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Adenosine receptors have been the target of intense research with respect to potential use of selective ligands in a variety of therapeutic areas. Caffeine and theophylline are adenosine receptor antagonists, and over the past three decades a wide range of selective agonists and antagonists for adenosine receptor subtypes have been developed. A complication to the therapeutic use of adenosine receptor ligands is the observation that the effects of acute administration of a particular ligand can be diametrically opposite to the chronic effects of the same ligand. This ‘effect inversion’ is discussed here by Ken Jecobson and colleagues, and has been observed for effects on cognitive processes, seizures and ischaemic damage.
Caffeine, the most widely consumed of all psychoactive drugs, is an adenosine receptor antagonist1-3. It is regularly ingested by more than half of the adult population in Western countries, mainly because of the mild stimulant effects of this methylxanthine. Acute and chronic effects of caffeine have been extensively studied, and the phenomena of tolerance and withdrawal are well-documented in animals and humans. The only known biochemical targets for caffeine, at which it has significant activity at concentrations achieved during normal human use, are A1 and A2 adenosine receptors. Xanthines were envisaged to have therapeutic potential as central stimulants and cognitive enhancers, as cardiac stimulants, as anti-asthmatics, as anti-Parkinson’s disease agents, anti-obesity agents, as analgesic adjuvants, and as diuretics. Many of the xanthines are selective for adenosine receptor subtypes, and many are more potent than caffeine. Conversely, a variety of adenosine analogues (Fig. 1) have been developed with envisaged therapeutic potential as analgesics, antipsychotics, anticonvulsants, or for treatment of stroke. However, over the past few years it has become apparent that the effect of acute or chronic treatment with caffeine, or other adenosine receptor ligands, can be qualitatively different. Thus, long-term treatment with adenosine receptor antagonists can have effects that resemble the acute effects of adenosine receptor agonists, and vice versa. Such diametrically opposed actions of chronic versus acute treatments have important implications in the development of adenosine receptor ligands as therapeutic agents.
The effects of chronic administration of caffeine on the amount of and function of central A1 and A2A adenosine receptors have been examined in many laboratories using a variety of dose regimens and different periods of time after the last administration2-8. The A1 receptors were found to be up-regulated in most studies, without any changes in adenosine A1 receptor mRNA (Ref. 4). This is consonant with the antagonism of an endogenous agonist that downregulates A1 receptors via a post-transcriptional mechanism. In contrast, in most, but not all, studies, A2A receptor binding and mRNA expression were unaltered.
Recently, a broad survey of biochemical alterations, following chronic caffeine ingestion by mice, was carried out;7,8 the effects on receptor levels were deduced from radioligand binding (Fig. 2). In addition to A1 adenosine receptors, 5-HT receptors, acetylcholine receptors, GABA receptors and δ-opioid receptors were up-regulated. Only β-adrenoceptors were dowsregulated. However, it should be noted that an apparent up-regulation of nicotinic acetylcholine receptors probably represents conversion to a desensitized, high-affinity state, and thus, actually represents a downregulation, as has been reported after chronic nicotine treatment9. Adenosine A2A receptors, α-adrenoceptors, dopamine receptors, and excitatory amino acid receptors were unaltered. Among other biochemical alterations, the apparent increase in levels of L-type Ca2+ channels after chronic caffeine administration is noteworthy.
There is now good evidence that treatment with adenosine receptor antagonists can alter immediate early gene expression and the expression of secondary gene products, such as neuropeptides. High doses of caffeine (>50mg kg−1) are known to induce c-fos, junB, c-jun, nerve growth factor-induced clone A (NGFI-A), and NGFI-B mRNA (Refs 10-12). The first three can form the AP-1 transcription factor, which also increases after caffeine administration11, and the latter two are transcriptional factors in their own right. Lower doses of caffeine can cause a decrease in the expression of certain gene products, particularly in the striatopallidal neurones in the cortex12. Thus, there are marked phenotypic changes in selected neurones following acute and long-term caffeine administration. It is reasonable to assume that these changes are also manifested in adaptations of their functional charactristics. Indeed, the various biochemical changes after chronic caffeine administration are paralleled by altered behavioural responses to caffeine and to other agents.
