First Occurrence of Megastigmane Glucosides in a Plant of Retama Genus.

Chemical investigation of Retama sphaerocarpa collected in Algeria resulted in the isolation of two megastigmane glucosides, compounds 1 and 2, along with a series of isoflavones and phenol derivatives. Compound 1, named retamoside, was new and its structure was determined by extensive application of spectroscopic methods, including HRMS, 1D and 2D NMR and CD. The anti-inflammatory properties of co-occurring main megastigmane, saurobaccioside B (2) and structurally related vomifoliol (3) on LPS-stimulated murine macrophages RAW 274.7 have been evaluated.

NMR Profiling of Exhaled Breath Condensate Defines Different Metabolic Phenotypes of Non-Cystic Fibrosis Bronchiectasis.

Nuclear-magnetic-resonance (NMR) profiling of exhaled breath condensate (EBC) provides insights into the pathophysiology of bronchiectasis by identifying specific biomarkers. We evaluated whether NMR-based metabolomics discriminates the EBC-derived metabolic phenotypes ("metabotypes") of 41 patients with non-cystic fibrosis (nCF) bronchiectasis of various etiology [24 subjects with Primary Ciliary Dyskinesia (PCD); 17 patients with bronchiectasis not associated with PCD (nCF/nPCD)], who were compared to 17 healthy subjects (HS). NMR was used for EBC profiling, and Orthogonal Projections to Latent Structures with partial least-squares discriminant analysis (OPLS-DA) was used as a classifier. The results were validated by using the EBC from 17 PCD patients not included in the primary analysis. Different statistical models were built, which compared nCF/nPCD and HS, PCD and HS, all classes (nCF/nPCD-PCD-HS), and, finally, PCD and nCF/nPCD. In the PCD-nCF/nPCD model, four statistically significant metabolites were able to discriminate between the two groups, with only a minor reduction of the quality parameters. In particular, for nCF/nPCD, acetone/acetoin and methanol increased by 21% and 18%, respectively. In PCD patients, ethanol and lactate increased by 25% and 28%, respectively. They are all related to lung inflammation as methanol is found in the exhaled breath of lung cancer patients, acetone/acetoin produce toxic ROS that damage lung tissue in CF, and lactate is observed in acute inflammation. Interestingly, a high concentration of ethanol hampers cilia beating and can be associated with the genetic defect of PCD. Model validation with 17 PCD samples not included in the primary analysis correctly predicted all samples. Our results indicate that NMR of EBC discriminates nCF/nPCD and PCD bronchiectasis patients from HS, and patients with nCF/nPCD from those with PCD. The metabolites responsible for between-group separation identified specific metabotypes, which characterize bronchiectasis of a different etiology.

Obesity Affects the Microbiota-Gut-Brain Axis and the Regulation Thereof by Endocannabinoids and Related Mediators.

The hypothalamus regulates energy homeostasis by integrating environmental and internal signals to produce behavioral responses to start or stop eating. Many satiation signals are mediated by microbiota-derived metabolites coming from the gastrointestinal tract and acting also in the brain through a complex bidirectional communication system, the microbiota-gut-brain axis. In recent years, the intestinal microbiota has emerged as a critical regulator of hypothalamic appetite-related neuronal networks. Obesogenic high-fat diets (HFDs) enhance endocannabinoid levels, both in the brain and peripheral tissues. HFDs change the gut microbiota composition by altering the Firmicutes:Bacteroidetes ratio and causing endotoxemia mainly by rising the levels of lipopolysaccharide (LPS), the most potent immunogenic component of Gram-negative bacteria. Endotoxemia induces the collapse of the gut and brain barriers, interleukin 1ß (IL1ß)- and tumor necrosis factor α (TNFα)-mediated neuroinflammatory responses and gliosis, which alter the appetite-regulatory circuits of the brain mediobasal hypothalamic area delimited by the median eminence. This review summarizes the emerging state-of-the-art evidence on the function of the "expanded endocannabinoid (eCB) system" or endocannabinoidome at the crossroads between intestinal microbiota, gut-brain communication and host metabolism; and highlights the critical role of this intersection in the onset of obesity.

Orexin-A represses satiety-inducing POMC neurons and contributes to obesity via stimulation of endocannabinoid signaling.

