一氧化氮與內皮素之間於新生鼠心臟細胞的交互作用

Abstract

內皮素會造成心肌細胞的肥大，而於 此過程中，心血管系統所釋放的一氧化氮 扮演著某種程度的對抗作用。本研究，在 於探討內生性一氧化氮對抗內皮素所誘發 c-fos 基因表現的作用機轉。實驗結果顯 示：內皮素經由活化其乙型接受器，會刺 激一氧化氮合成酵素，造成一氧化氮生成 增加。經由一氧化氮貢限劑如SNAP 、SIN-1 及一氧化氮清除劑PTIO 的運用，以北方式 點墨法搭配報告者基因轉染的方法，顯示 一氧化氮可對抗內皮素所誘發c-fos 的基因 表現；另外，以不同的負向突變基因Ras (RasN17), Raf-1 (Raf301), 或mERK ，轉染 入心肌細胞皆可對抗內皮素所誘發c-fos 的 基因表現，顯示內皮素誘發c-fos 的基因表 現是經由Ras/Raf/ERK 的訊息路徑；直接 觀查一氧化氮對內皮素所激活ERK 的作 用，一氧化氮亦可對抗內皮素的激活ERK 的活性；猶有甚者，一氧化氮尚且可對抗 內皮素所增加乙型重鍊肌蛋白基因啟動子 活性的作用。縱合觀之，內皮素會造成心 肌細胞一氧化氮生成增加，而一氧化氮可 進一步對抗內皮素的激活ERK 的活性，造成對抗內皮素所誘發c-fos 的基因表現。

Endothelin-1 (Et-1) treatment of cardiac myocytes (CM) induces cardiac hypertrophy. Cardiovascular release of nitric oxide (NO) may play a role during cardiac hypertrophy. The present study examined the protection mechanism of endogenously released NO on c-fos induction by Et-1 in neonatal rat CM. CM exposed to Et-1 induced NO release. Et-1 stimulated nitric oxide synthase (NOS) activity led to NO production that was attenuated by treating CM with an antagonist to endothelin B receptor. CM treated with a NO donor, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1), inhibited Et-1–induced c-fos expression. Conversely, CM treated with a NO scavenger, 2-phenyl-4,4,5,5,-tetramethyl-imidazoline-l-oxyl- 3-oxide (PTIO), augmented Et-1-induced c-fos expression. The attenuation of NO on c-fos expression was shown by reducing either c-fos mRNA levels or c-fos promoter activities using a chimera containing the c-fos promoter region (-2.25 kb) ligated to a reporter gene CAT. In contrast to the enhanced promoter activity in CM after PTIO treatment, attenuated Et-1-induced c-fos promoter activity was shown in CM treated with a NO donor. CM cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or a catalytically inactive mutant of extracellular signal–regulated kinase (ERK)– 2 (mERK) inhibited Et-1–induced c-fos promoter activity, indicating Ras/Raf/ERK pathway was involved. NO modulation of this signaling pathway was shown by its inhibitory effect on Et-1–induced ERK activity. CM treated with NO resulted in a decrease of Et-1-induced binding of nuclear proteins to the AP-1 binding sequences. Furthermore, CM treated with a NO donor significantly suppressed Et-1-induced b-myosin heavy chain promoter activities. These results indicate that CM under Et-1 treatment increases NO levels and the increased NO attenuated Et-1-induced c-fos expression via the ERK signaling pathway. These findings support the importance of NO as a negative regulator in Et-1–induced gene expression and cardiac hypertrophy.