Resveratrol and Quercetin as Regulators of Inflammatory and Purinergic Receptors to Attenuate Liver Damage Associated to Metabolic Syndrome.

Nonalcoholic fatty liver disease (NAFLD) is considered a manifestation of metabolic syndrome (MS) and is characterized by the accumulation of triglycerides and a varying degree of hepatic injury, inflammation, and repair. Moreover, peroxisome-proliferator-activated receptors (PPARs) play a critical role in the pathophysiological processes in the liver. There is extensive evidence of the beneficial effect of polyphenols such as resveratrol (RSV) and quercetin (QRC) on the treatment of liver pathology; however, the mechanisms underlying their beneficial effects have not been fully elucidated. In this work, we show that the mechanisms underlying the beneficial effects of RSV and QRC against inflammation in liver damage in our MS model are due to the activation of novel pathways which have not been previously described such as the downregulation of the expression of toll-like receptor 4 (TLR4), neutrophil elastase (NE) and purinergic receptor P2Y2. This downregulation leads to a decrease in apoptosis and hepatic fibrosis with no changes in hepatocyte proliferation. In addition, PPAR alpha and gamma expression were altered in MS but their expression was not affected by the treatment with the natural compounds. The improvement of liver damage by the administration of polyphenols was reflected in the normalization of serum transaminase activities.

Involvement of ectonucleotidases and purinergic receptor expression during acute Chagas disease in the cortex of mice treated with resveratrol and benznidazole.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi. CD affects people worldwide, primarily in tropical areas. The central nervous system (CNS) is an essential site for T. cruzi persistence during infection. The protozoan may pass through the blood-brain barrier and may cause motor and cognitive neuronal damage. Once in the CNS, T. cruzi triggers immune responses that the purinergic system can regulate. Treatment for CD is based on benznidazole (BNZ); however, this agent has negative side-effects and is toxic to the host. For this reason, we investigated whether resveratrol (RSV), a potent antioxidant and neuroprotective molecule, would modulate purinergic signaling and RSV alone or in combination with BNZ would prevent changes in purinergic signaling and oxidative damage caused by T. cruzi. We infected mice with T. cruzi and treated them with RSV or BNZ for 8 days. Increases in ATP and ADP hydrolysis by NTPDase in the total cortex of infected animals were observed. The treatment with RSV in infected group diminished ATP, ADP, and AMP hydrolysis compared to infected group. The combination of RSV + BNZ decreased AMP hydrolysis in infected animals compared to the INF group, exerting an anti-inflammatory effect. RSV acted as a neuroprotector, decreasing adenosine levels. Infected animals presented an increase of P2X7 and A2A density of purine receptors. RSV reduced P2X7 and A2A and increased A1 density receptors in infected animals. In addition, infected animals showed higher TBARS and reactive oxygen species (ROS) levels than control. RSV diminished ROS levels in infected mice, possibly due to antioxidant properties. In short, we conclude that resveratrol could act as a neuroprotective molecule, probably preventing inflammatory changes caused by infection by T. cruzi, even though the mice experienced high levels of parasitemia.

Autocrine and paracrine purinergic signaling in the most lethal types of cancer.

Cancer comprises a collection of diseases that occur in almost any tissue and it is characterized by an abnormal and uncontrolled cell growth that results in tumor formation and propagation to other tissues, causing tissue and organ malfunction and death. Despite the undeniable improvement in cancer diagnostics and therapy, there is an urgent need for new therapeutic and preventive strategies with improved efficacy and fewer side effects. In this context, purinergic signaling emerges as an interesting candidate as a cancer biomarker or therapeutic target. There is abundant evidence that tumor cells have significant changes in the expression of purinergic receptors, which comprise the G-protein coupled P2Y and AdoR families of receptors and the ligand-gated ion channel P2X receptors. Tumor cells also exhibit changes in the expression of nucleotidases and other enzymes involved in nucleotide metabolism, and the concentrations of extracellular nucleotides are significantly higher than those observed in normal cells. In this review, we will focus on the potential role of purinergic signaling in the ten most lethal cancers (lung, breast, colorectal, liver, stomach, prostate, cervical, esophagus, pancreas, and ovary), which together are responsible for more than 5 million annual deaths.

