Effect of Xanthohumol, a Bioactive Natural Compound from Hops, on Adenosine Pathway in Rat C6 Glioma and Human SH-SY5Y Neuroblastoma Cell Lines.

Xanthohumol (Xn) is an antioxidant flavonoid mainly extracted from hops (Humulus lupulus), one of the main ingredients of beer. As with other bioactive compounds, their therapeutic potential against different diseases has been tested, one of which is Alzheimer's disease (AD). Adenosine is a neuromodulatory nucleoside that acts through four different G protein-coupled receptors: A1 and A3, which inhibit the adenylyl cyclases (AC) pathway, and A2A and A2B, which stimulate this activity, causing either a decrease or an increase, respectively, in the release of excitatory neurotransmitters such as glutamate. This adenosinergic pathway, which is altered in AD, could be involved in the excitotoxicity process. Therefore, the aim of this work is to describe the effect of Xn on the adenosinergic pathway using cell lines. For this purpose, two different cellular models, rat glioma C6 and human neuroblastoma SH-SY5Y, were exposed to a non-cytotoxic 10 µM Xn concentration. Adenosine A1 and A2A, receptor levels, and activities related to the adenosine pathway, such as adenylate cyclase, protein kinase A, and 5'-nucleotidase, were analyzed. The adenosine A1 receptor was significantly increased after Xn exposure, while no changes in A2A receptor membrane levels or AC activity were reported. Regarding 5'-nucleotidases, modulation of their activity by Xn was noted since CD73, the extracellular membrane attached to 5'-nucleotidase, was significantly decreased in the C6 cell line. In conclusion, here we describe a novel pathway in which the bioactive flavonoid Xn could have potentially beneficial effects on AD as it increases membrane A1 receptors while modulating enzymes related to the adenosine pathway in cell cultures.

Effect of chronic maternal L-Glu intake during gestation and/or lactation on oxidative stress markers, AMPA Glu1 receptor and adenosine A1 signalling pathway from foetal and neonatal cerebellum.

L-Glutamate (L-Glu) is an amino acid present in the diet that plays a fundamental role in the central nervous system, as the main excitatory neurotransmitter participating in learning and memory processes. In addition, the nucleoside adenosine has a crucial role in L-Glu metabolism, by regulating the liberation of this neurotransmitter through four different receptors: A1, A2A, A2B and A3, which activate (A2A and A2B) or inhibit (A1 and A3) adenylate cyclase pathway. L-Glu at high concentrations can act as a neurotoxin and induce oxidative stress. The study of the oxidative stress correlated with an excess of L-Glu consumption during maternity is key to understand its effects on foetuses and neonates. Previous studies have shown that there is a change in the receptor levels in the brain of pregnant rats and their foetuses when mothers are administered L-Glu during gestation; however, its effect on the cerebellum is unknown. Cerebellum is known to be responsible for motor, cognitive and emotional functions, so its possible involvement after L-Glu consumption is an important issue to study. Therefore, the aim of the present work was to study the effect of L-Glu exposure during gestation and lactation on oxidative stress biomarkers and neurotransmitter receptors from the cerebellum of foetuses and neonates. After maternal L-Glu intake during gestation, oxidative stress was increased, as the ionotropic L-Glu receptors, and GluR1 AMPA subunit levels were altered in foetuses. A1 adenosine receptor suffered changes after L-Glu treatment during gestation, lactation or both, in lactating neonate cerebellum, while adenylate cyclase activity remain unaltered. Further studies will be necessary to elucidate the importance of L-Glu intake and its possible excitotoxicity in the cerebellum of Wistar rats during the pregnancy period and their involvement in long-term neurodegeneration.

Maternal Caffeine Consumption during Gestation and Lactation Abolishes Cortical Oxidative Stress and Restores Na+/K+-ATPase Activity in Neonates Exposed to Hyperthermia-Induced Seizures.

