Zhang Qian, Church Jarrod E, Jagnandan Davin, Catravas John D, Sessa William C, Fulton David
Vascular Biology Center, Department of Pharmacology, Medical College of Georgia, Augusta, GA 30912, USA.
Arterioscler Thromb Vasc Biol. 2006 May;26(5):1015-21. doi: 10.1161/01.ATV.0000216044.49494.c4. Epub 2006 Mar 2.
We have previously shown in COS-7 cells that targeting of endothelial NO synthase (eNOS) to the Golgi or plasma membrane (PM) regulates the mechanism and degree of eNOS activation. However, little is known about the functional significance of eNOS targeting in endothelial cells (ECs). The goal of the current study was to isolate these 2 pools of enzyme in ECs and determine their functional significance in response to agonist stimulation and manipulation of membrane cholesterol levels.
Using an RNA interference strategy, we generated stable populations of ECs that had >90% inhibition of eNOS expression and lacked the ability to produce NO. Reconstitution of these eNOS "knockdown" ECs with Golgi- and PM-targeted eNOS restored the ability of ECs to produce NO. Calcium-dependent agonists were the more efficient stimulus for the PM-restricted eNOS in ECs. In contrast, Golgi eNOS was less responsive to both calcium- and Akt-dependent agonists. eNOS restricted to the PM was more sensitive to manipulation of membrane cholesterol levels and was significantly attenuated by modified low-density lipoprotein.
Within ECs, the PM is the most efficient location to produce NO but is more vulnerable to cholesterol levels and modified low-density lipoprotein.
我们之前在COS-7细胞中发现，将内皮型一氧化氮合酶（eNOS）靶向至高尔基体或质膜（PM）可调节eNOS激活的机制和程度。然而，关于eNOS靶向在内皮细胞（ECs）中的功能意义知之甚少。本研究的目的是在ECs中分离这两部分酶，并确定它们在激动剂刺激和膜胆固醇水平调控下的功能意义。
利用RNA干扰策略，我们构建了稳定的ECs群体，其eNOS表达抑制率>90%且无法产生一氧化氮。用靶向高尔基体和质膜的eNOS重建这些eNOS“敲低”的ECs，恢复了其产生一氧化氮的能力。钙依赖性激动剂对ECs中质膜限制的eNOS是更有效的刺激物。相比之下，高尔基体eNOS对钙依赖性和Akt依赖性激动剂的反应较弱。局限于质膜的eNOS对膜胆固醇水平的调控更敏感，且会被氧化型低密度脂蛋白显著减弱。
在ECs内，质膜是产生一氧化氮的最有效部位，但对胆固醇水平和氧化型低密度脂蛋白更敏感。
