茶鹼衍生物 KMUP-1 對於肺動脈高血壓之藥理研究

Pharmacologic study of theophylline-based KMUP-1 on pulmonary artery hypertension

鍾慧萱(Hui-Hsuan Chung)

指導教授 ： 陳英俊

高雄醫學大學/醫學院/醫學研究所/博士(2010年)

摘要

肺高壓是一致死率極高的疾病，主要特徵就是肺動脈收縮與肺動脈平滑肌增生而造成肺動脈壓上升，本研究的目的為探討 KMUP-1 在治療肺高壓時抑制肺動脈收縮以及肺動脈平滑肌增生的藥理機轉。在 U46619 誘導急性肺高壓實驗中，KMUP-1 抑制肺動脈壓升高與血中氧氣消耗；提高大鼠血漿中 cGMP 與 cAMP 的含量；除此之外，KMUP-1 增加離體肺動脈 eNOS (endothelial nitric oxide synthase) sGC (soluble guanylyl cyclase) 和 PKG (protein kinase G)；以及抑制 PDE (phosphodiesterase)-5A、MYPT1 (myosin phosphatase target subunit 1) 磷酸化 (phosphorylation) 和 RhoA/ROCK (Rho kinase) 的蛋白質表現；而 KMUP-1 在離體肺動脈對於 ROCK 的抑制作用則被 sGC 抑制劑 ODQ、 eNOS 抑制劑 L-NAME 或 PKG 抑制劑 Rp-8-CPT-cGMPS 所拮抗。KMUP-1 拮抗由 PE (phenylephrine)、5-HT (serotonin) 以及 U46619 所引起的肺動脈收縮，而 KMUP-1 對於 U46619 在肺動脈的舒張作用則被 ODQ、L-NAME 與 AC (adenylyl cyclase) 抑制劑 SQ22536 所減弱。在 MCT (monocrotaline) 誘導慢性肺高壓實驗中，長期投予 KMUP-1 21 天後增加肺部 eNOS、sGC 和 PKG；抑制 RhoA/ROCK、5-HTT (serotonin transporter) 的蛋白質表現；降低血漿中 5-HT 的濃度，抑制肺動脈增生與右心室肥大。
在離體肺動脈平滑肌細胞實驗中，KMUP-1 抑制 thapsigargin 所引起的內鈣釋放及 5-HT 與 angiotension II 所誘導的外鈣內流。KMUP-1 抑制5-HT 所誘導的 RhoA/ROCK、5-HTT 、磷酸化 AKT 及 ERK1/2 的蛋白質表現，而同樣不屬於 5-HTT 抑制劑的 Y27632 和 simvastatin 也同樣抑制 5-HT 所誘導的 5-HTT 在肺動脈平滑肌細胞的表現。KMUP-1 (1-100 ?嵱) 增加人類肺動脈內皮細胞的 5-HT2B 受體 (5-HT2B receptor) 與 eNOS 的表現。經由藥物受體結合實驗證實 KMUP-1 對於血清素受體的結合能力為 5-HT2B 受體 > 5-HT 2A 受體 (5-HT2A receptor) > 5-HT2C 受體 (5-HT2C receptor)，KMUP-1 較有選擇性的作用在 5-HT2B 受體　上。5-HT2B 受體在內皮細胞中刺激 eNOS 生成釋放 NO 而使肺動脈舒張； KMUP-1 增加人類肺動脈平滑肌細胞 5-HT2B 受體 與 eNOS 的表現與 NO 釋放，且 5-HT2B 受體抑制劑 SB200646 減弱 KMUP-1 對人類肺動脈平滑肌細胞的 eNOS 表現，我們推論 KMUP-1 可能經由 5-HT2B 受體增加 eNOS 表現與 NO 釋放而使肺動脈的舒張。
經由以上實驗證明 KMUP-1 藉由 eNOS/PKG依賴性抑制RhoA/ROCK 的表現；KMUP-1 作用在5-HT受體與5-HTT　使血漿中 5-HT　含量減少，抑制5-HT所誘導的 RhoA/ROCK 與 AKT/ERK1/2 磷酸化。KMUP-1 提高大鼠血漿與肺動脈平滑肌 cAMP 與 cGMP 的含量，抑制細胞內鈣離子濃度 ([Ca2+]i) 上升，因此可治療 U46619 與 MCT 引起的急性或慢性肺高壓，抑制肺動脈增生、狹窄、收縮與右心室肥大。經由以上所述 ，KMUP-1 提供肺高壓治療的一項新選擇。

