Dynamic Receptor-dependent Activation of Inducible Nitric-oxide Synthase by ERK-mediated Phosphorylation of Ser745

Abstract

Nitric oxide (NO) is a pleiotropic regulator of vascular function, and its overproduction by inducible nitric-oxide synthase (iNOS) in inflammatory conditions plays an important role in the pathogenesis of vascular diseases. iNOS activity is thought to be regulated primarily at the level of expression to generate "high output" NO compared with constitutive NO synthases. Here we show iNOS activity is acutely up-regulated by activation of the B 1 -kinin receptor ( to Asp resulted in a basally hyperactive iNOS whose activity was not further increased by B 1 R agonist. ERK and phospho-ERK (after B 1 R activation) were co-localized with iNOS as determined by confocal fluorescence microscopy. Furthermore, ERK co-immunoprecipitated with iNOS. The discovery that iNOS can be phosphorylated by ERK and acutely activated by receptor-mediated signaling reveals a new level of regulation for this isoform. These findings provide a novel therapeutic target to explore in the treatment of vascular inflammatory diseases. Endothelial cells are a key source of nitric oxide (NO),2 an important mediator of vascular function (1-4). In general, NO serves a protective function at low concentrations under normal conditions, but in inflammatory conditions and at high levels, NO may contribute to tissue damage, especially after reaction with superoxide to form peroxynitrite (5-7). Under normal conditions, the primary NO synthase (NOS) generating NO in the vasculature is endothelial NOS (eNOS). The activity of this constitutive isoform is closely regulated in a variety of ways, including changes in intracellular Ca 2ϩ levels, Ser or Thr phosphorylation, S-nitrosylation, and by interaction with other proteins (4, 8 -11). Under inflammatory conditions, endothelial cells can also express inducible NOS (iNOS) (12-14). In contrast to eNOS, iNOS is considered to be regulated primarily at the level of expression (15). Once expressed, iNOS is thought to continuously generate NO in the presence of sufficient cofactors and substrate until the protein is degraded (15, 16). These properties have led to the concept that iNOS generates high output NO in an unregulated fashion with primarily cytotoxic functions, for example in the host defense response (4). We showed that activation of the inducible kinin B 1 receptor (B 1 R) with peptide agonists or angiotensin-converting enzyme inhibitors directly stimulates high output NO production in cytokinetreated human lung microvascular endothelial cells (HLMVEC) (13,17). Surprisingly, the NO produced in response to B 1 R activation was generated primarily by iNOS (13), indicating the possibility of its acute, post-translational activation. We report here that B 1 R activation results in ERK1/2 activation and phosphorylation of Ser 745 in iNOS, resulting in a profound activation and generation of "super-high output" NO. Receptor-dependent activation of iNOS via phosphorylation reveals a new layer of complexity in the regulation of this enzyme that can play an important role in inflammatory conditions. EXPERIMENTAL ...

Carboxypeptidase M and Kinin B1 Receptors Interact to Facilitate Efficient B1 Signaling from B2 Agonists

“…However, B1R signaling also has numerous beneficial effects, such as promoting angiogenesis and neovascularization during wound healing (62)(63)(64), protecting kidneys from renal fibrosis and attenuating cardiac remodeling in stroke-prone spontaneously hypertensive rats (65,66), and reducing lethality in a porcine model of endotoxic shock (67). B1R stimulation also results in high output NO production in human endothelial cells (21,22) via activation of extracellular signal-regulated kinase and acute stimulation of inducible nitric-oxide synthase activity via phosphorylation of Ser 745 (23). One outcome of high output NO production in endothelial cells is inhibition of protein kinase C⑀ activity (24).…”

“…This is of potential importance in inflammatory or pathological responses. For example, B1R activation stimulates extracellular signal-regulated kinase phosphorylation, prostaglandin production (20), and inducible nitric-oxide synthase-mediated high output NO production (21)(22)(23), which inhibits protein kinase C⑀ activity in endothelial cells (24).…”

Endothelial Nitric-oxide Synthase Activation Generates an Inducible Nitric-oxide Synthase-like Output of Nitric Oxide in Inflamed Endothelium

“…Moreover, it is generally held that vascular inflammation leads to reduced bioavailability of eNOS-derived NO and eNOS "uncoupling," accompanied by induction of endothelial iNOS expression, which is the source of high, uncontrolled NO that further exacerbates the disease process (10). However, we have found that iNOS activity in inflamed human endothelium can be increased 3-5-fold beyond its basal level by G protein-coupled receptor signaling (11)(12)(13), and accumulating evidence indicates that eNOS could also play an "iNOS"-type role in inflammation, but the mechanisms have not been defined (14).…”

“…In healthy endothelium, the kinin B2R activates eNOS resulting in a short burst of Ca 2ϩ -dependent NO production (20,21). Under inflammatory conditions, endothelial B1R stimulation leads to acute activation of iNOS via ERK1/2-dependent phosphorylation at Ser 745 and prolonged and high output NO production (11)(12)(13). However, signaling pathways for endothelial B2R-dependent NO production under inflammatory conditions have not been investigated.…”

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases

“…Jones et al have indicated the importance of calcium/ calmodulin-dependent protein kinase II in regulating iNOS trafficking and activity in VSM cells [77]. Activation of the inducible kinin B 1 receptor stimulates the production of NO in cytokine-treated human lung microvascular endothelial cells [131] and Zhang and coworkers have recently identified ser745 as a physiologically relevant -ERK-dependentphosphorylation site on human iNOS that enhances NO production [174]. These studies would indicate acute regulation of iNOS activity in human cells that express low levels of iNOS.…”