Defining Their Bioactivity
Nitric oxide (NO) is an endogenously generated diffusible messenger that mediates a multitude of physiological and pathological processes. Normally, NO is produced in low concentrations and acts both as a messenger and cytoprotective factor via direct interactions with transition metals and other free radicals. However, in the setting of inflammation or shock, the substantial amounts of NO released may lead to the formation of cytotoxic species. When NO is transformed into a nitrating or nitrosating species it can readily react with many other factors potentially modulating their biological activity. These NO-dependent interactions are central in the regulation of many processes affecting vascular and atherothrombotic disease.
Previous studies have shown that NO reacts with both lipids and lipoproteins. The interaction of NO with oxidizing lipids can be either protective to the vasculature or enhance inflammatory-mediated vascular injury. In certain situations, low levels of NO generated by endothelial NO synthase (eNOS) can terminate lipid radical chain propagation reactions.1 Conversely, prooxidant reactions can occur after superoxide reacts with NO and leads to the formation of potent secondary oxidants, such as peroxynitrite and nitrogen dioxide, that can enhance inflammatory injury to vascular cells.2
Unsaturated lipids of membranes and lipoproteins can be critical targets of reactive oxygen and nitrogen species, suggesting potential relevance for nitrogen-containing lipid products. When NO is transformed into a nitrating and nitrosating species, it has been shown to react with unsaturated lipids.3,4⇓ Recently, such nitrated lipids have been demonstrated to be formed in vivo.5 The oxidation product of NO, NO2, has been shown to react with arachidonic acid generating biologically active nitrohydroxyeicosatrienoic acids. These products appeared to be endogenous mediators of vascular tone, stimulating smooth muscle relaxation and leading to soluble guanylate cyclase activation.5
Although there is limited information concerning the bioactivity of NO-derived nitrated lipids, products of the reaction of ONOO− with linoleic acid have been shown to have characteristics of nitrolinoleate, a synthetic nitrated lipid. The effect of nitrolinoleate on human platelets has been studied to examine the impact of these species on thrombosis.6 Nitrolinoleate, but not linoleate or 3-nitrotyrosine, inhibited thrombin-induced platelet aggregation and significantly decreased thrombin-stimulated Ca2+ elevations. Interestingly, nitrolinoleate did not elevate platelet cGMP and its effects were not blocked with inhibitors of NO signaling such as oxyhemoglobin. However, exposure to nitrolinoleate led to increased platelet cAMP and inhibitors of adenylyl cyclase partially restored thrombin-stimulated aggregation. In platelet lysates, nitrolinoleate also inhibited cAMP hydrolysis to AMP. This study was particularly interesting as an NO-derived species had been shown to inhibit platelet function via a cGMP-independent mechanism. The precise mechanism for these effects is not well understood and may be due to the metabolism of the nitrated lipid or alteration in expression of eicosanoid receptor–activated adenylate cyclase. Although nitrated lipids could mediate signal transduction and platelet inhibition via direct NO donation or transnitrosation, the lack of elevation in cGMP suggests other potential mechanism(s).
In this issue of Circulation Research, Coles et al7 again examined nitrolinoleate to determine its effect on human neutrophil activity. Previously, it has been shown that administration of tumor necrosis factor-α as well as interleukin-8 inhibit neutrophil migration by stimulating endogenous NO production and these effects could be blocked with inhibitors of NO synthase.8 Although the significance of NO generated by neutrophils during inflammatory reactions is incompletely understood, exogenous NO has been clearly shown to exert regulatory effects on neutrophil function. Depending on dose and experimental conditions, NO may either inhibit or enhance neutrophil function. Low concentrations of NO have been mostly found to be stimulatory to neutrophils, whereas high concentrations appear inhibitory. It has been shown that NO, through increased cyclic GMP, inhibits the activation of human neutrophils and may thus act as a local modulator in the inflammatory process.9 Nitric oxide inhibits neutrophil-myocyte adhesion mainly by acting on neutrophils and without quantitatively affecting the upregulation of CD11b/CD18.10 Conversely, NO has been shown to increase endothelial-neutrophil adhesion through protein kinase G–mediated P-selectin mobilization to the cell surface as well as enhance platelet-activated factor synthesis.11
Coles and colleagues7 extend their previous platelet findings by demonstrating that a nitrated unsaturated fatty acid, specifically nitrolinoleate, also inhibits neutrophil function in vitro. In this study, nitrolinoleate inhibited generation of superoxide as well as cellular degranulation. The expression of CD11b was also attenuated, suggesting inhibition of adhesion and demonstrating inhibitory actions on signaling processes involved in neutrophil activation. Nitrolinoleate, but not linoleic acid or nitrated amino acid 3-nitrotyrosine, inhibited Ca2+ influx, superoxide release, and elastase release after stimulation. Incubation with nitrolinoleate without stimulation of the neutrophils did not alter superoxide release.
