Steinhour Emily, Sherwani Shariq I, Mazerik Jessica N, Ciapala Valorie, O'Connor Butler Elizabeth, Cruff Jason P, Magalang Ulysses, Parthasarathy Sampath, Sen Chandan K, Marsh Clay B, Kuppusamy Periannan, Parinandi Narasimham L
Lipid Signaling and Lipidomics Laboratory, Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Department of Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA.
Mol Cell Biochem. 2008 Aug;315(1-2):97-112. doi: 10.1007/s11010-008-9793-6. Epub 2008 May 22.
We have earlier reported that the redox-active antioxidant, vitamin C (ascorbic acid), activates the lipid signaling enzyme, phospholipase D (PLD), at pharmacological doses (mM) in the bovine lung microvascular endothelial cells (BLMVECs). However, the activation of phospholipase A(2) (PLA(2)), another signaling phospholipase, and the modulation of PLD activation by PLA(2) in the ECs treated with vitamin C at pharmacological doses have not been reported to date. Therefore, this study aimed at the regulation of PLD activation by PLA(2) in the cultured BLMVECs exposed to vitamin C at pharmacological concentrations. The results revealed that vitamin C (3-10 mM) significantly activated PLA(2) starting at 30 min; however, the activation of PLD resulted only at 120 min of treatment of cells under identical conditions. Further studies were conducted utilizing specific pharmacological agents to understand the mechanism(s) of activation of PLA(2) and PLD in BLMVECs treated with vitamin C (5 mM) for 120 min. Antioxidants, calcium chelators, iron chelators, and PLA(2) inhibitors offered attenuation of the vitamin C-induced activation of both PLA(2) and PLD in the cells. Vitamin C was also observed to significantly induce the formation and release of the cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites and to activate the AA LOX in BLMVECs. The inhibitors of PLA(2), COX, and LOX were observed to effectively and significantly attenuate the vitamin C-induced PLD activation in BLMVECs. For the first time, the results of the present study revealed that the vitamin C-induced activation of PLD in vascular ECs was regulated by the upstream activation of PLA(2), COX, and LOX through the formation of AA metabolites involving oxidative stress, calcium, and iron.
我们之前报道过，氧化还原活性抗氧化剂维生素C（抗坏血酸）在药理剂量（毫摩尔）下可激活牛肺微血管内皮细胞（BLMVECs）中的脂质信号酶磷脂酶D（PLD）。然而，另一种信号磷脂酶磷脂酶A2（PLA2）的激活以及在药理剂量维生素C处理的内皮细胞中PLA2对PLD激活的调节，迄今为止尚未见报道。因此，本研究旨在探讨在药理浓度维生素C作用下，培养的BLMVECs中PLA2对PLD激活的调节作用。结果显示，维生素C（3 - 10毫摩尔）在30分钟时显著激活PLA2；然而，在相同条件下处理细胞120分钟时才导致PLD激活。利用特异性药理试剂进行了进一步研究，以了解在维生素C（5毫摩尔）处理120分钟的BLMVECs中PLA2和PLD的激活机制。抗氧化剂、钙螯合剂、铁螯合剂和PLA2抑制剂可减弱细胞中维生素C诱导的PLA2和PLD激活。还观察到维生素C能显著诱导环氧化酶（COX）和脂氧合酶（LOX）催化的花生四烯酸（AA）代谢产物的形成和释放，并激活BLMVECs中的AA LOX。观察到PLA2、COX和LOX抑制剂可有效且显著减弱维生素C诱导的BLMVECs中PLD激活。本研究结果首次揭示，血管内皮细胞中维生素C诱导的PLD激活受PLA2、COX和LOX的上游激活调节，通过涉及氧化应激、钙和铁的AA代谢产物形成来实现。
