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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.

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.

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.

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.endothelial NOS ͉ neuronal NOS ͉ blood pressure I t has been Ͼ150 years since the effect of nitroglycerin as a potent vasodilator was first reported (1). Since then, intense research has led to extensive and well documented literature regarding nitroglycerin's physiological effects (2-4) and the identity of its derived metabolites in cells, tissues, laboratory animals, and humans (5, 6). Although much of nitroglycerin's pharmacology is known, the mechanisms through which nitroglycerin acts on the endothelium and the heart as well as the enzymatic pathways leading to its bioactivation are still controversial and under intense investigation. A number of hypotheses for nitroglycerin bioconversion in vivo have been formulated, implicating a multitude of enzymes such as GST (6, 7), oxidoreductases (8), and mitochondrial aldehyde dehydrogenase (9, 10) in the bioconversion of nitroglycerin to NO and/or other vasoactive compounds. For example, GST has been shown to catalyze the transnitration of lower thiols in the presence of nitroglycerin (6, 7). Xanthine oxidase and mitochondrial aldehyde dehydrogenase (which are closely related oxidoreductases) have been found to mediate nitroglycerin reduction to nitrite (11) and NO itself (8, 10). Several intermediate compounds, such as partially nitrated glycerin, nitrite (11), and nitrosothiols (6, 7), have been indicated as precursors of nitroglycerin-derived NO, which is ultimately responsible for the observed effects on the vasculature. Collectively, these studies contributed to establishing nitroglycerin as a metabolismdependent NO donor. Although some pathways have received more attention than others, none of the above-mentioned mechanisms seems to satisfactorily delineate nitroglycerin's peculiar kinetic and pharmacological behavior, which is distinct from that of other well characterized NO donors such as sodium nitroprusside (12). For instance, the nitrate groups of nitroglycerin are chemically resistant to rapid reduction because they are esters of nitrate. Also, minute doses of nitroglycerin [maximum plasma concentration Ϸ6 nM for 0.5 mg of nitroglycerin administered sublingually (13)], which are comparable to the basal levels of free NO [Ϸ5 nM as free NO (14)], result in...

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 mol/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.Septic shock, the most severe complication of sepsis, is a serious disorder with significant morbidity and mortality even after the appropriate antibiotic and supportive therapy are initiated. The poor outcome is considered to be a consequence of an overactive systemic inflammatory response elicited by microbial products, mainly lipopolysaccharide. The disease state is characterized by hypotension, hyporeactivity to vasoconstrictor agents, vascular damage and disseminated intravascular coagulation, which leads to multiple organ failure and death (Karima et al., 1999). Because the mortality rate after sepsis is in excess of 50%, it is clear that the present pharmacotherapy is inadequate.Inflammatory stimuli such as lipopolysaccharide activate a pathway that leads to expression, among other proinflammatory proteins, of the inducible nitric-oxide synthase (NOS-2). Once expressed, this enzyme is active for several hours and produces large amounts of nitric oxide (Alderton et al., 2001). It is well established that overproduction of NO in sepsis is one of the main causes of excessive vasodilation and reduced contractile response to vasoconstrictor agents (Titheradge, 1999). Regarding the molecular mechanism of NO-mediated vascular collapse in shock, the role of cGMP-dependent mechanism seems to be well established (Fleming et al., 1991;Paya et al., 1993;Keaney et al., 1994;Silva-Santos et al., 2002). NO activates solu...

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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.