Chronic ingestion of caffeine by humans can lead to tolerance, and withdrawal syndromes can occur, including apathy, drowsiness, headaches, nausea and anxiety13. True dependence, that is addiction, probably does not occur in humans and has been difficult to demonstrate in animals. In rats, chronic caffeine administration appears to result in almost complete tolerance to the motor stimulant effects of caffine14, an adaptation difficult to rationalize solely on the basis of an up-regulation of adenosine receptors because this should not cause complete tolerance to a competitive antagonist, such as caffeine. One explanation of complete tolerance to the behavioural stimulant effects of caffeine may be based on the biphasic concentration-response curve for caffeine. At low doses of caffeine, there is an increase in locomotor activity, presumed to be the result of antagonism at adenosine receptors, while higher doses cause depression. An increase in adenosine receptor function, after chronic administration of caffeine, might be expected to increase the threshold for the stimulatory action of caffeine to the point where the depressant action, which occurs at a different site (passibly inhibition of phosphodiesterases), overrides the stimulatory effects of adenosine receptor antagonism. It is also possible that alteration in other receptors, in pathways modulated by adenosinc receptors, accounts for the tolerance to caffeine.
The depressant locomotor effects of selective A1 receptor analogues in mice are somewhat enhanced after chronic caffeine administration15,16, consonant with an up-regulation of A1 receptors. However, responses to selective A2A receptor agonists are also enhanced, despite the fact that A2A receptors are not up-regulated. This may be the result of locomotor synergism between A1 and A2A receptors17.
Alterations in behavioural responses to cholinomimetic agents after chronic caffeine administration in mice can be explained in terms of desensitization of nicotinic acetylcholine receptors, that is, a tolerance to locomotor effects of nicotine, and in terms of up-regulation of muscarinic receptors – an increase in the threshold for locomotor stimulant effects of the muscarinic acetylcholine receptor antagonist, scopolamine15,16. The lack of alteration in striatal dopamine receptors7,8 and the responses to drugs such as amphetamine or cocaine, which affect dopamine systems15,16, after chronic caffeine administration is remarkable, as there is considerable evidence implicating dopamine systems as a major target for the pharmacological effects of caffeine and theophylline18. A possible clue to both the tolerance to caffeine and the apparent lack of alteration in dopamine-mediated responses has now been forthcoming. Thus, rats rendered tolerant to caffeine are also tolerant to agonists selective for either D1 and D2 receptors, but remain responsive to agonists that activate both D1 and D2 receptors19.
Further correlative studies on behavioural and biochemical alterations after chronic caffeine administration may lead to a more integrated view of the central sites of action of this agent. Although complete, or nearly complete, tolerance to stimulant locomotor effects of caffeine occurs in rats14,20, it does not occur in NIH Swiss strain mice15,16 or CD-1 mice21. Instead, chronic caffeine ingestion results in a behavioural depression of activity in the mice, in marked contrast to the behavioural stimulation caused by acute administration of caffeine.
Chronic effects of xanthines, other than caffeine and theophylline, have not been extensively studied, in contrast to the plethora of data on acute effects of various xanthines. Similarly, chronic effects of adenosine analogues have not been extensively studied, while acute effects are well documented. Chronic treatment with an adenosine analogue results in an increase in the behavioural stimulant effect of caffeine22. Three clinically relevant effects of adenosine receptor ligands, namely cognition enhancement (by xanthines), neuroprotection from seizures and from ischaemia (by adenosine analogues), have now been investigated following acute and chronic administration. In most cases, a regimen-dependent ‘effect inversion’ was observed following chronic administration. For example, acute administration of adenosine analogues have neuroprotective effects, while chronic treatment exacerbates neuronal injury. Such ‘effect inversions’ may depend on drug administration method, frequency, dose, and washout time before measuring functional changes.
Adenosine receptors may play a significant role in the process of cognition. Indeed, caffeine ingestion is commonly thought to improve intellectual performance in humans, although cognitive performance is often difficult to gauge both in humans and in animals1.
The effects of selective A1 receptor agonists and antagonists on cognition, when administered acutely, have been inconsistent. Thus, although the A1 receptor agonists N6-cyclopentyladenosine (CPA) and N6-(1-methyl-2-phenylethyl)adenosine (r-PIA) have been shown to impair retention in passive avoidance tests23,24, r-PIA has no effect on the working memory of rats25. Similarly, although the selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine(CPX) has no effect on responses in passive avoidance test23, another selective A1 receptor antagonist, KFM19, produces an increase in memory acquisition in the Y-maze26; another A1 receptor antagonist, MDL102503, reverses scopolamine-induced memory deficit in rats subjected to water-maze tests27.
Prolonged exposure of A1 receptors to their ligands can result in adaptive changes, hence it is not surprising that chronic exposure to either selective agonists or antagonists results in cognitive responses that differ from those observed following acute administration of these compounds. Thus, contrary to the expected pro-cognitive effects following acute administration of A1 receptor antagonists, chronic administration of the A1 receptor antagonist CPX in C57B] mice is entirely ineffective, or may even slightly impair memory acquisition in the water maze28. However, chronic treatment with the A1 receptor agonist CPA significantly improves both memory acquisition and retention. Chronic treatment with the selective A1 receptor agonist r-PIA prevented loss of memory in trained rats, while chronic caffeine treatment had no effect29.
