The antioxidant resveratrol acts as a non-selective adenosine receptor agonist.

Resveratrol (RSV) is a natural polyphenolic antioxidant with a proven protective role in several human diseases involving oxidative stress, although the molecular mechanism underlying this effect remains unclear. The present work tried to elucidate the molecular mechanism of RSV's role on signal transduction modulation. Our biochemical analysis, including radioligand binding, real time PCR, western blotting and adenylyl cyclase activity, and computational studies provide insights into the RSV binding pathway, kinetics and the most favored binding pose involving adenosine receptors, mainly A2A subtype. In this study, we show that RSV target adenosine receptors (AdoRs), affecting gene expression, receptor levels, and the downstream adenylyl cyclase (AC)/PKA pathway. Our data demonstrate that RSV activates AdoRs. Moreover, RSV activate A2A receptors by directly binding to the classical orthosteric binding site. Intriguingly, RSV-induced receptor activation can stimulate or inhibit AC activity depending on concentration and exposure time. Such subtle and multifaceted regulation of the AdoRs/AC/PKA pathway might contribute to the protective role of RSV. Our findings suggest that RSV molecular action is mediated, at least in part, by activation of adenosine receptors and create the opportunity to interrogate the therapeutic use of RSV in pathological conditions involving AdoRs, such as Alzheimer.

Resveratrol Modulates and Reverses the Age-Related Effect on Adenosine-Mediated Signalling in SAMP8 Mice.

Resveratrol (RSV) is a natural compound present in berries, grapes and red wine that has shown some neuroprotective properties, but the mechanism by which RSV exhibits its protective role is not very well understood yet. Little is known about the effect of RSV on adenosinergic system, a system regulated in an age-dependent manner in SAMP8 mice, widely considered as an Alzheimer's model. Therefore, the aim of the present work was to assess whether RSV intake was able to modulate the adenosine-mediated signalling in SAMP8 mice. Data showed herein clearly demonstrate the ability of RSV to modulate adenosine receptor gene expression as well as transduction pathway mediated by receptors expressed on plasma membrane. Interestingly, this polyphenol was able to reverse the age-related loss of adenosine A1 receptors and its corresponding signalling pathway. Moreover, adenosine A2A receptors were not modulated by aging or RSV, but A2A-mediated signalling was completely desensitized after RSV treatment compared to untreated mice. Enzymes involved on adenosine metabolism, such as 5'-nucleotidase and adenosine deaminase, were found to be reduced after RSV treatment, but adenosine levels remained unchanged. Nevertheless, an age-related decrease on 5'-nucleotidase activity and adenosine and related metabolite levels was observed. In conclusion, our data show that RSV modulates adenosine-mediated signalling, strongly suggesting that the role of RSV via adenosine receptor signalling and its modulation of neurotransmission in neurodegenerative diseases should be considered as new therapeutic target for RSV neuroprotective effect.

Maternal glutamate intake during gestation and lactation regulates adenosine A1 and A(2A) receptors in rat brain from mothers and neonates.