關鍵字

KMUP-1 ； 肺高壓

並列摘要

Pulmonary artery hypertension (PAH) is a debilitating disease, the hallmark of PAH is pulmonary arterial smooth muscle cells (PASMCs) proliferation and pulmonary pressure increasing. This study investigated the mechanisms of KMUP-1 inhibited pulmonary artery (PA) contraction and proliferation　in curing PAH. In the acute model of PAH induced by thromboxane A2 (TXA2)-mimetic U46619, KMUP-1 restored blood oxygenation and relaxed vasoconstriction by enhancing endothelial nitric oxide synthase (eNOS), soluble guanylate cyclase (sGC) and protein kinase G (PKG), and inhibited phosphodiesterase-5A (PDE-5A), RhoA/ROCK expression. The inhibition of KMUP-1 on ROCK expression was waken by ODQ、L-NAME and SQ22536. In the absence or presence of U46619 (0.5 ?嵱). Similarly, in isolated pulmonary artery (PA), KMUP-1 relaxed phenylephrine (10?n?嵱)- serotonin (5-HT)- and U46619 (0.5 ?嵱)-induced vasoconstriction, the relaxation effects of KMUP-1 are waken by ODQ、L-NAME and SQ22536. In the chronic model of PAH induced by monocrotaline (MCT), KMUP-1 prevented MCT-induced PAH over long-term administration. increased eNOS expression and reduced MYPT1 (myosin phosphatase target subunit 1) phosphorylation, RhoA/ROCK, 5-HT transporter (5-HTT) expression in lung tissue, reduced plasma 5-HT increasing, PA wall thickening, proliferation and right ventricular hypertrophy (RVH). Incubating PASMCs with KMUP-1 inhibited thapsigargin-induced Ca2+ efflux and angiotensin II-, 5-HT-induced Ca2+ influx. KMUP-1 (1-100 ?嵱) inhibited 5-HT-induced RhoA/ROCK expression, while KMUP-1, Y27632 and simvastatin at 10 ?嵱 inhibited 5-HT-induced 5-HTT expression and KMUP-1 inhibited 5-HT-induced phosphorylation of AKT and ERK1/2 in PASMCs. KMUP-1 (1-100 ?嵱) concentration-dependently increased the expression of eNOS and 5-HT2B and production of NO in human pulmonary arterial endothelial cells (HPAEC), 5-HT2B receptor antagonist SB200646 decreased the effects of KMUP-1 on eNOS. In radioligand binding, binding ability of KMUP-1 was 5-HT2B > 5-HT2A > 5-HT2C. KMUP-1 inhibits MCT-induced PA proliferation by binding to 5-HT2A, 5-HT 2B and 5-HT 2C receptors, increasing endothelial eNOS/5-HT2B receptor expression and inhibiting 5-HTT/RhoA/ROCK expression and AKT/ERK phosphorylation.
These findings suggest that KMUP-1 relieves and prevents PAH via enhancement of eNOS to releases NO and create a cGMP-dependent inhibition of RhoA/ROCK and Ca2+ desensitization in PASMCs. KMUP-1 inhibits MCT-induced PA proliferation by binding to 5-HT2A, 5-HT2B and 5-HT2C receptors, increasing endothelial eNOS/5-HT2B receptor expression and inhibiting 5-HTT/RhoA/ROCK expression and AKT/ERK phosphorylation. In conclusion, KMUP-1 has the potential to reduce vascular resistance, remodeling, hyperplasia and RVH in treating PAH.

並列關鍵字

KMUP-1 ； pulmonary hypertension

參考文獻

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