Acute administration of adenosine analogues have anticonvulsant effects, while xanthines including caffeine are proconvulsant, and at sufficiently high doses are convulsants1-3. Adenosine A1 receptors appear to be involved. Acute administration of A1 receptor agonists ameliorates, or even prevents, seizures elicited by chemical and electrical stimuli in a wide range of animal models30,31. The anti-epileptic effects both of adenosine and its analogues appear to be related to the inhibition of seizure initiation and propagation. It should be noted that proconvulsant effects of adenosine receptor agonists and anticonvulsant effects of adenosine receptor antagonists have been noted in some paradigms32.
Involvement of A2A receptors in the control of epileptic discharge is unclear at present. The selective A2A receptor agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA) antagonized seizures induced by methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM)32. However, the selective A2A receptor agonist CGS21680 had only a limited ability to antagonize bicuculline-induced seizures31. Further studies, in particular with selective A2A receptor antagonists, are required to delineate the role of such receptors in the various seizure paradigms.
It is unclear as to whether acute stimulation of the A3 receptor directly antagonizes seizure generation. A significant protective effect of acutely administered N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine (IB-MECA), a selective A3 receptor agonist, against NMDA- and pentylenetetrazole-induced seizures has been demonstrated recently33. Acute administration of IB-MECA was, however, entirely ineffective in ameliorating tonic convulsions produced by electroshock. Hence, there is a possibility that for chemically induced seizures, the protection afforded by IB-MECA is caused by phenomena unrelated to the direct interruption of neuronal hyperexcitability by this compound. Since agonist stimulation of A3 receptors results in arteriolar constriction34, it is possible that administration of IB-MECA constricts cerebral arterioles, resulting in subconvulsant amounts of the chemical convulsants reaching their target within the brain.
The effect of A1 receptor ligands on seizures appears to be highly regimen-dependent. Thus, while the acute administration of an A1 receptor agonist usually results in amelioration, and acute administration of an A1 receptor antagonist has pro-convulsive effects, chronic treatment produces diametrically opposite consequences (Table 1). Chronic administration of either a nonselective adenosine antagonist (such as caffeine or theophylline35,36) or a selective A1 receptor antagonist (such as CPX37) leads to a significant protection against NMDA-induced seizures. Chronic administration of theophylline leads to a prolongation of refractory periods for amygdala-kindled seizures in rats38. Long-term, low-dose caffeine treatment also protects against bicuculline- and pentylenetetrazole-induced seizures37, as does long-term, high-dose theophylline treatment36. The high-dose xanthine treatment is accompanied by significant changes in the amount of A1 receptors, the low-dose treatment is not. The protective effect of caffeine treatment against NMDA-induced seizures was observed both during and after treatment with low doses of caffeintis35. Chronic treatment with CPA results in a pronounced increase in seizure intensity and seizure-associated mortality37, a result diametrically opposite to the prolective effects observed following acute treatment. The effects of chronic administration of selechve A2A receptor agonists and antagonists on seizure susceptibility need to be studied.
Chronic treatment with the A3 receptor agonist IB-MECA results in almost complete protection against NMDA-induced seizures and a significant reduction of mortality accompanying both pentylenetetrazole- and electroshock-induced convulsions33. Acute administration of IB-MECA was ineffective versus electroshock-induced seizures, but did ameliorate chemically induced seizures. It should be noted that selective antagonists to A3 receptors have yet to be developed.
Adenosine analogues, particularly selective A1 receptor agonists, following acute administration have wellknown neuroprotective actions during ischaemia39. Hence, adenosine-based therapies represent an approach to treating seizures, stroke and neurodegenerative diseases40. In contrast, antagonists such as caffeine, exacerbate ischaemic damage. Acute administration of A1 receptor agonists, such as CPA, has been shown to protect neurones against the damage induced both by focal and by global ischaemia39,40. Acute treatment with an A1 receptor agonist is effective even when administered up to 30 min post-ischaemia41. The critical role of adenosine as an endogenous neuroprotective agent is demonstrated by the very significant increase in neuronal destruction following pre-ischaemic administration of either nonseltive (caffeine or theophylline) or selective A1 receptor (CPX) antagonists39-42.