In the hypothalamic arcuate nucleus (ARC), proopiomelanocortin (POMC) neurons and the POMC-derived peptide α-melanocyte-stimulating hormone (α-MSH) promote satiety. POMC neurons receive orexin-A (OX-A)-expressing inputs and express both OX-A receptor type 1 (OX-1R) and cannabinoid receptor type 1 (CB1R) on the plasma membrane. OX-A is crucial for the control of wakefulness and energy homeostasis and promotes, in OX-1R-expressing cells, the biosynthesis of the endogenous counterpart of marijuana's psychotropic and appetite-inducing component Δ(9)-tetrahydrocannabinol, i.e., the endocannabinoid 2-arachidonoylglycerol (2-AG), which acts at CB1R. We report that OX-A/OX-1R signaling at POMC neurons promotes 2-AG biosynthesis, hyperphagia, and weight gain by blunting α-MSH production via CB1R-induced and extracellular-signal-regulated kinase 1/2 activation- and STAT3 inhibition-mediated suppression of Pomc gene transcription. Because the systemic pharmacological blockade of OX-1R by SB334867 caused anorectic effects by reducing food intake and body weight, our results unravel a previously unsuspected role for OX-A in endocannabinoid-mediated promotion of appetite by combining OX-induced alertness with food seeking. Notably, increased OX-A trafficking was found in the fibers projecting to the ARC of obese mice (ob/ob and high-fat diet fed) concurrently with elevation of OX-A release in the cerebrospinal fluid and blood of mice. Furthermore, a negative correlation between OX-A and α-MSH serum levels was found in obese mice as well as in human obese subjects (body mass index > 40), in combination with elevation of alanine aminotransferase and γ-glutamyl transferase, two markers of fatty liver disease. These alterations were counteracted by antagonism of OX-1R, thus providing the basis for a therapeutic treatment of these diseases.

Role of Orexin-A in Hypertension and Obesity.

PURPOSE OF REVIEW: Hypertension is one of the most challenging health problems inducing cerebrovascular disease and high percentage of death when associated with diabetes, dyslipidemias, and obesity. Orexin/hypocretin is a peptide expressed by a small number of neurons of the dorsolateral hypothalamus, a brain feeding and autonomic "fight-or-flight" regulatory center. According to this function, orexin has been demonstrated to evoke cardiovascular responses, heart rate, hypertension, hyperarousal, hyperphagia, and obesity. The focus of this review is to provide an overview about the mechanism through which orexin regulates food intake and cardiovascular responses and its role in the pathogenesis of obesity and hypertension which could be of great interest to establish possible new therapies. RECENT FINDINGS: In normal rats and mice, central administration of orexin increases food intake, blood pressure, and sympathetic nerve activity and these effects are blocked by selective orexin receptor antagonist SB-334867 or almorexant. Moreover, upregulation of orexin signaling, in combination with elevation of epinephrine and norepinephrine circulating levels, occurs in rats exposed to chronic stress, in models of spontaneous hypertension (SHR and BPH/2J Schlager mice) and in obese mice (ob/ob or mice fed with high fat diet). Therefore, hyperactivity of orexinergic neurons could be a factor in the development of obesity and essential hypertension. Because of their widespread projections to the brain regions involved in appetite and cardiovascular responses, as far down as sympathetic preganglionic neurons in the spinal cord, orexin evokes sympathetically mediated cardiovascular responses. Lasting upregulation of orexin signaling can lead to hyperphagia, obesity, and hypertensive state. Dual orexin receptor antagonists (DORAs) and selective orexin receptor antagonists (SORAs) have antihypertensive effects that could be of clinical use for regulation of food intake and hypertension, supporting the role of orexinergic neurons as critical checkpoint in the neurogenic control of metabolic and cardiovascular functions.

Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons.

The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB1 receptor activity. Here we investigated the possibility of a negative regulation by CB1 receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2'-chloroethylamide (ACEA) in a CB1-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL(-/-) mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB1 activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.

Formation of OX-1R/CB1R heteromeric complexes in embryonic mouse hypothalamic cells: Effect on intracellular calcium, 2-arachidonoyl-glycerol biosynthesis and ERK phosphorylation.

