Calcium and calmodulin regulate mercury-induced phospholipase D activation in vascular endothelial cells.

Earlier, we reported that mercury, the environmental risk factor for cardiovascular diseases, activates vascular endothelial cell (EC) phospholipase D (PLD). Here, we report the novel and significant finding that calcium and calmodulin regulated mercury-induced PLD activation in bovine pulmonary artery ECs (BPAECs). Mercury (mercury chloride, 25 microM; thimerosal, 25 microM; methylmercury, 10 microM) significantly activated PLD in BPAECs. Calcium chelating agents and calcium depletion of the medium completely attenuated the mercury-induced PLD activation in ECs. Calmodulin inhibitors significantly attenuated mercury-induced PLD activation in BPAECs. Despite the absence of L-type calcium channels in ECs, nifedipine, nimodipine, and diltiazem significantly attenuated mercury-induced PLD activation and cytotoxicity in BPAECs. This study demonstrated the importance of calcium and calmodulin in the regulation of mercury-induced PLD activation and the protective action of L-type calcium channel blockers against mercury cytotoxicity in vascular ECs, suggesting mechanisms of mercury vasculotoxicity and mercury-induced cardiovascular diseases.

Redox-active antioxidant modulation of lipid signaling in vascular endothelial cells: vitamin C induces activation of phospholipase D through phospholipase A2, lipoxygenase, and cyclooxygenase.

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.