Proc. Nati. Acad. Sci. USAVol. 77, No. 11, pp. 6892-6896, November 1980Neurobiology

Biochemical characterization of putative central purinergicreceptors by using 2-chloro[3H]adenosine, a stable analog ofadenosine

(radioligand binding/central neuromodulators/methylxanthines)

MICHAEL WILLIAMS AND EDWIN A. RISLEYNeuropsychopharmacology Section, Merck Institute for Therapeutic Research, West Point, Pennsylvania 19486

Communicated by P. Roy Vagelos, August 4,1980

ABSTRACT After pretreatment of rat brain synapticmembranes with adenosine deaminase to remove endogenousadenosine, 2-chloroeHladenosine, a stable analog of adenosine,binds to two sites with Kd values of 1.3 and 16 nM and corre-sponding Bm.. values of 207 and 380 fmol/mg of protein.Binding is reversible, and the highest density of sites occurs inenriched synaptosomal fractions. In peri'heral tissue, negligiblebinding is observed in heart, kidney, and liver, while testicle has11 fmol of binding sites/mg of protein. In brain, caudate andhippocampus have the highest ensity of sites, and spinal cordand hypothalamus have the lowest. This high-affinity bindingis stereospecific; the L diasteromer of NA-phenylisopropylade-nosine is approximately 30times more potent as a isplacer of2-chloro3Hjadenosine than the D isomer and is also sensitiveto theophylline (ICso = 8.8 pM) and other purine-related com-pounds. Several putative neurotransmitters, neurotransmitterantagonists, and other centrally active compounds have no ef-fect on binding The data are consistent with the hypothesis that2-chloro[3Hjadenosine is binding to central purinergic recep-tors.

The concept that adenosine (Ado) and ATP can function asmodulators or neurotransmitters in mammalian tissues has beendeveloped by Burnstock (1) from pharmacological data gen-erated in peripheral systems; it is postulated that the nucleosideproduces its effects via specific P1 purinergic receptors and thenucleotide produces its effects via P2 type receptors. Bio-chemical, electrophysiological and, to a lesser extent, behavioraland autoradiographical data have suggested a similar neuro-modulatory-neurotransmitter role for these purines in centralnervous system function (2, 3).Ado is an effective regulator of cyclic AMP formation in

brain slices (4, 5) and homogenates (6), acting via membrane-localized extracellular receptor mechanisms that are susceptibleto blockade by alkylxanthines such as theophylline (7). It is alsoa potent inhibitor of neurotransmitter release, modulating therelease of acetylcholine, norepinephrine, and dopamine incentral and peripheral nervous tissue (8-10). It is especiallyactive in striatal synaptosomal preparations, where the nucle-oside inhibits dopamine release [50% inhibition concentration(IC5o) = 10 nM; 10].

Both Ado and ATP depress cell firing in the mammaliancentral nervous system (3, 11, 12), acting via alkylxanthine-sensitive receptor mechanisms. Conversely, theophylline andother adenosine antagonists increase cell firing (11). Althoughthe effects of the purines on cell firing may be attributed toinhibition of excitatory neurotransmitter release, it is thoughtthat a direct inhibitory effect via purinergic PI receptors mayalso occur; however, there is controversy about the evidence

for the existence of P2 receptors in the central nervous system(3).

Behaviorally, Ado has sedative, hypnotic, and anticonvulsantactivity (13-16), in contrast to caffeine and theophylline, whichare central stimulants (17).

Evidence for depolarization-evoked, calcium-dependentrelease of Ado and ATP from brain slices (18, 19) and synap-tosomal preparations (12, 20) suggests a potential physiologicalrole for purine-modulated alterations in cell firing, transmitterrelease and cyclic AMP metabolism, and their sequelae.

Demonstration of high-affinity binding of a suitable ra-dioligand to brain membranes has become an additional cri-terion for the identification of an endogenous substance as aputative neurotransmitter. For Ado and ATP, their involvementin nearly all facets of cell function (21) has made finding asuitable radioligand difficult due to the likelihood of extensivemetabolism and binding to cellular components uninvolved incellular communication. Despite these considerations, [3H]-adenosine (22, 23), [3H]adenosine cyclopropyl carboxamide (24)and [3H]-5'-adenylyl imidodiphosphate (p[NH]ppA; 25) havebeen examined as potential ligands for P1 and P2 purinergicreceptors. However, unusually large numbers of binding sites,low affinities (Kd - 10-6 M), low specific binding, andequivocal pharmacology have however made these assays un-satisfactory for the study of purinergic receptors.

