Ana M. Sebastião

Adenosine receptors in the nervous system: pathophysiological implications

Adenosine is a ubiquitous homeostatic substance released from most cells, including neurones and glia. Once in the extracellular space, adenosine modifies cell functioning by operating G-protein-coupled receptors (GPCR; A(1), A(2A), A(2B), A(3)) that can inhibit (A(1)) or enhance (A(2)) neuronal communication. Interactions between adenosine receptors and other G-protein-coupled receptors, ionotropic receptors and receptors for neurotrophins also occur, and this might contribute to a fine-tuning of neuronal function. Manipulations of adenosine receptors influence sleep and arousal, cognition and memory, neuronal damage and degeneration, as well as neuronal maturation. These actions might have therapeutic implications for neurodegenerative diseases such as Parkinsons disease, Alzheimers disease, as well as for other neurological situations such as epilepsy, idiopathic pain or even drug addition. Peripheral side effects associated with adenosine receptor agonists limit their usefulness in therapeutics; in contrast, adenosine receptor antagonists appear to have less side effects as it is the case of the well-known non-selective antagonists theophylline (present in tea) or caffeine (abundant in coffee and tea), and their emerging beneficial actions in Parkinsons disease and Alzheimers disease are encouraging. A(1) receptor antagonism may also be useful to enhance cognition and facilitate arousal, as well as in the periphery when deficits of neurotransmitter release occur (e.g. myasthenic syndromes). Enhancement of extracellular adenosine levels through drugs that influence its metabolism might prove useful approaches in situations such as neuropathic pain, where enhanced activation of inhibitory adenosine A(1) receptors is beneficial. One might then consider adenosine as a fine-tuning modulator of neuronal activity, which via subtle effects causes harmonic actions on neuronal activity. Whenever this homeostasis is disrupted, pathology may be installed and selective receptor antagonism or agonism required.

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

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.

Adenosine : does it have a neuroprotective role after all?

A neuroprotective role for adenosine is commonly assumed. Recent studies revealed that adenosine may unexpectedly, under certain circumstances, have the opposite effects contributing to neuronal damage and death. The basis for this duality may be the activation of distinct subtypes of adenosine receptors, interactions between these receptors, differential actions on neuronal and glial cells, and various time frames of adenosinergic compounds administration. If these aspects are understood, adenosine should remain an interesting target for therapeutical neuroprotective approaches after all.

Caffeine and Adenosine

Caffeine causes most of its biological effects via antagonizing all types of adenosine receptors (ARs): A1, A2A, A3, and A2B and, as does adenosine, exerts effects on neurons and glial cells of all brain areas. In consequence, caffeine, when acting as an AR antagonist, is doing the opposite of activation of adenosine receptors due to removal of endogenous adenosinergic tonus. Besides AR antagonism, xanthines, including caffeine, have other biological actions: they inhibit phosphodiesterases (PDEs) (e.g., PDE1, PDE4, PDE5), promote calcium release from intracellular stores, and interfere with GABA-A receptors. Caffeine, through antagonism of ARs, affects brain functions such as sleep, cognition, learning, and memory, and modifies brain dysfunctions and diseases: Alzheimers disease, Parkinsons disease, Huntingtons disease, Epilepsy, Pain/Migraine, Depression, Schizophrenia. In conclusion, targeting approaches that involve ARs will enhance the possibilities to correct brain dysfunctions, via the universally consumed substance that is caffeine.

Evidence for high-affinity binding sites for the adenosine A2A receptor agonist [3H] CGS 21680 in the rat hippocampus and cerebral cortex that are different from striatal A2A receptors

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 Kd of 58 nM and a Bmax of 353 fmol/mg protein in the hippocampus, and with a Kd of 58 nM and a Bmax of 264 fmol/mg protein in the cortex; in the striatum, the single high-affinity [3H] CGS 21680 binding site had a Kd of 17 nM and a Bmax 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), N6-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 Kd of 20 nM and a Bmax of 56 fmol/mg protein in the hippocampus and with a Kd of 22 nM and a Bmax 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 Kd of 97 nM and a Bmax of 255 fmol/mg protein in the hippocampus and with a Kd of 112 nM and a Bmax 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.

Inhibition of NMDA receptor-mediated currents in isolated rat hippocampal neurones by adenosine A1 receptor activation.

&NA; The effect of the stable adenosine analogue, 2‐chloroadenosine (CADO), on the currents elicited by iontophoretic application of N‐methyl‐d‐aspartate (NMDA) to pyramidal cells acutely dissociated from the CA1 area of the rat hippocampus was studied using the patch‐clamp technique in the whole‐cell configuration. CADO (3‐300 nM) reversibly inhibited NMDA receptor‐mediated currents (maximal effect: 54.2 ± 6.6% decrease, EC50 = 10.3 nM). This effect was prevented by the adenosine A1 receptor antagonist, 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX)(50 nM). CADO (100 nM) inhibited the conductance induced by iontophoretic application of NMDA, without changing its reversal potential, in both the absence and the presence of Mg2+ (30 &mgr;M). Adenosine may contribute to the regulation of the NMDA receptor function, particularly under conditions, like hypoxia and ischaemia, leading to excessive NMDA receptor activation.

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.

Activation of Adenosine A2A Receptor Facilitates Brain-Derived Neurotrophic Factor Modulation of Synaptic Transmission in Hippocampal Slices

Both brain-derived neurotrophic factor (BDNF) and adenosine influence neuronal plasticity. We now investigated how adenosine influences the action of BDNF on synaptic transmission in the CA1 area of the rat hippocampal slices. Alone, BDNF (20–100 ng/ml) did not significantly affect field EPSPs (fEPSPs). However, a 2 min pulse of high-K+ (10 mm) 46 min before the application of BDNF (20 ng/ml) triggered an excitatory action, an effect blocked by the TrkB receptor inhibitor K252a (200 nm), by the adenosine A2A receptor antagonist ZM 241385 (50 nm), and by the protein kinase A inhibitor H-89 (1 μm). Presynaptic, rather than postsynaptic depolarization was required to trigger the BDNF action because after K+ depolarization BDNF also increased EPSCs recorded from pyramidal neurons voltage-clamped at –60 mV, and transient postsynaptic depolarization was unable to unmask the BDNF action. A weak theta burst stimulation of the afferents could elicit potentiation of synaptic transmission only when applied in the presence of BDNF. Activation of adenosine A2A receptors with CGS 21680 (10 nm), or the increase in extracellular adenosine levels induced by 5-iodotubercidin (100 nm) triggered the excitatory action of BDNF, a process prevented by ZM 241385 and by H-89. In the presence of dibutyryl-cAMP (0.5 mm), BDNF also increased fEPSPs but postsynaptic cAMP (0.5 mm) was unable to trigger the BDNF action. It is concluded that presynaptic activity-dependent release of adenosine, through activation of A2A receptors, facilitates BDNF modulation of synaptic transmission at hippocampal synapses.