Endothelial-transcytosed myeloperoxidase activates endothelial nitric oxide synthase via a phospholipase C-dependent...

During vascular inflammation, the leukocyte-derived enzyme myeloperoxidase (MPO) is transcytosed across the endothelium and into the sub-endothelial extracellular matrix, where it promotes endothelial dysfunction by catalytically consuming nitric oxide (NO) produced by endothelial NO synthase (eNOS). In the presence of chloride ions and hydrogen peroxide (H₂O₂), MPO forms the oxidant hypochlorous acid (HOCl). Here we examined the short-term implications of HOCl produced by endothelial-transcytosed MPO for eNOS activity. Incubation of MPO with cultured aortic endothelial cells (ECs) resulted in its transport into the sub-endothelium. Exposure of MPO-containing ECs to low micromolar concentrations of H₂O₂ yielded enhanced rates of H₂O₂ consumption that correlated with HOCl formation and increased eNOS enzyme activity. The MPO-dependent activation of eNOS occurred despite reduced cellular uptake of the eNOS substrate l-arginine, which involved a decrease in the maximal activity (V_max), but not substrate affinity (K_m), of the major endothelial l-arginine transporter, cationic amino acid transporter-1. Activation of eNOS in MPO-containing ECs exposed to H₂O₂ involved a rapid elevation in cytosolic calcium and increased eNOS phosphorylation at Ser-1179 and de-phosphorylation at Thr-497. These signaling events were attenuated by intracellular calcium chelation, removal of extracellular calcium and inhibition of phospholipase C. This study shows that stimulation of endothelial-transcytosed MPO activates eNOS by promoting phospholipase C-dependent calcium signaling and altered eNOS phosphorylation at Ser-1179 and Thr-497. This may constitute a compensatory signaling response of ECs aimed at maintaining eNOS activity and NO production in the face of MPO-catalyzed oxidative stress.