This paper presents the results of a study of the binding oflabeled 2-chloroadenosine (Cl[3H]Ado), which is not a substratefor the catabolic enzyme adenosine deaminase (ADase) (5), inrat brain membranes.

MATERIALS AND METHODSTissue Preparation. Crude synaptic membranes were pre-

pared from brain as described (26) and stored at -80'C for atleast 18 hr before use. Frozen tissue samples were stable for upto 1 month.

Subcellular fractions from rat brain were prepared by themethod of Whittaker (27); the nuclear, crude synaptosomal,myelin, enriched synaptosomal, mitochondrial, and microsomalsubfractions were homogenized by using a Polytron (Brink-mann PT 20-ST generator, setting 5.5 for 10 sec) in distilledwater; centrifuged at 48,000 X g for 10 min; and stored at-80°C for at least 18 hr before use. After dissection of rat brainregions (28), synaptosomal fractions were prepared from tissuepooled from 3-4 rats. Peripheral tissues were homogenized byusing a Polytron in 0.32M sucrose and centrifuged at 2500 X

Abbreviations: Ado, adenosine; ADase, adenosine deaminase;p[NHlppA, 5'-adenylyl imidodiphosphate; ClAdo, 2-chloroadenosine;EHNA, erythro-9-[3-(2-hydroxynonyl)]adenine; IBMX, isobutyl-methylxanthine.

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Proc. Natl. Acad. Sci. USA 77 (1980) 6893

g for 10 min. The resultant pellets were washed twice by cen-trifugation and stored at -80'C. Aliquots of all tissue prepa-rations were assayed for protein by the method of Lowry et al.(29).

Binding. Binding of Cl[3H]Ado was measured in tissuepreparations pretreated with ADase to remove endogenous Ado(30; see below). Frozen tissue preparations were resuspendedat 1.0mg protein/ml in buffer (50mM Tris-HCl, pH 7.4) usingthe Polytron (setting 5.5; 20 sec), and ADase was added to a finalconcentration of 2 units per ml. After incubation at 370C for30 min, the suspension was held on ice for 10 min, centrifugedat 48,000 X g for 10 min, and resuspended in fresh buffer foruse in the binding assay. Assay tubes contained 0.1 ml ofCl[3H]Ado (specific activity 12 Ci/mmol; 1 Ci = 3.7 X 1010becquerels) to final concentrations of 1 nM for routine assaysand of 0.3-300 nM for saturation studies, ADase-pretreatedtissue to a final protein concentration of 0.65-1.0 mg/ml, andbuffer to a final volume of 2 ml. Specific binding, which was90-95% of total binding at 1 nM Cl[3H]Ado, was determinedin the presence of 10 AuM unlabeled ClAdo. Concentrations ofClAdo up to 1000AM produced no further significant increasein specific binding. Drugs were added in a final volume of 0.1ml, all dilutions being made in buffer. Binding was initiated bythe addition of tissue and continued at 230C for 60 min. Re-ceptor-ligand complexes were isolated by vacuum filtrationon Whatman GF/B glass fiber filters; unbound radioactivitywas removed by two 5-ml washes with ice-cold buffer. Sampleswere counted by conventional liquid scintillation spectros-copy.

Purity of the radioligand was checked on a biweekly basisagainst a reference standard of unlabeled ClAdo by the high-performance liquid chromatography method of Anderson andMurphy (31) and was consistently greater than 99%. Exami-nation of bound radioligand after elution from filters into ab-solute alcohol and lyophilization showed a single peak of ra-dioactivity that had a k' identical to that of the original radio-active material and unlabeled ClAdo.

Cl[3H]Ado was purchased from Moravek Biochemicals (Cityof Industry, CA). With the exception of the following com-pounds, all commercial compounds were from Sigma; 8-phenyltheophylline and Tris (Ultrol-grade) from Calbio-chem-Behring, La Jolla, CA), 8-bromoadenosine from P-LBiochemicals, cyclic 8-methylthioAMP from ICN, L-N6-phenylisopropyladenosine from Boehringer-Mannheim, EHNA

6

05

0.~~~~~~03

L202

o 00~~~~~~

10 20 30 40 50Cl[3H]Ado, nM

FIG. 1. Saturation isotherms for Cl[3H]Ado binding in rat braincrude synaptic membranes. Binding was measured over a radioligandconcentration range of 0.1-50 nM. Nonspecific binding was deter-mined in the presence of 10 ,M ClAdo. Membranes were incubatedat a final concentration of 1 mg protein/ml for 60 min at 230C. Re-ceptor-ligand complexes were isolated and counted. X, Total countsbound; 0, specific counts bound; 0, nonspecific counts bound. Theseisotherms are representative of eight determinations.

