A2 adenosine receptors: their presence and neuromodulatory role in the central nervous system

Gen. Pharmac. Vol. 27, No. 6, pp. 925-933, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA.

ISSN 0306-3623/96 $15.00 + .00 PlI S0306-3623(96)00044-4 All rights reserved ELSEVIER

REVIEW

A2 Adenosine Receptors: Their Presence and Neuromodulatory Role in the Central Nervous System Serena Latini, Marta Pazzagli, Giancarlo Pepeu and Felicita Pedata* DEPARTMENT OF PRECL1NICALAND CLINICAL PHARMACOLOGY,

UNIVERSITYO¢ FmRENCE, VIALE MORGAGNI65, 50134 FLORENCE,ITALY [TEL: 39 55 4237411-4237446; FAX:39 55 4361613; E-MAIL: [email protected]] ABSTRACT. 1. Adenosine is an endogenous neuromodulator that exerts its depressant effect on neurons by acting on the A, adenosine receptor subtype. Excitatory actions of adenosine, mediated by the activation of the Az adenosine receptor subtype, have also been shown in the central nervous system. 2. Adenosine Az, receptors are highly localized in the striatum, as demonstrated by the binding assay of the Aza selective agonist, CGS2680, and by analysis of the A2 receptor mRNA localization with in situ hybridization histochemistry. However, adenosine Az, receptors, albeit at lower levels, are also localized in other brain regions, such as the cortex and the hippocampus. 3. In the striatum, adenosine A2, receptors are implicated in the control of motor activity. Evidences exists of an antagonistic interaction between adenosine A2, and dopamine Dz receptors. 4. Utilizing selective agonists and antagonists for adenosine Az, receptors, their role in the modulation of the release of several neurotransmitters (acetylcholine, dopamine, glutamate, GABA) has been extensively studied in the brain (striatum, cortex, hippocampus). Controversial results have been obtained and, because the overall effect of endogenous adenosine in the brain is that of an inhibitory tonus, the physiological meaning of the excitatory A2 receptor remains to be clarified. GEN PHARMAC 27;6:925--933, 1996 KEY WORDS. Adenosine, central nervous system, A:~, receptors INTRODUCTION Adenosine is an important endogenous modulator that has been shown to play a role in the regulation of physiological activity in several organs and tissues (Daly, 1982; Williams, 1989). In the central nervous system, endogenous adenosine exerts a potent depressant effect on neurons (Dunwiddie, 1985; Fredhohn and Dunwiddie, 1988) by reducing transmitter release from presynaptic nerve terminals (Phillis et al., 1979; Okada and Ozawa, 1980; Corradetti et al., 1984) and increasing potassium conductance in the postsynaptic cell (Trussel and Jackson, 1985; Stone and Bartrup, 1991). However, all evidence cited above is indirect and only more recently has the inhibitory action of adenosine on central synapses been demonstrated by a quantal transmission analysis, which detects the suppression of neurotransmitter release from hippocampal synapses (Yamamoto et al., 1993). This inhibitory tonus on excitatory neurotransmission is exerted by endogenous adenosine, which accumulates in the extracellular space during neuronal activity (Pull and McIlwain, 1972; Pedata et al., 1990; White and Hoehn, 1991), and interacts with specific membrane-bound receptors. In addition to the inhibitory role, excitatory actkms of adenosine have also been demonstrated in the central nervous system. It has been reported that, at low concentrations, adenosine produces an excitatory response in central synapses, resulting in increased release of glutamate, with a mechanism that seems not to involve adenosine receptors (Okada et al., 1992; Hirai and Okada, 1994). On the other hand, a receptor-mediated excitatory action of adenosine on *To whom correspondence should be addressed. Received 7 November 1995; accepted 15 December 1995.

acetylcholine release has recently been demonstrated in central and peripheral nervous systems (Brown et al., 1990; Correira-de-Sa et al., 1991). Over the last 5 years, the role of the A: adenosine receptor subtype in mediating this excitatory action of adenosine on synaptic functions has been extensively studied. Selective agonists and antagonists have recently been developed that are useful in the study of distribution of A: receptors in the brain and their modulatory role on neurotransmission. However, because the net effect of endogenous adenosine on neurons is an inhibitory tonus, the functional rote of excitatory A2 adenosine receptors in the brain is not yet understood. ADENOSINE RECEPTOR SUBTYPES Adenosine exerts its effects by acting on specific receptors located on the external cell membrane (Londos et al., 1980), which have been subdivided (Van Calker et al., 1979) into two classes: Al receptors, coupled by a G protein with the inhibition of adenylate cyclase, and A: receptors, which mediate the stimulation of the enzyme. However, because adenosine receptors have also been reported to modulate other second messenger systems (Morgan, 1991), the current classification is based mainly on the rank-order potency of agonists in eliciting physiological responses. In general, the Al receptor shows an affinity constant for adenosine and its derivatives in the nanomolar range, whereas micromolar concentrations are required for activation of the Ae receptor subtype. The potency rank of agonists for the a l adenosine receptor is: N~"cyclopentyladenosine (CPA)>NC'-cyclohexyladenosine (CHA)> N6-(R-phenylisopropyl)adenosine (R-PIA) > 2-chloroadenosine

