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Notes: Hippocampal metabotropic glutamate 5 receptors (mGlu5Rs) regulate both physiological and pathological responses to glutamate. Because mGlu5R activation enhances NMDA-mediated effects, and given the role played by NMDA receptors in synaptic plasticity and excitotoxicity, modulating mGlu5R may influence both the physiological and the pathological effects elicited by NMDA receptor stimulation. We evaluated whether adenosine A2A receptors (A2ARs) modulated mGlu5R-dependent effects in the hippocampus, as they do in the striatum. Co-application of the A2AR agonist CGS 21680 with the mGlu5R agonist (RS)-2-chloro-s-hydroxyphenylglycine(CHPG) synergistically reduced field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices. Endogenous tone at A2ARs seemed to be required to enable mGlu5R-mediated effects, as the ability of CHPG to potentiate NMDA effects was antagonized by the selective A2AR antagonist ZM 241385 in rat hippocampal slices and cultured hippocampal neurons, and abolished in the hippocampus of A2AR knockout mice. Evidence for the interaction between A2ARs and mGlu5Rs was further strengthened by demonstrating their co-localization in hippocampal synapses. This is the first evidence showing that hippocampal A2ARs and mGlu5Rs are co-located and act synergistically, and that A2ARs play a permissive role in mGlu5R receptor-mediated potentiation of NMDA effects in the hippocampus.

Notes: Arachidonic acid (AA) is proposed to be a facilitatory retrograde messenger in hippocampal glutamatergic synapses. In this study, we found that AA (10 μM) increased the basal outflow (19 ± 4%) and the K+-evoked release of [3H]GABA (38 ± 3%) from rat hippocampal synaptosomes. This effect is likely to be a direct action of AA, as it was not mimicked by arachidic acid (10 μ M) and was not modified by inhibition of either lipooxygenase with nordihydroguaiaretic acid (50 μM) or cyclooxygenase with indomethacin (100 μM). Activation of protein kinase C may be involved, as chelerythrine (6 μM), a protein kinase C inhibitor, attenuated the AA (10 μM)-facilitation of K+-evoked [3H]GABA release by 58 ± 5%. Phospholipase A2 (2 U/mL), an enzyme that releases AA, and melittin (1 μM), a phospholipase A2 activator, mimicked the AA-facilitation of evoked [3H]GABA release (70 ± 6% and 76 ± 7% facilitation, respectively). These results show that exogenously added and endogenously produced AA increased basal outflow and K+-evoked release of [3H]GABA from rat hippocampal synaptosomes. Thus, AA can no longer be considered solely a facilitatory neuromodulator in the hippocampus, as this AA-facilitation of the release of the main inhibitory neurotransmitter may predominate under certain circumstances.

Notes: Abstract The effect of guanine nucleotide-binding protein (G protein) modifiers on the binding of the adenosine A2A receptor agonist 2-[4-(2-p-carboxyethyl[3H])phenyl-amino]-5’-N-ethylcarboxamidoadenosine ([3H]CGS 21680) and of the adenosine A1 receptor agonist [3H]R-phenylisopropyladenosine ([3H]R-PIA) to rat cortical and striatal membranes was studied. Guanosine 5’-(β,γ-imido)triphosphate (1–300 μM), which uncouples all G proteins, more effectively inhibited [3H]CGS 21680 (30 nM) binding in the cortex than [3H]R-PIA (2 nM) binding to cortical or striatal membranes or [3H]CGS 21680 (30 nM) binding in the striatum. N-Ethylmaleimide (1–300 μM) or pertussis toxin (1–100 μg/ml), which uncouple Gi/Go protein-coupled receptors, more effectively inhibited [3H]R-PIA binding to cortical or striatal membranes and [3H]CGS 21680 binding in the cortex than [3H]CGS 21680 binding in the striatum. Cholera toxin (2.5–250 μg/ml), which uncouples Gs protein-coupled receptors, more effectively inhibited [3H]CGS 21680 binding in the striatum than [3H]CGS 21680 binding in the cortex and less effectively inhibited [3H]R-PIA binding to cortical or striatal membranes. Treatment of solubilised cortical membranes with pertussis toxin (50 μg/ml) decreased [3H]CGS 21680 (30–100 nM) binding which almost fully recovered after reconstitution with Gi/Go proteins. The K i for displacement of [2-3H]-(4-{2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl}phenol) ([3H]ZM 241385, 1 nM) by CGS 21680 was 110 nM (95%CI: 98–122 nM) in non-treated, 230 (167–292) nM in pertussis toxin (25 μg/ml)-treated and 222 (150–295) nM in cholera toxin (50 μg/ml)-treated cortical membranes; in contrast, the K i for displacement of [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine ([3H]SCH 58261, 1 nM) by CGS 21680 was 74 (57–91) nM in non-treated, 71 (44–100) nM in pertussis toxin-treated and 147 (100–193) nM in cholera toxin-treated cortical membranes. Finally, CGS 21680 displaced monophasically the binding of the A1 antagonist, [3H]8-cyclopentyl-1,3-dipropylxanthine (2 nM), and the A1 agonist, [3H]R-PIA (2 nM), in 2 or 10 mM Mg2+-medium, either at 25°C or 37°C, in cortical or striatal membranes. These results indicate that CGS 21680 does not bind to A1 receptors and that limbic CGS 21680 binding sites differ from striatal-like A2A receptors since they couple to Gi/Go proteins, as well as to Gs proteins.