(erythro-9-[3-(2-hydroxynonyl)]adenine) from BurroughsWellcome (Research Triangle Park, NC), and pronase from E.Merck, MCB Biochemicals, Norwood, OH). The following weregifts: adenosine cyclopropyl carboxamide and adenosine ethylcarboxamide from David Brodie (Abbott) and D-N6-phenyl-isopropyladenosine from John Daly (National Institutes ofHealth). Reference drugs were from the sample collection,Department of Pharmacology, Merck Institute.

Statistical Treatment of Data. ICso values for the variouscompounds were determined by using 3-5 concentrations ofeach compound in triplicate and linear regression analysis ofthe data. After a least-squares regression analysis (32) to de-termine the biphasic nature of the Scatchard plots (33), Kdvalues for Cl[3H]Ado binding from equilibrium binding ex-periments were determined by using linear regression analysis.All Scatchard data were corrected for zone behavior (34). As-sociation-dissociation data were fitted by using the nonlinearleast-squares regression program of the Cyber computer system,MSDRL, Rahway, NJ.

RESULTSSpecific binding of Cl[3H]Ado in untreated rat brain mem-branes was only 50% of the total counts bound. However, re-moval of endogenous Ado by pretreatment with ADase resultedin an increase in specific binding to 90-95% of total countsbound with no significant change in nonspecific binding (datanot shown).When 1 nM of Cl[3H]Ado was used, specific binding reached

equilibrium after approximately 50 min at 230C and remainedat equilibrium for up to 150 min, the longest time studied.Binding was completely reversible, with a large excess (1000,MM) of unlabeled ligand (data not shown).

Specific binding was saturable over the range 0.3-300 nMCl[3H]Ado (Fig. 1). Scatchard analysis (35) of saturation datasuggested the presence of two binding sites having Kd valuesof 1.31 4 0.27 nM (n = 8, mean + SD) and 16.2 + 4.10 nM (n= 7) and B. values of 207 I 36 and 380 ± 70 fmol/mg ofmembrane protein (Fig. 2). Analysis of saturation data by themethod of Hill (36) showed nH be 1.14 + 0.12 (n = 7), indi-cating no cooperative interaction between binding sites. De-termination of association (k+1) and dissociation (k-1) constantsfrom the "on-off" curves (35) gave k+1 = 5.03 X 105 M-1sec1and k-, = 5.10 X 10-4 sec-1 (n = 2). The Kd from the rela-tionship k_1/k+j = Kd was 1.11 nM for the high-affinity site.(Unless otherwise indicated, all further data relates to the

120

100 *

80

60-40

; 40-

20

0-0.1 0.2 0.3Bound, pmol/mg

FIG. 2. Scatchard plot of saturation data. Data from a typicalsaturation isotherm was subjected to Scatchard analysis. Site 1, Ki= 1.4 nM and Bm,, = 176 fmol/mg of membrane protein; site 2, Kd= 12 nM and Bm,= = 310 fmol/mg of membrane protein.

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6894 Neurobiology: Williams and Risley

Table 1. Subcellular distribution of high-affinityCl[3H]Ado binding

Specific bindingfmol/mg Totalprotein fmol %

FractionHomogenate 36.3 ± 2.4 9539 + 604 100Nuclear 26.2 ± 1.0 3300 + 116 35Crude synaptosomal 47.1 ± 7.0 4820 + 486 51Microsomal 24.5 ± 2.9 479 ± 28 5

Subfraction of crude synaptosomal fractionMyelin 8.6 I 4.9Synaptosomal 82.6 + 3.0Mitochondrial 5.8 + 1.5

Fractions were prepared as described and stored at -801C for atleast 18 hr before use. Binding was measured in the presence of 1 nMCl[2H]Ado, using 10AM ClAdo to determine specific binding. Boundcounts were isolated and determined as described. Results are mean± SD for three separate observations on pooled tissue from 3-4 wholerat brains.

high-affinity site using 1 nM Cl[3H]Ado in the presence of 10AM unlabeled ligand to define its binding characteristics.)