Aluminum-Induced Alterations in Purinergic System Parameters of BV-2 Brain Microglial Cells.

Aluminum (Al) is ubiquitously present in the environment and known to be a neurotoxin for humans. The trivalent free Al anion (Al3+) can cross the blood-brain barrier (BBB), accumulate in the brain, and elicit harmful effects to the central nervous system (CNS) cells. Thus, evidence has suggested that Al increases the risk of developing neurodegenerative diseases, particularly Alzheimer's disease (AD). Purinergic signaling has been shown to play a role in several neurological conditions as it can modulate the functioning of several cell types, such as microglial cells, the main resident immune cells of the CNS. However, Al effects on microglial cells and the role of the purinergic system remain elusive. Based on this background, this study is aimed at assessing the modulation of Al on purinergic system parameters of microglial cells. An in vitro study was performed using brain microglial cells exposed to Al chloride (AlCl3) and lipopolysaccharide (LPS) for 96 h. The uptake of Al, metabolism of nucleotides (ATP, ADP, and AMP) and nucleoside (adenosine), and the gene expression and protein density of purinoceptors were investigated. The results showed that both Al and LPS increased the breakdown of adenosine, whereas they decreased nucleotide hydrolysis. Furthermore, the findings revealed that both Al and LPS triggered an increase in gene expression and protein density of P2X7R and A2AR receptors, whereas reduced the A1R receptor expression and density. Taken together, the results showed that Al and LPS altered the setup of the purinergic system of microglial cells. Thus, this study provides new insights into the involvement of the purinergic system in the mechanisms underlying Al toxicity in microglial cells.

Using RNA Interference for Purinoceptor Knockdown In Vivo.

RNA interference (RNAi) is a powerful post-transcriptional gene silencing (PTGS) induced by small double-stranded RNA (dsRNA). The method allows silencing of genes of interest by translation inhibition or by mRNA degradation. In this chapter, we provide a brief overview of the mechanisms involved in each step of gene silencing. A nonviral infusion of short siRNA into ventricular system of rats was used to study purinoceptor in the rat brain.

Zebrafish models: Gaining insight into purinergic signaling and neurological disorders.

Zebrafish (Danio rerio) has been considered a complementary model for biomedical studies, especially due to advantages such as external and rapid development, and genetic manipulation. There is growing interest in this model in neuroscience research since the species has morphological and physiological similarities to mammals and a complex behavioral repertoire. The purinergic signaling has been described in zebrafish, and purinoceptors and nucleotide- and nucleoside-metabolizing enzymes have already been identified in the central nervous system (CNS) of this species. The involvement of the purinergic system in several models of neurological disorders, such as Alzheimers disease, Parkinson's disease, epilepsy, schizophrenia, and autism has been investigated in zebrafish. This mini review presents several studies describing purinergic signaling in the zebrafish CNS and the action of this neurotransmitter system in models of neurological disorders using this species as a biological model. The use of pharmacological approaches at different stages of development may be a useful tool for preclinical assays and the testing of purinergic compounds as new alternatives for the treatment of neurological disorders.

Purinergic system in psychiatric diseases.

Psychiatric disorders are debilitating diseases, affecting >80 million people worldwide. There are no causal cures for psychiatric disorders and available therapies only treat the symptoms. The etiology of psychiatric disorders is unknown, although it has been speculated to be a combination of environmental, stress and genetic factors. One of the neurotransmitter systems implicated in the biology of psychiatric disorders is the purinergic system. In this review, we performed a comprehensive search of the literature about the role and function of the purinergic system in the development and predisposition to psychiatric disorders, with a focus on depression, schizophrenia, bipolar disorder, autism, anxiety and attention deficit/hyperactivity disorder. We also describe how therapeutics used for psychiatric disorders act on the purinergic system.

Glioma sensitive or chemoresistant to temozolomide differentially modulate macrophage protumor activities.

