found between SHR and WKY in the dorsomedial-perifornical hypothalamic area, paraventricular nucleus, or nucleus tractus solitarius. The OX1R protein levels in these four regions did not differ between SHR and WKY. Injection of TCS-OX2-29 (50 pmole) into the RVLM produced a larger reduction of MAP in SHR than WKY. We concluded that elevated OX2R-mediated activity in the brain, especially the RVLM, may contribute to hypertension in SHR.
Unexpectedly, the level of OX2R protein in the RVLM was lower in SHR than in WKY. The mechanisms underlying the elevated OX2R activity within the RVLM in SHR were then examined and the following observation were found. (1) More orexin-A-immunoreactive (Orexin-A-IR) cells existed in SHR than WKY of either 4- (2217±43 vs. 1809±69) or 16-week-old (1829±59 vs. 1230±84). The number of Orexin-A-IR cells that project to the RVLM, identified by labeling with a retrograde tracer, was also higher in 16-week-old SHR than WKY (91±11 vs. 52±11). (2) Predominantly Orexin-A-IR cells and RVLM-projecting Orexin-A-IR cells were found in the dorsomedial hypothalamus (DMH) and perifornical hypothalamus (PeF) in 16-week-old SHR. (3) SHR had higher levels of orexin-A and orexin-B in the hypothalamus and higher levels of orexin-A, but not orexin B, in the RVLM than WKY. (4) Unilaterally applied OX2R agonists, orexin-A or [Ala11,D-Leu15]-orexin B (ALOXB) (50 pmol), into the RVLM induced a larger pressor response in SHR than in WKY (orexin-A: 32.7±6.2 vs. 14.6±1.8 mmHg; ALOXB: 25.8±3.9 vs. 11.8±1.6 mmHg). (5) Intra-RVLM pretreatment with a neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitro-indazole (2.5 pmol), or a soluble guanylate cyclase inhibitor, methylene blue (250 pmol), almost abolished intra-RVLM induced ALOXB-induced pressor response in both WKY and SHR. Contrastingly, a selective inhibitor of inducible NOS, aminoguanidine (100 pmol), was ineffective in both strains. (6) Intra-RVLM injection of ALOXB caused increases of nNOS protein and total nitrite/nitrate in the RVLM in both WKY and SHR and the increments were larger in SHR than in WKY. (7) nNOS is co-expressed with OX1R or with OX2R in RVLM neurons. We conclude that increased orexinergic input and enhanced OX2R-nNOS signaling may underlie elevated OX2R activity in the RVLM and contribute to OX2R-associated pathological mechanisms of hypertension in SHR. |