Binding in subcellular fractions of rat brain was highest inthe crude synaptosomal fraction (Table 1), which contained51% of the total binding sites in the homogenate. The nuclear(P1) and microsomal (P3) subfractions had, respectively, 35%and 5% of the total sites.

Subfractionation of the crude synaptosomal fraction gave a10-fold enrichment of high-affinity sites in the synaptosomalfraction, as compared with the myelin and mitochondrialfractions (see Table 1).

Analysis of regional binding by using crude synaptosomalfractions from pooled regions of rat brain showed highestbinding in the caudate and hippocampus and the lowest con-centration of high-affinity sites in the hypothalamus and spinalcord (Table 2). Binding studies in peripheral tissue under theassay conditions used to determine binding in the brain showednegligible binding in heart, liver, and kidney, but some bindingin testicle.

Binding was linear with protein concentration up to 2 mg/mland sensitive to boiling and pronase treatment (data notshown).

Table 2. Regional distribution of high-affinityCl[3H]Ado binding

Region

CaudateHippocampusCerebellumThalamusPons-medullaCortexHypothalamusSpinal cordTesticleLiverKidneyHeart

Specific binding,fmol/mgprotein

79 ± 573 ± 362 ± 1856 + 337 ± 434 ± 1530 ± 830 + 911 ± 31.7 ± 0.51.2+ 0.41.2 ± 0.7

Table 3. Pharmacology of high affinity Cl[3H]Ado bindingCompound IC50, nM

L-N6-Phenylisopropyladenosine 1.09 ± 0.17ClAdo 2.62 ± 0.56Adenosine cyclopropyl carboxamide 11.03 ± 1.27D-N6-Phenylisopropyladenosine 29.3 ± 0.7Adenosine ethyl carboxamide 42.5 ± 9.1EHNA 61 ± 175-Deoxy-5'-methylthioadenosine 66 ± 128-Phenyltheophylline 116 i 221-Methyladenosine 323 ± 21p[NH]ppA 515 + 143Cyclic AMP 751 + 115Dipyridamole 998 ± 545'-Guanylyl imidodiphosphate 1,167 ± 202GTP 1,765 ± 343Isobutylmethylxanthine 3,922 ± 325Theophylline 8,775 ± 2,947Ado 20,207 ± 5,694Caffeine 27,300 ± 8,2008-Bromoadenosine 51,000 ± 8,000Inosine 114,500 ± 17,700

Compounds without significant effect on Cl[3H]Ado binding at 100.uM included hypoxanthine, adenine, adenine sulfate, cytosine, thy-midine, D-ribose, D-AMP, 2-deoxy-3'-monophosphate, guanosine,guanine, adenosine-5'-monosulfate-3-deoxyadenosine, uracil, 2-deoxyadenosine, 2-deoxyadenosine-3',5'-cyclic AMP, cyclic 8-methylthioAMP, cyclic GMP, norepinephrine, dopamine, y-ami-nobutyric acid, serotonin, clonidine, diazepam, naloxone, phento-lamine, propranolol, yohimbine, atropine, methergoline, mianserine,methysergide, imipramine, haloperidol, ethanol, hydralazine, RO-20-1724, papaverine, ouabain, dithiothreitol, mercaptoethanol.Displacement of the binding of 1 nM of Cl[3H]Ado was measured

under routine assay conditions. All compounds were run at 3-7 con-centrations in the binding assay, and IC5os were determined by linearregression of the log-probit analysis for each compound.

Results are the mean + SD for 3-6 separate observations.

A number of purine-related compounds, alkylxanthines andcompounds that affect Ado function displace Cl[3H]Ado in aconcentration-dependent manner (Table 3). Other centrallyactive agents at 100 MM had no significant effect on binding.