Caffeine is a psychoactive substance that is widely consumed by individuals of various demographics, including pregnant women. It can readily cross the blood-brain and placental barriers, easily reaching the fetal brain. In addition, caffeine has also shown antioxidant properties, as its consumption reduces oxidative stress in various pathologies, including epilepsy. Febrile seizures (FS) are among the most common convulsive disorders in infants and young children. Here, we used an animal model of FS to learn whether maternal caffeine (1 g/L) intake consumption during gestation and lactation could exert beneficial effects on the rat cortex. Neonatal development was analyzed by measuring pinna opening, eye opening, righting reflex on the surface, and geotaxis reflex. Five and twenty days after HIS, the rats were euthanized, and plasma membranes and cytosolic fractions were isolated from their cortex brain. The enzymatic activities of glutathione reductase, glutathione S-transferase, Na+/K+-ATPase, and Mg2+-ATPase, as well as the levels of thiobarbituric acid reacting substances, were quantified. Results showed that maternal caffeine intake eliminates oxidative stress and normalizes Na+/K+-ATPase activity disrupted by HIS and also affects some parameters relating to the neurodevelopment of neonates. As FS in infants has been related to epilepsy in adults, the antioxidant properties of caffeine could prevent potential damage from hyperthermia.

Na+/K+- and Mg2+-ATPases and Their Interaction with AMPA, NMDA and D2 Dopamine Receptors in an Animal Model of Febrile Seizures.

Febrile seizures (FS) are one of the most common seizure disorders in childhood which are classified into short and prolonged, depending on their duration. Short FS are usually considered as benign. However, epidemiological studies have shown an association between prolonged FS and temporal lobe epilepsy. The development of animal models of FS has been very useful to investigate the mechanisms and the consequences of FS. One of the most used, the "hair dryer model", has revealed that prolonged FS may lead to temporal lobe epilepsy by altering neuronal function. Several pieces of evidence suggest that Na+/ K+-ATPase and Mg2+-ATPase may play a role in this epileptogenic process. In this work, we found that hyperthermia-induced seizures (HIS) significantly increased the activity of Na+/ K+-ATPase and Mg2+-ATPase five and twenty days after hyperthermic insult, respectively. These effects were diminished in response to AMPA, D2 dopamine A1 and A2A receptors activation, respectively. Furthermore, HIS also significantly increased the protein level of the AMPA subunit GluR1. Altogether, the increased Na+/ K+-ATPase and Mg2+-ATPase agree well with the presence of protective mechanisms. However, the reduction in ATPase activities in the presence of NMDA and AMPA suggest an increased propensity for epileptic events in adults.

Resveratrol enhances A1 and hinders A2A adenosine receptors signaling in both HeLa and SH-SY5Y cells: Potential mechanism of its antitumoral action.

Despite great efforts, effective treatment against cancer has not yet been found. However, natural compounds such as the polyphenol resveratrol have emerged as promising preventive agent in cancer therapy. The mode of action of resveratrol is still poorly understood, but it can modulate many signaling pathways related to the initiation and progression of cancer. Adenosinergic signaling may be involved in the antitumoral action of resveratrol since resveratrol binds to the orthosteric binding site of adenosine A2A receptors and acts as a non-selective agonist for adenosine receptors. In the present study, we measured the impact of resveratrol treatment on different adenosinergic pathway components (i.e. adenosine receptors levels, 5'-nucleotidase, adenosine deaminase, and adenylyl cyclase activities, protein kinase A levels, intracellular adenosine and other related metabolites levels) and cell viability and proliferation in HeLa and SH-SY5Y cell lines. Results revealed changes leading to turning off cAMP signaling such as decreased levels of A2A receptors and reduced adenylyl cyclase activation, increased levels of A1 receptors and increased adenylyl cyclase inhibition, and lower levels of PKA. All these changes could contribute to the antitumoral action of resveratrol. Interestingly, these effects were almost identical in HeLa and SH-SY5Y cells suggesting that resveratrol enhances A1 and hinders A2A adenosine receptors signaling as part of a potential mechanism of antitumoral action.

Neuroprotective Effects of Resveratrol by Modifying Cholesterol Metabolism and Aß Processing in SAMP8 Mice.

