Vitamin C-induced activation of phospholipase D in lung microvascular endothelial cells: Regulation by MAP kinases

Our earlier studies have shown that vitamin C at pharmacological doses (mM) induces loss of redox-dependent viability in bovine lung microvascular endothelial cells (BLMVECs) that is mediated by oxidative stress. Therefore, here, we investigated the vitamin C-induced activation of the lipid signaling enzyme, phospholipase D (PLD) in BLMVECs. Monolayer cultures of BLMVECs were treated with vitamin C (0-10 mM) for different time periods (0-2 h) and the activity of PLD was determined. Vitamin C induced activation of PLD in BLMVECs in a time- and dose-dependent fashion that was significantly attenuated by antioxidants, p38 mitogen-activated protein kinase (p38 MAPK)-specific inhibitor (SB203580), extracellular signal-regulated protein kinase (ERK)-specific inhibitor (PD98059), and transient transfection of cells with dominant-negative (DN)-p38 MAPK and DN-ERK1/ERK2. Vitamin C also induced phosphorylation and enhanced the activities of p38 MAPK and ERK in BLMVECs in a time-dependent fashion. It was also evident that vitamin C induced translocation of PLD₁ and PLD₂, association of p38 MAPK and ERK with PLD₁ and PLD₂, threonine phosphorylation of PLD₁ and PLD₂ and SB203580- and PD98059-inhibitable threonine phosphorylation of PLD₁ in BLMVECs. Transient transfection of BLMVECs with DN-p38 MAPK and DN-ERK1/ERK2 resulted in marked attenuation of vitamin C-induced phosphorylation of threonine in PLD₁ and PLD₂. We, for the first time, showed that vitamin C at pharmacological doses, activated PLD in the lung microvascular ECs through oxidative stress and MAPK activation.