臺灣博碩士論文加值系統

心房利鈉胜 (atrial natriuretic peptide, ANP) 是在1980年最早被發現的利鈉胜家族成員，是一種很強的血管擴張、利尿、利鈉及皮質醛酮分泌抑制的多胜。ANP 首先發現存在心房組織，在體液擴張狀態下，心房細胞被刺激而分泌ANP進入血液循環，引起利尿和利鈉作用，使體液回復平衡。ANP除了在心房細胞合成並分泌以外，研究發現心房以外的許多組織器官都可以合成及分泌ANP，如腦下垂體、心室、胸腺、主動脈、肺靜脈及神經節等。在1988年利鈉胜另一成員--腦利鈉胜 (brain natriuretic peptide, BNP)從豬腦萃取物中被分離出來，BNP與ANP類似，BNP主要存在於心臟組織，在體液平衡的維持亦擔負有重要的角色。研究指出，BNP除了主要在心臟可以合成及分泌之外，心臟以外之其它器官，亦皆被發現有BNP mRNA及蛋白質的存在，然而，對這些心臟以外的器官分泌利鈉胜的角色則所知有限。
由於腎上腺本身可能可以合成利鈉胜，且腎上腺皮質又是重要的利鈉胜作用部位，因此本研究假設腎上腺可能分泌利鈉胜以調節腎上腺機能，並參與體液及電解質平衡的維持。首先探討原發性皮質醛酮過多症病人腎上腺組織ANP及BNP合成的狀況。利用組織萃取及放射免疫分析發現相當多量的ANP及BNP免疫反應物質存在於原發性皮質醛酮過多症病人的正常非腫瘤腎上腺組織及捐腎者正常腎上腺組織，以高效率液態凝膠層析法(HP-GPC)分析ANP之分子形式發現主要為γ-hANP，也就是ANP的prehormone。以免疫組織化學方法利用特異性的BNP抗體染色研究發現腎上腺組織的BNP免疫反應物質主要存在於腎上腺髓質位置。進一步以原位雜交法，利用ANP 及BNP cRNA為探針，發現腎上腺髓質細胞含有ANP及BNP傳訊者核醣核酸(mRNA)，證實腎上腺髓質細胞可以合成及分泌ANP及BNP。以北方點墨法比較正常人腎上腺與原發性皮質醛酮過多症病人的非腫瘤腎上腺組織的ANP及BNP mRNA含量，發現在原發性皮質醛酮過多症病人的腎上腺ANP 及BNP mRNA有明顯的上升。利用反轉錄及多聚鏈鎖反應技術(RT-PCR)實驗發現人類腎上腺組織可以選殖出BNP互補去氧核醣核酸(cDNA)。以核酸定序法證實BNP cDNA序列與心房組織所發現者相同。再利用半定量多聚鏈鎖反應方法發現原發性皮質醛酮過多症病人非腫瘤腎上腺組織ANP及BNP mRNA表現皆比正常人的腎上腺組織者為高，其中ANP mRNA含量遠比BNP mRNA為高，但是ANP/BNP mRNA比值則較正常人的腎上腺組織者為低。本項研究結果是首次發現人類腎上腺髓質可以合成ANP及BNP，同時腎上腺ANP及BNP的合成在原發性皮質醛酮過多症有增加的現象。這些結果支持腎上腺髓質ANP及BNP的合成可能扮演以旁分泌方式調節體液平衡或腎上腺皮質功能的假說。
腎上腺髓質既可以合成利鈉胜，而腎上腺皮質就是利鈉胜作用的目標之一。在解剖學上，腎上腺皮質及髓質來自不同胚胎來源，卻合在同一器官，因此認為腎上腺利鈉胜以旁分泌方式作用於皮質或髓質本身。利用免疫組織化學方法、電子顯微鏡和免疫電子顯微鏡術的研究，發現腎上腺髓質細胞和皮質細胞有互相混合的現象，在皮質部位可發現有分泌ANP的髓質細胞存在，在皮質細胞附近有神經突觸終末，在其內亦可見到ANP反應顆粒存在。顯微鏡下發現人類腎上腺靜脈為含有特殊肥厚呈螺旋排列之平滑肌細胞，因此，腎上腺髓質ANP可能經由腎上腺靜脈逆流、神經突觸或直接接觸方式作用於腎上腺皮質細胞。
腎臟也是ANP合成器官之一，但是其調節機轉及生理作用則仍不清楚。為探討腎臟內利鈉胜的合成在維持體液平衡之角色，將大白鼠以DOCA-鹽水模式造成體液變動後，以反轉錄一多聚鏈鎖反應法及放射免疫分析觀察腎臟利鈉胜mRNA、血液利鈉胜濃度及尿液利鈉胜排泄之變化。在DOCA-鹽水組大白鼠尿液鹽份排泄量、尿量及尿液ANP排泄量從第二天起明顯地比正常組者為多，同時尿液ANP排泄量與尿液鈉排泄量呈正相關。血漿ANP濃度在DOCA-鹽水組明顯地上升，而血漿腎素活性則相反地呈顯著的下降。利用免疫組織化學方法，以抗ProANP（1-30），ProANP（31-67）及α-ANP抗體，實驗發現ANP免疫反應物質存在腎臟的遠端及近端腎小管細胞。以反轉錄多聚鏈鎖反應，選殖及定序方法，發現ANP cDNA存在於腎臟組織，且其序列與心房ANP cDNA相同。利用半定量多聚鏈鎖反應及南方點墨法，研究發現左心室及腎臟皮質組織的ANP mRNA表現明顯地比正常組者為多。我們的結果證實腎臟為ANP合成器官，而腎臟ANP的合成在體液擴張下會被刺激而上升，本項研究認為腎臟ANP合成可能在體液電解質平衡的調節或皮質醛酮脫逃現象上扮有相當的角色。
本研究的結果顯示腎上腺皮質及髓質之間有互相影響之關係。此外，腎臟ANP的合成及分泌參與調節腎臟鹽分的排泄，此有助於了解腎臟本身如何調節體液平衡的機轉。總結研究結果：利鈉胜可能在腎上腺及腎臟是經由旁分泌的方式來調節腎上腺及腎臟自身的功能以維持體液之平衡。

Atrial natriuretic peptide (ANP), a peptide with potent diuretic, natriuretic, vasorelaxant, and aldosterone inhibiting activities, was isolated and identified from rat and human atrial tissues. Originally found in mammalian atria, ANP is not exclusively a cardiac hormone. In 1988, brain natriuretic peptide (BNP) was isolated from the porcine brain by Sudoh et al. BNP, like ANP is a potent diuretic, natriuretic, vasorelaxant, and aldosterone inhibitory peptide. BNP was found mainly secreted from the atria and play some role in the fluid electrolyte homeostasis. BNP was also found synthesized in extra-atrial tissues. To date, the physiological role of extra-cardiac natriuretic peptide is still unknown.
There were evidence indicating that adrenal gland may be the site of natriuretic peptides synthesis and suggested that adrenal natriuretic peptides may act in a paracrine or endocrine manner to regulate water and electrolyte homeostasis. To test this hypothesis, the present study was designed to determine whether or not ANP and BNP are synthesized in the human adrenal gland and, if so, to investigate the ANP and BNP content of adrenal tissue and the mRNA changes in patients with primary aldosteronism. A considerable amount of alpha-hANP-like and