Orexin 1 (OX-1R) and cannabinoid receptor (CB1R) belong to the superfamily of G-protein-coupled receptors (GPCRs) and are mostly coupled to Gq and Gi/o proteins, respectively. In vitro studies in host cells over-expressing OX-1R and CB1R revealed a functional interaction between these receptors, through either their ability to form heteromers or the property for OX-1R to trigger the biosynthesis of 2-arachidonoylglycerol (2-AG), an endogenous CB1R ligand. Since: i) OX-1R and CB1R co-espression has been described at postsynaptc sites in hypothalamic circuits involved the regulation of energy homeostasis, and ii) increased orexin-A (OX-A) and 2-AG levels occur in hypothalamic neurons during obesity, we sought here to investigate the OX-1R/CB1R interaction in embryonic mouse hypothalamic NPY/AgRP mHypoE-N41 neurons which express, constitutively, both receptors. Treatment of mHypoE-N41 cells with OX-A (0.1-0.3µM), but not with the selective CB1R agonist, arachidonyl-2-chloroethylamide (ACEA; 0.1-0.3µM), transiently elevated [Ca(2+)]i. Incubation with a subeffective dose of OX-A (0.1µM)+ACEA (0.1µM) led to stronger and longer lasting elevation of [Ca(2+)]i, antagonized by OX-1R or CB1R antagonism with SB-334867 or AM251, respectively. FRET and co-immunoprecipitation experiments showed the formation of OX-1R/CB1R heteromers after incubation with OX-A (0.2µM), or OX-A (0.1µM)+ACEA (0.1µM), but not after ACEA (0.2µM), in a manner antagonized by SB-334867 or AM251. OX-A (0.2µM) or OX-A (0.1µM)+ACEA (0.1µM) also led to 2-AG biosynthesis. Finally, a stronger activation of ERK1/2(Thr202/185) phosphorylation in comparison to basal or each agonist alone (0.1-0.2µM), was induced by incubation with OX-A (0.1µM)+ACEA (0.1µM), again in a manner prevented by OX-1R or CB1R antagonism. We suggest that OX-A, alone at effective concentrations on [Ca(2+)]i, or in combination with ACEA, at subeffective concentrations, triggers intracellular signaling events via the formation of OX-1R/CB1R heteromers and an autocrine loop mediated by 2-AG.

Obesity-driven synaptic remodeling affects endocannabinoid control of orexinergic neurons.

Acute or chronic alterations in energy status alter the balance between excitatory and inhibitory synaptic transmission and associated synaptic plasticity to allow for the adaptation of energy metabolism to new homeostatic requirements. The impact of such changes on endocannabinoid and cannabinoid receptor type 1 (CB1)-mediated modulation of synaptic transmission and strength is not known, despite the fact that this signaling system is an important target for the development of new drugs against obesity. We investigated whether CB1-expressing excitatory vs. inhibitory inputs to orexin-A-containing neurons in the lateral hypothalamus are altered in obesity and how this modifies endocannabinoid control of these neurons. In lean mice, these inputs are mostly excitatory. By confocal and ultrastructural microscopic analyses, we observed that in leptin-knockout (ob/ob) obese mice, and in mice with diet-induced obesity, orexinergic neurons receive predominantly inhibitory CB1-expressing inputs and overexpress the biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol, which retrogradely inhibits synaptic transmission at CB1-expressing axon terminals. Patch-clamp recordings also showed increased CB1-sensitive inhibitory innervation of orexinergic neurons in ob/ob mice. These alterations are reversed by leptin administration, partly through activation of the mammalian target of rapamycin pathway in neuropeptide-Y-ergic neurons of the arcuate nucleus, and are accompanied by CB1-mediated enhancement of orexinergic innervation of target brain areas. We propose that enhanced inhibitory control of orexin-A neurons, and their CB1-mediated disinhibition, are a consequence of leptin signaling impairment in the arcuate nucleus. We also provide initial evidence of the participation of this phenomenon in hyperphagia and hormonal dysregulation in obesity.

The short-chain fatty acid acetate modulates orexin/hypocretin neurons: A novel mechanism in gut-brain axis regulation of energy homeostasis and feeding.

