Endothelial Nitric Oxide Synthase Activation Generates an Inducible Nitric Oxide Synthase-Like Output of Nitric Oxide in Inflamed Endothelium
journal contributionposted on 15.04.2014, 00:00 by Jessica L. Lowry, Viktor Brovkovych, Yongkang Zhang, Randal A. Skidgel
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 kinin B2 receptor (B2R) signaling that results in Ca2+ dependent activation of eNOS and transient NO. In inflamed HLMVEC(pretreated with interleukin-1 beta and interferon-gamma), 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 G alpha(i)-, MEK1/2-, and JNK1/2-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 JNK phosphorylation were inhibited with MEK1/2 and JNK inhibitors or MEK1/2 and JNK1/2 siRNA but not with ERK1/2 inhibitor. BK induced Ca2+-dependent eNOS phosphorylation at Ser(1177), Thr(495), and Ser(114) 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, G alpha(q/11), G(alpha i1,2), JNK, 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.