茶鹼衍生物影響人類臍靜脈內皮細胞一氧化氮合成酶;與血基質氧化酶的表現

本研究主要以黃嘌呤 (xanthines) 為基本骨架，在其相關分子結構上進行化學修飾，合成一系列新型黃嘌呤衍生物 KMUP-1, 3及4，並經由離體組織和細胞培養等實驗模式，來探討其藥理機轉。之前我們實驗室發表的文獻已間接證實在離體相關實驗中， KMUP-1, 3 及 4 會活化一氧化氮合成酶 (endothelial nitric oxide synthase , eNOS)的表現，進而促進一氧化氮(NO, Nitric Oxide)產生以達到血管鬆弛作用。為了確定一氧化氮的釋放是否經由KMUP-1, 3 及 4 活化一氧化氮合成酶的表現,而且NO/cGMP 與血基質氧化酶(heme oxygenase, HO) 系統是互相關連的，因此本實驗推測KMUP-1, 3 及 4可能具有調控血管內皮eNOS、HO-1與HO-2蛋白質表現作用。本實驗以西方墨點法 (western blot) 證實KMUP-1, 3 及 4可促使人類臍帶內皮細胞一氧化氮合成酶(eNOS) 之表現增加。另一方面也以西方墨點法證實KMUP-1, 3 及 4會增加HO-1與HO-2之蛋白質表現。而KMUP-1, 3 及 4 增加HO-1與HO-2蛋白質的表現會因為前處理L-NAME 100 ?M）與ODQ （guanylyl cyclase inhibitor, 10 ?M）而減弱。另外，在人類臍帶內皮細胞 (HUVECs)，以放射性免疫分析方法利用 [125I]cGMP 進行分析評估 cGMP 之釋放量，結果發現KMUP-1, 3 及 4 (10-9~10-5 M) 呈現劑量相關性增加細胞內 cGMP 的量。當分別前處理L-NAME (100 ?M), ODQ (10 ?M) 與ZnPPⅨ (Zinc protoporphyrin Ⅸ, 10 ?M) 三十分鐘後，具有意義的抑制KMUP-1, 3 及 4所釋放的cGMP含量。在人類臍靜脈離體實驗中，以5-Hydroxytryptamine (5-HT, 10 ?M)誘導收縮，KMUP-1, 3 及 4 (10-9~10-5 M) 累積投予均會產生劑量相關性的血管鬆弛作用。當前處理ODQ（10 ??M）之後，血管鬆弛作用則呈現明顯遞減。基於以上的實驗結果，顯示KMUP-1, 3 及 4 可以調控一氧化氮合成酶 (eNOS) 的表現，增加NO的釋放量造成血管鬆弛效果，因為NO的釋放亦可活化HO-1與HO-2 蛋白質表現。因此，KMUP-1, 3 及 4造成血管鬆弛效果的機轉包括活化eNOS 產生NO ，活化heme oxygenase 產生CO (Carbon monoxide) ，而NO與CO 皆能活化sGC 而促使GTP 轉變成 cGMP而達到血管的鬆弛作用。

關鍵字

KMUP-1 ； 3 and 4 ；一氧化氮合成酶血基質氧化酶

並列摘要

KMUP-1, 3 and 4, three new xanthine derivatives, we have demonstrated that they could activate the NO/sGC pathway. The present study try to identify the relationship between nitric oxide (NO) releasing effects of KMUP-1, 3 and 4 and associated expression of endothelial nitric oxide synthase (eNOS) and figure out the interaction of NOS/NO and heme oxygenase (HO)/CO pathways. We supposed that KMUP-1, 3 and 4 should have some direct effects on the expression and interaction of eNOS, HO-1 and HO-2 protein in vascular endothelial cells. Western blotting analysis indicated that KMUP-1, 3 and 4 caused a time-dependent increases of the expression of eNOS, HO-1 and HO-2 in human umbilical vein endothelial cells (HUVECs). KMUP-1, 3 and 4 at 10-9~10-5 ? also produce concentration-dependent increases of the expression of eNOS, HO-1 and HO-2. The increase of these protein expression were inhibited in the presence of a NOS inhibitor L-NAME (100 ?M) and guanylyl cyclase inhibitor ODQ (10 ?M), respectively. More over, KMUP-1, 3 and 4 increased the intracellular cGMP levels in HUVECs. These increases in the cGMP contents were abolished in the prescence with L-NAME, ODQ and HO inhibitor (Znpp-Ⅸ 10 ?M) as previously described. In isolated human umbilical vein strips precontrated with 5-HT(10 ?M), KMUP-1, 3 and 4 caused a concentration-dependent relaxation activities. These vasorelaxant effects of KMUP-1, 3 and 4 were inhibited by the pretretment with ODQ (10 ?M). These finding indicates that KMUP-1, 3 and 4 may enhance NO production through upregulating the expression of eNOS in endothelial cells. The associated NO/sGC/cGMP pathway also includes the expression of HO. It is suggested that the vessel relaxant effects of KMUP-1, 3 and 4 might be mediated by the stimulation of eNOS, HO-1 and HO-2 pathways.