BACKGROUND: Glioblastomas are the most devastating brain tumor characterized by chemoresistance development and poor prognosis. Macrophages are a component of tumor microenvironment related to glioma malignancy. The relation among inflammation, innate immunity and cancer is accepted; however, molecular and cellular mechanisms mediating this relation and chemoresistance remain unresolved. OBJECTIVE: Here we evaluated whether glioma sensitive or resistant to temozolomide (TMZ) modulate macrophage polarization and inflammatory pathways associated. The impact of glioma-macrophage crosstalk on glioma proliferation was also investigated. METHODS: GL261 glioma chemoresistance was developed by exposing cells to increasing TMZ concentrations over a period of 6months. Mouse peritoneal macrophages were exposed to glioma-conditioned medium or co-cultured directly with glioma sensitive (GL) or chemoresistant (GLTMZ). Macrophage polarization, in vitro and in vivo glioma proliferation, redox parameters, ectonucleotidase activity and ATP cytotoxicity were performed. RESULTS: GLTMZ cells were more effective than GL in induce M2-like macrophage polarization and in promote a strong immunosuppressive environment characterized by high IL-10 release and increased antioxidant potential, which may contribute to glioma chemoresistance and proliferation. Interestingly, macrophage-GLTMZ crosstalk enhanced in vitro and in vivo proliferation of chemoresistant cells, decreased ectonucleotidase activities, which was followed by increased macrophage sensitivity to ATP induced death. CONCLUSIONS: Results suggest a differential macrophage modulation by GLTMZ cells, which may favor the maintenance of immunosuppressive tumor microenvironment and glioma proliferation. GENERAL SIGNIFICANCE: The induction of immunosuppressive environment and macrophage education by chemoresistant gliomas may be important for tumor recovery after chemotherapy and could be considered to overcome chemoresistance development.

Nucleotide receptors control IL-8/CXCL8 and MCP-1/CCL2 secretions as well as proliferation in human glioma cells.

Glioma cells release cytokines to stimulate inflammation that facilitates cell proliferation. Here, we show that Lipopolysaccharide (LPS) treatment could induce glioma cells to proliferate and this process was dependent on nucleotide receptor activation as well as interleukin-8 (IL-8/CXCL8) secretion. We observed that extracellular nucleotides controlled IL-8/CXCL8 and monocyte chemoattractant protein 1 (MCP-1/CCL2) release by U251MG and U87MG human glioma cell lines via P2X7 and P2Y6 receptor activation. The LPS-induced release of these cytokines was also modulated by purinergic receptor activation since IL-8 and MCP-1 release was decreased by the nucleotide scavenger apyrase as well as by the pharmacological P2Y6 receptor antagonists suramin and MRS2578. In agreement with these observations, the knockdown of P2Y6 expression decreased LPS-induced IL-8 release as well as the spontaneous release of IL-8 and MCP-1, suggesting an endogenous basal release of nucleotides. Moreover, high millimolar concentrations of ATP increased IL-8 and MCP-1 release by the glioma cells stimulated with suboptimal LPS concentration which were blocked by P2X7 and P2Y6 antagonists. Altogether, these data suggest that extracellular nucleotides control glioma growth via P2 receptor-dependent IL-8 and MCP-1 secretions.

Infection with Leishmania amazonensis upregulates purinergic receptor expression and induces host-cell susceptibility to UTP-mediated apoptosis.

Nucleotides are released into the extracellular milieu from infected cells and cells at inflammatory sites. The extracellular nucleotides bind to specific purinergic (P2) receptors and thereby induce a variety of cellular responses including anti-parasitic effects. Here we investigated whether extracellular nucleotides affect leishmanial infection in macrophages, and found that UTP reduces strongly the parasite load in peritoneal macrophages. Ultrastructural analysis of infected cells revealed that UTP induced morphological damage in the intracellular parasites. Uridine nucleotides also induced dose-dependent apoptosis of macrophages and production of ROI and RNI only in infected macrophages. The intracellular calcium measurements of infected cells showed that the response to UTP, but not UDP, increased the sensitivity and amplitude of cytosolic Ca(2+) changes. Infection of macrophages with Leishmania upregulated the expression of P2Y(2) and P2Y(4) receptor mRNA. The data suggest indirectly that Leishmania amazonensis infection induces modulation and heteromerization of P2Y receptors on macrophages. Thus UTP modulates the host response against L. amazonensis infection. UTP and UTP homologues should therefore be considered as novel components of therapeutic strategies against cutaneous leishmaniasis.

P19 embryonal carcinoma cells as in vitro model for studying purinergic receptor expression and modulation of N-methyl-D-aspartate-glutamate and acetylcholine receptors during neuronal differentiation.

