Characterization of adenosine receptors in a model of cultured neurons from rat forebrain

The neuromodulator adenosine is acting through specific receptors coupled to adenylate cyclase via G-proteins. The expression of both adenosine receptors A₁ and A₂ as well as forkolin binding sites was investigated by radioligand binding techniques in 8-day-old neurons isolated from fetal rat forebrain and cultured in chemically-defined medium. Adenosine A₁ receptors were specifically labeled with [³H]chloro-N⁶-cyclopentyladenosine (CCPA), whereas [³H]CGS 21680 was used for the analysis of A₂ receptors. Cultured neurons exhibited high affinity binding sites for CCPA (Bmax=160 fmol/mg protein; Kd=2.9 nM), and for CGS 21680 (Bmax=14 fmol/mg protein; Kd=1.7 nM). These data correlate well with those obtained in crude membranes isolated from the newborn rat forebrain. The incubation of culture membranes in the additional presence of guanylyl-5-imidodiphosphate (Gpp(NH)p, a GTP analogue) led to significantly increased Kd-values, suggesting the association of adenosine receptors with G-proteins. Finally, cultured neurons also bound specifically [³H]forskolin with characteristics close to those found in the newborn brain, indicating that cultured neurons appear as an appropriate model for studying the neuromodulatory properties of adenosine.     

Ontogeny of Adenosine Binding Sites in Rat Forebrain and Cerebellum

The metabolically stable adenosine analogue N6-cyclohexyl [3H]adenosine ([3H]CHA) was used to label adenosine receptors in rat forebrain and cerebellum during development. [3H]CHA binding develops rather slowly, with adult binding levels obtained at 24 days in cerebellum and later in the forebrain. Ontogenic profiles in both areas are consistent with the onset of neuronal differentiation. High and low affinity sites appear to develop in parallel, since Scatchard analysis in forebrain tissue obtained from 5-day-old animals revealed both binding sites.     

Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide

The regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide (NEM) was investigated using the agonist ligand, [³H] cis methyldioxolane ([³H] CD). Characterization studies on rat forebrain homogenates showed that [³H] CD binding was linear with tissue concentration and was unaffected by a change in pH from 5.5 to 8.0. The regional variation in [³H] CD binding in the rat brain correlated generally with [³H] (?)3-quinuclidinyl benzilate ([³H] (?)QNB) binding, although the absolute variation in binding was somewhat less. At a concentration of 100 μM, the GTP analogue, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], caused a 43–77% inhibition of [³H] CD binding in the corpus striatum, ileum, and heart. The results of binding studies using several Gpp(NH)p concentrations demonstrated that the potency of this guanine nucleotide for inhibition of [³H] CD binding was greater in the heart than in the ileum. In contrast to its effects on [³H] CD binding, Gpp(NH)p caused an increase in [³H] (?)QNB binding in the heart heart and ileum and no change in [³H] (?)QNB binding in the corpus striatum. When measured by competitive inhibition of [³H] (?)QNB binding to the longitudinal muscle of the ileum, Gpp(NH)p (100 μM) caused an increase in the IC₅₀ values of a series of agonists in a manner that was correlated with the efficacy of these compounds. The results of binding studies on NEM treated forebrain homogenates revealed an enhancement of [³H] CD binding by NEM.     

Binding of [3H]forskolin to human platelet membranes. Regulation by guanyl-5'-yl imidodiphosphate, NaF, and prostaglandins E1 and D2