Cholesterol metabolism seems dysregulated and linked to amyloid-ß (Aß) formation in neurodegeneration, but the underlying mechanisms are poorly known. Resveratrol (RSV) is a polyphenol with antioxidant activity and neuroprotective properties. Here, we analyzed the effect of age and RSV supplementation on cholesterol metabolism in the brain and blood serum, and its potential link to Aß processing, in SAMP8 mice-an animal model of aging and Alzheimer's disease. In the brain, our results revealed an age-related increase in ApoE and unesterified cholesterol in the plasma membrane whereas LDL receptor, HMG-CoA reductase, HMG-CoA-C1 synthase, and ABCA1 transporter remained unaltered. Furthermore, BACE-1 and APP gene expression was decreased. This dysregulation could be involved in the amyloidogenic processing pathway of APP towards Aß formation. In turn, RSV exhibited an age-dependent effect. While levels of unesterified cholesterol in the plasma membrane were not affected by RSV, several participants in cholesterol uptake, release, and de novo synthesis differed, depending on age. Thus, RSV supplementation exhibited a different neuroprotective effect acting on Aß processing or cholesterol metabolism in the brain at earlier or later ages, respectively. In blood serum, HDL lipoprotein and free cholesterol were increased by age, whereas VLDL and LDL lipoproteins remained unaltered. Again, the protective effect of RSV by decreasing the LDL or increasing the HDL levels also seems to depend on the intervention's moment. In conclusion, age is a prominent factor for cholesterol metabolism dysregulation in the brain of SAMP8 mice and influences the protective effects of RSV through cholesterol metabolism and Aß processing.

Perceptions of Patients and Their Families Regarding Limitation of Therapeutic Effort in the Intensive Care Unit.

OBJECTIVE: Our objective was to determine and describe the opinion and attitudes of patients and their families regarding the limitation of therapeutic effort and advanced directives in critical patients and whether end-of-life planning occurs. Religious affiliation, education level, and pre-admission quality of life were also evaluated to determine whether they may influence decisions regarding appropriate therapeutic effort. METHODS: A prospective, observational and descriptive study, approved by the center's ethical committee, was carried out with 257 participants (94 patients and 163 family members) in the intensive care unit (ICU). A questionnaire regarding the opinions of patients and relatives about situations of therapeutic appropriateness in case of poor prognosis or poor quality of life was used. The questionnaire had three sections. In the first section, sociodemographic features were investigated. In the second section, information was collected on the quality of life and functional situation before ICU admission (taking as a reference the situation one month before admission) assessed by the Karnofsky scale, Barthel index, and the PAEEC scale (Project for the Epidemiological Analysis of Critical Care Patients). The third section aimed to determine whether the family knew the patient's opinion regarding his/her end of life. RESULTS: Of those interviewed, 62.2% would agree to limit treatment in case of poor prognosis or poor quality of future life. In contrast, 37.7% considered that they should fight for life, even if it is irretrievable. Only 1.6% had advanced directives registered, 43.9% of the participants admitted deterioration in their quality of life before ICU admission, 18.2% with moderate-severe deterioration. Our study shows that the higher the educational level, the lower the desire to fight for life when it is irretrievable and the greater the agreement to limit treatment. Besides, those participants not affiliated with a religion were significantly less likely to fight for life, including when irretrievable, than Catholics and were more likely to agree to limit treatment. CONCLUSIONS: More than half of the participants would agree to limit treatment in the case of a poor prognosis. Our results indicate that patients do not prepare for the dying process well in advance. Religion and educational level were determining factors for the choice of procedures at the end of life, both for patients and their families.

High-Fat and Resveratrol Supplemented Diets Modulate Adenosine Receptors in the Cerebral Cortex of C57BL/6J and SAMP8 Mice.

Neurodegenerative disorders are devastating diseases in which aging is a major risk factor. High-fat diet (HFD) seems to contribute to cognition deterioration, but the underlying mechanisms are poorly understood. Moreover, resveratrol (RSV) has been reported to counteract the loss of cognition associated with age. Our study aimed to investigate whether the adenosinergic system and plasma membrane cholesterol are modulated by HFD and RSV in the cerebral cortex of C57BL/6J and SAMP8 mice. Results show that HFD induced increased A1R and A2AR densities in C57BL/6J, whereas this remained unchanged in SAMP8. Higher activity of 5'-Nucleotidase was found as a common effect induced by HFD in both mice strains. Furthermore, the effect of HFD and RSV on A2BR density was different depending on the mouse strain. RSV did not clearly counteract the HFD-induced effects on the adenosinergic system. Besides, no changes in free-cholesterol levels were detected in the plasma membrane of cerebral cortex in both strains. Taken together, our data suggest a different modulation of adenosine receptors depending on the mouse strain, not related to changes in plasma membrane cholesterol content.

Antitumoral Action of Resveratrol Through Adenosinergic Signaling in C6 Glioma Cells.

