J.A. Ribeiro

Adenosine A2 receptor-mediated excitatory actions on the nervous system.

The distribution, molecular structure and role of adenosine A2 receptors in the nervous system, is reviewed. The adenosine A2a receptor subtype, identified in the nervous system with ligand binding, functional studies or genetic molecular techniques, has been demonstrated in the striatum and other basal ganglia structures, in the hippocampus, in the cerebral cortex, in the nucleus tractus solitarius, in motor nerve terminals, in noradrenergic terminals in the vas deferens, in myenteric neurones of the ileum, in the retina and in the carotid body. The A2b receptors have been identified in glial and neuronal cells, and may have a widespread distribution in the brain. Activation of adenosine A2a receptors can enhance the release of several neurotransmitters, such as acetylcholine, glutamate, and noradrenaline. The release of GABA might be either enhanced or inhibited by A2a receptor activation. The A2 receptor activation also modulates neuronal excitability, synaptic plasticity, as well as locomotor activity and behaviour. The ability of A2 receptors to interact with other receptors for neurotransmitters/neuromodulators, such as dopamine D2 and D1 receptors, adenosine A1 receptors, CGRP receptors, metabotropic glutamate receptors and nicotinic autofacilitatory receptors, expands the range of possibilities used by adenosine to interfere with neuronal function and communication. These A2 receptor-mediated adenosine actions might have potential therapeutic interest, in particular in movement disorders such as Parkinsons disease and Huntingtons chorea, as well as in schizophrenia, Alzheimers disease, myasthenia gravis and myasthenic syndromes.

Adenosine receptors and calcium: Basis for proposing a third (A3) adenosine receptor

Abbreviations

Inhibitory and excitatory effects of adenosine receptor agonists on evoked transmitter release from phrenic nerve endings of the rat

1 The effects of the adenosine analogues, 5′‐N‐ethyl‐carboxamide adenosine (NECA), R‐N6‐phenylisopropyladenosine (R‐PIA), 2‐chloroadenosine (CADO), and CGS 21680C on electrically evoked tritium outflow from preparations loaded with [3H]‐choline and on evoked endplate potentials (e.p.ps), as well as the ability of the xanthines, 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX) and PD 115,199 to antagonize the effects of the adenosine analogues, were investigated in phrenic nerve‐diaphragm preparations. 2 NECA, R‐PIA and CADO decreased, in a concentration‐dependent manner, the evoked tritium outflow from preparations loaded with [3H]‐choline. NECA and R‐PIA were about equipotent and more potent than CADO. 3 DPCPX shifted to the right in a near parallel fashion the concentration‐response curve for the inhibitory effect of R‐PIA on evoked tritium outflow. 4 In the presence of DPCPX, NECA increased, rather than decreased, evoked tritium outflow. PD 115,199 antagonized, in a concentration‐dependent manner, this excitatory effect of NECA. 5 CGS 21680C, in low nanomolar concentrations, increased evoked tritium outflow, an effect also antagonized by PD 115,199. 6 CGS 21680C increased, and R‐PIA decreased, the amplitude of e.p.ps recorded from preparations paralysed with tubocurarine. Both effects could be observed in the same endplate. 7 It is concluded that both inhibitory (probably A1) and excitatory (probably A2) adenosine receptors coexist at the rat neuromuscular junction, modulating the evoked release of acetylcholine.

Evidence for the presence of excitatory A2 adenosine receptors in the rat hippocampus

The A2 adenosine receptor agonist, CGS 21680 in nanomolar concentrations, reversibly increased in a concentration-dependent manner the amplitude of orthodromically-evoked population spikes recorded from the CA1 pyramidal cell layer of rat hippocampal slices. The adenosine receptor antagonist, 3,7-dimethyl-l-propargylxanthine (DMPX, 10 microM), which has selectivity for A2 adenosine receptors, prevented this excitatory effect of CGS 21680. These results suggest that A2 adenosine receptors are present in the rat hippocampus and that activation of these receptors enhance hippocampal excitability.

The inhibitory adenosine receptor at the neuromuscular junction and hippocampus of the rat: antagonism by 1,3,8-substituted xanthines.

