Kainic acid

Protection against neuronal damage is a major objective of current research in areas such as stroke medicine, Alzheimer's disease and other neurodegenerative conditions. Adenosine receptors are important modulators of cell survival, and thus agents targeting these receptors could be valuable therapeutic agents. Agonists at A^sub 1^ receptors and antagonists at A^sub 2A^ receptors are known to protect acutely against neuronal damage caused by toxins or ischemia-reperfusion, and these compounds can also protect against the cell damage inflicted by reactive oxygen species. Even endogenous adenosine may be neuroprotective, since its levels rise substantially in association with a period of ischemia-reperfusion. Unfortunately, there is growing evidence that the efficacy of adenosine receptor activation can be reduced by the concomitant activation of glutamate receptors responding to N-methyl-D-aspartate (NMDA), probably acting via the release of nitric oxide. Such problems will need to be resolved before adenosine receptor agonists can proceed far as neuroprotective agents. The use of receptor antagonists may prove a more valuable approach. [Neurol Res 2005; 27: 161-1681

Keywords: Glutamate; NMDA; ischemia; neurodegeneration; neuroprotection

INTRODUCTION

Prevention of the brain damage caused by stroke, mechanical trauma or neurodegenerative diseases, such as Alzheimer's and Parkinson's disease has become a major focus of neurological research. While there is a range of hypotheses to explain the mechanisms linking a cerebrovascular accident or genetic mutations to the production of cellular injury in the brain, a central role is often postulated for the excitatory transmitter glutamate. For example, one of the initial consequences of a cerebral thrombosis or haemorrhage is the efflux of massive quantities of glutamate from neurons and glial cells. Glutamate then activates ionotropic receptors including those sensitive to N-methyl-D-aspartate (NMDA) leading to a rise of intracellular calcium levels and the activation of pro-oxidant and proteolytic enzymes causing cell damage1.

Adenosine has been considered for almost 20 years as a possible neuroprotectant against such cell damage, since it was first shown by Phillis and his colleagues to depress neuronal excitability2 partly by the direct hyperpolarisation of neurons and partly by the inhibition of glutamate release from excitatory neurons, and it suppresses the rise of intracellular calcium levels, which trigger neuronal damage. Indeed, adenosine may be an important endogenous neuroprotectant. During and following a hypoxic or ischemic episode, the extracellular concentration of adenosine may rise from the normal baseline levels of around 1 μM, up to 100 μM or more3-5. The importance of adenosine as a neuroprotectant is strongly suggested by reports that 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and other A1 receptor blockers increase the neuronal damage resulting from ischemia or excitotoxins5.

In terms of therapeutic relevance, interest in adenosine and the roles of its several receptors-A^sub 1^, A^sub 2A^, A^sub 2B^ and A^sub 3^-is heightened by the possibility that not only might those receptors be potential targets for the development of novel synthetic compounds acting on them, but also that adenosine itself might be an important endogenous neuroprotectant. The latter concept in turn raises the possibility of modifying adenosine's effectiveness as a protectant, the pathophysiological activity of other endogenous molecules able to modify its effectiveness, or increasing the effectiveness of adenosine directly by raising its endogenous concentration or by amplifying its activation of receptors.

ADENOSINE AND NEUROPROTECTION

A1 receptors and neuroprotection

Following an early report by von Lubitz et al.6 that the A1 receptor agonist N6-cyclohexyladenosine (CHA) protected neurones against damage following an ischemic episode in gerbils, several other A1 receptor agonists were shown to be effective7-11. We and others demonstrated that the A1 receptor agonist R-phenylisopropyladenosine (PIA) was also protective against the excitotoxic effects of glutamate receptor agonists such as quinolinic acid and kainic acid12-16. Protection could be prevented by the A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). R-PIA fully protected against kainate-induced damage in parts of the hippocampus, the basolateral amygdaloid nuclei, the pyriform cortex and rhinal fissure14.

The A1 receptor agonist N6-cyclohexyladenosine (CHA) also protects against damage induced by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)17. This may suggest that an A1 receptor agonist may be of value in the treatment of Parkinson's disease. The mechanism of protection against MPTP, however, remains unclear, although an interference with oxidative stress and mitochondrial oxidation systems may be involved. Alternatively, since antagonists at the NMDA receptors are also able to reduce toxicity caused by MPTP, it is possible that the same mechanisms are operating for A1 receptor protection against MPTP and against kainate or NMDA receptor activation, including the suppression of glutamate release, and the direct inhibition of NMDA receptor activation18,19.