The short-chain fatty acids (SCFAs) acetate, propionate and butyrate, the major products of intestinal microbial fermentation of dietary fibres, are involved in fine-tuning brain functions via the gut-brain axis. However, the effects of SCFAs in the hypothalamic neuronal network regulating several autonomic-brain functions are still unknown. Using NMR spectroscopy, we detected a reduction in brain acetate concentrations in the hypothalamus of obese leptin knockout ob/ob mice compared to lean wild-type littermates. Therefore, we investigated the effect of acetate on orexin/hypocretin neurons (hereafter referred as OX or OX-A neurons), a subset of hypothalamic neurons regulating energy homeostasis, which we have characterized in previous studies to be over-activated by the lack of leptin and enhancement of endocannabinoid tone in the hypothalamus of ob/ob mice. We found that acetate reduces food-intake in concomitance with a reduction of orexin neuronal activity in ob/ob mice. This was demonstrated by evaluating food-intake behaviour and orexin-A/c-FOS immunoreactivity coupled with patch-clamp recordings in Hcrt-eGFP neurons, quantification of prepro-orexin mRNA, and immunolabeling of GPR-43, the main acetate receptor. Our data provide new insights into the mechanisms of the effects of chronic dietary supplementation with acetate, or complex carbohydrates, on energy intake and body weight, which may be partly mediated by inhibition of orexinergic neuron activity.

Orexin induces the production of an endocannabinoid-derived lysophosphatidic acid eliciting hypothalamic synaptic loss in obesity.

OBJECTIVE: Orexin-A (OX-A) is a neuropeptide produced selectively by neurons of the lateral hypothalamus. It exerts powerful control over brain function and physiology by regulating energy homeostasis and complex behaviors linked to arousal. Under conditions of chronic or acute brain leptin signaling deficiency, such as in obesity or short-term food deprivation, respectively, OX-A neurons become hyperactive and promote hyperarousal and food seeking. However, this leptin-dependent mechanism is still mostly unexplored. The endocannabinoid 2-arachidonoyl-glycerol (2-AG) is known to be implicated in food consumption by promoting hyperphagia and obesity, and we and others demonstrated that OX-A is a strong inducer of 2-AG biosynthesis. Here, we investigated the hypothesis that, under acute (6 h fasting in wt mice) or chronic (in ob/ob mice) hypothalamic leptin signaling reduction, OX-A-induced enhancement of 2-AG levels leads to the production of the 2-AG-derived 2-arachidonoyl-sn-glycerol-3-phosphate (2-AGP), a bioactive lipid belonging to the class of lysophosphatidic acids (LPAs), which then regulates hypothalamic synaptic plasticity by disassembling α-MSH anorexigenic inputs via GSK-3ß-mediated Tau phosphorylation, ultimately affecting food intake. METHODS: We combined cell-type-specific morphological (CLEM and confocal microscopy), biochemical, pharmacological, and electrophysiological techniques to dissect the leptin- and OX-A/2-AGP-mediated molecular pathways regulating GSK-3ß-controlled pT231-Tau production at POMC neurons of obese ob/ob and wild-type (wt) lean littermate mice and in an in vitro model of POMC neurons such as mHypoN41 neurons (N41). RESULTS: 2-AGP is overproduced in the hypothalamus of obese leptin-deficient, or lean 6 h food-deprived mice, and promotes food intake by reducing α-MSH-expressing synaptic inputs to OX-A neurons via lysophosphatidic acid type-1 receptor (LPA1-R) activation, and pT231-Tau accumulation in α-MSH projections. This effect is due to the activation of the Pyk2-mediated pTyr216-GSK3ß pathway and contributes to further elevating OX-A release in obesity. Accordingly, we found a strong correlation between OX-A and 2-AGP levels in the serum of obese mice and of human subjects. CONCLUSIONS: Hypothalamic feeding pathways are endowed with 2-AGP-mediated synaptic plasticity according to their inherent functional activities and the necessity to adapt to changes in the nutritional status. These findings reveal a new molecular pathway involved in energy homeostasis regulation, which could be targeted to treat obesity and related disturbances.

The biomarkers' landscape of post-COVID-19 patients can suggest selective clinical interventions.

