NOS-1-derived NO is an essential triggering signal for the development of systemic inflammatory responses.

Nitric oxide (NO) produced by the NO synthase type 2 (NOS-2) is known to have a prominent role in the course of the inflammatory response but less is known concerning the role of NO derived from the constitutive NOS isoforms. We have examined the role of NO derived from NOS-1 in the initiation of the systemic inflammatory response using sepsis models. Injection of LPS in rats induced an early hypotension, NOS-2 expression, increased lung myeloperoxidase activity and increased NO metabolite (NOx) levels in the skeletal muscle. Pre-treatment with 7-nitroindazol (7-NI) prevented all these changes, but its administration after LPS injection was ineffective. Septic (cecal ligation and puncture method, CLP) rats exhibited signs of organ failure, hyporesponsiveness to vasoconstrictors and 75% mortality over 3 days after surgery. Pre-treatment with 7-NI prevented or significantly reduced these alterations. Injection of 7-NI after sepsis onset was without effect. Wild type mice injected with LPS exhibited increased plasma NOx, NOS-2 and COX-2 expression and 80% mortality. NOS-1(-/-) mice injected with LPS exhibited smaller increase in plasma NOx, no NOS-2 and COX-2 expression and reduced mortality. Injection of an NO donor in CLP rats pre-treated with 7-NI or in NOS-1(-/-) mice returned the mortality rate to those of CLP in rats and LPS in mice. Our results demonstrate that NOS-1-derived NO acts as a signaling element and it is essential for the initiation of systemic inflammation as demonstrated by the reduction of the inflammatory response and mortality by both pharmacological inhibition and genetic deletion of NOS-1.

Sexually dimorphic actions of glucocorticoids provide a link to inflammatory diseases with gender differences in prevalence.

Males and females show differences in the prevalence of many major diseases that have important inflammatory components to their etiology. These gender-specific diseases, which include autoimmune diseases, hepatocellular carcinoma, diabetes, and osteoporosis, are largely considered to reflect the actions of sex hormones on the susceptibility to inflammatory stimuli. However, inflammation reflects a balance between pro- and anti-inflammatory signals, and investigation of gender-specific responses to the latter has been neglected. Glucocorticoids are the primary physiological anti-inflammatory hormones in mammals, and synthetic derivatives of these hormones are prescribed as anti-inflammatory agents, irrespective of patient gender. We explored the possibility that sexually dimorphic actions of glucocorticoid regulation of gene expression may contribute to the dimorphic basis of inflammatory disease by evaluating the rat liver, a classic glucocorticoid-responsive organ. Surprisingly, glucocorticoid administration expanded the set of hepatic sexually dimorphic genes. Eight distinct patterns of glucocorticoid-regulated gene expression were identified, which included sex-specific genes. Our experiments also defined specific genes with altered expression in response to glucocorticoid treatment in both sexes, but in opposite directions. Pathway analysis identified sex-specific glucocorticoid-regulated gene expression in several canonical pathways involved in susceptibility to and progression of diseases with gender differences in prevalence. Moreover, a comparison of the number of genes involved in inflammatory disorders between sexes revealed 84 additional glucocorticoid-responsive genes in the male, suggesting that the anti-inflammatory actions of glucocorticoids are more effective in males. These gender-specific actions of glucocorticoids in liver were substantiated in vivo with a sepsis model of systemic inflammation.

Generating diversity in glucocorticoid receptor signaling: mechanisms, receptor isoforms, and post-translational modifications.

Glucocorticoids are necessary for life after birth and regulate numerous homeostatic functions in man, including glucose homeostasis, protein catabolism, skeletal growth, respiratory function, inflammation, development, behavior, and apoptosis. In a clinical setting, they are widely used as anti-inflammatory agents to control both acute and chronic inflammation. Unfortunately, owing to their broad range of physiological actions, patients treated with glucocorticoids for long periods of time experience a variety of serious side effects, including metabolic syndrome, bone loss, and psychiatric disorders including depression, mania, and psychosis. Our understanding of how one hormone or drug regulates all of these diverse processes is limited. Recent studies have shown that multiple glucocorticoid receptor isoforms are produced from one gene via combinations of alternative mRNA splicing and alternative translation initiation. These isoforms possess unique tissue distribution patterns and transcriptional regulatory profiles. Owing to variation in the N-terminal and C-terminal length of glucocorticoid receptor isoforms, different post-translational modifications including ubiquitination, phosphorylation, and sumoylation are predicted, contributing to the complexity of glucocorticoid signaling. Furthermore, increasing evidence suggests that unique glucocorticoid receptor isoform compositions within cells could determine the cell-specific response to glucocorticoids. In this review, we will outline the recent advances made in the characterization of the transcriptional activity and the selective regulation of apoptosis by the various glucocorticoid receptor isoforms.

Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis.

Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression.

Constitutive nitric oxide synthase activation is a significant route for nitroglycerin-mediated vasodilation.

