Thai, T., Zhong, F., Dang, L., Chan, E., Ku, J., Malle, E., Geczy, C. L., Keaney., Jr, J. F., & Thomas, S. R. (2021). Endothelial-transcytosed myeloperoxidase activates endothelial nitric oxide synthase via a phospholipase C-dependent calcium signaling pathway. Free Radical Biology and Medicine, 166, 255-264.
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 (Vmax), but not substrate affinity (Km), 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 dephosphorylation 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.
endothelial dysfunction, endothelial nitric oxide synthase, hydrogen peroxide, hypochlorous acid, L-arginine, myeloperoxidase, nitric oxide, oxidative stress, reactive oxygen species, redox signaling
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