Endothelial nitric-oxide synthase activation generates an inducible nitric-oxide synthase-like output of nitric oxide in inflamed endothelium

Abstract

High levels of NO generated in the vasculature under inflammatory conditions are usually attributed to inducible nitric-oxide synthase (iNOS), but the role of the constitutively expressed endothelial NOS (eNOS) is unclear. In normal human lung microvascular endothelial cells (HLMVEC), bradykinin (BK) activates kiniii B2 receptor (B2R) signaling that results in Ca 2+-dependent activation of eNOS and transient NO. In inflamed HLMVEC (pretreated with interleukin-1β and interferon-y), we found enhanced binding of eNOS to calcium-calmodulin at basal Ca2+ levels, thereby increasing its basal activity that was dependent on extracellular L-Arg. Furthermore, B2R stimulation generated prolonged high output eNOS-derived NO that is independent of increased intracellular Ca2+ and is mediated by a novel Gαr, MEK1/2-, and JNKl/2-dependent pathway. This high output NO stimulated with BK was blocked with a B2R antagonist, eNOS siRNA, or eNOS inhibitor but not iNOS inhibitor. Moreover, B2R-mediated NO production and INK phosphorylation were inhibited with MEK1/2 and INK inhibitors or MEK1/2 and JNK1/2 siRNA but not with ERK1/2 inhibitor. BK induced Ca 2+-dependent eNOS phosphorylation at Ser1177, Thr 495, and Ser114 in cytokine-treated HLMVEC, but these modifications were not dependent on JNK1/2 activation and were not responsible for prolonged NO output. Cytokine treatment did not alter the expression of B2R, Gαq/11, Gαi1,2, INK, or eNOS. B2R activation in control endothelial cells enhanced migration, but in cytokine-treated HLMVEC it reduced migration. Both responses were NO-dependent. Understanding how JNK regulates prolonged eNOS-derived NO may provide new therapeutic targets for the treatment of disorders involving vascular inflammation. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.