1 The ability of 1,3,8‐substituted xanthines to antagonize the inhibitory effects of adenosine receptor agonists on the amplitude of nerve‐evoked twitches of the rat phrenic‐diaphragm and on the amplitude of orthodromically‐evoked population spikes, recorded from the CA1 pyramidal cells of rat hippocampal slices, was investigated. 2 1,3‐Dipropyl‐8‐cyclopenthylxanthine (DPCPX), 1,3‐dipropyl‐8‐(carboxymethyloxyphenyl)xanthine (XCC), 1,3‐dipropyl‐8‐(4‐((2‐aminoethyl)amino)carbonylmethyloxyphenyl)xanthine (XAC), 1,3‐dipropyl‐8‐(2‐amino‐4‐chlorophenyl)xanthine (PACPX), 8‐phenyltheophylline (8‐PT), 1,3‐diethyl‐8‐phenylxanthine (DPX) and PD 115,199, in concentrations virtually devoid of effect on neuromuscular transmission, shifted to the right in a near parallel manner the log concentration‐response curve for the inhibitory effect of 2‐chloroadenosine (CADO) on nerve‐evoked twitches of the phrenic‐diaphragm. Linear Schild plots with slopes near to unity were obtained for all the xanthines. 3 The order of potency of the xanthines as antagonists of the effect of CADO in the phrenic‐diaphragm was DPCPX (Ki = 0.54 nm) > XCC (Ki = 10 nm), XAC (Ki = 11 nm), PACPX (Ki = 13 nm) > DPX (Ki = 22 nm), 8‐PT (Ki = 25 nm) > PD 115,199 (Ki = 57 nm). The potency of DPCPX in antagonizing the inhibitory effects of R‐N6‐phenylisopropyladenosine (R‐PIA) and 5′‐N‐ethylcarboxamide adenosine (NECA) on nerve‐evoked twitch responses was not statistically different from its potency in antagonizing the inhibitory effect on CADO. 4 In the hippocampal slices, DPCPX, XCC and XAC, used in concentrations virtually devoid of effect on population spike amplitude, shifted to the right in a near parallel manner the log concentration‐response curve for the inhibitory effect of CADO on the amplitude of the population spikes. The Schild plots were linear with slopes near unity. 5 The potencies of DPCPX (Ki = 0.45 nm) and XAC (Ki = 11 nm) in antagonizing the inhibitory adenosine receptor at the hippocampus were similar to their potencies for antagonism of the inhibitory adenosine receptor at the phrenic‐diaphragm. XCC was only slightly more potent (Ki = 5.4 nm) as an antagonist of the adenosine receptor in the hippocampus than in the phrenic‐diaphragm. 6 The results suggest that the inhibitory adenosine receptors in the phrenic‐diaphragm and in the hippocampus of the rat are similar, and that according to the antagonist potencies, these receptors belong to the A1‐adenosine receptor subtype.

Ecto‐5′‐Nucleotidase Is Associated with Cholinergic Nerve Terminals in the Hippocampus but Not in the Cerebral Cortex of the Rat

Abstract: The extracellular catabolism of exogenously added AMP was studied in immunopurified cholinergic nerve terminals and in slices of the hippocampus and cerebral cortex of the rat. AMP (10 μM) was catabolized into adenosine and inosine in hippocampal cholinergic nerve terminals and in hippocampal slices, as well as in cortical slices. IMP formation from extracellular AMP was not detected. α,β‐Methylene ADP (100 μM) inhibited almost completely the extracellular catabolism of AMP in these preparations. The relative rate of catabolism of AMP was greater in hippocampal slices than in cortical slices. AMP was virtually not catabolized when added to immunopurified cortical cholinergic nerve terminals, although ATP could be catabolized extracellularly under identical conditions. The comparison of the relative rates of catabolism of exogenously added AMP, calculated from the amount of AMP catabolized after 5 min, in hippocampal cholinergic nerve terminals and in hippocampal slices revealed a nearly 50‐fold enrichment in the specific activity of ecto‐5′‐nucleotidase upon immunopurification of the cholinergic nerve terminals from the hippocampus. The results suggest that there is a regional variation in the subcellular distribution of ecto‐5′‐nucleotidase activity in the rat brain, the ecto‐5′‐nucleotidase in the hippocampus being closely associated with the cholinergic nerve terminals, whereas in the cerebral cortex ecto‐5′‐nucleotidase activity seems to be located preferentially outside the cholinergic nerve terminals.