The physiological effects of nitroglycerin as a potent vasodilator have long been documented. However, the molecular mechanisms by which nitroglycerin exerts its biological functions are still a matter of intense debate. Enzymatic pathways converting nitroglycerin to vasoactive compounds have been identified, but none of them seems to fully account for the reported clinical observations. Here, we demonstrate that nitroglycerin triggers constitutive nitric oxide synthase (NOS) activation, which is a major source of NO responsible for low-dose (1-10 nM) nitroglycerin-induced vasorelaxation. Our studies in cell cultures, isolated vessels, and whole animals identified endothelial NOS activation as a fundamental requirement for nitroglycerin action at pharmacologically relevant concentrations in WT animals.

Steroids and nitric oxide in sepsis.

Nitric oxide (NO) is produced by several cell types and has effects both detrimental and beneficial to the host. Sepsis and septic shock are conditions in which NO plays a central role in physiopathology. Stressful circumstances such as pathogens, toxins, and trauma elicit a wide variety of physiological changes. Steroid hormones and notably glucocorticoids are one of the main players in this orchestrated response. Although steroids have been used for sepsis some decades ago, their use in this condition was practically banned for several years following studies showing that high glucocorticoid doses were harmful to the host. Recently, the subject has been raised again since some studies demonstrated that adrenal insufficiency may happen in sepsis and that low dose/long-term regimen with cortisol may be beneficial to sepsis and septic shock. However, there are great gaps in our knowledge regarding the role played by steroids in sepsis, as well as the contribution of NO. In the present review, we will attempt to highlight the relationship among NO, sepsis and steroids, mainly glucocorticoids. A second purpose is to raise some unanswered questions that may provide better therapeutic alternatives to treat sepsis and septic shock.

Multiple glucocorticoid receptor isoforms and mechanisms of post-translational modification.

Glucocorticoids regulate diverse physiological effects in virtually every organ and tissue in the body. Glucocorticoid actions are mediated through the glucocorticoid receptor (GR), a ligand-dependent transcriptional factor that activates or represses gene transcription. Since, the cloning of the human GR in 1985, research efforts have been focused on describing the mechanism of action exerted by one of the GR isoforms, GRalpha. However, recent studies from our lab and others have suggested that multiple isoforms of hGR are generated from one single gene and one mRNA species by the mechanisms of alternative RNA splicing and alternative translation initiation. These isoforms display diverse cytoplasm-to-nucleus trafficking patterns and distinct transcription activities. In addition, this new information predicts that each hGR protein can be subjected to a variety of post-translational modifications, such as phosphorylation, sumoylation and ubiquitination. The nature and degree of post-translational modification, as well as subcellular localization, may differentially modulate stability and function among the GR isoforms in different tissues providing an additional important mechanism for regulation of GR action. We outline the recent advances made in identifying the processes that generate and modify multiple GR isoforms and the post-translational modifications that contribute to the increasing diversity in the glucocorticoid signaling pathway.

Nitric oxide-dependent reduction in soluble guanylate cyclase functionality accounts for early lipopolysaccharide-induced changes in vascular reactivity.

We investigated the role of soluble guanylate cyclase in lipopolysaccharide-induced hyporesponsiveness to phenylephrine. The effects of phenylephrine on the blood pressure of female Wistar rats were evaluated at 2, 8, and 24 h after lipopolysaccharide injection (12.5 mg/kg i.p.). Vasoconstrictive responses to phenylephrine were reduced 40 to 50% in all time periods. Methylene blue, a soluble guanylate cyclase inhibitor (15 micromol/kg i.v.) restored the reactivity to phenylephrine in animals injected with lipopolysaccharide 2 and 24 h earlier. However, it failed to do so in animals injected with lipopolysaccharide 8 h earlier. Incubation with sodium nitroprusside (SNP) increased lung and aorta cGMP levels in control animals and in tissues of rats treated with lipopolysaccharide 24 h earlier. However, SNP failed to increase tissue cGMP in rats injected 8 h earlier. Lipopolysaccharide reduced the vasodilatory response to NO donors 8 h after injection. This effect and the decreased lung cGMP accumulation in response to SNP were reversed by an NO synthase blocker. Guanylate cyclase protein levels were lower than controls in lungs harvested from rats injected 8 h earlier and were back to normal values in lungs of rats injected 24 h earlier with lipopolysaccharide. Thus, data indicate that there is a temporal window of 8 h after lipopolysaccharide injection in which soluble guanylate cyclase is not functional and that this loss of function is NO-dependent. Thus, the putative use of soluble guanylate cyclase inhibitors in the treatment of endotoxemia may be beneficial mainly at early stages of this condition.

Corticosterone modulates noradrenaline-induced melatonin synthesis through inhibition of nuclear factor kappa B.

