Role of Ca^<2+> signaling and reactive oxygen species in endothelial dysfunction induced by lysophosphatidylcholine

溶血磷脂酰胆碱(LPC)是一种氧化型低密度脂蛋白致动脉粥样硬化的化合物，可引起内皮功能障碍。在这项研究中，我们研究了钙离子和活性氧在低密度脂蛋白诱导内皮细胞(ECS)信号转导中的作用。LPC可以增加内皮细胞内钙离子浓度。用Fura-2法测定培养的人主动脉内皮细胞[Ca~(2+)]_i，用全细胞膜片钳方法测定膜电流。HMG-CoA还原酶抑制剂(他汀类)显著降低LPC引起的[Ca~(2+)&gt；]_i升高。这种抑制作用被香叶基香叶基焦磷酸(GGPP)迅速逆转，并被Rho和Rho-Kinase的抑制剂所模拟。LPC可在1min内诱导RhoA的GTP结合活性形式移位到细胞膜上，并降低细胞质中的RhoA水平，表明LPC能迅速激活RhoA。他汀类药物阻止gtp/gdp互换…GGPP可逆转LPC引起的RhoA及其膜转位的增多以及他汀类药物的上述作用。RhoA激活对他汀类药物和GGPP的反应与它们对钙离子动员的影响是一致的。LPC还在滞后后产生非选择性阳离子电流(NSC)。他汀类药物延长电流延迟，降低电流幅度，GGPP可阻断他汀类药物对电流的抑制作用。综上所述，LPC通过依赖Rho激活的PLC通路诱导内皮细胞钙动员和膜电流，他汀类药物通过阻止Rho依赖于GGPP的脂质修饰而阻断这些作用。本研究暗示Rho参与LPC对钙离子运动的刺激(Yokyama K，Ishibashi T等人)。发行量。2002年；105：962-967)。此外，我们还研究了LPC对心肌细胞膜电流及其信号转导的影响(Li，L等。莫尔·法玛科尔。2002：62；602-607)。我们还研究了在LPC刺激中ROS与钙离子内流的关系。LPC引起的[Ca~(2+)]_i升高可被超氧化物歧化酶和过氧化氢酶等自由基清除剂抑制。事实上，我们在LPC刺激60秒后检测到过氧化氢(H_2O_2)的产生。当有外源Ca~(2+)存在时，过氧化氢酶的抑制作用大于超氧化物歧化酶。过氧化氢酶可抑制LPC诱导的NSC的幅度，而SOD无此作用。H_2O_2确实能诱导ECS中的NSC。我们发现在LPC刺激中，H_2O_2参与了ECS的NSC，提示LPC产生的H_2O_2可能在NSC介导的钙信号转导中起作用。此外，我们还研究了RhoA激活在人巨噬细胞(动脉粥样硬化)组织因子(TF)和纤溶酶原激活物抑制物-1(PAI-1)基因表达中的作用。2002年；163：39-47，BBA。2002年；1590：123-130)。较少

Lysophosphatidylcholine (LPC) is an atherogenic compound of oxidized low density lipoprotein which evokes endothelial dysfunction. In this research project, we investigated the roles of Ca^<2+> and reactive oxygen species (ROS) in signal transduction induced by LPC in endothelial cells (Ecs).LPC is known to increase intracellular Ca^<2+> concentration ([Ca^<2+>]_i) Ecs. [Ca^<2+>]_i was determined in cultured human aortic Ecs by fura-2 assay and membrane current was measured by whole-cell patch-clamp. HMG-CoA reductase inhibitors (statins) markedly decreased the [Ca^<2+>]_i increase caused by LPC. This suppressive effect was quickly reversed by geranylgeranylpyrophosphate (GGPP) and was mimicked by inhibitors of Rho and Rho-kinase. LPC induced the translocation of the GTP-bound active form of RhoA into membranes within 1 minute as determined by pull-down assay and reduced the levels of RhoA in the cytoplasm, indicating that LPC quickly activates RhoA. Statins prevented the GTP/GDP excha … More nge of RhoA and its membrane translocation from the cytoplasm caused by LPC and these effects of statins were reversed by GGPP. The responses of RhoA activation to statins and GGPP concurred with their effects on Ca^<2+> mobilization. LPC also induced a nonselective cation current (NSC) after a lag. Statins prolonged the lag and decreased the current amplitude and GGPP abolished the inhibitory effect of statins on the current. In summary of this part, LPC induced Ca^<2+> mobilization and membrane current via a Rho activation-dependent PLC pathway in Ecs and statins blocked these effects by preventing the GGPP-dependent lipid modification of Rho. The present study implicates Rho in LPC stimulation of Ca^<2+> movement (Yokoyama K, Ishibashi T et al. Circulation. 2002 ; 105 : 962-967). In addition, the effect of LPC on membrane current and its signaling were examined in cardiomyocytes (Li L et al. Mol Pharmacol. 2002 : 62 ; 602-607).We also investigated the association of ROS with Ca^<2+> influx in LPC stimulation. The [Ca^<2+>]_i increase caused by LPC was suppressed by radical scavengers such as superoxide dismutase (SOD) and catalase. In fact, we detected the generation of hydrogen peroxide (H_2O_2) 60 seconds after LPC stimulation. When external Ca^<2+> was present, the suppressive effect of catalase was greater than that of SOD. Catalase, but not SOD, suppressed the amplitude of NSC induced by LPC. H_2O_2 indeed induced NSC in Ecs. We show that in LPC stimulation H_2O_2 contributes to NSC in Ecs, suggesting that H_2O_2 generated by LPC may play a role in Ca^<2+> signaling mediated via NSC.Furthermore, we studied the role of RhoA activation in the gene expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in human macrophages (Atherosclerosis. 2002 ; 163 : 39-47, BBA. 2002 ; 1590 : 123-130). Less