926 (2-CADO)>N-ethylcarboxamidoadenosine (NECA) > S-PIA > 2-(phenylamino)adenosine (CV1808), and for the A2 receptor is: 2-[[2-[4- (2-carboxyethyl)phenyl]ethyl]amino]-N-ethylcarboxamidoadenosine (CGS21680) = N E C A > 2-CADO>CV 1808 =R-PIA> C P A = C H A > S - P I A (Williams, 1990). Recently, new adenosine agonists, such as 2-chloro-N6-cyclopentyladenosine (CCPA), with high affinity (Ki=0.4 nM) and selectivity (about 10,000-fold) for A~ receptors (Lohse et al., 1988b), and 2-hexynyl-NECA with high affinity (/(,=3.8 nM) and selectivity for A2 vs AI receptors, on the order of 12-fold, have been described (Cristalli et al., 1992). Both classes of adenosine receptors can be antagonized by xanthine compounds, such as caffeine, theophylline and related 8-phenylxanthines. More recently, a number of selective xanthine antagonists, such as 8-cyclopenthyl-l,3-dipropylxanthine (DPCPX) for A~ receptors (Ki=0.5 nM; AdAl =700) (Bruns etal., 1987b; Lohse et al., 1987), (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837) (K,=I.0 nM; AI/A2=62) (Nonaka et al., 1994), and 8-(3-chlorostyryl)caffeine (CSC) (K=54 nM; AI/A:=518) (Jacobson et al., 1993) for A2 receptors, have been developed. Several non-xanthine antagonists selective for A2 receptors, but with low water solubility, such as 4-amino-8-chloro- 1-phenyl[1,2,4]triazolo[4,3-e~]quinoxaline (CP66713) (K,= 12 nM; AI/A2= 13.0) (Sarges eta/., 1990), and 9-chloro2-(2-furanyl)- 5,6-dihydro- 1,2,4-triazolo[1,5-c]quinazoline-5-imine (CGS15943A) (K=4 nM; At/A2=6) (Jarvis et al., 1991), have also recently been described. These compounds are useful in the characterization of A~ and A2 receptor subtypes. A sub-classification of the A~ receptor into A~.~,with high affinity for agonists and located in the central nervous system, and A~b, with low affinity and located in the peripheral nervous system, has been proposed (Gustafsson et al., 1990). However, there is currently no evidence at the molecular level to support the existence of A~, and A,~ receptors. A~ receptors have also been subclassified into A2,, a high-affinity receptor highly localized in the striatum, and A21~, a low-affinity receptor that exists in ahnost all areas of the brain (Bruns et al., 1986). Compounds such as CV1808 and CGS21680 have been developed that show high affinity for the rat striatal A,,, receptor (Bruns et al., 1986; Jarvis et al., 1989b), and could be used to differentiate A:, from either A> or A~ receptors. Adenosine A2~, and A2b receptors have also been cloned from several animal species (Linden et al., 1994). Recently, another adenosine receptor, termed A~, has been cloned in the rat brain (Zhou et al., 1992). Although this name was previously used by Ribeiro and Sebastiao (1986) to indicate a presynaptic adenosine receptor at the neuromuscular junction of the frog sartorius muscle, there seems to be no relationship between this and the cloned receptor. An adenosine receptor, termed A4, has been characterized in rat brain tissue using CV1808 (Cornfield et al., 1992), but it has not yet been cloned. Thus, the original AI and A., receptor classification has been extended, because to date 4 distinct adenosine receptors (AI, A~,~,,A2b, A~) have been cloned from a variety of mammalian species (see Table 1). LOCALIZATION OF ADENOSINE Az RECEPTORS IN THE BRAIN The distribution of adenosine receptor subtypes in the mammalian brain has been extensively studied (Goodman and Snyder, 1982; Jarvis, 1988). Adenosine A1 receptors have been well characterized by using binding assays (Bruns et al., 1980; Schwabe and Trost, 1980) and autoradiography (Goodman and Snyder, 1982; Jarvis,