Notes: Abstract  The binding of the adenosine A2A receptor selective agonist 2-[4-(2-p-carboxyethyl) phenylamino] -5′-N-ethylcarboxamidoadenosine (CGS 21680) to the rat hippocampal and cerebral cortical membranes was studied and compared with that to striatal membranes. [3H] CGS 21680, in the concentration range tested (0.2–200 nM), bound to a single site with a K d of 58 nM and a B max of 353 fmol/mg protein in the hippocampus, and with a K d of 58 nM and a B max of 264 fmol/mg protein in the cortex; in the striatum, the single high-affinity [3H] CGS 21680 binding site had a K d of 17 nM and a B max of 419 fmol/mg protein. Both guanylylimidodiphosphate (100 μM) and Na+ (100 mM) reduced the affinity of [3H] CGS 21680 binding in the striatum by half and virtually abolished [3H] CGS 21680 binding in the hippocampus and cortex. The displacement curves of [3H] CGS 21680 binding with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), N 6-cyclohexyladenosine (CHA), 5′-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) were biphasic in the hippocampus and cortex as well as in the striatum. The predominant [3H]CGS 21680 binding site in the striatum (80%) had a pharmacological profile compatible with A2A receptors and was also present in the hippocampus and cortex, representing 10–25% of [3H]CGS 21680 binding. The predominant [3H]CGS 21680 binding site in the hippocampus and cortex had a pharmacological profile distinct from A2A receptors: the relative potency order of adenosine antagonists DPCPX, 1,3-dipropyl-8-{4-[(2-aminoethyl)amino]carbonylmethyloxyphenyl} xanthine (XAC), 8-(3-chlorostyryl) caffeine (CSC), and (E)-1,3-dipropyl-8-(3,4-dimethoxystyryl)-methylxanthine (KF 17,837) as displacers of [3H] CGS 21680 (5 nM) binding in the hippocampus and cerebral cortex was DPCPX 〉 XAC ≫ CSC ≈ KF 17,837, and the relative potency order of adenosine agonists CHA, NECA, CADO, 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5′-N-ethylcarboxamidoadenosine (APEC), and 2-phenylaminoadenosine (CV 1808) was CHA ≈ NECA≥CADO〉 APEC ≈ CV1808 〉 CGS 21680. In the presence of DPCPX (20 nM), [3H] CGS 21680 (0.2–200 nM) bound to a site (A2A-like) with a K d of 20 nM and a B max of 56 fmol/mg protein in the hippocampus and with a K d of 22 nM and a B max of 63 fmol/mg protein in the cortex. In the presence of CSC (200 nM), [3H]CGS 21680 (0.2–200 nM) bound to a second high-affinity site with a K d of 97 nM and a B max of 255 fmol/mg protein in the hippocampus and with a K d of 112 nM and a B max of 221 fmol/mg protein in the cortex. Two pharmacologically distinct [3H]CGS 21680 binding sites were found in synaptosomal membranes of the hippocampus and cortex and in the striatum, one corresponding to A2A receptors and the other to the second high-affinity [3H]CGS 21680 binding site. In contrast, the pharmacology of [3H]CHA binding was similar in synaptosomal membranes of the three brain areas. The present results establish the existence of at least two high-affinity [3H]CGS 21680 binding sites in the CNS and demonstrate that the [3H]CGS 21680 binding site predominant in the hippocampus and cerebral cortex has different binding characteristics from the classic A2A adenosine receptor, which predominates in the striatum.

