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

本研究探讨了神经酰胺对胞质磷脂酶A_2 （cPLA_2）的调控机制，以及神经酰胺通过加速cPLA_2活化参与动脉粥样硬化和肾病的发病机制。所得结果如下：1)用Ca^<2+>离子载体刺激兔血小板或在血小板裂解液中加入Ca^<2+>离子，通过免疫印迹法测定cPLA_2蛋白的升高和血小板膜活性的升高，评价Ca^<2+>依赖性cPLA_2与血小板膜的关联。神经酰胺明显增加了这种关联。2)在抗原刺激的大鼠嗜碱性白血病细胞中，神经酰胺加速了原钒酸盐敏感蛋白酪氨酸磷酸酶的激活，导致细胞外信号调节激酶1/2的去磷酸化，从而减弱了cPLA_2的激活效力。此外，神经酰胺抑制抗原诱导的磷脂酶D的激活和随后磷脂酸的形成，磷脂酸更被认为是cPLA_2的底物。这些事件导致cPLA_2活性降低，因此，神经酰胺在激动剂刺激的cPLA_2激活中起负调节作用。3)巨噬细胞摄取氧化低密度脂蛋白刺激胆固醇酯的形成完全依赖于cPLA_2的活化。在此条件下，引发了神经酰胺的新合成。此外，鞘磷脂酶预处理细胞可促进酯的形成。这些结果表明，新合成的神经酰胺通过cPLA_2活化导致泡沫细胞形成，从而加速胆固醇酯的形成，并提示神经酰胺参与动脉粥样硬化的发生和发展。4)肿瘤坏死因子α (TNFα)刺激系膜细胞后，转录因子、核因子-κB活化后，环氧化酶-2的表达和pla2的分泌增加。鞘磷脂酶预处理细胞增强了这些反应，这种增强完全依赖于活性氧的产生。TNFα也激活鞘磷脂酶，使鞘磷脂产生神经酰胺。这些结果表明神经酰胺促进前列腺素的产生，并参与肾脏疾病的发病机制。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