Purinergic modulation of the evoked release of [3H]acetylcholine from the hippocampus and cerebral cortex of the rat: role of the ectonucleotidases.

Modulation by exogenous and endogenous adenine nucleotides and adenosine of [3H]acetylcholine release evoked by veratridine (10 μM) was compared in synaptosomal fractions from the hippocampus and the cerebral cortex of the rat. In both brain areas, exogenously added ATP or adenosine (10–100 μM) inhibited the evoked tritium release. In the hippocampus, ATPμS, an ATP analogue more resistant to catabolism than ATP, was virtually devoid of effect on tritium release, and the effect of ATP was prevented by the ecto‐5′‐nucleotidase inhibitor α,β‐methylene ADP (100 μM), by adenosine deaminase (2 U/ml) and by the A1 adenosine receptor antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX, 20 nM). In contrast, in the cerebral cortex, the effect of ATP on tritium release was not prevented by either α,β‐methylene ADP (100 μM) or adenosine deaminase (2 U/ml), and several ATP analogues (30 μM) inhibited release. The order of intensity of the inhibitory effects of the ATP analogues was: ATPγS > ATP > ñ,γ‐imido ATP > β,γ‐methylene ATP > 2‐methyl‐S‐ATP, α,β‐methylene ATP. The effect of ATPγS in the cerebral cortex was prevented by DPCPX (20 nM) and was not affected by the P2 purinoceptor antagonist suramin (100 μM). In the hippocampus, α,β‐methylene ADP (100 μM) increased the evoked release of tritium, and adenosine deaminase (2 U/ml) produced an even greater increase; when adenosine deaminase was added in the presence of α,β‐methylene ADP, adenosine deaminase still increased the evoked release of tritium. In the cerebral cortex, DPCPX (20 nM) and adenosine deaminase (2 U/ml) increased the evoked tritium release by a similar magnitude, but the effect of adenosine deaminase was smaller than in the hippocampus. It is concluded that in the cerebral cortex ATP as such presynaptically inhibits acetylcholine release, whereas in the hippocampus the role of adenine nucleotides is as a source of endogenous extracellular adenosine that tonically inhibits acetylcholine release. The results also show that besides formation of adenosine from adenine nucleotides, released adenosine as such contributes in nearly equal amounts to the pool of endogenous adenosine that presynaptically inhibits acetylcholine release in the hippocampus.

Triggering of BDNF facilitatory action on neuromuscular transmission by adenosine A2A receptors

Motor nerve terminals possess adenosine A(2A) receptors and brain derived neurotrophic factor (BDNF) TrkB receptors. In the present work we evaluated how BDNF actions on neuromuscular transmission would be influenced by adenosine A(2A) receptors activation. BDNF (20-100 ng/ml) on its own was devoid of effect on evoked endplate potentials (EPPs) recorded intracellularly from rat innervated diaphragms paralysed with tubocurarine. However, when BDNF was applied 45 min after a brief (2 min) depolarizing KCl (10 mM) pulse or when the adenosine A(2A) receptors were activated with CGS 21680 (10 nM), BDNF (20 ng/ml) increased EPPs amplitude without influencing the resting membrane potential of the muscle fibre. The action of BDNF was prevented by the adenosine A(2A) receptor antagonist, ZM 241385 (50 nM) as well as by the TrkB receptor phosphorylation inhibitor, K252a (200 nM). The PKA inhibitor, H-89 (1 microM), prevented the excitatory effect of CGS 21680 (10 nM) on EPPs as well as prevented its ability to trigger a BDNF effect. The PLCgamma inhibitor, U73122 (5 microM), did not prevent the excitatory action of CGS 21680 (10 nM) on neuromuscular transmission, but abolished the action of BDNF in the presence of the A(2A) receptor agonist. The results suggest the following sequence of events in what concerns cooperativity between A(2A) receptors and TrkB receptors at the neuromuscular junction: A(2A) receptor activates the PKA pathway, which promotes the action of BDNF through TrkB receptors coupled to PLCgamma, leading to enhancement of neuromuscular transmission.