Notes: Abstract The binding of the adenosine A2A receptor selective agonist 2-[4-(2-p-carboxyethyl) phenylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680) to the rat hippocampal and cerebral cortical membranes was studied and compared with that to striatal membranes. [3H] CGS 21680, in the concentration range tested (0.2–200 nM), bound to a single site with a K d of 58 nM and a B max of 353 fmol/mg protein in the hippocampus, and with a K d of 58 nM and a B max of 264 fmol/mg protein in the cortex; in the striatum, the single high-affinity [3H] CGS 21680 binding site had a K d of 17 nM and a B max of 419 fmol/mg protein. Both guanylylimidodiphosphate (100 μM) and Na+ (100 mM) reduced the affinity of [3H] CGS 21680 binding in the striatum by half and virtually abolished [3H] CGS 21680 binding in the hippocampus and cortex. The displacement curves of [3H] CGS 21680 binding with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), N 6-cyclohexyladenosine (CHA), 5′-N-ethyl-carboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) were biphasic in the hippocampus and cortex as well as in the striatum. The predominant [3H]CGS 21680 binding site in the striatum (80%) had a pharmacological profile compatible with A2A receptors and was also present in the hippocampus and cortex, representing 10–25% of [3H]CGS 21680 binding. The predominant [3H]CGS 21680 binding site in the hippocampus and cortex had a pharmacological profile distinct from A2A receptors: the relative potency order of adenosine antagonists DPCPX, 1,3-dipropyl8-{4-[(2-aminoethyl)amino]carbonylmethyloxyphenyl} xanthine (XAC), 8-(3-chlorostyryl) caffeine (CSC), and (E)-1,3-dipropyl-8-(3,4-dimethoxystyryl)-methylxanthine (KF 17,837) as displacers of [3H] CGS 21680 (5 nM) binding in the hippocampus and cerebral cortex was DPCPX 〉 XAC ≫ CSC ≈ KF 17,837, and the relative potency order of adenosine agonists CHA, NECA, CADO, 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5′-N-ethylcar-boxamidoadenosine (APEC), and 2-phenylaminoadenosine (CV 1808) was CHA ≈ NECA ⩾ CADO 〉 APEC ≈ CV1808 〉 CGS 21680. In the presence of DPCPX (20 nM), [3H] CGS 21680 (0.2-200 nM) bound to a site (A2A-like) with a K d of 20 nM and a B max of 56 fmol/mg protein in the hippocampus and with a K d of 22 nM and a B max of 63 fmol/mg protein in the cortex. In the presence of CSC (200 nM), [3H]CGS 21680 (0.2–200 nM) bound to a second high-affinity site with a K d of 97 nM and a B max of 255 fmol/mg protein in the hippocampus and with a K d of 112 nM and a B max of 221 fmol/mg protein in the cortex. Two pharmacologically distinct [3H]CGS 21680 binding sites were found in synaptosomal membranes of the hippocampus and cortex and in the striatum, one corresponding to A2A receptors and the other to the second high-affinity [3H]CGS 21680 binding site. In contrast, the pharmacology of [3H]CHA binding was similar in synaptosomal membranes of the three brain areas. The present results establish the existence of at least two high-affinity [3H]CGS 21680 binding sites in the CNS and demonstrate that the [3H]CGS 21680 binding site predominant in the hippocampus and cerebral cortex has different binding characteristics from the classic A2A adenosine receptor, which predominates in the striatum.

Notes: Summary Adenosine mono-, di- and triphosphates, inosine monophosphates, adenosine, inosine and hypoxanthine were separated in a single run by high-performance liquid chromatography with UV detection. AC-18 reversephase column was used. An isocratic elution with 100mM phosphate buffer (KH2PO4) pH6.5 and 1.2% methanol, followed by a 5 minute linear gradient up to 15% methanol after the 10th minute was performed during each run with a flow rate of 1.25ml/min. The time required for each analysis was 25 minutes. The detection limit for each substance ranged from 3 to 5 pmol. All the substances under study were detected in the incubation medium when adenosine triphosphate was exogenously applied to the innervated frog sartorius muscle.