Pregnant rats were treated daily with 1 g/L of L-glutamate in their drinking water during pregnancy and/or lactation. The effect on adenosine A1 receptor (A1R) and A(2A) receptor (A(2A)R) in brains from both mothers and 15-day-old neonates was assayed using radioligand binding and real time PCR assays. Mothers receiving L-glutamate during gestation, lactation, and throughout gestation and lactation showed a significant decrease in total A1R number (water+water, 302±49 fmol/mg; L-glutamate+water, 109±11 fmol/mg, P<0.01; water+L-glutamate, 52±13 fmol/mg, P<0.01; L-glutamate+L-glutamate, 128±33 fmol/mg, P<0.05). No variations were detected in the Kd parameter. Concerning adenosine A(2A)R, radioligand binding assays revealed that Bmax parameter remains unaltered in maternal brain in response to glutamate exposure. However, Kd parameter was significantly decreased in all L-glutamate-treated groups (water+water, 5.3±1.3 nM; L-glutamate±water, 0.5±0.1 nM; water+L-glutamate, 0.9±0.1 nM; L-glutamate±L-glutamate, 0.7±0.1 nM, P<0.01 in all cases). In both male and female neonates, A1R was also decreased after long-term glutamate exposure during gestation, lactation, and gestation plus lactation (male neonates: water+water, 564±68 fmol/mg; L-glutamate+water, 61±8 fmol/mg; water+L-glutamate, 95±20 fmol/mg; L-glutamate+L-glutamate, 111±15 fmol/mg; P<0.01 in all cases; female neonates: water+water, 216±35 fmol/mg; L-glutamate+water, 59±9 fmol/mg; water+L-glutamate, 139±16 fmol/mg; L-glutamate+L-glutamate, 97±14 fmol/mg; P<0.01 in all cases). No variations were found in mRNA level coding adenosine A(1)R in maternal or neonatal brain. Concerning adenosine A(2A)R, radioligand binding assays revealed that Bmax parameter was significantly increased in male and female neonates exposed to L-glutamate during lactation (male neonates: water+water, 214±23 fmol/mg; water+L-glutamate, 581±49 fmol/mg; P<0.01; female neonates: water+water, 51±10 fmol/mg; water+L-glutamate, 282±52 fmol/mg; P<0.05). No variations were found in mRNA level coding adenosine A(2A)R in maternal or neonatal brain. In summary, long-term L-glutamate treatment during gestation and lactation promotes a significant down-regulation of A1R in whole brain from both mother and neonates and a significant up-regulation of A(2A)R in neonates exposed to L-glutamate during lactation.

Maternal caffeine intake during gestation and lactation down-regulates adenosine A1 receptor in rat brain from mothers and neonates.

Even though caffeine can be excreted in breast milk, few studies have analyzed the effect of maternal caffeine consumption during lactation on neonatal brain. In the present work pregnant rats were treated daily with 1 g/L of caffeine in their drinking water during pregnancy and/or lactation and the effect on adenosine A(1) receptor in brains from both lactating mothers and 15 days-old neonates was assayed using radioligand binding and real time PCR assays. Mothers receiving caffeine during gestational period developed motor activation in gestational days 8-10 which was associated with a significant decrease of total adenosine A(1) receptor number (84%). A similar decrease was detected in mothers treated with caffeine during lactation (76%) and throughout gestation and lactation (73%); this was accompanied by a significant decrease in mRNA level coding adenosine A(1) receptor (28%). In male neonates, adenosine A(1) receptor was also decreased after chronic caffeine exposure during gestation (80%), lactation (76%) and gestation plus lactation (80%). In female neonates, adenosine A(1) receptor tended to decrease in response to caffeine exposure although no significant variations were found. No variation in the level of mRNA coding adenosine A(1) receptor was detected in neonates in any case. Concerning adenosine A(2A) receptor, radioligand binding assays revealed that this receptor remains unaltered in maternal and neonatal brain in response to caffeine exposure. However, caffeine consumption during gestation and lactation evoked a significant decrease in mRNA level coding A(2A) receptor (32%) in mothers' brain.

Reduced expression and desensitization of adenosine A1 receptor/adenylyl cyclase pathway after chronic (-)N6-phenylisopropyladenosine intake during pregnancy.