S. Latini et al. 1988), thanks to the wide availability of radiolabeled agonists and antagonists that are highly selective for this receptor subtype. It has been shown that A1 sites are widely distributed throughout the brain but, in particular, they are highly concentrated in the hippocampus, cerebellum and cortex. The distribution of adenosine A2 receptors has been determined only more recently, when pH]CGS21680, a radioligand with high affinity and selectivity for A2~ receptor subtype, has become available (Hutchison et al., 1989). Nevertheless, prior to the introduction of this compound, the A2 receptor had been studied by using two radioligands, the nonselective agonist pH]NECA and the nonselective antagonist 8-(p-sulfoamidophenyl)xanthine (pH]PD115119), both of which label AI and A2 sites equally well (Bruns et al., 1986, 1987a). Using these compounds to label A2 sites, A1 receptors need to be blocked with selective ligands, such as the antagonist DPCPX or the agonist CPA (Bruns et al., 1986). The binding of both pH]PDll5119 (+DPCPX) and pH]NECA (+CPA) is specifically concentrated in the striatum and olfactory tubercle of the rat brain (Jarvis et al., 1989a). However, the interpretation of these binding studies may be controversial because of marked species differences in the ratio of AdA2 binding for pH]PD115119 and the ability of pH]NECA to label also a nonadenosine riboside uronamide binding site (Lohse et al., 1988a). More recently, a novel 2-substituted analogue of NECA, CGS21680, has been described (Hutchison et al., 1989) as being 140 times more selective for A: sites than for the Al receptor. Furthermore, CGS21680 appears to specifically label the high-affinity Ae receptor (A2~,) and not the low-affinity A2b receptor subtype (Lupica et al., 1990). Consistent with the observations obtained with the nonselective radioligands [~H]NECA and pH]PD115119, it has been shown that pH]CGS21680 binding sites are highly localized in the striatal region of the rat brain, principally in the caudate-putamen, nucleus accumbens and olfactory tubercle (Jarvis and Williams, 1989; Jarvis et al., 198%). All binding sites in the rat striatum localized with pH]CGS21680 are coupled with G-proteins (Parkinson and Fredholm, 1990). Likewise, in the human brain the highest concentration of pH]CGS21680 binding sites has been found in the caudate nucleus, globus pallidus and putamen (Wan et al., 1990). Another study on the postmortem human brain reveals 2 subtypes of pH]CGS21680 binding sites, one of which is comparable to the rat A2~,receptor localized in the striatum, and the other is more widely distributed and intermediate between A~ and A2 receptors (James et al., 1992). The recent cloning of the cDNA encoding for A2 receptors (Maenhaut et al., 1990) has been useful in the study of cellular localization of the A2 receptor mRNA by in situ hybridization histochemistry. In agreement with binding studies, the A2 receptor mRNA is exclusively expressed in the caudate-putamen, nucleus accumbens and olfactory tubercle of the dog, rat and human brain (Schiffman et al., 1990, 1991a, b). It has also been demonstrated that A2 receptor mRNA is specifically expressed by medium-sized, but not by large, neurons of the rat striatum. In particular, the adenosine A2 receptor is expressed exclusively by the enkephalinergic striatal subpopulation but not by the substance P-containing and cholinergic neurons (Schiffman et al., 1990, 1991a). In agreement with these results, lesion studies have confirmed that A2 receptors are localized on intrinsic neurons of the striatum, rather than on the nerve terminal of fibers afferent to the striatum (Wojcik and Neff, 1983; MartinezMir et al., 1991; Moser et al., 1991). In a more recent work, James and Richardson (1993) showed a similar distribution of pH]CGS21680 binding sites and of the cho-

A2 Adenosine Receptors in the CNS

927

TABLE 1. Adenosine purinoceptors in the central nervous system Adenosine receptors AI

Agonist

A2~,

CHA, CPA, R-PIA, CCPA, NECA, 2-CADO CGS21680, CV1808, NECA

A21. A~

NECA, 2-CADO I-APNEA, N6-benzyl NECA

Antagonist DPCPX, CPT, KFM19 KF17837, CP66713, CSC, CGS15943A XAC, CPX BWA1433, I-ABOPX

Location Hippocampus, cerebellum, cortex Striatum Ubiquitous Wide; species differences

