Institute of Hepatobiliary Surgery, Chinese PLA General Hospital, Beijing 100853, China.
Prostaglandins Leukot Essent Fatty Acids. 2010 Jul;83(1):37-43. doi: 10.1016/j.plefa.2010.02.002. Epub 2010 Mar 4.
To determine whether DHA suppresses oxidative stress induced endothelial cell calcium influx by altering lipid composition and TRPC1 distribution in membrane rafts.
Endothelial cells (EC) were pretreated with DHA or stearic acid, then incubated for another 3h with media containing H(2)O(2). Membrane lipid rafts were isolated using the discontinuous sucrose density gradient ultracentrifugation method. Intracellular calcium was detected with laser scanning confocal microscope. TRPC1 protein in membrane fractions was detected by immunoblotting. Membrane fatty acids compositions were analyzed by gas chromatography; raft cholesterol level was assayed by an Amplex Red Cholesterol Assay kit, and DAG concentration was quantified by a DAG kinase assay.
DHA significantly reduced oxidative stress induced calcium influx; pretreated with DHA the n-3 PUFAs were significantly increased in raft fractions, as well as saturated myristic acid, palmitic acid content of membrane rafts in EC; while the stearic acid, monounsaturated oleic acid and cis-oleic acid were decreased. Incubation with DHA also significantly reduced the amount of SM and cholesterol levels in the raft. Interestingly, we fractioned plasma membrane subcellular compartments and discovered that certain amounts of TRPC1 existed in detergent-resistant plasma membrane fractions of EC. After DHA treatment, TRPC1 was partly displaced from lipid raft to detergent-soluble membrane fractions.
DHA significantly reduces oxidative stress induced endothelial calcium influx, this effect might be associated with, at least in part, altered raft lipid environment, and suppresses TRPC1-mediated calcium signaling pathway by partially displacing TRPC1 from membrane caveolar lipid rafts.
通过改变脂质组成和质膜筏 TRPC1 分布，确定 DHA 是否通过抑制氧化应激诱导的内皮细胞钙内流。
用 DHA 或硬脂酸预处理内皮细胞（EC），然后用含 H2O2 的培养基孵育 3 小时。采用不连续蔗糖密度梯度超速离心法分离质膜脂筏。用激光扫描共聚焦显微镜检测细胞内钙。用免疫印迹法检测膜部分的 TRPC1 蛋白。用气相色谱法分析膜脂肪酸组成；用 Amplex Red 胆固醇测定试剂盒测定筏胆固醇水平，用 DAG 激酶测定法定量测定 DAG 浓度。
DHA 显著降低氧化应激诱导的钙内流；用 DHA 预处理后，n-3PUFAs 显著增加，EC 质膜筏中的饱和豆蔻酸、棕榈酸含量也增加；而硬脂酸、单不饱和油酸和反式油酸含量降低。DHA 孵育也显著降低了筏中的 SM 和胆固醇水平。有趣的是，我们对质膜亚细胞区室进行了分级，发现 EC 的去污剂抗性质膜部分存在一定量的 TRPC1。DHA 处理后，TRPC1 部分从质膜筏转移到去污剂可溶膜部分。
DHA 显著降低氧化应激诱导的内皮细胞钙内流，这种作用可能与改变质膜筏脂质环境有关，通过部分将 TRPC1 从质膜小窝脂筏中置换出来，抑制 TRPC1 介导的钙信号通路。