Gliomas are the most common and aggressive primary tumors in the central nervous system. The nucleoside adenosine is considered to be one major constituent within the tumor microenvironment. The adenosine level mainly depends on two enzymatic activities: 5'-nucleotidase (5'NT or CD73) that synthesizes adenosine from AMP, and adenosine deaminase (ADA) that converts adenosine into inosine. Adenosine activates specific G-protein coupled receptors named A1, A2A, A2B, and A3 receptors. Resveratrol, a natural polyphenol present in grapes, peanuts, and berries, shows several healthy effects, including protection against cardiovascular, endocrine, and neurodegenerative diseases and cancer. However, the molecular mechanisms of resveratrol actions are not well known. Recently, we demonstrated that resveratrol acts as an agonist for adenosine receptors in rat C6 glioma cells. The present work aimed to investigate the involvement of adenosine metabolism and adenosine receptors in the molecular mechanisms underlying the antitumoral action of resveratrol. Results presented herein show that resveratrol was able to decrease cell numbers and viability and to reduce CD73 and ADA activities, leading to the increase of extracellular adenosine levels. Some resveratrol effects were reduced by the blockade of A1 or A3 receptors by DPCPX or MRS1220, respectively. These results suggest that reduced CD73 activity located in the plasma membrane in addition to a fine-tuned modulatory role of adenosine receptors could be involved, at least in part, in the antiproliferative action of resveratrol in C6 glioma cells.

Early Effects of the Soluble Amyloid ß25-35 Peptide in Rat Cortical Neurons: Modulation of Signal Transduction Mediated by Adenosine and Group I Metabotropic Glutamate Receptors.

The amyloid ß peptide (Aß) is a central player in the neuropathology of Alzheimer's disease (AD). The alteration of Aß homeostasis may impact the fine-tuning of cell signaling from the very beginning of the disease, when amyloid plaque is not deposited yet. For this reason, primary culture of rat cortical neurons was exposed to Aß25-35, a non-oligomerizable form of Aß. Cell viability, metabotropic glutamate receptors (mGluR) and adenosine receptors (AR) expression and signalling were assessed. Aß25-35 increased mGluR density and affinity, mainly due to a higher gene expression and protein presence of Group I mGluR (mGluR1 and mGluR5) in the membrane of cortical neurons. Intriguingly, the main effector of group I mGluR, the phospholipase C ß1 isoform, was less responsive. Also, the inhibitory action of group II and group III mGluR on adenylate cyclase (AC) activity was unaltered or increased, respectively. Interestingly, pre-treatment of cortical neurons with an antagonist of group I mGluR reduced the Aß25-35-induced cell death. Besides, Aß25-35 increased the density of A1R and A2AR, along with an increase in their gene expression. However, while A1R-mediated AC inhibition was increased, the A2AR-mediated stimulation of AC remained unchanged. Therefore, one of the early events that takes place after Aß25-35 exposure is the up-regulation of adenosine A1R, A2AR, and group I mGluR, and the different impacts on their corresponding signaling pathways. These results emphasize the importance of deciphering the early events and the possible involvement of metabotropic glutamate and adenosine receptors in AD physiopathology.

Modulation of Adenosine Receptors by Hops and Xanthohumol in Cell Cultures.

Adenosine receptors (ARs) have been involved in neurodegenerative diseases such as Alzheimer disease, where oxidative stress contributes to neurodegeneration and cell death. Therefore, there is increasing interest in developing antioxidative strategies to avoid or reduce neurodegeneration. We have previously described that different beer extracts modulate ARs and protect glioma and neuroblastoma cells from oxidative stress. The present work aimed to analyze the possible protective effect of hops (Humulus lupulus L.), a major component of beer, and xanthohumol on cell death elicited by oxidative stress and their modulation of ARs in rat C6 glioma and human SH-SY5Y neuroblastoma cells. Different extraction methods were employed in two hops varieties (Nugget and Columbus). Cell viability was determined by the XTT method in cells exposed to these hops extracts and xanthohumol. ARs were analyzed by radioligand binding and real-time PCR assays. Hops extract reverted the cell death observed under oxidative stress and modulated adenosine A1 and A2 receptors in both cell types. Xanthohumol was unable to revert the effect of oxidative stress in cell viability but it also modulated ARs similarly to hops. Therefore, healthy effects of beer described previously could be due, at least in part, to their content of hops and the modulation of ARs.

The Density of Group I mGlu5 Receptors Is Reduced along the Neuronal Surface of Hippocampal Cells in a Mouse Model of Alzheimer's Disease.