In COVID-19 clinical symptoms can persist even after negativization also in individuals who have had mild or moderate disease. We here investigated the biomarkers that define the post-COVID-19 clinical state analyzing the exhaled breath condensate (EBC) of 38 post COVID-19 patients and 38 sex and age-matched healthy controls via nuclear magnetic resonance (NMR)-based metabolomics. Predicted gene-modulated microRNAs (miRNAs) related to COVID-19 were quantified from EBC of 10 patients and 10 controls. Finally, clinical parameters from all post-COVID-19 patients were correlated with metabolomic data. Post-COVID-19 patients and controls showed different metabolic phenotype ("metabotype"). From the metabolites, by using enrichment analysis we identified miRNAs that resulted up-regulated (hsa-miR146a-5p) and down-regulated (hsa-miR-126-3p and hsa-miR-223-3p) in post-COVID-19. Taken together, our multiomics data indicate that post-COVID-19 patients before rehabilitation are characterized by persistent inflammation, dysregulation of liver, endovascular thrombotic and pulmonary processes, and physical impairment, which should be the primary clinical targets to contrast the post-acute sequelae of COVID-19.

Identification of biomarkers in COPD by metabolomics of exhaled breath condensate and serum/plasma.

Chronic obstructive pulmonary disease (COPD) is the third cause of death worldwide, presenting poor long-term outcomes and chronic disability. COPD is a condition with a wide spectrum of clinical presentations because its pathophysiological determinants relate to tobacco smoke, genetic factors, alteration of several metabolic pathways, and oxidative stress. Consequently, patients present different phenotypes even with comparable degrees of airflow limitation. Because of the increasing social and economic costs of COPD, a growing attention is currently paid to "omics" techniques for more personalized treatments and patient-tailored rehabilitation programs. In this regard, the systematic investigation of the metabolome (i.e., the whole set of endogenous molecules) in biomatrices, namely metabolomics, has become indispensable for phenotyping respiratory diseases. The metabolomic profiling of biological samples contains the small molecules produced during biological processes and their identification and quantification help in the diagnosis, comprehension of disease outcome and treatment response. Exhaled breath condensate (EBC), plasma and serum are biofluids readily available, with negligible invasiveness, and, therefore, suitable for metabolomics investigations. In this paper, we describe the latest advances on metabolomic profiling of EBC, plasma and serum in COPD patients.

Metabolomics of COPD Pulmonary Rehabilitation Outcomes via Exhaled Breath Condensate.

Chronic obstructive pulmonary disease (COPD) is characterized by different phenotypes and clinical presentations. Therefore, a single strategy of pulmonary rehabilitation (PR) does not always yield the expected clinical outcomes as some individuals respond excellently, others discreetly, or do not respond at all. Fifty consecutive COPD patients were enrolled. Of them, 35 starting a 5-week PR program were sampled at admission (T0), after 2 (T2W) and 5 (T5W) weeks, while 15 controls not yet on PR were tested at T0 and T5W. Nuclear magnetic resonance (NMR) profiling of exhaled breath condensate (EBC) and multivariate statistical analysis were applied to investigate the relationship between biomarkers and clinical parameters. The model including the three classes correctly located T2W between T0 and T5W, but 38.71% of samples partially overlapped with T0 and 32.26% with T5W, suggesting that for some patients PR is already beneficial at T2W (32.26% overlapping with T5W), while for others (38.71% overlapping with T0) more time is required. Rehabilitated patients presented several altered biomarkers. In particular, methanol from T0 to T5W decreased in parallel with dyspnea and fatigue, while the walk distance increased. Methanol could be ascribed to lung inflammation. We demonstrated that the metabolic COPD phenotype clearly evolves during PR, with a strict relationship between clinical and molecular parameters. Methanol, correlating with clinical parameters, represents a useful biomarker for monitoring personalized outcomes and establishing more targeted protocols.

Metabolomic Profiling of Exhaled Breath Condensate and Plasma/ Serum in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is an increasing cause of global morbidity and mortality, with poor long-term outcomes and chronic disability. COPD is a condition with a wide spectrum of clinical presentations, with different phenotypes being identified even among patients with comparable degrees of airflow limitation. Considering the burden of COPD in terms of social and economic costs, in recent years growing attention has been given to the need for more personalized approaches and patienttailored rehabilitation programs. In this regard, the systematic analysis of metabolites in biological matrices, namely metabolomics, may become an essential tool in phenotyping diseases. Through the identification and quantification of the small molecules produced during biological processes, metabolomic profiling of biological samples has thus been proposed as an opportunity to identify novel biomarkers of disease outcome and treatment response. Exhaled breath condensate (EBC) and plasma/serum are fluid pools, which can be easily extracted and analyzed. In this review, we discuss the potential clinical applications of the metabolomic profiling of EBC and plasma/serum in COPD.