2-CADO, 2-chloroadenosine; BWA1433, 1,3-dipropyl-8-(4-acrylate)phenylxanthine; CCPA, 2-chloro-N~'cyclopentyladenosine; CGS15943A, 9-chloro-2-(2-furanyl)-5,6-dihydro-l,2,4-triazolo[1,5,-c]quinazoline-5-imine; CGS21680, 2-[[2-[4-(2-carboxyethyl)phenyl]ethyl]amino]-N-ethylcarboxamido-adenosine; CHA, N~-cyctohexyladenosine; CP66713, 4-amino-8-chloro-l-phenyl[1,2,4]triaz*)lo[4,3-c~]quinoxaline; CPA, N~'-cyclopentyladenosine; CFq', cyclopentyltheophilline;CPX, 8-cyclopentyl-l,3-dipropylxanthine;CSC, 8-( 3-chlorostyryl)caffeine;CV1808, 2-phenylaminoadenosine; DPCPX, 2-hexynyl-NECA8-cyclopenthyl-l,3-dipropylxanthine;I-ABOPX, 1-propyl-3(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)-phenylxanthine; I-APNEA,N"-2-(3-iodo-4-aminophenyl)ethyladenosine; KF17837, (E)-8-(3,4-dimethoxystyryl)-l,3-dipropyl-7-methylxanthine; KFM19, (-+)-8-(3-oxocyclopentyl)-1,3-dipropylxanthine; NECA, N-ethylcarboxamidoadenosine;R-PIA, N~'-(R-phenylisopropyl)adenosine;XAC, xanthine amine congener. linergic nerve terminal marker, acetylcholinesterase. This result and the observation that pH]CGS21680 binding sites copurify with ChAT during the immunoaffinity purification of cholinergic nerve terminals, led the authors to conclude that there are also a2~ receptors on striatal cholinergic nerve terminals (James and Richardson, 1993). These data are in agreement with the observation that NECA-stimulated adenylate cyclase activity co-purified with choline acetyltransferase in the preparation of the cholinergic nerve terminals (Brown et al., 1990), but is not consistent with data of Schiffman et al. (1990, 1991a, b). A possible explanation for this discrepancy is that the level of Ae,, receptors is too low in the cholinergic neurons to be detected with the in situ hybridization technique. In summary, all these results have led to the conclusion that although A> receptors are widely distributed in the brain, the A2, receptor subtype exists only in the striatum, so that a specific neuromodulatory role of adenosine in basal ganglia function has been proposed. In addition to these observations on a selective localization of A2~, receptors in basal ganglia, there is recent evidence that A2, receptors may also have more widespread distribution in the central nervous system. Binding studies on human and rat brain have shown the existence of pH]CGS21680 binding sites, albeit at a lower level than in the striatum, also in the cortex and hippocampus (Wan et al., 1990; James et al., 1992; Johansson et al., 1993; Cunha et al., 1994b). Binding sites for pH]CGS21680 in the rat cortex differ from those in the striatum and its properties do not resemble any of the well-described adenosine receptor subtypes (Johansson et al., 1993). These binding sites may represent a modified A2, receptor, rather than a novel type of adenosine receptor. For instance, modifications of the binding of pH]CGS21680 to A2:, receptors in the rat striatum have been observed in the presence of mono- and divalent ions (Johansson et al., 1992). The expression of mRNA for the A2,,receptor has also been demonstrated in the hippocampus utilizing thermocycling analysis and in situ hybridization techniques, with a longer exposure time of the A2~,mRNA probe than that used in previous studies (Cunha et al., 1994b). Furthermore, the A2~,receptors appear to be present on the cholinergic nerve terminals in the hippocampus. Cunha et al. (1995) have, in fact, demonstrated co-expression of A:, receptors and choline acetyltransferase mRNAs in the nucleus of the diagonal band and the medial septum where the cholinergic cell bodies that proj-

ect to the hippocampus are located. Binding sites for pH]CGS21680 were also found in the hippocampus, utilizing a higher CGS21680 concentration and longer exposure time than in previous studies (Jarvis et al., 1989b). IMPLICATION OF A2a ADENOSINE RECEPTORS IN THE CONTROL OF MOTILITY AND INTERACTION WITH Dz DOPAMINE RECEPTORS The specific role of adenosine A2~ receptors in basal ganglia functions has not been entirely defined. However, the selective localization of this receptor subtype in the striatum, which is a critical component of subcortical circuits involved in the processing of motor activity, suggests a possible implication in the control of motility. It is well known that two main efferent pathways connect the striatal complex (Caudate-putamen, nucleus accumbens and olfactory tubercle) with the pallidal complex (globus pallidus and ventral pallidum) (Fig. 1): 1. A "direct" pathway rises from GABAergic-substance P striatal neurons and projects directly to the internal segment of globus pallidus/substantia nigra pars reticulata. The activation of this pathway results in inhibition of the internal segment of the globus pallidus. From this area, another GABAergic projection innervates thalamic excitatory neurons that project back to the cortex. Thus, an inhibition of the internal segment of the globus pallidus results in disinhibition of thalamic nuclei, thereby facilitating cortically initiated movements. 2. An "indirect" pathway arises from the GABAergic-enkephalin striatal neurons and projects to the external segment of the globus pallidus. From this area, GABAergic pathway innervates the subthalamic nucleus from which glutamatergic neurons project to the internal segment of the globus pallidus/substantia nigra pars reticulata. The activation of this pathway increases the inhibitory discharge of the internal segment of the globus pallidus on thalamic neurons, with a consequent reduction of locomotor activity (Alexander and Crutcher, 1990). A dopaminergic nigrostriatal pathway innervates the striatopallidal system and controls the two different pathways, by stimulating the former and inhibiting the latter (Graybiel, 1990). These actions are mediated by different dopamine receptor subtypes. From cloning of Di and D, receptors, it has emerged that D~ receptors are mainly

928

S. Latini et al. CORTEX

1

Thai

OPe

8Ne

D

@

GPi./SNr

FIGURE 1. Diagram showing the basal ganglia-thalamocortical circuitry (modified from Alexander and Crutcher, 1990). Excitatory neurons are shown as filled symbols, inhibitory as open symbols. Abbreviations: GPe, external segment of globus pallidus; GPi, internal segment of globus pallidus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus; Thai, thalamus.

