Studies on the mechanism for regulation of phospholipase A_2 activation by ceramide

本研究旨在探讨神经酰胺对胞浆型磷脂酶A_2（cPLA_2）的调节机制，以及神经酰胺通过促进cPLA_2活化在动脉粥样硬化和肾病发病中的作用。结果如下：（1）用Ca ^<2 +>离子载体刺激兔血小板，或在血小板溶解液中加入Ca ^<2 +>，通过免疫印迹分析测定血小板膜上cPLA_2蛋白的增加和膜上cPLA_2活性的增加，来评价血小板膜与cPLA_2的Ca ^<2 +>依赖性结合。2）在抗原刺激的大鼠嗜碱性白血病细胞中，神经酰胺促进原钒酸敏感蛋白酪氨酸磷酸酶的激活，使细胞外信号调节激酶1/2去磷酸化，从而减弱cPLA_2的激活能力。此外，神经酰胺抑制抗原诱导的磷脂酶D激活和随后的磷脂酸形成，这是一种特异性的抑制剂。 ...更多信息 鉴定为cPLA_2的底物。3）巨噬细胞摄取氧化型低密度脂蛋白刺激的胆固醇酯生成完全依赖于cPLA_2的激活。在此条件下，神经酰胺的从头合成被引发。此外，用鞘磷脂酶预处理细胞增强了酯的形成。提示神经酰胺通过激活cPLA_2促进胆固醇酯的形成，从而导致泡沫细胞的形成，提示神经酰胺参与了动脉粥样硬化的发生和发展。4）用肿瘤坏死因子α（TNF α）刺激系膜细胞，可激活核因子-κ B，增加环氧化酶-2的表达和分泌PLA_2。用鞘磷脂酶预处理细胞增强了这些反应，并且这种增强完全依赖于活性氧的产生。TNF α还激活鞘磷脂酶，从鞘磷脂产生神经酰胺。这些结果表明，神经酰胺促进了三尖杉酯碱的产生，并与肾脏疾病的发病机制有关。少

In the study, the regulatory mechanism of cytosolic phospholipase A_2 (cPLA_2) by ceramide, and the implication of ceramide in the pathogenesis of atherosclerosis and nephropathy via acceleration of cPLA_2 activation were investigated. The results obtained are as follows.1) When rabbit platelets were stimulated with Ca^<2+> ionophore or Ca^<2+> was added into platelet lysate, Ca^<2+>-dependent association of cPLA_2 with platelet membranes was assessed by increase in cPLA_2 protein using immunoblot analysis and in the activity in membranes. Ceramide apparently increased the association.2) In rat basophilic leukemia cells stimulated with antigen, ceramide accelerated orthovanadate-sensitive protein tyrosine phosphatase activation, which induces dephosphorylation of extracellular signal-regulated kinase 1/2 to attenuate the potency of cPLA_2 activation. Furthermore, ceramide suppressed antigen-induced phospholipase D activation and subsequent formation of phosphatidic acid, which is chara … More cterized to be substrate for cPLA_2. These events lead to decrease in cPLA_2 activity, hence, providing evidence that ceramide acts as a negative regulator in agonist-stimulated cPLA_2 activation.3) Cholesterol ester formation stimulated by the uptake of oxidized low-density lipoproteins in macrophages was fully dependent on cPLA_2 activation. Under the condition de novo synthesis of ceramide was elicited. Furthermore, pretreatment of the cells with sphingomyelinase enhanced the ester formation. These results indicate that de novo-synthesized ceramide accelerates cholesterol ester formation via cPLA_2 activation leading to foam cell formation, and suggest that ceramide is implicated in the initiation and development of atherosclerosis.4) Stimulation of mesangial cells with tumor necrosis factor α (TNFα) increased expression of cyclooxygenase-2 and secretory PLA_2 following activation of transcription factor, nuclear factor-κB. Pretreatment of the cells with sphingomyelinase enhanced these responses and the enhancement was fully dependent on the production of reactive oxygen species. TNFα also activated sphingomyelinase to produce ceramide from sphingomyelin. These results suggest that ceramide facilitates production of prostaglandins and is implicated in the pathogenesis of renal diseases. Less