[3H]Forskolin binds to human platelet membranes in the presence of 5 mM MgCl2 with a Bmax of 125 fmol/mg of protein and a Kd of 20 nM. The Bmax for [3H]forskolin binding is increased to 455 and 425 fmol/mg of protein in the presence of 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and 10 mM NaF, respectively. The increase in the Bmax for [3H]forskolin in the presence of Gpp(NH)p or NaF is not observed in the absence of MgCl2. The EC50 values for the increase in the number of binding sites for [3H]forskolin by Gpp(NH)p and NaF are 600 nM and 4 mM, respectively. The EC50 value for Gpp(NH)p to increase the number of [3H]forskolin binding sites is reduced to 35 mM and 150 nM in the presence of 50 microM PGE1 or PGD2, respectively. The increase in the number of [3H]forskolin binding sites observed in the presence of NaF is unaffected by prostaglandins. The binding of [3H]forskolin to membranes that are preincubated with Gpp(NH)p for 120 min or assayed in the presence of PGE1 reaches equilibrium within 15 min. In contrast, a slow linear increase in [3H]forskolin binding is observed over a period of 60 min when Gpp(NH)p and [3H]forskolin are added simultaneously to membranes. A slow linear increase in adenylate cyclase activity is also observed as a result of preincubating membranes with Gpp(NH)p. In human platelet membranes, agents that activate adenylate cyclase via the guanine nucleotide stimulatory protein (Ns) increase the number of binding sites for [3H]forskolin in a magnesium-dependent manner. This is consistent with the high affinity binding sites for [3H]forskolin being associated with the formation of an activated complex of the Ns protein and adenylate cyclase. This state of the adenylate cyclase may be representative of that formed by a synergistic combination of hormones and forskolin.     

Characterization of [3H]forskolin binding sites in the iris-ciliary body of the albino rabbit

[3H]Forskolin binding sites were identified using membranes prepared from the iris-ciliary body of adult, albino rabbits. Scatchard analysis of saturation binding experiments demonstrated that [3H]forskolin bound to a single population of high affinity sites. The Kd and Bmax values were 8.7 +/- 0.9 nM and 119.0 +/- 30.9 fmol/mg prot. using membranes prepared from frozen tissue and 17.0 +/- 6.2 nM and 184.4 +/- 47.2 fmol/mg prot. using fresh tissue. The binding of [3H]forskolin was magnesium-dependent. The Bmax was enhanced by sodium fluoride and Gpp(NH)p, a nonhydrolyzable guanine nucleotide analog. Forskolin was the most potent inhibitor of [3H]forskolin binding; two commercially-available analogs were weaker inhibitors. In an adenylate cyclase assay, there was the same rank order of potency to enhance enzyme activity. Based upon binding affinities, magnesium-dependence, sensitivity to sodium fluoride and Gpp(NH)p, rank order of potencies of analogs and correlation of binding with adenylate cyclase activity, these studies suggest that the [3H]forskolin binding site in the iris-ciliary body is similar to the binding site in other tissues.     

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [³H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [³H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent K_D for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 M) with no change in B_max. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the B_max was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [³H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [³H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [³H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The B_max values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [³H]SR 95531 binding whereas the treatment increases apparent affinity of [³H]SR 95531 and [³H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein. These results also suggest that properties of cerebellar GABA_A receptors are different from those in other regions.Abbreviations used GABA -aminobutyric acid - FC frontal cortex - CB cerebellum - ST striatum - SN substantia nigra     

Differential Binding Properties of Adenosine Receptor Agonists and Antagonists in Brain