DISCUSSIONAfter pretreatment with ADase to remove endogenous Ado (5,30), crude synaptic membranes from rat brains showed twobinding sites for Cl[3H]Ado. Binding to the high-affinity site(Kd = 1.3 nM and B,,. = 207 fmol/mg membrane protein) wassaturable, indicating a finite number of binding sites, and re-versible (data not shown); the highest number of binding sitesoccurred in the enriched synaptosomal fraction. These physicaland anatomical observations are consistent with four of thecriteria proposed by Cuatrecasas and Hollenberg (37) for areceptor.The tissue distribution of binding is also consistent with a role

unique to nervous tissue. Negligible binding was observed inheart, kidney, and liver, tissues in which purinergic innervationis either absent or occurs only in defined areas (1). The bindingin testicle (and the associated vas deferens), a tissue rich inpurinergic receptors (1), was -10-fold higher than that in theother peripheral tissues, while brain tissue was 3-7 times richerin high-affinity binding sites than testicle. Binding was highestin the caudate and hippocampus and lowest in spinal cord andhypothalamus. This was reflected in differences in the numberof binding sites (Bma) in cortex, caudate, and liver (data notshown), rather than in affinity (Kd), indicating that the regionaldistribution observed is not due to the presence of differing

Crude synaptosomal regions were prepared by using tissue pooledfrom the brain regions of 3-4 rats. Peripheral tissues were treated ina similar manner. Binding was measured in the presence of 1 nMCl[3H]Ado using 10AM ClAdo to determine specific binding. Resultsare mean + SD for three separate observations.

Proc. Natl. Acad. Sci. USA 77 (1980)

Proc. Natl. Acad. Sci. USA 77 (1980) 6895

amounts of residual endogenous adenosine in the regionsstudied.The Hill coefficient (nH) for Cl[3H]Ado binding was not

significantly different from 1.0, indicating that there is no co-operative interaction between binding sites and that, under theassay conditions used, there is only one class of high-affinitysites.

Binding was sensitive to the proteolytic enzyme, pronase, andto boiling, suggesting that part of the binding site for Cl[3H]Adois protein in nature. Dithiothreitol (100MM) and mercapto-ethanol (100MM), sulfhydryl group reagents, had no effect onbinding, suggesting, although not proving, that disulfide bridgesare not involved in the interaction of the radioligand with itsbinding site.The pharmacology of Cl[3H]Ado binding is, in general,

consistent with that previously reported for the biochemical andelectrophysiological effects of Ado in the central nervous system(3-6, 11). Most important, binding is sensitive to theophylline.This Ado-antagonist was a weak inhibitor of high-affinitybinding (IC50 = 8.8 MM), in agreement with its previously re-ported effects on Ado-evoked iontophoretic and adenylatecyclase-related responses (5, 6, 11, 12). 8-Phenyl theophylline,which is 10-17 times more active as an antagonist of the brainslice cyclase system than theophylline (38), was 75 times moreactive than the parent alkylxanthine in the binding assay. Iso-butylmethylxanthine (IBMX), although more potent thantheophylline, was also a weak antagonist of binding (IC50 = 3.9MiM). Similarly, caffeine, which is less active than either theo-phylline and IBMX in antagonizing the effects of the purinein guinea pig neocortical slices (7) was 14-25% as active in thebinding assay.The D-isomer of phenylisopropyladenosine was t4% as ac-

tive in displacing Cl[3H]Ado as the L isomer, in agreement withthe stereospecific effects reported for the two isomers on mousemotor activity, guinea pig heart rate and coronary flow (16),cyclic AMP production (39), and evoked excitatory postsynapticpotential inhibition in rat hippocampal slices (11, 39). Adenosinecyclopropyl and ethyl carboxamides were, respectively, 25%and 6% as active as ClAdo, suggesting that modifications to theribose moiety alter the binding characteristics of the purine.Ado itself was a very weak inhibitor of high-affinity binding,

a fact that may be attributed to the presence of residual ADaseactivity in the pretreated membrane preparation. This sup-position was supported by the marked effects of the potentADase inhibitor, EHNA (40), on binding, which are presumablydue to endogenous Ado formation, and by the fact that ma-nipulation of ADase-pretreated tissue can dramatically enhancethe effectiveness of Ado in displacing Cl[3H]Ado. Thus, incu-bating Ado in the presence of 50 nM EHNA under equilibriumconditions at 4°C gave an IC5o for the parent purine that wasvirtually identical to that obtained for ClAdo under the sameassay conditions, while 8-phenyltheophylline gave approxi-mately the same IC50 under these conditions as it did underroutine assay conditions, indicating that, to the limited extentstudied, the EHNA at 4°C assay conditions do not appreciablyalter the pharmacology of binding (data not shown). Further-more, by using washed ADase-pretreated crude synapticmembrane preparations in which EHNA had no concentra-tion-dependent effect on binding, Ado was found to have anIC50 of 1 nM.Although 50% inhibition of binding was routinely obtained

for AMP, ADP, and ATP over the range 1-20,uM, the biphasicand triphasic displacement curves observed, coupled with theobservation that the slowly metabolizable analog of ATP,p[NH]ppA, gave consistent results indicated that, under theassay conditions used, the nucleotides were undergoing meta-bolic degradation.

