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Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium

Kalwa, Hermann, Sartoretto, Juliano L., Martinelli, Roberta, Romero, Natalia, Steinhorn, Benjamin S., Tao, Ming, Ozaki, C. Keith, Carman, Christopher V., Michel, Thomas
Proceedings of the National Academy of Sciences of the United States of America 2014 v.111 no.9 pp. 3383-3388
AMP-activated protein kinase, adenosine diphosphate, antagonists, biosensors, cardiovascular diseases, cytosol, drug therapy, endothelial cells, endothelial nitric oxide synthase, endothelium, energy, fluorescence, guanosinetriphosphatase, hydrogen peroxide, image analysis, microfilaments, mitochondria, neoplasms, phospholipids, phosphorylation, receptor protein-tyrosine kinase, receptors, signal transduction, transcriptional activation, tyrosine
ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H ₂O ₂) in mediating cellular responses activated by the G protein-coupled P2Y1 receptor for ADP. We found that ADP-dependent phosphorylation of key endothelial signaling proteins—including endothelial nitric oxide synthase, AMP-activated protein kinase, and the actin-binding MARCKS protein—was blocked by preincubation with PEG-catalase, which degrades H ₂O ₂. ADP treatment promoted the H ₂O ₂-dependent phosphorylation of c-Abl, a nonreceptor tyrosine kinase that modulates the actin cytoskeleton. Cellular imaging experiments using fluorescence resonance energy transfer-based biosensors revealed that ADP-stimulated activation of the cytoskeleton-associated small GTPase Rac1 was independent of H ₂O ₂. However, Rac1-dependent activation of AMP-activated protein kinase, the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate, and the c-Abl–interacting protein CrkII are mediated by H ₂O ₂. We transfected endothelial cells with differentially targeted HyPer2 H ₂O ₂ biosensors and found that ADP promoted a marked increase in H ₂O ₂ levels in the cytosol and caveolae, and a smaller increase in mitochondria. We performed a screen for P2Y1 receptor-mediated receptor tyrosine kinase transactivation and discovered that ADP transactivates Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase expressed in these cells. Our observation that P2Y1 receptor-mediated responses involve Flt3 transactivation may identify a unique mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors promotes vascular dysfunction. Taken together, these findings establish a critical role for endogenous H ₂O ₂ in control of ADP-mediated signaling responses in the vascular wall.