In this study, the authors also begin to define the mechanism(s) for these interesting observations. The findings suggest that incubation with a nitrated lipid leads to alteration of multiple neutrophil functions including degranulation as well as adhesion. As opposed to previous studies examining the direct effects of NO on neutrophils, nitrolinoleate altered Ca2+ flux but did not increase cyclic GMP, and inhibition of guanylate cyclase did not restore neutrophil responses. However, incubation with nitrolinoleate led to elevations in cyclic AMP. Additionally, the effects did not appear to be due to a direct effect on NADPH oxidase–dependent superoxide generation. Neutrophils activated with N-formyl-Met-Leu-Phe (fMLP) were inhibited more by nitrolinoleate than when phorbol 12-myristate-13-acetate (PMA) stimulated, suggesting that this nitrated species likely has multiple sites of action. These experiments, however, are nonspecific and more information is needed to understand the precise signaling pathways affected by nitrated lipids. It is also unclear why the findings in neutrophils differ from those in myocytes where previously a nitrated lipid was shown to moderate vascular reactivity via soluble guanylate cyclase activation.5 Whether this is due to differences in metabolism of the nitrated lipid or receptor-specific events remains to be determined.
Although these findings are intriguing, further studies documenting the formation of nitrated lipids in relevant quantities in vivo are needed. Levels of nitrated lipids formed in vivo are too low to allow for purification and use in experimental studies; however, further identification of these isomers and examination of their bioactivity will be important. The form of nitrated lipid used in this study also raises questions. Although it is structurally similar to nitrated lipids observed in vivo, its bioactivity may not necessarily be the same. The lack of neutrophil inhibition by 3-nitrotyrosine or linoleic acid of fMP-stimulated cells indicates that the effect of nitrolinoleate is not common to all nitro compounds or 18:2 fatty acids; however, it still does not address the issue of similarity to other nitrated lipids. The preparation used was a mixture of 4 positional isomers. Although formation of nitrated fatty acids in vivo is not likely to be isomer specific, activation of adenylate cyclase through receptor-dependent pathways may be isomer specific.
The assumption from this study is that nitrated lipid species would be formed during sepsis when leukocyte release of NO would be greatest; however, the biological reason for neutrophil inhibition in this setting is not clear. Impairment of neutrophil migration has been shown present in an animal model of sepsis and the leukocyte inhibition was NO dependent.12 In contrast to the general findings of the present study, leukocytes from septic individuals have been shown to have an increase in adhesion.13
Nitrated lipids found in plasma and select tissue may have properties beneficial in the atherothrombotic process, including platelet inhibition and vascular smooth muscle relaxation. This study adds to these findings by showing that nitrolinoleate inhibits neutrophil activation in response to stimulation. This potentially antiinflammatory effect would be beneficial in the setting of vascular disease; however, its role in the setting of sepsis is less clear. Further characterization of its formation and role in inflammatory diseases as well as the mechanism(s) for these findings will be important in establishing the bioactivity of nitrated lipids.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- ↵Rubbo H, Radi R, Anselmi D, Kirk M, Barnes S, Butler J, Eiserich JP, Freeman BA. Nitric oxide reaction with lipid peroxyl radicals spares α-tocopherol during lipid peroxidation: greater oxidant protection from the pair nitric oxide/α-tocopherol than α-tocopherol/ascorbate. J Biol Chem. 2000; 275: 10812–10818.
- ↵Rubbo H, Radi R, Trujillo M, Telleri R, Kalyanaraman B, Barnes S, Kirk M, Freeman BA. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation: formation of novel nitrogen-containing oxidized lipid derivatives. J Biol Chem. 1994; 269: 26066–26075.
- ↵Balazy M, Iesaki T, Park JL, Jiang H, Kaminski PM, Wolin MS. Vicinal nitrohydroxyeicosatrienoic acids: vasodilator lipids formed by reaction of nitrogen dioxide with arachidonic acid. J Pharmacol Exp Ther. 2001; 299: 611-619.
- ↵Coles B, Bloodsworth A, Eiserich JP, Coffey MJ, McLoughlin RM, Giddings JC, Lewis MJ, Haslam RJ, Freeman BA, O’Donnell VB. Nitrolinoleate inhibits platelet activation by attenuating calcium mobilization and inducing phosphorylation of vasodilator-stimulated phosphoprotein through elevation of cAMP. J Biol Chem. 2002; 277: 5832–5840.
- ↵Coles B, Bloodsworth A, Clark SR, Lewis MJ, Cross AR, Freeman BA, O’Donnell VB. Nitrolinoleate inhibits superoxide generation, degranulation, and integrin expression by human neutrophils: novel antiinflammatory properties of nitric oxide–derived reactive species in vascular cells. Circ Res. 2002; 91: 375–381.
- ↵Benjamim CF, Ferreira SH, Cunha FQ. Role of nitric oxide in the failure of neutrophil migration in sepsis. J Infect Dis. 2000; 182: 214–223.