Mechanisms of A1 receptor neuroprotection

As noted above, while the mechanisms of A1 receptor neuroprotection are not known with certainty, it is likely that they include suppression of the release of a variety of neurotransmitters, including glutamate20-21 acetylcholine22 and dopamine (Figure 1)23. On the other hand, the extracellular concentrations of glutamate do not always correlate with the degree of neuronal damage in the hippocampus24. The systemic administration of A1 receptor agonists was also reported not to suppress the elevation of glutamate levels caused by ischemia11,25,26, although cyclopentyladenosine (CPA) decreased glutamate release in a rat four-vessel occlusion model27.

A1 receptors can also reduce calcium influx in neuronal and cardiac tissues8,28,29, possibly secondary to the modulation of potassium conductances including the ATP-sensitive potassium channels in heart and hippocampal neurons30,31. Since it is believed that the rise of intracellular calcium levels represents the final common path of several injurious stimuli and that it is the attainment of a critical calcium concentration that is responsible for triggering the pro-oxidant and protease systems that ultimately cause neuronal death1, the lowering of intracellular calcium would be a key response to the presence of adenosine or its mimetic agonists.

A^sub 2A^ RECEPTORS

The highest density of A^sub 2A^ receptors is in the striatum, mainly located on GABA-releasing striopallidal projection neurons and cholinergic neurons32-34. There is also a major presence of these receptors in the nucleus accumbens, and olfactory tubercle with lesser amounts in the hippocampus and cerebral cortex34. In human brain, A^sub 2A^ receptors were reported to be confined largely to striatal regions35, but later work has claimed their presence more widely36.

A2 receptor agonists and neuroprotection

Agonists acting at A^sub 2A^ receptors can protect against ischemia37,38 and excitotoxins such as kainate39,40. The protection was not prevented by the A^sub 1^ receptor antagonist DPCPX, but was largely prevented by 8-(p-sulphophenyl)-theophylline (8PST), a non-selective A1 and A2 adenosine receptor antagonist, which does not penetrate the blood-brain barrier. This demonstration suggests that protection by A2A agonists involves a peripheral site of action. Consistent with this hypothesis, the direct injection of CGS 21680 into the hippocampus failed to afford protection.

A2 receptor antagonists and neuroprotection

One of the most surprising discoveries relating to adenosine receptors and neuroprotection has been the observation of neuroprotection by an A^sub 2A^ receptor antagonist, as reported by Gao & Phillis41. This group found that the relatively non-selective A2 receptor antagonist 5-amino-9-chloro-2-(2-furyl)-1,2,4-triazolo[1,5-c]quinazoline (CGS 15943) could protect against cerebral ischemia in a gerbil model. This result was subsequently confirmed using other compounds and ischemic models42,43. We extended this work to show protection against excitotoxins using the kainate and quinolinic acid models of excitotoxicity39,40. The A^sub 2A^ receptor antagonist 4-{2-[7-amino-2-(2-furyl) (1,2,4)-triazolo(2,3-a)-(1,3,5)triazin-5-yl-amino]ethyl} phenol (ZM 241385), which is 80-fold selective for A^sub 2A^ versus A^sub 2B^ receptors, and 500-1000-fold selective for A^sub 2A^ versus A^sub 1^ receptors, can protect the hippocampus against damage produced by kainate. A combination of the agonist CGS21680 and ZM241385 produced an additive effect with complete preservation of all hippocampal neurones39,40, rather than the anticipated antagonistic interaction. The mutual potentiation of these ligands may indicate that they protection by different mechanisms.

The use of knockout models present a valuable means of confirming that the effects of compounds, such as A^sub 2A^ receptor antagonists is indeed due to the blockade of receptors, rather than to an unrecognised or non-specific action of the compounds. Thus, Chen er al.44 have demonstrated that animals lacking A^sub 2A^ receptors are resistant to neuronal damage following ischemia. Blockade of A^sub 2A^ receptors can also protect against dopaminergic neurotoxins such as MPTP, and Ongini et al.45,46 have demonstrated a reduced neurotoxic effect of MPTP in mice lacking A^sub 2A^ receptors.

Mechanisms of A^sub 2A^ receptor antagonist protection

The A^sub 2A^ receptor agonist CGS 21680 has been reported to increase the efflux of glutamate in response to ischemia in the rat27,47, consistent with evidence for an excitatory action of A^sub 2A^ receptor activation on transmitter release. The A^sub 2A^ receptor antagonist CGS 15943 can depress glutamate release, possibly contributing to its neuroprotective effects by blocking the effects of endogenous adenosine at A^sub 2A^ receptors27.

Protection in neurodegenerative disorders

Although much of the interest in the therapeutic value of purine receptor ligands has centered on protection following strokes, there remains the possibility that over-activation of glutamate receptors may contribute to neurodegenerative disorders such as Alzheimer's disease and Huntington's disease. This possibility is the rationale for studying the protective effects of agents against excitotoxins. It has already been noted that A1 agonists and A^sub 2A^ receptor agonists and antagonists can protect against kainic acid induced damage12,39,40. One of the excitotoxins of greatest relevance, however, is quinolinic acid, a tryptophan metabolite for which the evidence for a role in some degenerative disorders is substantial48,49. Reggio et al.50 have reported that an A2 receptor antagonist, DMPX, can protect against neuronal loss induced by quinolinic acid injected into the striatum-a frequently used model of Huntington's disease.