Little is known about the G protein-coupled receptor desensitization process during pregnancy. Wistar pregnant rats were treated with (-)N(6)-phenylisopropyladenosine (R-PIA), an adenosine A(1) receptor (A(1)R) agonist, in their drinking water during pregnancy, and the effect on A(1)R/adenylyl cyclase system was studied in both maternal and fetal brain. In maternal brain, binding assays revealed a significant decrease in total receptor numbers in plasma membranes (27%, P<0.05), with no significant changes in receptor affinity. The effect of R-PIA on plasma membranes from fetal brains was more marked, with approximately 42% (P<0.05) of the total receptors detected in control fetuses. Real time reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that chronic R-PIA treatment during the whole gestational period only decreased significantly mRNA level coding A(1)R in maternal brain (P<0.05). alpha Gi(1,2) and alpha Gi(3) subunits were not affected in mothers or fetuses as revealed by immunoblotting. mRNA levels coding these subunits were also unaffected in mothers and fetuses. On the other hand, forskolin- and forskolin-plus guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S)-stimulated adenylyl cyclase activity was decreased in maternal (P<.01) and fetal brain (P<.001). Furthermore, adenylyl cyclase inhibition elicited by N(6)-cyclohexyladenosine (CHA), a selective A(1)R agonist, was significantly decreased in both maternal (P<0.05) and fetal brain (P<.01), suggesting a desensitization of the A(1)R/adenylyl cyclase pathway. Therefore, these results suggest that R-PIA intake during pregnancy causes desensitization of the A(1)R-mediated inhibitory transduction pathway in both maternal and fetal brain, probably due to the decreased density of A(1)R at the cell surface.

Age-related expression of adenosine receptors in brain from the senescence-accelerated mouse.

Senescence-accelerated mice (SAM) are used as a model of aging and age-associated diseases. SAMP8 are prone strains that show shortened life span and deficits in learning and memory processes, while SAMR1 are strains of accelerated senescence-resistant, long-lived mice. Due to their abnormal APP (amyloid precursor protein) metabolism in brain, SAMP8 may be an Alzheimer-type model. Adenosine receptors are G-protein coupled receptors which are altered in brain from Alzheimer disease (AD) cases. The analysis of adenosine receptors in brain from young (21 days old) and middle-aged (180 days old) SAMP8 as compared with SAMR1 mice revealed differences between these strains associated with age. The age-related increase in mRNA coding A1 and A2B receptors observed in SAMR1 was absent in SAMP8. A1 receptors were significantly decreased with age in SAMR1, while no differences were observed in SAMP8. However, the levels of A1 receptors in young SAMP8 were even lower than those obtained in middle-aged SAMR1. In addition, A2A receptors were significantly increased only in aged SAMR1. A similar age-related decrease in A1 receptors level was also observed in brain from male Wistar rats. These results suggest different age-related effects on adenosine receptors in SAMR1 and SAMP8 strains. Since A1 receptors are mainly neuroprotective, their important loss in very young SAMP8 strain suggests the involvement of these receptors in the pathogenesis of neurodegenerative diseases associated with aging.

Expression levels of adenosine receptors in hippocampus and frontal cortex in argyrophilic grain disease.

Expression of adenosine receptors of the A1, A2A and A2B type has been examined in the post-mortem frontal cortex and hippocampus in argyrophilic grain disease (AGD), a tauopathy affecting the hippocampus but usually not the frontal cortex, in an attempt to learn about the modulation of the adenosine pathway in this disorder. Significant increased levels of A1, but not of A2A and A2B, have been observed in AGD in the hippocampus but not in the frontal cortex, when compared with age-matched controls. This is accompanied by increased levels of adenylyl cyclase (AC), an effector of A1, and by increased (although not significant) percentage of inhibition of forskolin-stimulated AC by the A1 agonist cyclohexyladenosine in the hippocampus in AGD. These findings indicate sensitization of A1/AC in the hippocampus in AGD, and support a putative activation of the A1/AC pathway that may facilitate protection of this preferentially involved region in AGD.

Metabotropic glutamate receptor/phospholipase C pathway is increased in rat brain at the end of pregnancy.