In addition to this effect of adenosine on spontaneous locomotor activity, behavioral evidence suggests that adenosine agonists inhibit, whereas adenosine antagonists enhance, the locomotor activity mediated by the activation of dopamine D2 receptors (Ferr~ et al., 1991a, b). On the other hand, stimulation of Dz receptors counteracts the A>-mediated inhibition of motor activity (Ferr~ et al., 1991c). There is also biochemical evidence of a functional interaction between adenosine A2;, and dopamine D2 receptors. It has been shown that the stimulation of A2~, receptors with CGS21680 decreases the affinity of D2 agonists for their binding sites (Ferrd et al., 1991d; Fuxe et al., 1993), and decreases the signal from the D2 receptor to the guanine nucleotide-binding regulatory protein (G protein) in rat striatal membranes (Ferrd et al., 1993b). An in vivo microdialysis study provides direct functional evidence for the antagonistic intramembrane interaction between adenosine and dopamine receptors, h has been shown that the inhibitory action on GABA release from globus pallidus of the D2 agonist pergolide, infused in the striatum, was completely counteracted by the co-infusion of the Ae~,agonist CGS21680 and enhanced by the co-infusion of the adenosine antagonist theophylline (Ferrd et al., 1993a). Furthermore, a functional interaction is also provided by the observation that adenosine Aa, antagonists enhance the D2 dopaminereceptor dependent regulation of c-fos in the striatopallidal pathway (Pollak and Fink, 1995). These reports, and the observation that the density of A2,,sites is decreased in the basal ganglia of patients with Huntington's chorea (Martinez-Mir et al., 1991), suggest that adenosine, acting on A2.,receptors, plays a role in the pathogenesis of movement disorders. New therapeutic strategies, including specific Ae, agonists and antagonists, may be useful in the treatment of basal ganglia disorders such as Parkinson's disease and Huntington's chorea.

confined to the GABAergic-substance P-dynorphin neurons, and D2 receptors are localized in GABAergic-enkephalin neurons (Gerfen et al., 1990; Le Moine et al., 1990, 1991). Thus, endogenous dopamine release from neurons arising from the substantia nigra, by acting on D~ receptors, stimulates the "direct" pathway and, by acting on D2 receptors, inhibits the "indirect" pathway; in both cases resulting in a facilitation of locomotor activity. The selectivity of expression of A2~,adenosine receptors in the ROLE OF A2 ADENOSINE RECEPTORS GABAergic-enkephalin subpopulation of striatal efferent neurons, IN THE M O D U L A T I O N OF N E U R O T R A N S M I T T E R and their co-localization with D2 dopamine receptors, suggest an im- RELEASE A N D SYNAPTIC F U N C T I O N plication of adenosine A2~,receptors in the basal ganglia functions Following the earliest evidence supporting the hypothesis that and an interaction with dopamine D2 receptors. adenosine plays a role in the modulation of neurotransmitter release In addition to these anatomical observations, some experimental and in synaptic functions (Vizi and Knoll, 1976; Harms et al., 1979; evidence indicates that the striatopallidal pathway is the main locus Pedata et al., 1983), it has been repeatedly demonstrated that the for adenosine A2a receptor-dopamine D2 receptor interaction. ability of adenosine to inhibit the release of several neurotransmitBehavioral studies have shown that adenosine agonists produce ters, such as dopamine (Harms et al., 1979; Myers and Pugsley, a marked inhibition of locomotor activity in rodents (for review, see 1986), serotonin (Harms et al., 1979; Feuerstein et al., 1985), norFerr~ et al., 1992). On the basis of the potencies of different adenoepinephrine (Harms et al., 1979; Jonzon and Fredhohn, 1984), acesine agonists, this effect has been attributed to activation of the A2~, tylcholine (Pedata et al., 1983; Jackisch et al., 1984), and glutamate receptor subtype (Durcan and Morgan, 1989). Adenosine antago(Dolphin and Archer, 1983, Corradetti et al., 1984), appears to be nists, such as caffeine and theophylline, inhibit the psychomotor demediated by the activation of the AL adenosine receptor subtype. pression evoked by adenosine analogues and, when administered On the other hand, a receptor-mediated excitatory role of adenoalone, produce locomotor activation, probably by antagonizing the sine on neurotransmitter release has been observed in the central inhibitory action of endogenous adenosine (Snyder et al., 1981). An (Pedata et al., 1983; Spignoli et al., 1984; Brown et al., 1990) and implication of A2,, receptors in mediating the behavioral effects of peripheral nervous systems (Correira-de-Sa et al., 1991). This excitadenosine has also been shown by the observation that, although the AJA2-receptor antagonist CGS15943A increases the locomotor atory action of adenosine has been attributed to the activation of activity of mice, the highly selective adenosine A~ receptor antago- the low-affinity adenosine receptor subtype (A2 receptor), because nist, DPCPX, does not significantly modify the locomotor behavior it was observed that a high concentration of nonselective adenosine of mice (Griebel et al., 1991). On the basis of these observations, it agonists, which affect both A~ and A2 receptor subtypes, is much may be assumed that the inhibitory action of adenosine on motility more effective in increasing the release of neurotransmitters rather could result from the activation of A2~,adenosine receptors localized than in inhibiting it (Spignoli et al., 1984). Furthermore, when AI on GABAergic-enkephalin striatal neurons, with a consequent in- receptors are blocked with selective antagonists, the mixed A~/A2 crease of the inhibitory discharge from the internal segment of the adenosine receptor agonist, NECA, shows an excitatory action on neurotransmission (Brown et al., 1990). globus pallidus to thalamic neurons (Fig. 1).