As yet, little is known abut the effects of acute administration of A2A receptor ligands on the extent and nature of ischaemic damage. The selective A2A receptor agonist 2-[(2-aminoethylamino)-carbonylethylphenyl-ethylamino]-5′-N-ethylcarboxamidoadenosine (APEC) improved recovery of post-ischaemic blood flow and survival of gerbils, but had no effect on loss of hippocampal neurones43. Pretreatment with an A2 receptor antagonist CGSI5943 was found to reduce the extent of morphological and neurological impairment following brief cerebral ischaemia in gerbils44. The selective A2A receptor antagonist 8-(3-chlorostyryl)caffeine (CSC) protected hippocampal neurones and improved survival during the first ten days post-ischaemia43; however, deaths occurred at later times, and the mortality endpoint following acute administration of CSC was not significantly different from control.
A regimen-dependent inversion of effects of exposure to adenosine receptor ligands (Table 1) has been observed with respect to consequences of cerebral ischaemia. Rudolphi and co-workers were the first to describe the protective effect of chronic caffeine treatment on post-ischaemic morphology of the gerbil brain45. Recently, chronic administration of CPX in gerbils was found to afford a large amount of protection against brain ischaemia, while chronic administration of CPA resulted in damage and mortality significantly exceeding that of the controls42. A similar neuroprotective effect was observed in neonatal rats that had received low doses of caffeine in their mother’s milk46. The neuroprotective effect was not associated with any significant change in the numbers of A1 and A2A receptors.
Chronic treatment of gerbils with CSC leads to a slight, but statistically insignificant, protection against ischaemic damage; chronic administration of APEC, however, leads to a significant reduction of ischaemic damage43. However, acute administration of CSC also protects against ischaemic damage, but acute administration of APEC does not. Thus, the evidence for a regimen-dependent ‘effect inversion’ appears to be much weaker for A2A receptors than for A1 receptors. Since stimulation of A2A receptors causes vasodilation, chronic administration of CSC may induce adaptive changes of vascular A2A receptors, resulting in an enhanced normalization of post-ischaemic blood flow. Indeed, such improvement has been recently observed following chronic treatment with CSC and forebrain ischaemia in gerbils43. A rapid restoration of normal blood perfusion may be expected to lead to a better outcome from an ischaemic insult. It should be noted that the tolerance to the hypotensive effects of selective A2A receptor agonists has been reported after continuous infusion47, but not after chronic injections48.
The role of the A3 receptors in ischaemia is poorly understood49, and the involvement of mast-cell activation50 complicates the interpretation of findings. It is known, however, that acute pre-ischaemic stimulation of A3 receptors with IB-MECA results in a significant increase in morphological damage and enhancement of post-ischaemic mortality47. As mentioned previously, stimulation of A3 receptors results in arteriolar constriction. Hence, it is not surprising that the pre-ischaemic treatment with IB-MECA leads to a substantial delay in the normalization of post-ischaemic blood flow. Chronic treatment with IB-MECA results in a significant reduction of post-ischaemic cerebral damage and mortality following 10 min and 20 min occlusion of both carotid arteries in gerbils49. This effect is the opposite of that seen following the acute administration of the compound.
The presence of the regimen-dependent ‘effect inversion’ for adenosine receptor agonists and antagonists leads to questions concerning the extent to which up- or downregulation participates in these phenomena. Many studies have demonstrated that chronic exposure to non-selective A1 receptor antagonists, such as caffeine or theophylline, results in up-regulation of A1 receptors. Indeed, this may be the explanation of the protective effect of chronic administration of caffeine prior to cerebral ischaemia in gerbils. However, in at least one study, the regimen of chronic administration of caffeine administration failed to produce any detectable up-regulation of A1 receptors35. In another study, chronic exposure to selective agonists or antagonists at A1 receptors appeared not to have any effect on A1 receptor density42. In spite of the lack of changes in A1 receptors ‘effect inversion’ occurred in both studies. Thus, the role of up- or down-regulation of adenosine receptors in the processes of regimen-dependent ‘effect inversion’ remains an enigma. The presence or absence of ‘effect inversion’ may be directly dependent upon the duration of exposure, withdrawal, and nature of the non-selective and selective adenosine receptor ligands, and further studies are required. At present, for caffeine and other adenosine receptor ligands, evidence for such inversions has been obtained with respect to behavioural activity, cognition, seizure lability, and neuroprotection during ischaemia. The ‘effect inversion’ for such agents is highly relevant in the development of behavioural stimulants, cognitive enhancers, anticonvulsants, and possible therapies in the treatment of stroke.
Kenneth A. Jacobson, Molecular Recognition Section.
John W. Daly, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of health, Bethesda, MD 20892, USA.
Bertil B. Fredholm, Department of Physiology and Pharmacology, Section of Molecular Neuropharmacology, Karolinska Institue, S-17177, Stockholm, Sweden.