Wistar pregnant rats were sacrificed at the end of pregnancy and the status of metabotropic glutamate receptors/phospholipase C (mGluR/PLC) pathway was studied in brain from pregnant and non-pregnant female rats. Pregnancy causes a significant increase in metabotropic glutamate receptors number, determined by radioligand binding assay, without significant changes on receptor affinity. Similar increase in mGluR(1) type was obtained by immunoblotting assay using specific anti-mGluR(1) antibody. However, no significant differences were observed in mGluR(5) type, suggesting that the increase detected by radioligand assays could be due to mGluR(1) up-regulation. On the other hand, a significant increase in the alpha subunit of G(q) protein was also detected in pregnant rats by immunoblotting assays. Real-time PCR experiments revealed a significant increase in gene expression of metabotropic glutamate receptors and G(q) proteins. Neither protein level nor gene expression of phospholipase C beta(1) isoform was altered in pregnant rats. However, an increase in basal and agonist-stimulated phospholipase C activity was observed in membranes from pregnant rats. These results suggest that gestational period causes the up-regulation of both metabotropic glutamate receptors and coupled G(q)-protein and, in turn, an increase in phospholipase C activity.

Chronic intake of caffeine during gestation down regulates metabotropic glutamate receptors in maternal and fetal rat heart.

Caffeine is the most widely consumed substance in the world which antagonizes adenosine effects. Adenosine acting through A(1) receptors inhibits glutamate release which binds to metabotropic glutamate receptors (mGluRs). Recently, we have shown that maternal caffeine intake during gestation causes down-regulation of A(1) and metabotropic glutamate receptors in the brain of both rat mothers and fetuses. In the present work we provide evidence that caffeine also affects receptors in hearts, causing a decrease in mGluRs from both maternal and fetal hearts. A decrease in G(q/11) and PLC beta(1) proteins level was also observed in both tissues. However, phospholipase C activity was only affected in fetal heart, being significantly decreased. These results suggest an in vivo cross-talk mechanism between adenosine and glutamate receptors in peripheral tissues. Therefore, special attention should be paid to caffeine ingestion during gestation.

Effect of chronic gestational treatment with caffeine or theophylline on Group I metabotropic glutamate receptors in maternal and fetal brain.

Pregnant rats were treated throughout the gestational period with either caffeine or theophylline, and its effect on the metabotropic glutamate receptor (mGluRs) signal transduction pathway was studied in both maternal and fetal brain. In maternal brain, radioligand binding assays showed that chronic treatment with methylxanthines caused a significant decrease in the total number of mGluRs. This decrease was accompanied by an increase in receptor affinity. Immunodetection showed that mGluR1a and phospholipase C beta1 (PLCbeta1) were significantly decreased in response to chronic methylxanthine treatment, whereas alphaG(q/11) was not affected. A loss was also detected of PLC stimulation mediated by (S)-3,5-dihydroxyphenylglycine (DHPG), a selective Group I mGluR agonist, suggesting desensitization of the mGluR/PLC pathway. In fetal brain, a loss in total mGluRs was observed in fetuses from mothers treated with caffeine or theophylline, without variation in receptor affinity. A decrease in mGluR1a, alphaG(q/11) and PLCbeta1 levels was also observed in response to treatment. However, changes detected in this immature tissue were not associated with variations in PLC activity. These results suggest that chronic caffeine or theophylline treatment down-regulates several mGluR/PLC transduction pathway components in both maternal and fetal brain, causing a loss of receptor responsiveness only in maternal brain.

Chronic caffeine or theophylline intake during pregnancy inhibits A1 receptor function in the rat brain.

The aim of this work was to study whether caffeine or theophylline chronically consumed during pregnancy affect inhibitory adenylyl cyclase pathway mediated by adenosine, in rat brain of both mothers and full-term fetuses. Immunoblotting analysis revealed a significant decrease in alphaGi(1,2) subunit level (27-29% in mothers, 15-18% in fetuses), associated with a significant increase in the mRNA level coding alphaGi(1) in both maternal and fetal rat brain (12-22%) after methylxanthine intake. No significant differences in alphaGi(3) level were detected in any case. On the other hand, forskolin- and forskolin plus guanosine-5'-O(3-thiotriphosphate) tetralithium salt-stimulated adenylyl cyclase activity was significantly decreased (30-36%) in maternal brain. Moreover, adenylyl cyclase inhibition elicited by N(6)-cyclohexyladenosine, specific adenosine A(1) receptor agonist, was also significantly decreased in caffeine- (40.5%) and theophylline- (55.0%) treated mothers, suggesting a desensitization of adenosine A(1) receptor/adenylyl cyclase pathway in maternal brain. However, no significant differences were detected in fetal brain between control and treated animals. Therefore, caffeine or theophylline chronic intake during pregnancy differently modulates inhibitory adenylyl cyclase pathway mediated by adenosine in maternal and fetal brain causing a loss of the system responsiveness only in maternal brain but down-regulating Gi(1) protein in both mother and fetus brain.

