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.

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.

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.

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.

Gender-specific desensitization of group I metabotropic glutamate receptors after maternal l-glutamate intake during lactation.

In the present work we have studied the effect of maternal intake of l-Glutamate (l-Glu) (1 g/L) during lactation on group I mGluR transduction pathway in brain plasma membrane from 15 days-old neonates. Results obtained have shown that maternal l-glutamate intake did not significantly affect neither weights of pups nor negative geotaxis reflex, an index of neurobehavioral development, but increased l-Glu plasma level in both male and female neonates. In male neonates, maternal l-Glu intake evoked a loss of mGluR1 whereas no variation on mGluR5 was observed as revealed by Western-blotting assay. The loss of mGlu1R was accompanied by a decrease on l-Glu-stimulated phospholipase C activity suggesting, therefore, a loss of group I mGluR functionality. Concerning female neonates, no variations were detected neither mGluR1 nor mGluR5 and group I mGluR functionality was also preserved.

Cerebellar oxidative stress and fine motor impairment in adolescent rats exposed to hyperthermia-induced seizures is prevented by maternal caffeine intake during gestation and lactation.

Febrile seizures (FS) is one of the most common convulsive disorders in infants and young children that only occurs during the first years of life in humans, when the cerebellum is still developing. Several works have shown that maternal caffeine consumption during gestation and lactation can exert protective effects on developing brain under pathological conditions. Here, we have used an animal model of FS to know whether maternal caffeine consumption during gestation and lactation exhibited protective effects on rat cerebellum. Pregnant rats were allowed to freely drink water or caffeine (1 g/l) during gestation and lactation. At PD13, neonates were submitted to hyperthermia-induced seizures (HIS) whereas pups not subject to hyperthermic stimulus were used as controls. 48 h, 5 and 20 days after HIS, rats were killed and plasma membranes and cytosolic fractions were isolated from cerebella. The enzymatic activities of glutathione reductase, glutathione S-transferase, caspase-3, 5´-nucleotidase and the levels of thiobarbituric acid reacting substances, adenosine A1 and A2A receptors were studied in these preparations. Furthermore, rats were tested in balance beam test and footprint test 20 days after HIS (PD33) in order to investigate the effect on fine motor coordination and gait patterns. Results obtained suggest that maternal caffeine consumption during gestation and lactation exerts two kinds of beneficial effects on cerebellum from rats submitted to HIS: a) at short term, maternal caffeine abolishes hyperthermic seizures induced-oxidative stress and caspase-3 activation and b) in adolescent rats (PD33), maternal caffeine prevents fine motor coordination impairment and gait disturbances.

Chronic oral administration of MPEP, an antagonist of mGlu5 receptor, during gestation and lactation alters mGlu5 and A2A receptors in maternal and neonatal brain.

Antidepressant and anxiolytic drugs are widely consumed even by pregnant and lactating women. The metabotropic glutamate receptor 5 (mGlu5) antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) exerts antidepressant- and anxiolytic-like actions. Given that treatment for anxiety and depression use to be prolonged in time, it is conceivable a possible modulation of metabotropic glutamate receptors (mGlu receptors) after prolonged MPEP exposure, which could also modify adenosine A2A receptors (A2AR) since functional cross-talk between them has been reported. Here we report that MPEP crosses placental barrier and reaches neonatal brain through maternal milk using LC-MS/MS methods. Therefore, we analyzed mGlu receptors, mainly mGlu5, and A2AR in both maternal and fetal brain after chronic maternal consumption of MPEP during gestation and/or lactation using radioligand binding, Western-blotting, real-time PCR and phospholipase C (PLC) activity assays. In maternal brain, chronic MPEP consumption caused a significant loss of mGlu, including mGlu5, and A2AR receptors level in plasma membrane. PLC activity assays showed that mGlu5 signaling pathway was desensitized. No variations on mRNA level coding A2AR, A1R and mGlu5 were found after MPEP treatments. In female neonatal brain, maternal consumption of MPEP caused a significant increase in mGlu, including mGlu5, and A2AR receptors level. Neither mGlu receptors nor A2AR were modified in male neonatal brain after maternal MPEP intake. Finally, neither molecular nor behavioral changes (anxiety- and depression-like behavior) were observed in 3-month-old female offspring. In summary, mGlu5 and A2AR are altered in both maternal and female neonatal brain after chronic maternal consumption of MPEP during gestation and/or lactation.

Hyperthermia-induced seizures alter adenosine A1 and A2A receptors and 5'-nucleotidase activity in rat cerebral cortex.

Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia-induced seizures on adenosine A1 and A2A receptors and 5'-nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real-time PCR, and 5'-nucleotidase activity assays. Hyperthermic seizures were induced in 13-day-old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A1 receptor density was observed using [(3) H]DPCPX as radioligand, and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density was detected, using [(3) H]ZM241385 as radioligand, 48 h after hyperthermia-evoked convulsions. These short-term changes in A1 and A2A receptors were also accompanied by a loss of 5'-nucleotidase activity. No significant variations either in A1 or A2A receptor density or 5'-nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A1 and A2A receptors and loss of 5'-nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures. Febrile seizure is one of the most common convulsive disorders in children. The consequences of hyperthermia-induced seizures (animal model of febrile seizures) on adenosine A1 and A2A receptors and 5'-nucleotidase activity have been studied at different periods in cerebral cortical area. A significant increase in A1 receptor density and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density and 5'-nucleotidase activity was detected 48 h after convulsions evoked by hyperthermia. These changes suggest the possible existence of a neuroprotective mechanism against seizures.

Interacción entre receptores de glutamato y receptores de nucleótidos en neuronas granulares de cerebelo en cultivo / Cross-talk between glutamate and nucleotide receptors in cerebellar granule neurons in culture

ATP elicits Ca2+ transients in cultured cerebellar granule neuronsacting through specific ionotropic (P2X) and metabotropic (P2Y)purinergic receptors. In these neurons, application of L-Glutamate(L-Glu) immediately before ATP induced a prolonged reductionof ATP-mediated responses that remains at least 5 minutes afterL-Glu wash out. alpha-amino-3-hydro-5-methyl-4-isoxazolpropionicacid (AMPA), N-methyl-D-aspartate (NMDA) and 3,5-dihydroxyphenyl-glycine (DHPG), selective agonists of ionotropic non-NMDA,NMDA and Group I metabotropic glutamate receptors respectively,mimicked Glu-induced attenuating effects. The activity of calciumcalmodulindependent protein kinase II (CaMKII) seems to beinvolved, at least at long term, because inhibitors of CaMKII, 1-[N,Obis(5-isoquinolinesulfonyl)-N-methyl-L-(KN-62) and N-[2-[[[3-(4'-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4'-methoxybenzenesulfonamide(KN-93), abolished the inhibitory effect of L-Glu on ATP-mediatedresponses. However, it is likely that other protein kinases could beinvolved in the cross-talk process between both groups of receptorsat short term. Therefore, these results demonstrate that the activationof glutamate receptors is able to modulate nucleotide responses incerebellar granule neurons(AU)

Interacción entre receptores de glutamato y receptores denucleótidos en neuronas granulares de cerebelo en cultivoEl ATP induce un incremento de Ca2+ en neuronas granulares decerebelo en cultivo actuando a través de receptores purinérgicos específicosionótropicos (P2X) y metabotrópicos (P2Y). En estas neuronas,la aplicación de L-Glutamato (L-Glu) inmediatamente antes del ATPinduce una prolongada disminución de las respuestas mediadas porATP que se mantiene al menos durante cinco minutos tras el lavadodel L-Glu. Los agonistas selectivos de los receptores ionotrópicosde glutamato no-NMDA, NMDA y del Grupo I, el ácido alfa-amino-3-hidro-5-metil-4-isoxazolpropiónico (AMPA), el N-metil-D-aspartato(NMDA) y el 3,5-dihidroxifenil-glicina (DHPG), respectivamente, mimetizanlos efectos atenuantes inducidos por el glutamato. La actividadde la proteína calcio-calmodulina quinasa II (CaMKII) pareceestar implicada en este proceso, al menos a largo plazo, puesto quelos inhibidores de la CaMKII, 1-[N,O-bis(5-isoquinolinesulfonil)-Nmetil-L-tirosil]-4fenilpiperazine (KN-62) y N-[2-[[[3-(4'-clorofenil)-2-propenil]metilamino]metil]fenil]-N-(2-hidroxietil)-4'-metoxibenzenosulfonamida(KN-93), revierten el efecto inhibitorio del L-Glu sobrelas respuestas mediadas por ATP. Sin embargo, es probable que puedanestar implicadas otras proteín quinasas en los procesos de interacciónentre ambos grupos de receptores a corto plazo. Por lo tanto,estos resultados demuestran que la activación de los receptores deglutamato son capaces de modular las respuestas a nucleótidos enneuronas granulares de cerebelo(AU)