The binding properties of N6-cyclohexyl [3H]adenosine ( [3H]CHA) and 1,3-diethyl-8-[3H]phenylxanthine ( [3H]DPX) in rat forebrain membrane are compared. The kinetic parameters of binding for each ligand are quite distinct, with [3H]CHA displaying two populations of binding sites (KD = 0.4 +/- 0.05 nM and 4.2 +/- 0.3 nM; Bmax = 159 +/- 17 and 326 +/- 21 fmol/mg protein), whereas [3H]DPX yielded monophasic Scatchard plots (KD = 13.9 +/- 1.1 nM; Bmax = 634 +/- 27 fmol/mg protein). The metals copper, zinc, and cadmium are potent inhibitors of [3H]CHA binding, with respective IC50 concentrations of 36 microM, 250 microM, and 70 microM. Copper is a much less potent inhibitor of [3H]DPX binding (IC50 = 350 microM). The inhibitory effect of copper on both [3H]CHA and [3H]DPX binding is apparently irreversible, as membranes pretreated with copper cannot be washed free of its inhibitory effect. The inhibitory effect of both copper and zinc on [3H]CHA binding was reversed by the guanine nucleotide Gpp(NH)p. [3H]DPX binding is only partially inhibited by zinc and cadmium (60% of specific binding remains unaffected), suggesting that this adenosine receptor ligand binds to two separate sites. Guanine nucleotides had no effect on the inhibition of [3H]DPX binding by either copper or zinc. Differential thermal and proteolytic denaturation profiles are also observed for [3H]CHA and [3H]DPX binding, with the former ligand binding site being more labile in both cases. Stereospecificity is observed in the inhibition of both [3H]CHA and [3H]DPX binding, with L-N-phenylisopropyladenosine (PIA) being 50-fold more potent than D-PIA in both cases. Evidence is therefore provided that adenosine receptor agonists and antagonists have markedly different binding properties to brain adenosine receptors.     

Dissociation of the plasticity of 5-HT1A sites and 5-HT transporter sites

To study the early effects of neonatal 5,7-dihydroxytryptamine lesions on 5-hydroxytryptamine_1A (5-HT_1A) receptors, we measured regional [³H]8-OH-DPAT-labeled 5-HT_1A sites in binding assays and compared them to our previous studies of [³H]paroxetine-labeled 5-HT transporter sites during the first month in the same rats. While there were significant time- and dose-dependent effects of 5,7-DHT on 5-HT transporter sites, there were no significant changes in 5-HT_1A sites in cortex, hippocampus, diencephalon, brainstem, cerebellum, or spinal cord. 5,7-DHT lesions also did not alter the K_i of Gpp(NH)p at brainstem 5-HT_1A sites or the K_i of 5-HT in cortex or brainstem in the presence or absence of GTPS or Gpp(NH)p. There were significant regional differences between the density of 5-HT_1A sites and 5-HT transporter sites. The ontogeny of brainstem 5-HT_1A sites was a pattern of increases until three weeks postnatal, and 5,7-DHT lesions did not alter the ontogeny of 5-HT_1A sites. These data suggest differential plasticity of 5-HT_1A and 5-HT transporter binding sites during the first month after neonatal 5,7-DHT lesions.     

Upregulation of (+)-7-Hydroxy-N,N-di-n-[3H]Propyl-2-Aminotetralin Binding Following Intracerebroventricular Administration of a Nitric Oxide Generator

Nitric oxide modulation of dopamine D₂ and D₃ receptor binding was examined using [¹²⁵I]epidepride (D₂) and (+)7-hydroxy-N,N-di-n-[³H]propyl-2-aminotetralin([³H](+)-7-OH-DPAT, D₃). Nitric oxide, generated by i.c.v. injection of S-nitroso-N-acetyl-penicillamine (SNAP; 5 g or 10 g), significantly increased the density of [³H](+)-7-OH-DPAT binding sites (39% and 134%, respectively) in the striatum 24 hours post-injection in the absence of Gpp(NH)p, representing an upregulation of either D₃, receptors or high affinity D₂ receptors. In the presence of 10 M Gpp(NH)p, D₃ receptor upregulation was maintained in both the 5 g (increased 35%) and 10 g SNAP (increased 44%) groups. [³H](+)-7-OH-DPAT binding was reduced in both striatum and nucleus accumbens in the presence of 10 M Gpp(NH)p compared to binding in the absence of Gpp(NH)p, suggesting an upregulation of D₃ receptors. Administration of SNAP did not alter total specific [¹²⁵I]epidepride binding in either brain region. These data suggest that; 1) D₃ receptor density is modified following nitric oxide generation, and 2) the density of high affinity D₂ receptors identified by [³H](+)-7-OH-DPAT increases in the striatum, but decreases in the nucleus accumbens.     