Effect of chronic glutamate administration to pregnant rats during gestation on metabotropic glutamate receptors from mothers and full-term fetuses brain.

Chronic glutamate treatment during gestational period caused a significant decrease in total metabotropic glutamate receptors (mGluR) number. Similar results were observed on the steady-state level of mGlu(1) receptor detected by immunoblotting assays, suggesting that this is the main receptor subtype modulated by agonist exposure. Furthermore, no variations on mRNA coding mGlu(1) receptor were found, suggesting post-transcriptional modulation as a possible mechanism of the lost of receptor detected at the membrane surface. On the other hand, western-blotting to determine level of G(q/11) protein and phospholipase C beta(1) revealed a significant decrease of both proteins in mothers brain. This decrease was associated with significant variation in glutamate and DHPG-stimulated phospholipase C activity. No significant differences on mGluR transduction pathway components were observed in fetuses brain. These results suggest that glutamate intake during pregnancy causes a down-regulation of different proteins involved in glutamate response mediated by mGluR only in mothers brain without significantly affecting fetuses brain.

Abnormal metabotropic glutamate receptor expression and signaling in the cerebral cortex in diffuse Lewy body disease is associated with irregular alpha-synuclein/phospholipase C (PLCbeta1) interactions.

Diffuse Lewy body disease (DLBD) is a degenerative disease of the nervous system, involving the brain stem, diencephalic nuclei and cerebral cortex, associated with abnormal a-synuclein aggregation and widespread formation of Lewy bodies and Lewy neurites. DLBD presents as pure forms (DLBDp) or in association with Alzheimer disease (AD) in the common forms (DLBDc). Several neurotransmitter abnormalities have been reported including those of the nigrostriatal and mesocorticolimbic dopaminergic system, and central noradrenergic, serotoninergic and cholinergic pathways. The present work examines metabotropic glutamate receptor (mGluR) expression and signaling in the frontal cortex of DLBDp and DLBDc cases in comparison with age-matched controls. Abnormal L-[3H]glutamate specific binding to group I and II mGluRs, and abnormal mGluR1 levels have been found in DLBD. This is associated with reduced expression levels of phospholipase C beta1 (PLCbeta1), the effector of group I mGluRs following protein G activation upon glutamate binding. Additional modification in the solubility of PLCbeta1 and reduced PLCbeta1 activity in pure and common DLBD further demonstrates for the first time abnormal mGluR signaling in the cerebral cortex in DLBD. In order to look for a possible link between abnormal mGluR signaling and a-synuclein accumulation in DLBD, immunoprecipitation studies have shown alpha-synuclein/PLCbeta1 binding in controls and decreased alpha-synuclein/PLCbeta1 binding in DLBD. This is accompanied by a shift in the distribution of a-synuclein, but not of PLCbeta1, in DLBD when compared with controls. Together, these results support the concept that abnormal a-synuclein in DLBD produces functional effects on cortical glutamatergic synapses, which are associated with reduced alpha-synuclein/PLCbeta1 interactions, and, therefore, that mGluRs are putative pharmacological targets in DLBD. Finally, these results emphasize the emergence of a functional neuropathology that has to be explored for a better understanding of the effects of abnormal protein interactions in degenerative diseases of the nervous system.