A, Adenosine Receptors in the CNS

Neuromodulatory role of A2 adenosine receptors in the cortex We have shown that the inhibitory effect of the Aj adenosine agonist C H A on acetylcholine release evoked by electrical stimulation of cortical slices was reduced by increasing its concentration from 10 nM to 100 I~M, and that the mixed A J A 2 adenosine receptor agonist NECA at high concentration (100 btM) brought about a marked increase in acetylcholine release. Aminophylline antagonizes both inhibitory and excitatory effects of adenosine analogues (Spignoli et al., 1984). We have also previously demonstrated a biphasic effect, both stimulatory and inhibitory, of methylxanthines on acetylcholine release, depending on their concentration (Pedata et al., 1984). On the basis of these observations, we hypothesized the existence in the rat cortex of an inhibitory A~ type receptor, and a stimulatory receptor, possibly of the A: type (Spignoli et al., 1984). Unfortunately, the hypothesis of an excitatory role of adenosine A:~,receptors in the cortex has not been confirmed by our recent experiments, in which the effect of selective ligands for A:, receptors has been evaluated on acetylcholine release. We have observed that neither the A:;, agonist CGS21680 (250 nM, 1 IzM) nor the A:~,antagonist CP66713 (10 nM, 5 gM), significant modified the electrically-evoked release (10 Hz) of acetylcholine from rat cortical slices suggesting that Ae, receptors are not involved in the modulation of acetylcholine release from the cortex. Conversely, an excitatory action of A> receptors on neurotransmitter release has been demonstrated in the cortex under pathological conditions, such as ischemia. O'Regan et al. (1992a), utilizing the in vivo cortical cup technique, have shown that CGS21680 significantly enhances the ischemia-evoked release of excitatory amino acids and that the high concentration of endogenous adenosine reached in brain tissue after ischemia may activate A::, receptors and, then, enhance fitrther the release of excitatory amino acids. It has been shown that, in these experimental conditions, the A, receptor antagonist, CGS15943A, inhibits the ischemia-evoked release of aspartate and glutamate (Simpson et al., 1992). Thus, under pathological conditions, the excitatory effect resulting from the activation of A,, receptors could prevail over the inhibitory effect mediated by A~ receptors. In this regard, it has also been observed that stimulation of A:, receptors with CGS21680 inhibits GABA release during reperfusion after ischemia (O'Regan et al., 1992b). However, a lack of effect of CGS21680 on ischemia-evoked release of acetylcholine (Phillis et al., 1993c) and on K+-evoked release of acetylcholine and excitatory amino acids (Phillis et al., 1993a, b) has been reported. These controversial results ,nay be explained when considering that a low level of expression of A::, receptors has been observed in the cerebral cortex compared to A, receptors (Wan et al., 1990; James et al., 1992) and that the binding site fi~r [~H]CGS21680 in the rat cortex does not show exactly the same properties as an a , , adenosine receptor (Johansson et al., 1993). The numerical dominance of Aj receptors over a.,~ receptors may contribute to the dominance of inhibitory effects of adenosine, especially when its concentration in the tissue is not high enough to activate the lowaffinity A,~, receptor subtype.

Neuromodulatory role of A2 adenosine receptors in the striatum As already reported in this review, adenosine A2~ receptors are highly concentrated in the striatum (Jarvis and Williams, 1989; Martinez-Mir et al., 1991); thus, allowing this region of the brain to