Metabotropic glutamate receptor subtype 5 (mGlu5) is implicated in the pathophysiology of Alzheimer´s disease (AD). However, its alteration at the subcellular level in neurons is still unexplored. Here, we provide a quantitative description on the expression and localisation patterns of mGlu5 in the APP/PS1 model of AD at 12 months of age, combining immunoblots, histoblots and high-resolution immunoelectron microscopic approaches. Immunoblots revealed that the total amount of mGlu5 protein in the hippocampus, in addition to downstream molecules, i.e., Gq/11 and PLCß1, was similar in both APP/PS1 mice and age-matched wild type mice. Histoblots revealed that mGlu5 expression in the brain and its laminar expression in the hippocampus was also unaltered. However, the ultrastructural techniques of SDS-FRL and pre-embedding immunogold demonstrated that the subcellular localisation of mGlu5 was significantly reduced along the neuronal surface of hippocampal principal cells, including CA1 pyramidal cells and DG granule cells, in APP/PS1 mice at 12 months of age. The decrease in the surface localisation of mGlu5 was accompanied by an increase in its frequency at intracellular sites in the two neuronal populations. Together, these data demonstrate, for the first time, a loss of mGlu5 at the plasma membrane and accumulation at intracellular sites in different principal cells of the hippocampus in APP/PS1 mice, suggesting an alteration of the excitability and synaptic transmission that could contribute to the cognitive dysfunctions in this AD animal model. Further studies are required to elucidate the specificity of mGlu5-associated molecules and downstream signalling pathways in the progression of the pathology.

Adenosine Metabolism in the Cerebral Cortex from Several Mice Models during Aging.

Adenosine is a neuromodulator that has been involved in aging and neurodegenerative diseases as Alzheimer's disease (AD). In the present work, we analyzed the possible modulation of purine metabolites, 5'nucleotidase (5'NT) and adenosine deaminase (ADA) activities, and adenosine monophosphate (AMP)-activated protein kinase (AMPK) and its phosphorylated form during aging in the cerebral cortex. Three murine models were used: senescence-accelerated mouse-resistant 1 (SAMR1, normal senescence), senescence-accelerated mouse-prone 8 (SAMP8, a model of AD), and the wild-type C57BL/6J (model of aging) mice strains. Glutamate and excitatory amino acid transporter 2 (EAAT2) levels were also measured in these animals. HPLC, Western blotting, and enzymatic activity evaluation were performed to this aim. 5'-Nucleotidase (5'NT) activity was decreased at six months and recovered at 12 months in SAMP8 while opposite effects were observed in SAMR1 at the same age, and no changes in C57BL/6J mice. ADA activity significantly decreased from 3 to 12 months in the SAMR1 mice strain, while a significant decrease from 6 to 12 months was observed in the SAMP8 mice strain. Regarding purine metabolites, xanthine and guanosine levels were increased at six months in SAMR1 without significant differences in SAMP8 mice. In C57BL/6J mice, inosine and xanthine were increased, while adenosine decreased, from 4 to 24 months. The AMPK level was decreased at six months in SAMP8 without significant changes nor in SAMR1 or C57BL/6J strains. Glutamate and EAAT2 levels were also modulated during aging. Our data show a different modulation of adenosine metabolism participants in the cerebral cortex of these animal models. Interestingly, the main differences between SAMR1 and SAMP8 mice were found at six months of age, SAMP8 being the most affected strain. As SAMP8 is an AD model, results suggest that adenosinergic metabolism is involved in the neurodegeneration of AD.

Analysis of Ion Pairing in Solid State and Solution in p-Cymene Ruthenium Complexes.