Adenosine A1 receptor agonist treatment up-regulates rat brain metabotropic glutamate receptors.

Chronic R-N(6)-phenylisopropiladenosine (R-PIA) subcutaneous injection for 6 days significantly increased total glutamate receptor number (180% of control) in rat brain synaptic plasma membranes (SPM), without affecting receptor affinity. A higher increase in metabotropic glutamate (mGlu) receptor number (258% of control) was also detected, indicating that mGlu is the main type of glutamate receptor affected by this treatment. On the other hand, the observed increase in basal and calcium- and Gpp(NH)p-stimulated phospholipase C (PLC) activity after treatment was associated with a significant increase in PLC beta(1) isoform, detected in SPM by immunoblotting assays. Moreover, an increase in PLC activity stimulation with trans-ACPD, in the absence and in the presence of Gpp(NH)p, was detected after R-PIA treatment. These results show that mGlu receptors and its effector system, PLC activity, are up-regulated by chronic exposure to an adenosine A(1) receptor agonist and suggest the existence of a cross-talk mechanism between both signal transduction pathways in rat brain.

Cross-talk between beta-adrenergic and metabotropic glutamate receptors in rat C6 glioma cells.

Exposure of rat C6 glioma cells to the beta-adrenergic receptor agonist isoproterenol potentiates basal and metabotropic glutamate receptor-stimulated phospholipase C activity in rat C6 glioma cells. After treatment of cells for 24 h with 10 microM isoproterenol, metabotropic glutamate receptors and phospholipase C activity were determined in C6 plasma membranes. Isoproterenol treatment caused an increase of 67% in the total number of binding sites (Bmax=12.1+/-1. 8 pmol/mg protein versus Bmax=20.27+/-0.88 pmol/mg protein) with Kd values of the same order (Kd=1250+/-101 nM versus Kd=1401+/-211 nM), using l-[3H]glutamate as radioligand. On the other hand, basal, guanylyl imidodiphosphate (Gpp[NH]p)- and trans-aminocyclopentane-1, 3-dicarboxylic acid (trans-ACPD)-stimulated phospholipase C activities were also significantly increased in membranes from isoproterenol-treated cells compared to control cells, by 337%, 33% and 40% respectively. Moreover, a significant increase of 94% in the steady-state level of phospholipase C beta1 in membranes from isoproterenol-treated cells compared to control was also detected by immunoblot. These results show that metabotropic glutamate receptors and its effector system, phospholipase C, are affected by isoproterenol treatment, showing the existence of cross-talk between these signal transduction pathways.

Characterization of metabotropic glutamate receptors in rat C6 glioma cells.

Metabotropic glutamate receptors in rat C6 glioma cells have been characterized by pharmacological and kinetic binding experiments, using both L-[3H]glutamate and [3H(+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid ([3H](+/-)-trans-ACPD) radioligands. Saturation experiments revealed a single binding site with a Kd = 1250 +/- 101 nM and Bmax = 12.1 +/- 1.8 pmol/mg protein when the assays were performed with L-[3H]glutamate as radioligand in the presence of AMPA, kainate, NMDA and DL-threo-beta-hydroxyaspartic acid. When [3H](+/-)-trans-ACPD was used as radioligand, the kinetic parameters obtained were Kd = 2605 +/- 1042 nM and Bmax = 13.66 +/- 5.01 pmol/mg protein. Pharmacological characterization indicated that specific binding of L-[3H]glutamate was sensitive to different agonists of mGlu receptors, showing a rank order of affinity L-glutamate > L-quisqualic acid > (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) > ibotenic acid >>> (2S, 'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Specific binding of L-[3H]glutamate to mGlu receptors is regulated by guanine nucleotides. Guanylyl imidodiphosphate (Gpp(NH)p) causes an affinity shift on the L-glutamate dose-response curve, increasing the IC50 value. These results support the evidence that metabotropic glutamate receptors are present in rat C6 glioma cells and they are coupled to a G-protein.