Interference with reactive oxygen species

Quinolinic acid is an endogenous metabolite of tryptophan and is a selective agonist at N-methyl-D-aspartate (NMDA) receptors48,49. Its neurotoxic effects can be potentiated by the presence of free radicals generated by a mixture of xanthine and xanthine oxidase, a well-recognised superoxide and hydroxylradical generating system. Interestingly, toxicity produced by the quinolinate/free radical combination could be suppressed by three different A^sub 2A^ receptor antagonists-CSC, ZM 241385 and SCH 58261 (Figure 2)51. This result emphasises that A^sub 2A^ receptor antagonists may have a potentially wide use as neuroprotectants against cell injury in many situations where damage is produced by either glutamate receptor activation or oxygen radical generation, or their combination. It is presumably the latter that accounts for neuroprotection during ischemia, when glutamate and free radical levels are known to rise. The generation of endogenous quinolinic acid is increased in glia and macrophages in response to inflammation or immune activation48,49, and in parallel with increased levels of nitric oxide and superoxide. The mutual potentiation between these agents could therefore account for a significant fraction of the overall cell damage, which occurs with neurodegeneration and its associated inflammation (Figure 4). Since immune-activated macrophages can pass across the blood-brain barrier, part of the neuroprotective activity of A^sub 2A^ adenosine receptors may well involve the suppression of free radical generation by white cells and microglia activated by immune stimulation52,53.

RECEPTOR INTERACTIONS

Biochemical54 and electrophysiological work55 has supported the view that interactions occur between adenosine receptors. For example, A^sub 2A^ receptors can suppress the activity of A1 receptors. The A^sub 2A^ receptor agonist CGS 21680 reduces neuronal sensitivity to CPA55 and induces a low-affinity receptor site for 2-chloro-N6-cyclopentyladenosine (CCPA)54, possibly via protein kinase C. If this interaction operates in vivo, then the presence of an A^sub 2A^ receptor antagonist should release or 'unmask' A1 receptors, and allow the neuroprotective effect of these receptors to be seen. This was the conclusion drawn to explain the ability of an A1 receptor antagonist to reduce the neuroprotective effects of A^sub 2A^ antagonism39,40. Electrophysiological extracellular and intracellular studies have confirmed the inhibitory effect of A^sub 2A^ receptors on A1 receptor activation55.

In parallel with the direct studies of neuronal damage, we have developed an interest in endogenous molecules, which might modify the potential of endogenous adenosine to act as a neuroprotectant. As noted in the introduction, there is increasing evidence for interactions between adenosine and N-methyl-D-aspartate (NMDA) receptors. Our first observations in this area were that the inhibitory effect of adenosine on hippocampal population spikes and the decrease of paired-pulse inhibition were suppressed in magnesium-free medium, or by superfusing the slices with NMDA56. Agonists at the other glutamate-activated ionotropic receptors (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid [AMPA] and kainite) did not reduce responses to adenosine and NMDA did not modify responses to the GABAB receptor agonist baclofen. Even the physiological activation of NMDA receptors associated with long-term potentiation reduced significantly the effects of adenosine. Consistent with these data was the weaker ability of adenosine receptor activation to suppress neuronal firing induced by microiontophoretically applied NMDA compared with firing induced by acetylcholine or quisqualate57. The interaction between NMDA and adenosine appeared to involve the enhancement of responses mediated by A^sub 2A^ receptors, since NMDA did not modify the inhibitory effect of the selective adenosine A^sub 1^ receptor agonist N6-cyclopentyladenosine, but did enhance the excitatory effect of the adenosine A2A receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Furthermore, the response to a mixture of NMDA and CGS21680 was prevented by the adenosine A^sub 2A^ receptor selective antagonist 4-{2-[7-amino-2-(2-furyl)(1,2,4)triazolo-(2,3a)(1,3,5)triazin-5-ylamino]ethyl}phenol (ZM241385). This study indicated that the activation of NMDA receptor can suppress neuronal sensitivity to adenosine, probably via an increase in the action of adenosine A2A receptors58. However, as discussed above, the activation of A^sub 2A^ receptors can, in turn, suppress the activation of A1 receptors and this sequence may account for the apparent inhibitory effect of NMDA on A1 adenosine receptors.

