第二型血管張力素第二型受器對醛固酮分泌和腎上腺細胞自毀的影響及機制之探討

The Impact and Mechanism of Angiotensin II Type 2 Receptor（AT2） on Aldosterone Secretion and Adrenocortical Cell Apoptosis

朱培倫(Pei-Lun Chu)

指導教授 ： 吳寬墩

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01759

摘要

背景與目的：Renin-angiotensin-aldosterone system (RAA system)是體內調控血流動力、體液及電解質平衡非常重要的系統。腎素（renin）使血管張力素原（angiotensinogen）轉換成第一型血管張力素（angiotensin I），再經過血管張力素轉換酵素 (angiotensin converting enzyme，ACE)將第一型血管張力素轉成第二型血管張力素(angiotensin II， ANGII)。ANGII已有不同的受器亞型被發現，主要有第一型及第二型(AT1 & AT2)。當ANGII作用在AT1時，會促使血管收縮使血壓上升，並促進腎上腺增加醛固酮（aldosterone）的產生和分泌。目前已知AT2在胚胎發育時期有廣泛表現,但在出生後卻迅速退化或消失除了在一些特定的組織外。目前對AT2的一般生理功能瞭解包括，抑制細胞生長、促進細胞分化、促進細胞凋亡、促進神經細胞再生、抑制血管新生、促進血壓下降及情緒穩定等，這些大部份似乎都在拮抗AT1的生理功能。在人類腎上腺，有40%的AT2表現在zona glomerulosa中，但這些AT2的生理功能不若AT1研究的透徹，尤其是在aldosterone的分泌及對細胞凋亡的影響，甚少被提出。因此吾人希望能針對此部分作進一步的探討，以瞭解AT2在人類腎上腺對aldosterone分泌及細胞凋亡所扮演的角色。
方法：首先，我們建立了穩定的H295R細胞株，並經由反轉錄聚合酵素連鎖反應（Reverse transcriptional polymerase chain reaction,RT-PCR）及西方墨點（Western blot）證實H295R細胞上確實有AT2的表現。並利用免疫放射分析法（radioimmunoassay,RIA）分析H295R細胞aldosterone基礎分泌量、ANGII對aldosterone分泌影響之時間與濃度效應、AT阻斷劑和AT2刺激劑對H295R細胞aldosterone分泌之影響。RT-PCR測定aldosterone synthase （CYP11B2）mRNA、西方墨點測定AT2之蛋白質在不同條件下之表現。最後並以流式細胞儀（Flow cytometry）進行細胞凋亡（cell apoptosis）分析。
結果：在加入10-6M ANGII後24小時測得aldosterone分泌明顯的增加4.8倍（p=0.03）。加入valsartan時，ANGII刺激的aldosterone分泌量會降低61%；而PD123319則對ANGII刺激的aldosterone分泌沒有影響，但若與valsartan合併使用，則可以使ANGII刺激aldosterone分泌的作用完全抑制，且分泌量下降達80%。利用RT-PCR則可發現在經過1μM ANGII處理後，在第2個小時偵測到CYP11B2 mRNA之表現開始增加，並於第8個小時達到最大量，約增加25倍。Valsartan可以減低CYP11B2 mRNA的表現量達35%，而同時加入valsartan及PD123319則減低CYP11B2 mRNA的表現量59.8%。雖然CGP42112A也會增加CYP11B2 mRNA的表現，但並不影響aldosterone的分泌。而在H295R細胞株上則並未看到刺激AT2所引起的細胞凋亡。
結論：ANGII增加aldosterone分泌主要是經由AT1的作用，且是藉由CYP11B2 mRNA的表現增加來達成，這個作用並無法以AT2的阻斷劑抑制。吾人進一步發現，同時阻斷AT1和AT2可以完全抑制ANGII刺激細胞分泌aldosterone，這個情形在CYP11B2的轉譯層次也有相同的結果。因此，AT1和AT2在腎上腺細胞也有“相互對話“的現象。這個相互對話的訊息傳遞有待進一步研究。而AT2的表現，目前認為並不影響腎上腺細胞之細胞凋亡。

關鍵字

第二型血管張力素第二型受器（AT2） ； 腎素-血管加壓素-醛固酮系統 ； 第二型血管張力素（ANGII） ； 人類腎上腺皮質腫瘤細胞（H295R cell） ； 醛固酮 ； 細胞凋亡 ； 醛固酮合成酵素（CYP11B2）

並列摘要

Renin-Angiotensin-Aldosterone system (RAA system) controls the hemodynamic status, body fluid and electrolyte balance in human body. In the system, ANGII is a key hormone which has 2 main receptor subtypes, AT1 and AT2. Most physiological effects of ANGII were mediated by AT1. It constricts arteries, elevates blood pressure and also stimulates adrenal gland to secret aldosterone. However, the role of AT2 in adrenal gland is not well studied as AT1.
AT2, a G-protein coupled receptor has 7 transmembrane domains, is widely expressed during fetal development. It regresses or even disappears rapidly in early postnatal period except some limited tissue like limbic system, several thalamic nucleus, adrenal medulla, zona glomerulosa and uterine myometrium etc. The general understanding of its functions are inhibition of cell growth, promotion of cell differentiation or cell apoptosis, mediates neural cell remodeling, inhibition of angiogenesis and counteracts AT1’s effects. In adult adrenal gland, 40% of AT2 is presented in zona glomerulosa; however, the significance of its persistent expression is unknown.
We established stable H295R cell line and aldosterone concentration was detected by radioimmunoassay (RIA) which was corrected by total protein amount. CYP11B2 mRNA and AT2 protein expression were detected by RT-PCR and Western blot later. Cell apoptosis was analyzed by flow cytometry.
After ANGII stimulation, aldosterone was detectable since 24th hour and lasted up to 72 hours. The ANGII induced aldosterone secretion was 61% suppressed by AT1 blocker, valsartan; but AT2 blocker, PD123319 has no effect on it. However, co-treatment with valsartan and PD123319 could suppress aldosterone secretion almost totally.
CYP11B2 mRNA expression could be detected since 2nd hour and maxially expressed at 8th hour after 1μM ANGII treatment. Valsartan suppressed CYP11B2 mRNA expression, whereas PD123319 alone has no impact on it. If pretreated with valsartan and PD123319 simultaneously, the expression of CYP11B2 mRNA was suppressed to basal level as control group.The mRNA change seems to correlate with the aldosterone secretion change. Although CGP42112A up-regulates CYP11B2 mRNA expression, it does not interfer aldosterone secretion as PD123319 does. By Western blot, AT2 expression was not altered by every tested condition and the flow cytometry showed no obvious apoptosis.
In conclusion, AT1, is the main receptor for aldosterone secretion through CYP11B2；AT2 is involved too, but there might be a “cross talk”between AT1 and AT2. It does not function via CYP11B2 totally or even with post-transcription, translation modification by AT1 or other pathways. So far, AT2 mediated cell apoptosis was not observed in H295R cell.

並列關鍵字

Renin-Angiotensin-Aldosterone system (RAA system) ； angiotensin II（ANGII） ； human adrenocortical carcinoma cell（H295R cell） ； angiotensin II type 2 receptor（AT2） ； aldosterone ； aldosterone synthase（CYP11B2） ； apoptosis

參考文獻

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