Abstract-The stimulation of endothelium-dependent NO release by angiotensin-(1-7) ] has been indirectly shown in terms of vasodilation, which was diminished by NO synthase inhibition or removal of the endothelium. However, direct measurement of endothelium-derived NO has not been analyzed. With a selective porphyrinic microsensor, NO release was directly assessed from single primary cultured bovine aortic endothelial cells. Ang-(1-7) caused a concentration-dependent release of NO of 1 to 10 mol/L, which was attenuated by NO synthase inhibition.[D-Ala 7 ]Ang-(1-7) (5 mol/L), described as a selective antagonist of Ang-(1-7) receptors, inhibited Ang-(1-7)-induced NO release only by Ϸ50%, whereas preincubation of bovine aortic endothelial cells with the angiotensin II subtype 1 and 2 receptor antagonists EXP 3174 and PD 123,177 (both at 0.1 mol/L) led to an inhibition of 60% and 90%, respectively. A complete blockade of the Ang-(1-7)-induced NO release was observed on preincubation of the cells with 1 mol/L concentration of the bradykinin subtype 2 receptor antagonist icatibant (HOE 140), suggesting an important role of local kinins in the action of Ang-(1-7). Simultaneous direct measurement of superoxide (O 2 Ϫ ) detected by an O 2 Ϫ -sensitive microsensor revealed that the moderately Ang-(1-7)-stimulated NO release was accompanied by a very slow concomitant O 2 Ϫ production with a relative low peak concentration in comparison to the O 2 Ϫ production of the strong NO releasers bradykinin and, especially, calcium ionophore. Thus, Ang-(1-7) might preserve the vascular system, among others, due to its low formation of cytotoxic peroxynitrite by the reaction between NO and O 2 Ϫ . (Hypertension. 2001;37:72-76.)

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 ...

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO(-/-) mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO(-/-) mice unexpectedly had improved survival compared with wild-type (WT) mice within 5-12 h after intraperitoneal E. coli challenge. Lungs of MPO(-/-) mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO(-/-) mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO(-/-) mice. Inhibition of iNOS in MPO(-/-) mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO(-/-) mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.

Inducible nitric oxide synthase (iNOS) is a major source of nitric oxide during inflammation whose activity is thought to be controlled primarily at the expression level. The B1 kinin receptor (B1R) post-translationally activates iNOS beyond its basal activity via extracellular signal regulated kinase (ERK)-mediated phosphorylation of Ser745. Here we identified the signalling pathway causing iNOS activation in cytokine-treated endothelial cells or HEK293 cells transfected with iNOS and B1R. To allow kinetic measurements of nitric oxide release, we used a sensitive porphyrinic microsensor (response time = 10 msec.; 1 nM detection limit). B1Rs signalled through Gαi coupling as ERK and iNOS activation were inhibited by pertussis toxin. Furthermore, transfection of constitutively active mutant Gαi Q204L but not Gαq Q209L resulted in high basal iNOS-derived nitric oxide. G-βγ subunits were also necessary as transfection with the β-adrenergic receptor kinase C-terminus inhibited the response. B1R-dependent iNOS activation was also inhibited by Src family kinase inhibitor PP2 and trans-fection with dominant negative Src. Other ERK-MAP kinase members were involved as the response was inhibited by dominant negative H-Ras, Raf kinase inhibitor, ERK activation inhibitor and MEK inhibitor PD98059. In contrast, PI3 kinase inhibitor LY94002, calcium chelator 1,2-bis-(o-Aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), protein kinase C inhibitor calphostin C and protein kinase C activator PMA had no effect. Angiotensin converting enzyme inhibitor enalaprilat also directly activated B1Rs to generate high output nitric oxide via the same pathway. These studies reveal a new mechanism for generating receptor-regulated high output nitric oxide in inflamed endothelium that may play an important role in the development of vascular inflammation.