Positive association between plasmatic levels of orexin A and the endocannabinoid-derived 2-arachidonoyl lysophosphatidic acid in Alzheimer's disease.

A regular sleep-wake cycle plays a positive function that preserves synaptic plasticity and brain activity from neuropathological injuries. The hypothalamic neuropeptide orexin-A (OX-A) is central in sleep-wake regulation and has been found to be over-expressed in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD) suffering from sleep disturbances. OX-A promotes the biosynthesis of 2-arachidonoylglycerol (2-AG), which, in turn, could be phosphorylated to 2-arachidonoyl lysophosphatidic acid (2-AGP). The reorganization of the actin cytoskeleton during neurite retraction is one of the best-characterized effects of lysophosphatidic acids. However, less information is available regarding the reorganization of the neuronal microtubule network in response to OX-A-induced 2-AG and, possibly consequent, 2-AGP production in AD patients. This is of special relevance also considering that higher 2-AG levels are reported in the CSF of AD patients. Here, we found a positive correlation between OX-A and 2-AGP concentrations in the plasma, and an increase of 2-AGP levels in the CSF of AD patients. Furthermore, a negative correlation between the plasmatic 2-AGP levels and the mini-mental state examination score is also revealed in AD patients. By moving from the human patients to in vitro and in vivo models of AD we investigated the molecular pathway linking OX-A, 2-AG and 2-AGP to the phosphorylation of pT231-Tau, which is a specific early plasma biomarker of this disorder. By LC-MS analysis we show that OX-A, via OX-1R, induces 2-AG biosynthesis via DAGLα, and in turn 2-AG is converted to 2-AGP in primary hippocampal neurons. By confocal microscopy and western blotting assay we found an OX-A- or 2-AGP-mediated phosphorylation of Tau at threonine 231 residue, in a manner prevented by LPA1R (2-AGP receptor) or OX1R (OX-A receptor) antagonism with AM095 or SB334867, respectively. Finally, by patch-clamp recording we documented that 2-AGP-mediated pT231-Tau phosphorylation impairs glutamatergic transmission in the mouse hippocampus. Although further additional research is still required to clarify the potential role of orexin signaling in neurodegeneration, this study provides evidence that counteraction of aberrant OX-A signaling, also via LPA-1R antagonism, may be beneficial in the mild-to-moderate age-related cognitive decline associated with sleep disturbances.

Arachidonic acid inhibits neuronal nitric oxide synthase elicited by proinflammatory stimuli and promotes astrocyte survival with both exogenous and endogenous peroxynitrite via different mechanisms.

Cytosolic phospholipase A(2)-inhibited astrocytes respond to the cocktail lipopolysaccharide/interferon-gamma with an immediate formation of peroxynitrite (ONOO(-)) and a delayed lethal response. Low concentrations of arachidonic acid (ARA; i.e., <0.1 microM) cause tyrosine kinase-dependent inhibition of neuronal nitric oxide synthase (nNOS) activity, thereby suppressing formation of ONOO(-) and the ensuing lethal response. ARA promoted its effects only when given to the cultures just prior to, or in parallel with, the proinflammatory mixture. High concentrations of ARA, i.e., >3 microM, promoted cytoprotection when applied to the cultures up to 50 min after the formation of endogenous ONOO(-) had been completed or up to 30 min after addition of exogenous ONOO(-). The mechanism(s) involved in these responses was, however, independent of tyrosine kinase activation and was in fact mediated by ARA metabolites of the lipoxygenase pathway. These results are consistent with a scenario in which astrocytes respond to low or high amounts of ARA with the triggering of different pathways involved in the inflammatory response. Early nNOS inhibition mediated by very low levels of ARA is indeed critical for nuclear factor-kappaB activation, which is otherwise effectively inhibited by constitutive nitric oxide, and for preventing early formation of ONOO(-). Greater ARA concentrations promote survival in astrocytes committed to death by ONOO(-), a species extensively released under inflammatory conditions, via a mechanism dependent on lipoxygenase metabolism and inhibition of downstream events leading to cell demise.