The importance of ion pairing in different fields of chemistry is widely recognized. In this work, we have synthesized a set of cationic p-cymene ruthenium complexes of general formula [(p-cym)Ru(L')(κ2-O^N-L)]X (p-cym = p-cymene; L' = N-methylimidazole (MeIm), N-ethylpiperidylimidazole (EpipIm), 1,3,5-triaza-7-phosphaadamantane (PTA); L = 2-(1H-benzimidazol-2-yl)phenolato (L1), 2-(1,3-benzothiazol-2-yl)phenolato (L2); X = Cl-, BF4-, OTf-, BPh4-). X-ray diffraction studies on selected complexes revealed relatively strong anion-cation interactions in the solid state mainly based on N-H···X (X = Cl, F, O) and C-H···π interactions, also observed in the DFT-modeled complexes in the gas phase. Moreover, NMR studies showed that they exist as intimate ion pairs in solution and, remarkably, as head-to-tail quadruples in the particular case of the cation [(p-cym)Ru(MeIm)(κ2- O^N-L1)]+ ([1]+) with Cl- and BPh4- as counteranions. Furthermore, a value of ΔG = -2.9 kcal mol-1 at 299 K has been estimated for the equilibrium {[1]BPh4···[1]BPh4} ⇆ 2{[1]+···BPh4-} in concentrated CDCl3 solutions. In addition, preliminary studies concerning the cytotoxic properties against HeLa cell lines of the derivatives suggested a positive effect derived from the presence of the lipophilic BPh4- anion and also from the NH group of the benzimidazolyl fragment.

Adenosine and Metabotropic Glutamate Receptors Are Present in Blood Serum and Exosomes from SAMP8 Mice: Modulation by Aging and Resveratrol.

Adenosine (ARs) and metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors (GPCRs) that are modulated in the brain of SAMP8 mice, an animal model of Alzheimer's disease (AD). In the present work, it is shown the presence of ARs and mGluRs in blood serum and derived exosomes from SAMP8 mice as well as its possible modulation by aging and resveratrol (RSV) consumption. In blood serum, adenosine A1 and A2A receptors remained unaltered from 5 to 7 months of age. However, an age-related decrease in adenosine level was observed, while 5'-Nucleotidase activity was not modulated. Regarding the glutamatergic system, it was observed a decrease in mGluR5 density and glutamate levels in older mice. In addition, dietary RSV supplementation caused an age-dependent modulation in both adenosinergic and glutamatergic systems. These GPCRs were also found in blood serum-derived exosomes, which might suggest that these receptors could be released into circulation via exosomes. Interestingly, changes elicited by age and RSV supplementation on mGluR5 density, and adenosine and glutamate levels were similar to that detected in whole-brain. Therefore, we might suggest that the quantification of these receptors, and their corresponding endogenous ligands, in blood serum could have predictive value for early diagnosis in combination with other distinctive hallmarks of AD.

Resveratrol Differently Modulates Group I Metabotropic Glutamate Receptors Depending on Age in SAMP8 Mice.

Glutamate homeostasis is critical for neurotransmission as this excitatory neurotransmitter has a relevant role in cognition functions through ionotropic and metabotropic glutamate receptors in the central nervous system. During the last years, the role of the group I metabotropic glutamate receptors (mGluRs) in neurodegenerative diseases such as Alzheimer's disease has been intensely investigated. Resveratrol (RSV) is a natural polyphenolic compound that is thought to have neuroprotective properties for human health. However, little is known about the action of this compound on mGluR signaling. Therefore, the aim of this study was to investigate the possible modulation of group I mGluRs in SAMP8 mice five and seven months of age supplemented with RSV in the diet. Data reported herein show that RSV plays a different modulatory action on group I mGluRs: mGluR5 is downregulated as age increases, independently of RSV presence, and mGluR1 is upregulated or downregulated by RSV treatment depending on age (i.e., depending on mGluR5 levels). In addition, a neuroprotective role can be inferred for RSV as lower glutamate levels, higher synapsin levels, and unchanged caspase-3 activity were detected after RSV treatment. In conclusion, our findings indicate that RSV treatment modifies the group I mGluR-mediated glutamatergic system in SAMP8 mice, which could contribute to the beneficial effects of this natural polyphenol.

Hyperthermia-induced seizures produce long-term effects on the functionality of adenosine A1 receptor in rat cerebral cortex.

Febrile seizures are one of the most frequent childhood neurological disorders; they are classified into simple and prolonged, depending on their duration. Prolonged FS lasts more than 15 min and may evoke neurological sequelae in a process in which molecular alterations seem to play an important role. Adenosine is a purine nucleoside that exerts anticonvulsant effects through binding to adenosine A1 receptor (A1 R). This receptor belongs to the GPCR superfamily and is negatively coupled to adenylyl cyclase (AC) activity through Gi proteins. In the present study, we analyzed the functionality of A1 R, measured as the inhibition of forskolin-stimulated AC activity, 48 hr after hyperthermia-induced seizures (HIS). Surprisingly, the results obtained show that the activation of A1 R increased forskolin-stimulated cAMP production instead of decreasing it. This alteration was not accompanied by changes in αG protein levels. The functionality of A1 R remained altered two months after HIS. However, this alteration was abolished when AC assays were carried out in the presence of anti αGs subunit-specific antibody, suggesting that HIS can switch A1 R coupling from Gi to Gs proteins. Finally, radioligand binding assays revealed that density and affinity of A1 R were not significantly altered by HIS. In summary, the results obtained show that HIS induces long-term changes in the A1 R/AC signaling pathway in rat brain cortex.

