J. Alexandre Ribeiro

Preferential release of ATP and its extracellular catabolism as a source of adenosine upon high- but not low-frequency stimulation of rat hippocampal slices

Abstract: The release of adenosine and ATP evoked by electrical field stimulation in rat hippocampal slices was investigated with the following two patterns of stimulation: (1) a brief, high‐frequency burst stimulation (trains of stimuli at 100 Hz for 50 ms applied every 2 s for 1 min), to mimic a long‐term potentiation (LTP) stimulation paradigm, and (2) a more prolonged (3 min) and low‐frequency (5 Hz) train stimulation, to mimic a long‐term depression (LTD) stimulation paradigm. The release of ATP was greater at a brief, high‐frequency burst stimulation, whereas the release of [3H]adenosine was slightly greater at a more prolonged and low‐frequency stimulation. To investigate the source of extracellular adenosine, the following two pharmacological tools were used; α,β‐methylene ADP (AOPCP), an inhibitor of ecto‐5′‐nucleotidase, to assess the contribution of the catabolism of released adenine nucleotides as a source of extracellular adenosine, and S‐(4‐nitrobenzyl)‐6‐thioinosine (NBTI), an inhibitor of adenosine transporters, to assess the contribution of the release of adenosine, as such, as a source of extracellular adenosine. At low‐frequency stimulation, NBTI inhibited by nearly 50% the evoked outflow of [3H]adenosine, whereas AOPCP inhibited [3H]adenosine outflow only marginally. In contrast, at high‐frequency stimulation, AOPCP inhibited by 30% the evoked release of [3H]adenosine, whereas NBTI produced a 40% inhibition of [3H]adenosine outflow. At both frequencies, the kinetics of evoked [3H]adenosine outflow was affected in different manners by AOPCP and NBTI; NBTI mainly depressed the rate of evoked [3H]adenosine outflow, whereas AOPCP mainly inhibited the later phase of evoked [3H]adenosine accumulation. These results show that there is a simultaneous, but quantitatively different, release of ATP and adenosine from rat hippocampal slices stimulated at frequencies that can induce plasticity phenomena such as LTP (100 Hz) or LTD (5 Hz). The source of extracellular adenosine is also different according to the frequency of stimulation; i.e., at a brief, high‐frequency stimulation there is a greater contribution of released adenine nucleotides for the formation of extracellular adenosine than at a low frequency with a more prolonged stimulation.

Evidence for functionally important adenosine A2a receptors in the rat hippocampus

Adenosine A2a receptors are not confined to dopamine-rich areas of the brain, since thermocycling analysis shows that adenosine A2a receptor mRNA is expressed also in the hippocampus (CA1, CA3 and dentate gyrus) and cerebral cortex. The expression of A2a mRNA in three main areas of the hippocampus was confirmed by in situ hybridization; A2a mRNA expression was mainly localized in the pyramidal and granular cells, the same hippocampal regions that showed adenosine A1 receptor mRNA expression. Receptor autoradiographic studies with [3H]CGS 21680 (30 nM), a selective adenosine A2a receptor agonist, showed specific binding sites in the hippocampus. The density of [3H]CGS 21680 binding was greatest in the stratum radiatum of the CA1 area, followed by the stratum oriens of the cornu Ammonis, stratum radiatum of the CA3 are and supra-granular layer of the dentate gyrus. This anatomical distribution of [3H]CGS 21680 binding was similar to the pattern of [3H]CHA binding in the hippocampus. Electrophysiological studies in the Schaffer fibers/CA1 pyramids showed that upon activation of the A2a receptors with CGS 21680 (10 nM) the ability of the adenosine A1 receptor agonist, CPA, to inhibit neuronal activity was significantly attenuated. These results show functionally important co-expression and co-localization of adenosine A2a and A1 receptors in the hippocampus. The results also suggest that adenosine A2a receptor-mediated neuromodulation is not confined to the basal ganglia, but is more widespread throughout the nervous system.

Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides

1 In the present work, we investigated the action of adenosine originating from extracellular catabolism of adenine nucleotides, in two preparations where synaptic transmission is modulated by both inhibitory A1 and excitatory A2a‐adenosine receptors, the rat hippocampal Schaffer fibres/CA1 pyramid synapses and the rat innervated hemidiaphragm. 2 Endogenous adenosine tonically inhibited synaptic transmission, since 0.5‐2 u ml−1 of adenosine deaminase increased both the population spike amplitude (30 ± 4%) and field excitatory post‐synaptic potential (f.e.p.s.p.) slope (27 ± 4%) recorded from hippocampal slices and the evoked [3H]‐acetylcholine ([3H]‐ACh) release from the motor nerve terminals (25 ± 2%). 3 a, b̊‐Methylene adenosine diphosphate (AOPCP) in concentrations (100–200 μm) that almost completely inhibited the formation of adenosine from the extracellular catabolism of AMP, decreased population spike amplitude by 39 ± 5% and f.e.p.s.p. slope by 32 ± 3% in hippocampal slices and [3H]‐ACh release from motor nerve terminals by 27 ± 3%. 4 Addition of exogenous 5′‐nucleotidase (5 u Ml−1) prevented the inhibitory effect of AOPCP on population spike amplitude and f.e.p.s.p. slope by 43–57%, whereas the P2 antagonist, suramin (100 μm), did not modify the effect of AOPCP. 5 In both preparations, the effect of AOPCP resulted from prevention of adenosine formation since it was no longer evident when accumulation of extracellular adenosine was hindered by adenosine deaminase (0.5‐2 u ml−1). The inhibitory effect of AOPCP was still evident when A1 receptors were blocked by 1,3‐dipropyl‐8‐cyclopentylxanthine (2.5‐5 nM), but was abolished by the A2 antagonist, 3,7‐dimethyl‐1‐propargylxanthine (10 μm). 6 These results suggest that adenosine originating from catabolism of released adenine nucleotides preferentially activates excitatory A2 receptors in hippocampal CA1 pyramid synapses and in phrenic motor nerve endings.

ZM241385 is an antagonist of the facilitatory responses produced by the A2A adenosine receptor agonists CGS21680 and HENECA in the rat hippocampus.

1 In the present study, we investigated the ability of a recently introduced non‐xanthine A2A receptor antagonist, ZM241385 (4‐(2‐[7‐amino‐2‐(2‐furyl{1,2,4}‐triazolo{2,3‐a{1,3,5}triazin‐5‐yl‐aminoethyl)phenol) to displace binding of the prototypical A2A adenosine receptor agonist [3H]CGS21680 (2‐[4‐(2‐p‐carboxyethyl)phenylamino]‐5′‐N‐ethylcarboxamidoadenosine) and to modify the facilitatory responses caused by the A2A selective agonists, CGS21680 and HENECA (2‐hexynl‐5′‐N‐ethylcarboxamidoadenosine) in rat hippocampal preparations. 2 ZM241385 was nearly equipotent to displace [3H]CGS21680 (30 nM) binding to hippocampal (Ki of 0.52 nM) and to striatal membranes (Ki of 0.35 nM), whereas HENECA was a more potent displacer of [3H]CGS21680 binding to striatal (Ki of 4.5 nM) than to hippocampal membranes (Ki of 19 nM). 3 HENECA (3–30 nM) was equipotent with CGS21680 to facilitate veratridine‐evoked [3H]acetylcholine release from superfused hippocampal synaptosomes and ZM241385 (20 nM) inhibited the facilitatory effects of both HENECA (30 nM) and CGS21680 (30 nM); this antagonism was mimicked by CSC (250 nM). 4 In contrast, CGS21680 (10–30 nM) was more potent than HENECA (10–30 nM) to facilitate synaptic transmission in Schaffer fibres/CA1 pyramid synapses of hippocampal slices and the facilitatory effect of CGS21680 (10 nM) was blocked by ZM241385 (20 nM) whereas CSC (250 nM) caused a 40% attenuation of this CGS21680‐induced facilitation. 5 These results indicate that ZM241385 is the first A2A antagonist with equal potency to displace [3H]CGS21680 binding to striatal and limbic regions, and with general efficiency to antagonize HENECA‐ or CGS21680‐mediated facilitatory responses in the hippocampus.

Adenosine A2A receptors stimulate acetylcholine release from nerve terminals of the rat hippocampus

The nature of the adenosine receptors involved in the enhancement of acetylcholine release in the hippocampus was studied. The A2A agonist, CGS 21680, increased the veratridine-evoked release of [3H]acetylcholine from hippocampal synaptosomes. This presynaptic effect of CGS 21680 was greater at 3-30 nM than at 100 nM. The excitatory effect of CGS 21680 was antagonised by the A2 antagonist, DMPX (10 microM), and by the A2A antagonist, CSC (200 nM), but not by the A1 antagonist, DPCPX (20 nM). We also found co-expression of A2A and choline acetyltransferase mRNAs in the nucleus of the diagonal band and the medial septum, where the cholinergic cell bodies that project into the hippocampus are located. These results indicate that A2A adenosine receptors are present in cholinergic nerve terminals in the hippocampus and that activation of these receptors enhances acetylcholine release.