p[NH]ppA and cyclic AMP were t~0.3-0.5% as active asClAdo, while the guanine nucleotides 5'-guanylyl imidodi-phosphate and GTP were weaker still in inhibiting binding.These observations are consistent with the Cl[3H]Ado bindingsite being specific for adenine nucleosides rather than for nu-cleotides, similar to the specificity of the pharmacologicallydefined P1 purinergic receptor (1). Binding is also specific foradenine rather than guanine nucleotides, guanosine itself beingwithout effect. Adenine and D-ribose, the separate moieties ofAdo, had no effect on binding, while inosine and hypoxanthine,the catabolic products of ADase activity, were weak inhibitors.Inosine, which is without effect on slice cyclic AMP production(5), had an IC50 of 114 MM (Table 3), while hypoxanthine wastotally inactive. The finding that cyclic AMP was t0.5% asactive as ClAdo, while cyclic 8-methylthioAMP and cyclic GMPwere inactive, eliminates the possibility that binding is to acyclic AMP binding protein. The affinity of binding and thepharmacology are, however, consistent with data on the in-hibitory (A1) Ado receptor in fetal mouse brain (41) and othercells (42).

Dipyridamole, an Ado-uptake inhibitor (4), was reasonablyeffective in displacing the radioligand, while 8-bromoadenos-ine, 2-deoxyadenosine, and 3-deoxyadenosine (cordecypin)were very weak inhibitors, which agrees with their lack ofAdo-agonist activity in guinea pig neocortical slice preparations(6). 1-Methyladenosine, which is inactive on the cyclase (43),was reasonably active, while the 5-deoxy-5'-methylthio de-rivative was the most active Ado analog examined [except forthe ADase-resistant (6, 16) N6-substituted phenylisopropyl and2-chloro analogs].

Although these data would appear to be indicative of thestructure-activity requirements for the Cl[3H]Ado binding site,it must be born in mind that activity under our assay conditionsis a result of two separate factors; resistance to ADase activityand recognition of the high-affinity site. Thus, although ribosemodification, as in the carboxamides, alters binding affinity,this may be indicative of an effect on metabolic stability ratherthan of site specificity (44).The lack of effect of a number of centrally active compounds

on binding further supports the specificity of the Cl[3H]Adosite. The compounds tested that were inactive included a- and,B-adrenergic, opiate, serotonergic, dopaminergic, and mus-carinic cholinergic blocking agents; the neurotransmittersnorepinephrine, dopamine, a-aminobutyric acid, and serotonin;the central depressant, alcohol; the antidepressant, imipramine;and the anxiolytic agent, diazepam. RO 20-1724, a phospho-diesterase inhibitor that has some effect on adenosine uptake(6) was likewise inactive, as were ouabain, an ATPase inhibitor;papaverine, also a weak phosphodiesterase inhibitor (43); hy-dralazine, a compound suggested to have purine-agonist activity(45); and clonidine, a putative Ado antagonist (46).

During the course of our work, Phillis et al. (47) reportedCl[3H]Ado binding to a single high-affinity (Kd = 23 nM)binding site in untreated rat neocortical membranes. Bindingwas theophylline sensitive (IC50 = 350 nM) and showed regionaldistribution. It was sensitive to inosine, hypoxanthine, and ad-enine, in contrast to the binding reported here and that reportedby Bruns et al. (30), who used [3H]cyclohexyladenosine as li-gand.

In conclusion, the data presented here describe a reversible,high-affinity binding of a stable analog of Ado to rat brainsynaptic membranes. The pharmacology and the regional andsubcellular distribution of binding are, in general, consistentwith the concept that the binding site(s) is a central purinergicreceptor through which Ado may evoke its effects on centralnervous system function.

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6896 Neurobiology: Williams and Risley

We would like to thank James Totaro for performing the high-pressure liquid chromatography analyses; Dr. William Randall forperforming the computer analysis of binding data; Drs. John Daly, JohnPhillis, and Clement Stone for helpful discussions, and Ms. Dianne E.McDonald for excellent secretarial assistance.

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