929 be the preferred site for investigating the fimctional role of adenosine A2., receptors. Before the introduction of selective agonists and antagonists for A2 adenosine receptors, it was suggested that cholinergic nerve terminals in the rat striatum contain both a high-affinity inhibitory At receptor and a lower-affinity stimulatory A, receptor. This was concluded on the basis of the observation that, when adenosine A, receptors were blocked by DPCPX, the mixed agonist NECA brought about a stimulation of adenylate cyclase activity and an increase in pH]-acetylcholine release (Brown et al., 1990). By evaluating the effect of the more selective agonist CGS21680 and antagonists CP66713 and CGS15943A on the veratridine-ew~ked release of pH]-acetylcholine from rat striatal synaptosomes, the existence of A,, receptors on striatal cho[inergic nerve terminals and their stimulatory action on pH]-acetylcholine release (Kirkpatrick and Richardson, 1993) was confirmed. The authors discussed the discrepancy between their data and the lack of expression of A:., adenosine receptor mRNA in cholinergic neurons (Schiffman et al., 1991a), suggesting the existence of two A:~,-likestriatal receptors, one expressed by GABAergic-enkephalin neurons, and the other localized in cholinergic neurons and not detectable by the in situ hybridization technique. This idea is also supported by the observation that the stimulation of A+, receptors with the agonist CGS21680 has opposite effects on the K+-ew~kedrelease of [~H]-acetylcholine and pH]-GABA from striatat nerve terminals (i.e., by stimulating the former and inhibiting the latter) (Kirk and Richardson, 1994; Kurokawa et al., 1994). In agreement with these results, two A:~,-likebinding sites with different properties have been reported in human (James et al., 1992) and rat brain (Johansson et al., 1993). An opposite effect of A:~,agonist CGS 21680 on electrically-ew~ked release of endogenous GABA, has been demonstrated in rat globus pallidus (Mayfield et al., 1993). The discrepancy between results of Kirk and Richardson (1994) and Mayfield et al. (1993) may be due to the different preparations utilized: synaptosomes and slices, respectively. A possibility is that the activation of A,:, receptors, by decreasing the D,-dopamine receptor-mediated actions, such as the ability of dopamine to inhibit GABA release, results in the enhancement of GABA release from slices of the globus pallidus. This possibility should not be realizable in the synaptosomal preparation utilized by Kirk and Richardson (1994). With the aim of further examining the role of Ae, receptors in the modulation of acetylcholine release from the striatum, we have studied the effect of the A, agonist 2-hexynyl-NECA, the A:+,selective agonist CGS21680 and the A, selective antagonist CP66713 on electrically-ew~ked acetylcholine release from striatal slices. We ~bserved that the A: antagonist CP66713 (0.1 ~M) brought about a significant reduction in acetylcholine release ew~ked by electrical stimulation at a frequency of 2 Hz, but was without effect at a higher concentration (1 IxM) and when striatal slices were stimulated at a higher frequency (10 Hz) (Fig. 2). On the other hand, neither 2-hexynyl-NECA (250 nM) nor CGS21680 at different concentrations (10-250 nM, 1 IzM) affect the acetylcholine release evoked from striatal slices by electrical stimulation at different (0.2, 2, 10 Hz) frequencies. The lack of effect of 2-hexynyl-NECA and CGS21680 may indicate that adenosine levels reached in striatal tissue after electrical stimulation are sufficient to activate m o s t A~:, receptors. Because a:, receptors have been shown to stimulate GABA release in the striatum (Mayfield et al., 1993), we also investigated the possibility that an increase in acetylcholine release ew~ked by CGS21680 could be overcome by increasing inhibitory tonus on acetylcholine release mediated by GABA. A GABAergic modulation of striatal cholinergic interneurons has

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FIGURE 2. Effect of the selective Az antagonist CP66713 on electrically-evoked release of acetylcholine from rat striatal slices. Data are expressed as nanograms per gram per minute of superfusion (mean _+ SEM). Acetylcholine release was evoked by two 5-min periods of electrical stimulation (S1 and $2) at 2 and 10 Hz of stimulation frequency, as described by Pedata et al. (1984). Drugs were added to the perfusion fluid 25 rain before the second cycle of stimulation. Acetylcholine was extracted from superfusate sample, as described by Vannucchi et al. (1990), and assayed by HPLC technique, with an electrochemical detector according to the procedure described by Giovannini et al. (1991). *P < 0.03 Student's paired t-test, $2 vs corresponding S1.

been demonstrated in an in vivo microdialysis study (DeBoer and Westerink, 1994). In this work, it has been shown that stimulation of GABAA and GABAB receptors with selective agonists decreases the striatal acetylcholine outflow, whereas endogenous GABA, via GABAA, but not GABAB receptors, tonically inhibits the output of acetylcholine from the striatum. However, in our experiments, neither the GABAA antagonist bicuculline (10 IxM) alone, nor in the presence of CGS21680 (250 nM), significantly modified acetylcholine release evoked from striatal slices by electrical stimulation