Modulation of Adenosine Receptors and Antioxidative Effect of Beer Extracts in in Vitro Models.

The fight against neurodegenerative diseases is promoting the searching of nutrients, preferably of wide consumption, with proven effects on health. Beer is widely consumed and has potential benefits on health. In this work, three different extracts from dark beer (DB), non-alcoholic beer (NAB), and lager beer (LB) were assayed at 30 min and 24 h in rat C6 glioma and human SH-SY5Y neuroblastoma cells in order to study their possible protective effects. Cell viability and adenosine A1, A2A, A2B, and A3 receptor gene expression and protein levels were measured in control cells and in cells challenged with hydrogen peroxide as an oxidant stressor. Among the three extracts analyzed, DB showed a greater protective effect against H2O2-induced oxidative stress and cell death. Moreover, a higher A1 receptor level was also induced by this extract. Interestingly, A1 receptor level was also increased by NAB and LB extracts, but to a lower extent, and the protective effect of these extracts against H2O2 was lower. This possible correlation between protection and A1 receptor level was observed at 24 h in both C6 and SH-SY5Y cells. In summary, different beer extracts modulate, to a different degree, adenosine receptors expression and protect both glioma and neuroblastoma cells from oxidative stress.

Functional Cross-Talk between Adenosine and Metabotropic Glutamate Receptors.

G-protein coupled receptors are transmembrane proteins widely expressed in cells and their transduction pathways are mediated by controlling second messenger levels through different G-protein interactions. Many of these receptors have been described as involved in the physiopathology of neurodegenerative diseases and even considered as potential targets for the design of novel therapeutic strategies. Endogenous and synthetic allosteric and orthosteric selective ligands are able to modulate GPCRs at both gene and protein expression levels and can also modify their physiological function. GPCRs that coexist in the same cells can homo- and heteromerize, therefore, modulating their function. Adenosine receptors are GPCRs which stimulate or inhibit adenylyl cyclase activity through Gi/Gs protein and are involved in the control of neurotransmitter release as glutamate. In turn, metabotropic glutamate receptors are also GPCRs which inhibit adenylyl cyclase or stimulate phospholipase C activities through Gi or Gq proteins, respectively. In recent years, evidence of crosstalk mechanisms between different GPCRs have been described. The aim of the present review was to summarize the described mechanisms of interaction and crosstalking between adenosine and metabotropic glutamate receptors, mainly of group I, in both in vitro and in vivo systems, and their possible use for the design of novel ligands for the treatment of neurodegenerative diseases.

Early-life hyperthermic seizures upregulate adenosine A2A receptors in the cortex and promote depressive-like behavior in adult rats.

Febrile seizures (FS) represent one of the most frequent convulsive disorders in children which can be classified into simple and prolonged depending on the duration. Although simple FS are generally considered as benign, there is controversy about the outcome of prolonged FS. Here, we have used an animal model of prolonged FS to investigate persistent neurochemical and behavioral alterations in adult rats. Hyperthermic seizures were induced in 12-day-old rats using a warmed air stream from a hair dryer. Neonates exhibited arrest of heat-induced hyperkinesis followed by body flexion and rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. After 48 days, the animals were assayed on dark-light box and forced swim tests in order to detect if rats will show signs of anxiety or depression. Finally, animals were sacrificed 56 days after hyperthermia-induced seizures (HIS), and their effects on adenosine A2A receptor signaling and 5'-nucleotidase activity were studied in plasma membranes from the cerebral cortex by using radioligand-binding assay and by measuring the activities of adenylate cyclase and 5'-nucleotidase. Results obtained have shown that adult rats submitted to HIS during the neonatal period showed depressive-like behavior. Furthermore, animals exposed to hyperthermic insult showed an increase in A2A receptor level which was also accompanied by an increase in A2A receptor functionality.