BNP-like immunoreactive substances were extracted from the remnant adrenal glands of patients with primary aldosteronism and the adrenal glands of donors for transplantation. High-performance gel permeation chromatography analysis coupled with a radioimmunoassay of the tissue extract showed that the molecular form of ANP in the adrenal gland was the precursor form, ie gamma-hANP. Immunohistochemical study with a specific anti-human BNP antibody revealed that BNP-like immunoreactivity was localized in the adrenal medullary area. An in situ hybridization study using ANP and BNP cRNA probes indicated that the ANP and BNP mRNAs were localized mainly in the medullary area of the gland. Northern blot analysis detected ANP and BNP mRNAs in the adrenal gland. Furthermore, the level of ANP and BNP mRNAs in the adrenal gland of patients with primary aldosteronism were obviously elevated as compared to those of the kidney donors. Using reverse transcription and polymerase chain reaction (RT-PCR) technique, BNP cDNA was cloned from the human adrenal gland, and the sequence was identical to that of BNP identified in the atria. Quantitative PCR measurements of BNP and ANP mRNA showed that both of the adrenomedullary natriuretic peptide gene transcriptions were enhanced in patients with primary aldosteronism, but the amount of ANP mRNA was far higher than that of BNP mRNA in the human adrenal gland.
Our results are the first to indicate that ANP and BNP are synthesized in the human adrenal medulla, and that such medullary ANP and BNP synthesis increased in patients with primary aldosteronism. These facts support the proposal that adrenomedullary natriuretic peptides may play some role in water and electrolyte homeostasis or act in a paracrine manner to regulate adrenocortical functions.
As the adrenal medulla is in close contact with adrenal cortex, a site of ANP action, adrenal medullary ANP is considered through paracrine manner to regulate cortical aldosterone biosynthesis. To investigate the paracrine roles of adrenal medullary ANP, we studied the possible route of action of medullary ANP in the adrenal gland. Immunohistochemical study with specific anti-prepro-human ANP antibody showed that ANP immunoreactive material was present mainly in the medullary area. ANP immunoreactive chromaffin cells, identified by using chromogranin-A antibody immunostaining, were found interwoven or in close apposition with cortical cells and some of which were localized in the zona glomerulosa area. Electron microscopical study revealed that there are secretory granules containing nerve terminals around the zona glomerulosa cells. By using immunocytochemical study with immunogold technique, ANP immunoreactivity was identified in the secretory granule of chromaffin cells, furthermore, the secretory granules of the nerve terminals were also found to contain immunoreactive ANP material. Microscopically, there were specialized spiral bundles of mainly longitudinal smooth muscle fibers in the wall of adrenal vein. Contraction of these muscle bundles may occlude the medullary veins and lead to damming up the blood in the medulla and adjacent cortex with a reversal of blood flow toward the cortex. Our results suggest that adrenal medullary ANP may through direct contact with cortical cells, the nerve route, or blood flow to regulate adrenal cortical steroidogenesis.
