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      The Effect of the Nitric Oxide Synthase Inhibitor N-Nitro- L-Arginine-Methyl Ester on Neuropeptide-Induced Vasodilation and Protein Extravasation in Human Skin

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          Abstract

          Endogenous neuropeptides released from nociceptors can induce vasodilation and enhanced protein extravasation (neurogenic inflammation). The role of nitric oxide (NO) in the induction of neurogenic inflammation is controversial. In this study, dermal microdialysis was used in awake humans (n = 39) to deliver substance P (SP; 10<sup>–7</sup> and 10<sup>–6</sup> M) or calcitonin gene-related peptide (CGRP; 5 × 10<sup>–7</sup> M and 2 × 10<sup>–6</sup> M). Neuropeptide-induced local and axon reflex erythema was assessed by laser Doppler imaging. Total protein concentration in the dialysate was measured to quantify local protein extravasation. The responses were assessed in the absence and the presence of the nitric oxide synthase inhibitor, N-nitro- L-arginine-methyl ester ( L-NAME) added to the perfusate at concentrations of 5, 10 or 20 m M. L-NAME (5 m M) applied via the dialysis catheters reduced local blood flow by approximately 30%. In addition, L-NAME inhibited SP-induced vasodilation by about 40% for 10<sup>–7</sup> M SP and 30% for 10<sup>–6</sup> M SP (n = 11, p < 0.01). In contrast, CGRP-induced vasodilation was only marginally inhibited by L-NAME. SP, but not CGRP, provoked a dose-dependent increase in protein extravasation. L-NAME (5 m M) inhibited this increase by up to 40% for both SP concentrations used (n = 11, p < 0.01). Higher concentrations of L-NAME did not further reduce SP- or CGRP-induced vasodilation or SP-induced protein extravasation. Exogenously applied SP induces vasodilation and protein extravasation, which is partly NO mediated, whereas CGRP-induced vasodilation appears to be NO independent.

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          Most cited references 17

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          Inducible nitric-oxide synthase generates superoxide from the reductase domain.

          In the absence of L-arginine, the heme center of the oxygenase domain of neuronal nitric-oxide synthase reduces molecular oxygen to superoxide (O-2). Our recent work has provided evidence that inducible NOS (iNOS) may also catalyze O-2 formation in macrophages. However, there has been a lack of direct evidence of superoxide generation from the purified iNOS, and it was previously hypothesized that significant O-2 production does not occur. Moreover, the mechanism and enzyme site responsible for O-2 generation is unknown. To determine whether iNOS produces O-2 and to identify the mechanism of this process, we performed electron paramagnetic resonance measurements on purified iNOS using the spin trap 5,5-dimethyl-1-pyrroline N-oxide. In the presence of NADPH, prominent O-2 adduct signals were detected from iNOS. These signals were totally abolished by superoxide dismutase but not affected by catalase. High concentrations of L-arginine decreased this O-2 formation, whereas its enantiomer D-arginine did not. Pre-incubation of iNOS with the flavoprotein inhibitor diphenyleneiodonium totally blocked these O-2 signals. Conversely, pretreatment of the enzyme with the heme blocker cyanide had no effect on O-2 generation. Furthermore, strong O-2 generation was directly detected from the isolated iNOS reductase domain. Together, these data demonstrate that iNOS does generate O-2, and this mainly occurs at the flavin-binding sites of the reductase domain.
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            Superoxide generation by endothelial nitric oxide synthase: The influence of cofactors

            The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of l -arginine, N G -hydroxy- l -arginine, l -thiocitrulline, and l - N G -monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous–dioxygen complex and that occupation of the l -arginine binding site does not inhibit this process. However, the concomitant addition of l -arginine and tetrahydrobiopterin (BH 4 ) abolishes superoxide generation by eNOS. Under these conditions, l -citrulline production is close to maximal. Our data indicate that BH 4 fully couples l -arginine oxidation to NADPH consumption and prevents dissociation of the ferrous–dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH 4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.
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              Calcitonin gene-related peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat.

              Systemic blood pressure is controlled by changes in the resistance of the peripheral vascular bed for example in the mesenteric blood vessels. The tone of peripheral blood vessels is primarily maintained by sympathetic vasoconstrictor nerves. Although vasodilator innervation has been identified in certain isolated elastic arteries, it is not known whether vasodilator nerves contribute to the regulation of the peripheral resistance vessels. We present pharmacological evidence for the existence of nonadrenergic, noncholinergic (NANC) vasodilator nerves in the mesenteric resistance vessel of the rat and that the resistance is controlled by not only sympathetic vasoconstrictor nerves but also NANC vasodilator nerves. We also show that the neurogenic vasodilation was selectively abolished by depleting endogenous calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide, from perivascular nerves. This indicates that CGRP is a novel vasodilator neurotransmitter and may play a role in control of the total peripheral resistance of systemic circulation through a local reflex mechanism.
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                Author and article information

                Journal
                JVR
                J Vasc Res
                10.1159/issn.1018-1172
                Journal of Vascular Research
                S. Karger AG
                1018-1172
                1423-0135
                2003
                April 2003
                19 June 2003
                : 40
                : 2
                : 105-114
                Affiliations
                Departments of aPhysiology and Experimental Pathophysiology, bPediatrics, University of Erlangen/Nürnberg, Erlangen, and cAnesthesiology Mannheim, University of Heidelberg, Heidelberg, Germany; dDepartment of Allergy and Inflammation Sciences, University of Southampton, Southampton, UK
                Article
                70707 J Vasc Res 2003;40:105–114
                10.1159/000070707
                12808346
                © 2003 S. Karger AG, Basel

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                Page count
                Figures: 4, References: 56, Pages: 10
                Categories
                Research Paper

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