(2 Hz). J in et al. (1993) provide evidence against the presence of an A2~ receptor-mediated control of either dopamine or acetylcholine release from rat striatal slices because no excitatory effect of CGS21680 on their evoked release was found. Conversely, an inhibitory action of both CHA and CGS21680 on dopamine and acetylcholine release evoked by electrical stimulation was found. Although this effect was antagonized by DPCPX, because the difference in potency between CGS21680 and CHA was much lower than the difference in their relative affinity for A~ receptors, the authors suggest that the inhibitory effect of CGS 21680 may be mediated by a receptor different from the typical A~ adenosine receptor. Neuromodulatory

role of

Az adenosine receptor in the hippocampus In support of the hypothesis of the existence of a neuromodulatory role of Az~,adenosine receptors in the hippocampus are the observations that [~H]CGS2t680 binding sites are found on hippocampal membranes (Wan et al., 1990; James et al., 1992), albeit at a lower

level than in the striatum, and that CGS21680 in nanomolar concentrations increases the amplitude of orthodromically-evoked population spikes recorded from the CA1 pyramidal cell layer of rat hippocampal slices (Sebastiao and Ribeiro, 1992). More recently, as already reported in this review, molecular evidence for the presence of A2, adenosine receptors in the hippocampus has been provided (Cunha et al., 1994b). On the other hand, further studies with pH]CGS21680 revealed a binding site distinct from the typical striatal Ae, receptor in the hippocampus, with a nanomolar affinity for DPCPX (Cunha et al., 1994a). An excitatory effect of A2, adenosine receptor activation has been demonstrated on the electrically evoked [~H]-acetylcholine release from rat hippocampal slices. However, a differential modulation by A,, adenosine receptors of the electrically evoked pH]-acetylcholine release from different areas of hippocampal slices (CAI, CA3 and dentate gyrus) has been shown. An excitatory action of CGS21680 on pH]-acetylcholine release has been demonstrated in the CA3 and dentate gyrus areas, but not in the CA1 area. Furthermore, in the CA1 area, endogenous adenosine exerts only an inhibitory tonus, whereas in the CA3 area adenosine interacts with both inhibitory and excitatory adenosine receptors. On the contrary, in the dentate gyrus, endogenous adenosine seems to be unable to activate either the A1 or A2 adenosine receptors (Cunha et al., 1994c). Thus, although the final effect of adenosine on acetylcholine release is a tonic inhibition, adenosine may modulate in a different way the release of acetylcholine from different areas of the hippocampus. In support of the hypothesis that A2, receptors involved in these effects are located in cholinergic nerve terminals, is the observation that CGS21680 also increases the evoked release of acetylcholine from hippocampal synaptosomes and that this effect is antagonized by the A2 antagonist DMPX (Sebastiao et al., 1995). In the hippocampus, there is the highest density of adenosine A~ receptors in the central nervous system (Fastbom et al., 1987) and acetylcholine release is inhibited by both endogenous and exogenous adenosine (Jackisch et al., 1984; Pedata et al., 1986). However, the demonstration that CGS21680 attenuates the inhibitory actions of the A~ receptor agonist CPA on population spike amplitude (Cunha et al., 1994b) suggests that there is a cross-talk between A~inhibitory and A2,-excitatory adenosine receptors, and that the net modulation of synaptic activity in the hippocampus is the result of the summation of excitatory and inhibitory receptor-mediated actions. In the hippocampus, both endogenous and exogenous adenosine and adenosine analogues exert a tonic inhibitory effect on long-term potentiation (LTP). This effect has been shown to be mediated by AI adenosine receptors, because DPCPX, at a concentration that selectively antagonizes the a I adenosine receptor, facilitates LTP (de Mendonga and Ribeiro, 1994). On the other hand, the activation of adenosine A:, receptors by CGS21680 facilitates LTP and the effect is antagonized by the selective A2 receptor antagonist, CSC. The stimulation of A2 receptors by endogenous adenosine is unable to modulate induction of LTP, however, because CSC alone did not modify LTP (de Mendonga and Ribeiro, 1994). On the contrary, by utilizing the uroderately selective A2 receptor antagonist CP66713, it was shown that endogenous adenosine is involved, via A_, receptors, in the induction of LTP of evoked synaptic potentials (EPSPs) but not of population spikes (Sekino et al., 1991). These results are further evidence of the excitatory effect of adenosine in the hippocampus mediated through the activation of A:~, receptors. CONCLUSION Following the first suggestion in 1984 by Spignoli et al. of the existence of A2 excitatory adenosine receptors in cortical slices, the ad-

A2 Adenosine Receptors in the C N S vent of more selective A2 receptor agonists and antagonists, has led to an improvement in research on this subject. Much evidence reported in this review is consistent with the hypothesis of an excitatory role of A:~, adenosine receptors in modulating neurotransmission. A l t h o u g h the excitatory role of A2, receptors has clearly been demonstrated in the hippocampus, controversial findings have been obtained in the striatum, where A2,~receptors are highly concentrated. O n the other hand, the role of A2~, adenosine receptors in the cortex has only been shown under pathological conditions, such as ischemia. In conclusion, because Aj and A2~,receptors are often localized in the same brain areas, further work is required to clarify the physiological meaning of A~-mediated inhibitory and A2smediated stimulatory actions of adenosine in the central nervous system. The authors wish to thank Dr. G. Cristalli for providing 2-hexynyl-NECA, Dr. R. Sarges (PFIZER) fi~r providing CP66713 and Mary Forrest for correction of English. This work was supported by a grant from the University of Florence.

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