The in vitro differentiation of P19 murine embryonal carcinoma cells to neurons resembles developmental stages which are encountered during neuronal development. Three days following induction to neuronal differentiation by retinoic acid, most cells of the P19 population lost expression of the stage specific embryonic antigen (SSEA-1) and expressed the neural progenitor cell specific antigen nestin. Beginning from day 4 of differentiation nestin expression was down-regulated, and expression of neuron-specific enolase as marker of differentiated neurons increased. The molecular mechanisms underlying neuronal differentiation are poorly understood. We have characterized the participation of purinergic ionotropic (P2X) and metabotropic (P2Y) receptors at mRNA transcription and protein levels as well as ATP-induced Ca2+ transients during neuronal differentiation of P19 cells. Gene and protein expression of P2X2, P2X6, P2Y2, and P2Y6 receptors increased during the course of differentiation, whereas P2X3, P2X4, P2Y1 and P2Y4 receptor expression was high in embryonic P19 cells and then decreased following induction of P19 cells to differentiation. P2X1 receptor protein expression was only detected on days 2 and 4 of differentiation. Although P2X5 and P2X7 mRNA transcription was present, no protein expression for this receptor subunit could be detected throughout the differentiation process. In undifferentiated cells, mainly ionotropic P2X receptors contributed to the ATP-induced Ca2+-response. In neuronal-differentiated P19 cells, the ATP-induced Ca2+-response was increased and the metabotropic component predominated. Purinergic receptor function is implicated to participate in neuronal maturation, as cholinergic and glutamate-N-methyl-D-aspartate (NMDA) induced calcium responses were affected when cells were differentiated in the presence of purinergic receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin or reactive blue-2. Our data suggest that inhibition of P2Y1 and possibly P2X2 receptors led to a loss of NMDA receptor activity whereas blockade of possibly P2X2 and P2Y2 purinergic receptors during neuronal differentiation of P19 mouse led to inhibition of cholinergic receptor responses.

Inosine released after hypoxia activates hepatic glucose liberation through A3 adenosine receptors.

Inosine, an endogenous nucleoside, has recently been shown to exert potent effects on the immune, neural, and cardiovascular systems. This work addresses modulation of intermediary metabolism by inosine through adenosine receptors (ARs) in isolated rat hepatocytes. We conducted an in silico search in the GenBank and complete genomic sequence databases for additional adenosine/inosine receptors and for a feasible physiological role of inosine in homeostasis. Inosine stimulated glycogenolysis (approximately 40%, EC50 4.2 x 10(-9) M), gluconeogenesis (approximately 40%, EC50 7.8 x 10(-9) M), and ureagenesis (approximately 130%, EC50 7.0 x 10(-8) M) compared with basal values; these effects were blunted by the selective A3 AR antagonist 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino][1,2,4]-triazolo[1,5-c]quinazoline (MRS 1220) but not by selective A1, A2A, and A2B AR antagonists. In addition, MRS 1220 antagonized inosine-induced transient increase (40%) in cytosolic Ca2+ and enhanced (90%) glycogen phosphorylase activity. Inosine-induced Ca2+ mobilization was desensitized by adenosine; in a reciprocal manner, inosine desensitized adenosine action. Inosine decreased the cAMP pool in hepatocytes when A1, A2A, and A2B AR were blocked by a mixture of selective antagonists. Inosine-promoted metabolic changes were unrelated to cAMP decrease but were Ca2+ dependent because they were absent in hepatocytes incubated in EGTA- or BAPTA-AM-supplemented Ca2+-free medium. After in silico analysis, no additional cognate adenosine/inosine receptors were found in human, mouse, and rat. In both perfused rat liver and isolated hepatocytes, hypoxia/reoxygenation produced an increase in inosine, adenosine, and glucose release; these actions were quantitatively greater in perfused rat liver than in isolated cells. Moreover, all of these effects were impaired by the antagonist MRS 1220. On the basis of results obtained, known higher extracellular inosine levels under ischemic conditions, and inosine's higher sensitivity for stimulating hepatic gluconeogenesis, it is suggested that, after tissular ischemia, inosine contributes to the maintenance of homeostasis by releasing glucose from the liver through stimulation of A3 ARs.