We have recently returned to this problem in view of the increasing evidence that many effects of NMDA are mediated by the production of nitric oxide (NO). We have therefore examined the possibility that NO might mediate the suppressant effects of NMDA receptors on response to adenosine59. Field excitatory post-synaptic potentials (fEPSPs) were recorded extracellularly from the CA1 region of rat hippocampal slices and paired-pulse interactions were studied to localise the observed interactions to the pre-synaptic terminals. A NO donor, S-nitroso-N-acetylpenicillamine (SNAP) was applied to generate locally high concentrations of NO. SNAP reduced the pre-synaptic inhibitory effect of adenosine selectively, with no effect on responses to baclofen. The involvement of A1 receptors was indicated by the fact that responses to the selective agonist N^sup 6^-cyclopentyladenosine were also depressed by NO (Figure 1).

In order to determine whether this action of NO was mediated by the molecule itself or its free radical properties, we also tested the superoxide generating system of xanthine/xanthine oxidase (X/XO) (Figure 3). This combination also inhibited responses to adenosine and the inhibition was prevented by superoxide dismutase. The effects of superoxide were not affected by NO synthase inhibition. Interestingly, both SNAP and the X/XO produced a long-term potentiation of the fEPSP slope. When LTP was induced directly (by electrical high-frequency stimulation), it also reduced the responses to adenosine, but this effect was prevented by inhibiting NO synthesis using L-nitroarginine methyl ester, indicating the mediation of this effect by local NO generation. The testing of paired-pulse interactions indicated that the inhibition of the inhibitory effects of adenosine by NO and X/XO occurred pre-synaptically.

Since NO is believed to exert many of its actions through the activation of guanylate cyclase, we also examined the effects of the cyclase inhibitor (ODQ). This compound was able to prevent the inhibition of adenosine responses by both SNAP and X/XO but, surprisingly, it could not prevent the inhibition of adenosine by NMDA itself59.

Overall, this study showed that the pre-synaptic effects of adenosine can be inhibited by NO or superoxide, but that neither of these can fully account for the reduction of adenosine responses by NMDA. In contrast, electrically-induced LTP reduces sensitivity to adenosine via the generation of NO59. The pharmacological importance of these results for neurodegeneration, however, lies in the fact that NO or other reactive oxygen species generated during reperfusion injury in the heart of brain may compromise the potential protective actions of endogenous adenosine. Any approach to treatment of degeneration using adenosine receptor ligands should therefore be accompanied by measures to suppress free radical generation. These two approaches together would be expected to have a substantially synergistic neuroprotective activity.

THERAPEUTIC IMPLICATIONS

These various findings have aroused great interest in the search for new drugs, which could be used to slow or prevent the neuronal damage, which characterises neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. That interest is attributable not only to the efficacy of the compounds available, but also to the fact that they should be relatively free of major side effects. Whereas the use of A1 receptor agonists would lead to a suppression of transmitter release at many sites within the central and peripheral nervous system, A^sub 2A^ receptor antagonists should not be limited in this way. The A^sub 2A^ receptors are relatively low affinity sites for adenosine, and the levels of adenosine present normally in most biological fluids are probably insufficient to activate them. Activation would only be expected under conditions in which there is a large increase in the generation and release of adenosine under pathological conditions.

In practice, this means that A^sub 2A^ antagonists have little effect on heart rate, blood pressure or other vital signs under normal conditions. During ischemia in the brain, however, the levels of adenosine may rise to levels at which A^sub 2A^ receptors are activated. Stimulation of A^sub 2A^ receptors is known to increase the release of the excitotoxic amino acid glutamate, which would tend to cause or facilitate the occurrence of damage. Under these circumstances, A^sub 2A^ receptor antagonists should reduce the enhanced release of glutamate and thus decrease the extent of neuronal damage. Their beneficial activity would therefore be restricted to those areas of brain experiencing ischemia, with little or no effect on other areas of the body.

A particularly exciting aspect of A^sub 2A^ receptor protection is that, following an early brief report of symptomatic relief by theophylline60, receptor blockade may contribute to the long-term benefits of treating patients with Parkinson's disease with A^sub 2A^ receptor antagonists. It is clear that A^sub 2A^ receptors potently modulate cell sensitivity to dopamine receptors, accounting for the beneficial effects of adenosine antagonists in this disease60-62. This phenomenon has led to clinical trials with A^sub 2A^ receptor antagonists in Parkinson's disease with promising, though as yet unpublished, results. Certainly, in animal models (including primates), A^sub 2A^ antagonists are effective against toxin-induced models of the disorder17,63,64. If A^sub 2A^ receptor antagonists can not only treat the symptoms of Parkinson's disease, but also slow the underlying neurodegeneration and thus disease progression, these compounds could become major additions to the anti-Parkinsonian pharmacopoeia.

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Trevor W. Stone

Institute of Biomedical & Life Sciences, West Medical Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK

Correspondence and reprint requests to: T. W. Stone, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK. [t.w. stone@bio.gla.ac.uk] Accepted for publication December 2004.

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