Prostaglandin E2 signals monocyte/macrophage survival to peroxynitrite via protein kinase A converging in bad phosphorylation with the protein kinase C alpha-dependent pathway driven by 5-hydroxyeicosatetraenoic acid.

Monocytes/macrophages committed to death by peroxynitrite nevertheless survive with a signaling response promoting Bad phosphorylation, as well as its cytosolic localization, via upstream activation of cytosolic phospholipase A(2), 5-lipoxygenase, and protein kinase C alpha. We now report evidence for an alternative mechanism converging in Bad phosphorylation when the expression/activity of the above enzymes are suppressed. Under these conditions, also associated with peroxynitrite-dependent severe inhibition of Akt, an additional Bad kinase, Bad dephosphorylation promoted its accumulation in the mitochondria and a prompt lethal response. PGE(2) prevented toxicity via EP(2) receptor-mediated protein kinase A-dependent Bad phosphorylation. This notion was established in U937 cells by the following criteria: 1) there was a strong correlation between survival and cAMP accumulation, both in the absence and presence of phosphodiesterase inhibitors; 2) direct activation of adenylyl cyclase afforded cytoprotection; and 3) PGE(2) promoted loss of mitochondrial Bad and cytoprotection, mimicked by EP(2) receptor agonists, and prevented by EP(2) receptor antagonists or protein kinase A inhibitors. Finally, selected experiments performed in human monocytes/macrophages and in rat peritoneal macrophages indicated that the above cytoprotective pathway is a general response of cells belonging to the monocyte/macrophage lineage to both exogenous and endogenous peroxynitrite. The notion that two different pathways mediated by downstream products of arachidonic acid metabolism converge in Bad phosphorylation emphasizes the relevance of this strategy for the regulation of macrophage survival to peroxynitrite at the inflammatory sites.

Nitric Oxide and Hydrogen Sulfide: A Nice Pair in the Respiratory System.

Nitric Oxide (NO) is internationally regarded as a signal molecule involved in several functions in the respiratory tract under physiological and pathogenic conditions. Hydrogen Sulfide (H2S) has also recently been recognized as a new gasotransmitter with a diverse range of functions similar to those of NO. Depending on their respective concentrations, both these molecules act synergistically or antagonistically as signals or damage promoters. Nevertheless, available evidence shows that the complex biological connections between NO and H2S involve multiple pathways and depend on the site of action in the respiratory tract, as well as on experimental conditions. This review will provide an update on these two gasotransmitters in physiological and pathological processes.

Role of 2-Arachidonoyl-Glycerol and CB1 Receptors in Orexin-A-Mediated Prevention of Oxygen-Glucose Deprivation-Induced Neuronal Injury.

Orexin-A (OX-A) protects the brain against oxidative stress-mediated ischemic injury. Since the endocannabinoid 2-arachidonoylglycerol (2-AG) and cannabinoid type-1 (CB1) receptors were previously shown to mediate some of the effects of OX-A exerted through the orexin-1 receptor (OX-1R), we investigated the involvement of 2-AG in OX-A-induced neuroprotection following oxygen and glucose deprivation (OGD) in mouse cortical neurons. OGD-induced reactive oxygen species (ROS) accumulation and neuronal death were prevented by both OX-A and arachidonyl-2'-chloroethylamide (ACEA), a synthetic CB1 receptor agonist, in a manner sensitive to OX-1R and CB1 receptor antagonists, SB334867 and AM251. OX-A stimulated 2-AG biosynthesis in cortical neurons. In neurons isolated from monoacylglycerol lipase (MAGL, a 2-AG hydrolyzing enzyme) null mice, 10-fold higher 2-AG concentrations were found and OGD failed to induce ROS production and cell death, whereas AM251 restored these noxious effects. OX-A-induced neuroprotection was mediated by the phosphoinositide-3-kinase/Akt (PI3K/Akt) survival pathway since both OX-A and ACEA induced phosphorylation of Akt and prevented OGD-induced cytochrome c release from the mitochondria, in a manner counteracted by SB334867 or AM251. Administration of OX-A reduced infarct volume and elevated brain 2-AG levels in a mouse model of transient ischemia. These results suggest that 2-AG and CB1 receptor mediate OX-A prevention of ischemia-induced neuronal apoptosis.