On the adenosine receptor and adenosine inactivation at the rat diaphragm neuromuscular junction

1 The effects of adenosine and adenosine analogues 2‐chloroadenosine (CADO), l‐N6‐phenylisopropyladenosine (l‐PIA), d‐N6‐phenylisopropyladenosine (d‐PIA), N6‐cyclohexyladenosine (CHA) and 5′‐N‐ethylcarboxamide adenosine (NECA) on evoked endplate potentials (e.p.ps) and on twitch tension were investigated in innervated diaphragms of the rat. 2 Adenosine and its analogues decreased, in a concentration‐dependent manner, the amplitude of both the e.p.ps and the twitch responses evoked by nerve stimulation. The order of potency in decreasing the twitch tension was CHA, l‐PIA, NECA > d‐PIA > CADO > adenosine. l‐PIA was about 8 times more potent than d‐PIA. Neither adenosine nor the adenosine analogues affected the twitch responses of directly stimulated tubocurarine‐paralysed muscles. 3 8‐Phenyltheophylline (8‐PT), theophylline and isobutylmethylxanthine (IBMX), in concentrations virtually devoid of effect on neuromuscular transmission, antagonized the inhibitory effect of 2‐chloroadenosine. The order of potency of the alkylxanthines as antagonists of the adenosine receptor at the rat diaphragm neuromuscular junction was 8‐PT > IBMX > theophylline. The antagonism by these xanthines was shown to be competitive, the pA2 value for 8‐PT being 7.16. In concentrations slightly higher than those used to test its ability to antagonize the adenosine receptor, IBMX and 8‐PT increased the amplitude of e.p.ps without modifying their decay phase or the resting membrane potential of the muscle fibre. 4 The adenosine uptake inhibitor, nitrobenzylthioinosine (NBI) and the adenosine deaminase inhibitor, erythro‐9(2‐hydroxy‐3‐nonyl)adenine (EHNA), in concentrations virtually devoid of effect on neuromuscular transmission, potentiated the inhibitory effect of adenosine at the rat diaphragm neuromuscular junction. The potentiation factors were about 2.6 for NBI (5 μm), 2.2 for EHNA (25 μm) and 4.6 for the combination of NBI (5 μm) and EHNA (25 μm). 5 It is concluded that both uptake and deamination contribute to the inactivation of adenosine at the rat diaphragm neuromuscular junction and that in this preparation the inhibitory effect of adenosine on transmission is mediated by a xanthine‐sensitive adenosine receptor with an agonist profile which does not fit the criteria for its classification either as an A1 or A2‐adenosine receptor.

Evidence that the presynaptic A2a-adenosine receptor of the rat motor nerve endings is positively coupled to adenylate cyclase

The action of the A2a-adenosine analogue, CGS 21680C, on electrically evoked [3H]acetylcholine ([3H]-ACh) release, and its interaction with forskolin (an activator of adenylate cyclase), MDL 12,330A (an irreversible inhibitor of adenylate cyclase), rolipram (an inhibitor of cyclic AMP specific phosphodiesterase), dibutyryl- (db-cAMP) and 8-bromo- (8-Br-cAMP) cyclic AMP analogues (substances that mimic intracellular actions of cyclic AMP), were investigated using rat phrenic nerve-hemidiaphragm preparations.CGS 21680C facilitated [3H]ACh release. Forskolin (but not 1,9-dideoxy forskolin), rolipram, db-cAMP and 8-Br-cAMP also increased evoked neurotransmitter release in a concentration-dependent manner. When the evoked [3H]-ACh release that is dependent on stimulation of the adenylate cyclase/cyclic AMP transduction system was supramaximally stimulated by these compounds, CGS 21680 C (3 μmol/l) could not further increase [3H]-ACh release. Phosphodiesterase inhibition with low concentrations (⩽ 30 μmol/l) of rolipram significantly potentiated the augmenting effect of CGS 21680C (1 μmol/l) on evoked [3H]ACh release. MDL 12,330A (an irreversible inhibitor of adenylate cyclase) decreased evoked [3H]-ACh release. The irreversible blocking action of MDL 12,330A on [3H]-ACh release was overcome by by-passing cyclase activation with db-cAMP and 8-Br-cAMP, but could not be overcome with FSK or CGS 21680 C. The inhibitory effect of MDL 12,330A on evoked [3H]-ACh release was not mimicked by nifedipine.It is concluded that the increase in [3H]-ACh release caused by CGS 21680C results from activation of an A2a-adenosine receptor positively linked to the adenylate cyclase/cyclic AMP system.