In chronically inflamed animals, adrenal hormones exert a positive control on the secretion of melatonin by the pineal gland. In this paper, the mechanism of corticosterone as a modulator of melatonin and N-acetylserotonin (NAS) was determined. Rat pineal glands in culture, stimulated for 5 hr with noradrenaline (10 nm), were previously incubated with corticosterone (1.0 nm-1.0 microm) for 48 hr in the presence or absence of the glucocorticoid receptor (GR) antagonist, mifepristone (1.0 microm), the proteasome inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN, 12.5 microm) or the antagonist of the nuclear factor kappa B (NFkappaB), pyrrolidinedithiocarbamate (PDTC, 12.5 microm). Corticosterone potentiated noradrenaline-induced melatonin and NAS production in a bell-shaped manner. The increase in NAS (12.9 +/- 2.7, n=6 versus 34.3 +/- 8.3 ng per pineal) and melatonin (16.3 +/- 2.0, n=6 versus 44.3 +/- 12.9 ng per pineal) content induced by 1 microm corticosterone was blocked by mifepristone, and mimicked by ALLN and PDTC. The presence of GRs was shown by [3H]-dexamethasone binding (0.30 +/- 0.09 pmol/mg protein) and corticosterone inhibition of NFkappaB nuclear translocation was demonstrated by electromobility shift assay. Therefore, corticosterone potentiates noradrenaline-induced melatonin and NAS production through GR inhibition of NFkappaB nuclear translocation. To the best of our knowledge, this is the first time that this relevant pathway for passive and acquired immune response is shown to modulate melatonin production in pineal gland.

Caffeic acid derivatives: in vitro and in vivo anti-inflammatory properties.

Caffeic acid and some of its derivatives such as caffeic acid phenetyl ester (CAPE) and octyl caffeate are potent antioxidants which present important anti-inflammatory actions. The present study assessed the in vitro and in vivo effects of five caffeic acid derivatives (caffeic acid methyl, ethyl, butyl, octyl and benzyl esters) and compared their actions to those of CAPE. In the model of LPS-induced nitric oxide (NO) production in RAW 264.7 macrophages, the pre-incubation of all derivatives inhibited nitrite accumulation on the supernatant of stimulated cells, with mean IC50 (microM) values of 21.0, 12.0, 8.4, 2.4, 10.7 and 4.80 for methyl, ethyl, butyl, octyl, benzyl and CAPE, respectively. The effects of caffeic acid derivatives seem to be related to the scavenging of NO, as the compounds prevented SNAP-derived nitrite accumulation and decreased iNOS expression. In addition, butyl, octyl and CAPE derivatives significantly inhibited LPS-induced iNOS expression in RAW 264.7 macrophages. Extending the in vitro results, we showed that the pre-treatment of mice with butyl, octyl and CAPE derivatives inhibited carrageenan-induced paw edema and prevented the increase in IL-1beta levels in the mouse paw by 30, 24 and 36%, respectively. Butyl, octyl and CAPE derivatives also prevented carrageenan-induced neutrophil influx in the mouse paw by 28, 49 and 31%, respectively. Present results confirm and extend literature data, showing that caffeic acid derivatives exert in vitro and in vivo anti-inflammatory actions, being their actions mediated, at least in part by the scavenging of NO and their ability to modulate iNOS expression and probably that of other inflammatory mediators.

Inhibition of glucocorticoid receptor binding by nitric oxide in endotoxemic rats.

OBJECTIVE: Nitric oxide is an important participant in septic shock. For example, it causes profound vasodilation and hypotension. Despite their potent antiinflammatory properties, glucocorticoids are not routinely used in septic shock. Some studies show that antiinflammatory doses of glucocorticoids can be beneficial, but other studies do not indicate their use in this situation. We have previously shown the inhibitory effect of nitric oxide on glucocorticoid receptor binding in vitro. Nitric oxide donors decreased the binding of immunoprecipitated glucocorticoid receptor obtained from mouse L929 fibroblasts. These in vitro findings prompted us to study whether in vivo manipulations of the nitric oxide system would interfere with the glucocorticoid receptor binding. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university. SUBJECTS: Female Wistar rats. INTERVENTIONS: Injection of bacterial lipopolysaccharide, anesthesia, cardiovascular perfusion, and organ removal for biochemical assays. MEASUREMENTS AND MAIN RESULTS: Following lipopolysaccharide injection, plasma nitrate + nitrite increased and inducible nitric oxide synthase activity was stimulated in several organs, the highest rates being in the lung and spleen. If dexamethasone was injected before lipopolysaccharide, it completely blocked inducible nitric oxide synthase induction and the increase in plasma nitrate + nitrite. On the other hand, if dexamethasone was injected after lipopolysaccharide, it failed to affect both inducible nitric oxide synthase induction and increased plasma nitrate + nitrite levels. Lipopolysaccharide also caused an inhibition of glucocorticoid receptor binding in lung and spleen. Previous administration of a nitric oxide synthase inhibitor prevented both lipopolysaccharide-induced decrease in glucocorticoid receptor binding and the increase in plasma nitrate + nitrite. Injection of a nitric oxide donor into naive animals significantly decreased glucocorticoid receptor binding activity and prevented dexamethasone-induced increase in liver tyrosine aminotransferase activity. CONCLUSIONS: The results indicate that the failure of glucocorticoids to exhibit their antiinflammatory effects when administered to endotoxemic rats may be explained, at least in part, by the nitric oxide-induced inhibition of glucocorticoid receptor binding ability, thus precluding the expression of the antiinflammatory effects of both exogenous and endogenous corticosteroids.