There were evidence indicating that kidney is a site of ANP synthesis and renal ANP synthesis play a role in renal pathophysiology. To investigate the role of renal synthesis of ANP, as contributors to the water-sodium homeostasis, we studied the effects of electrolyte-water imbalance on renal natriuretic peptides mRNA levels, plasma natriuretic peptide concentrations and urinary natriuretic peptide excretion rates by using RT-PCR and radioimmunoassay. The urinary sodium excretion rate and urine volume were significantly elevated at two days and remained elevated for the entire 10-day study period in the DOCA-salt rats. Using PCR cloning and sequencing technique, ANP cDNA was cloned from the rat kidney, and the sequences were identical to that of ANP identified in the atria. By semi-quantitative PCR technique, expression of ANP mRNA in the ventricle, and renal cortex tissues were significantly enhanced in the DOCA-salt rats. The urinary ANP excretion rate was also significantly increased in DOCA-salt rats at 2 days after treatment and was well correlated to the urinary sodium excretion rate. Plasma ANP level was elevated in the DOCA-salt rats on the day of sacrifice. In contrast, plasma renin activities were markedly suppressed in DOCA-salt rats and increased in Low-salt rats. By immunohistochemical study with specific ANP antibodies, immunoreactive ANP materials were localized mainly in the proximal and distal cortical tubules of the kidney. Our results confirm that rat kidney is a site of ANP synthesis and indicates that renal ANP synthesis is enhanced in volume expansion state. These findings support the proposal that renally synthesized atrial natriuretic peptide participates in the intra-renal regulation of water-electrolyte homeostasis, and may contribute to renal adaptation during the mineralocorticoid escape phenomenon.
In conclusion: extra-atrial (adrenal gland and kidney) natriuretic peptides synthesis are up-regulated in volume expansion state and may indicated that extra-atrial natriuretic peptide, through an autocrine or paracrine manner, play a role in fluid-electrolyte homeostasis.

封面
目錄
誌謝（Acknowledgments）
I. 中文摘要（Abstract in Chinese)
II. 英文摘要（Abstract in English)
第一章　緒論（General lntroduction)
1.1.　心房利鈉胜□（Atrial natriuretic peptide, ANP）的發現-
1.2.　心房利鈉胜□分泌與調節
1.3. 心房利鈉胜□與體液平衡
1.4. 心房外心心房利鈉胜□之合成及作用
1.5. 腎上腺心房利鈉胜□之合成
1.6. 腎臟心房心房利鈉胜□之合成
1.7. 腦利鈉胜□及C-型利鈉胜□
1.8. 研究背景
1.9. 研究目的
1.10. 研究步驟
第二章 原發性皮質醛酮過多症腎上腺髓質心房利鈉胜□之合成增加
（Increased adrenal medullary atrial natriuretic polypeptide synthesis in patients with primary aldosteronism）
2.1. 前言
2.2. 材料與方法
2.3. 結果
2.4. 討論
2.5. 圖表
第三章 腎上腺髓質可以合成腦利鈉胜□，在原發性皮質醛酮過多症腎上髓質腦利鈉胜□及心房利鈉胜□傳訊者核酸之表現增加
（Brain natriuretic peptide is synthesized int he human adrenal medulla and its messenger ribonucleic acid expression with along that of atrial natriuretic peptide are enhanced inpatients with primary aldosteronism）
2.1. 前言
2.2. 材料與方法
2.3. 結果
2.4. 討論
2.5. 圖表
第四章 腎上腺髓質利鈉胜□於腎上腺作用途徑之探討（A Study of Action Routes of Adrenal Medullary
Atrial Natriuretic Peptide on Adrenal Gland）
2.1. 前言
2.2. 材料與方法
2.3. 結果
2.4. 討論
2.5. 圖表
第五章 DOCA-鹽水處理大白鼠增加腎臟心房利鈉胜□之合成（Increased renal atrial natriuretic peptide
synthesis in rats with deoxycorticosterone acetate-salt treatment）
2.1. 前言
2.2. 材料與方法
2.3. 結果
2.4. 討論
2.5. 圖表
第六章 綜合討論與未來研究方向（Conclusions and future researches）
參考文獻 （References）
附錄 I 英文名稱縮寫及全名
附錄 II 發表相關論文之摘要

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