Inhibition of [3H]γ-aminobutyric acid release by kainate receptor activation in rat hippocampal synaptosomes

We studied the modulation of gamma-aminobutyric acid (GABA) release by activation of kainate receptor in rat whole hippocampal synaptosomes. Kainate (10-300 microM) inhibited [3H]GABA release in a concentration-dependent manner with an EC50 of 25 microM. This effect of kainate (30 microM) was prevented by the ionotropic non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and by the selective kainate receptor antagonist 5-nitro-6,7,8,9-tetrahydrobenzo(g)indole-2,3-dione-3-oxime (NS-102, 10 microM), but not by the selective non-competitive AMPA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5 H-2,3-benzodiazepine (GYKI 52466, 100 microM). Other kainate receptor agonists, such as domoic acid (0.3-10 microM) and (2S,4R)-4-methylglutamic acid (MGA, 0.3-3 microM), also inhibited [3H]GABA release in a concentration-dependent manner with EC50 values of 4.0 microM and 0.90 microM, respectively, whereas alpha-amino-3-hydroxy-5-methyl-4-oxazolepropionate (AMPA, 10-100 microM) was devoid of effect. These inhibitory effects of both domoic acid (3 microM) and MGA (1 microM) were antagonized by CNQX (10 microM). These results indicate that GABA release can be modulated directly by presynaptic high-affinity kainate heteroreceptors.

2-Chloroadenosine decreases long-term potentiation in the hippocampal CA1 area of the rat.

The effect of the adenosine (ADO) analogue 2-chloroadenosine (CADO) on frequency-induced long-term potentiation (LTP) of the responses evoked by stimulation of the Schaffer fibres and recorded in CA1 area was studied in hippocampal slices of the rat. CADP significantly decreased LTP of the population spikes (PS) (EC50 = 0.28 microM), and LTP of the field excitatory postsynaptic potentials (f.e.p.s.p) (EC50 = 0.33 microM). These effects were reversed by the ADO receptor antagonist 8-phenyltheophylline (8-PT) (2.5 microM). It is concluded that CADO decreases LTP through activation of a xanthine-sensitive ADO receptor.

G protein coupling of CGS 21680 binding sites in the rat hippocampus and cortex is different from that of adenosine A1 and striatal A2A receptors.

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 Ki 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 Ki 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.

Adenosine A2A receptor facilitation of synaptic transmission in the CA1 area of the rat hippocampus requires protein kinase C but not protein kinase A activation

Adenosine is a neuromodulator in the hippocampus acting mainly via inhibitory A(1) receptors but also via facilitatory A(2A) receptors. We now investigated the transducing system operated by hippocampal A(2A) receptors. The selective A(2A) receptor agonist, CGS 21680 (10 nM), facilitated synaptic transmission by 14%, an effect not modified by the phosphodiesterase IV inhibitor, rolipram (30 microM), or by the adenylate cyclase activator, forskolin (3 microM), or by the protein kinase A inhibitor, HA-1004 (10 microM), but nearly abolished by the protein kinase C inhibitors, chelerythrine (6 microM) or bisindolylmaleimide I (1 microM). Inhibition of protein kinase C also prevented the A(2A) receptor-induced attenuation of A(1) receptor-mediated inhibition of hippocampal synaptic transmission. These results indicate that adenosine A(2A) receptor facilitation of hippocampal synaptic transmission involves protein kinase C rather than protein kinase A activation.

1,3-Dipropyl-8-cyclopentylxanthine attenuates the NMDA response to hypoxia in the rat hippocampus

Excitatory amino acids may cause neuronal damage and death in cerebral hypoxia and ischemia, through the activation of different subtypes of glutamate receptors, in particular of the N-methyl-D-aspartate (NMDA) receptor. In the present work, the effect of hypoxia on the component of the field excitatory postsynaptic potential (fepsp) mediated by the NMDA receptor was studied in the hippocampal CA1 area of the rat. A period of 15 min of hypoxia induced virtual abolition of the NMDA receptor-mediated fepsp and a 94.8 +/- 0.7% maximal decrease in the fepsp. A period of 3 min of hypoxia induced a 89.3 +/- 12.3% maximal decrease in the NMDA receptor-mediated component of the fepsp and only a 50.8 +/- 11.5% maximal decrease in the fepsp. Both periods of hypoxia thus induced a more pronounced depression of the NMDA receptor-mediated component of the fepsp than of the fepsp. We found that 48.5 +/- 9.1% decrease (about half of the total decrease) in the NMDA receptor-mediated fepsp, and 51.6 +/- 19.6% decrease (approximately all decrease) in the fepsp induced by hypoxia (3 min) were reversed in the presence of the selective adenosine A1 receptor antagonist, 1,3-dipropyl-8- cyclopentylxanthine (DPCPX) (50 nM), and thus likely to be mediated by endogenous adenosine, through the activation of adenosine A1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)