An [3H]oxotremorine binding method reveals regulatory changes by guanine nucleotides in cholinergic muscarinic receptors of cerebral cortex

A rapid, reliable filtration method for [³H]oxotremorine binding to membranes of the cerebral cortex that allows the direct study of regulation by guanine nucleotides of muscarinic receptors was developed. [³H]Oxotremorine binds to cerebral cortex membranes with high affinity (K _D, 1.9 nM) and low capacity (B _max, 187 pmol/g protein). These sites, which represent only about 18% of those labeled with [³H]quinuclidinyl benzilate, constitute a population of GTP-sensitive binding sites. Association and dissociation binding experiments revealed a similar value ofK _D (2.3 nM). Displacement studies with 1–4000 nM oxotremorine showed the existence of a second binding site of low affinity (K _D, 1.2 M) and large capacity (B _max, 1904 pmol/g protein). Gpp(NH)p, added in vitro, produced a striking inhibition of [³H]oxotremorine binding with an IC 50 of 0.3 M. Saturation assays, in the presence of 0.5 M Gpp(NH)p, revealed a non-competitive inhibition of the binding with little change in affinity. These results are discussed from the viewpoint of conflicting reports in the literature about guanine nucleotide regulation of muscarinic receptors in reconstituted systems and membranes from different tissues.     

MgCl2-sensitive and Gpp(NH)p-sensitive antagonist binding states of rat heart muscarinic receptors: preferential detection at ambient temperature assay and location in two subcellular fractions

Summary Some novel observations dealing with antagonist binding to cardiac particulate muscarinic receptors are described. Gpp(NH)p increased (2–3 fold) the specific binding of [³H]-QNB or [³H]-NMS, both potent muscarinic antagonists, to washed particles (WP), but not microsomes (MIC), when the binding was conducted at 30°C. Magnesium, on the other hand, increased (2–3 fold) the binding of these antagonists to MIC, but not to WP, under the same condition. The treatment of subcellular fractions with 0.2 mM N-ethylmaleimide (NEM), a sulfhydryl reagent, failed to significantly modify the respective stimulatory actions of either Gpp(NH)p on WP binding or of magnesium on MIC binding of these antagonists; treatment with dithiothreitol (1 mM) was also ineffective in this regard. Gpp(NH)p decreased K_d (WP) while magnesium increased K_d (MIC) for [³H]-QNB. Repeated freezing/thawing of isolated subcellular fractions abolished the stimulatory effect of magnesium on onist binding to MIC but not of Gpp(NH)p on WP antagonist binding; the freeze/thaw procedure per se increased MIC binding but not WP binding of these antagonists. When the binding was conducted at 4°C (24 hr), the stimulatory effect of Gpp(NH)p on [³H]-QNB binding was enhanced (6-fold) in the case of WP and was detectable (80%) in the case of MIC. Under this condition, the stimulatory effect of magnesium on [³H]-QNB binding was also enhanced (5-fold) in the case of MIC and became evident (200%) in the case of WP. The results of this work support the following views: (a) antagonist-occupied cardiac muscarinic receptors are capable of interaction with guanine nucleotide binding proteins (G protein like G₁,G_o) and such interaction influences antagonist binding properties (e.g. increased affinity) of the cardiac membrane-associated muscarinic receptors (b) magnesium influences (decreased affinity) antagonist binding properties by interacting with multiple sites of which some are likely associated with components other than G proteins of the particulate fractions (c) a pool of NEM-sensitive sulfhydryls involved in the regulation of Gpp(NH)p-sensitive agonist binding to cardiac muscarinic receptors is not involved in the regulation by either Gpp(NH)p or magnesium of antagonist binding in these subcellular fractions and (d) membrane fluidity and microenvironment surrounding the receptor and G proteins contribute to the actions of Gpp(NH)p and magnesium on antagonist binding.     

ADP- and epinephrine-elicited release of [3H]guanylylimododiphosphate from platelet membranes: Implications for receptor-Ni stoichiometry

Epinephrine-promoted release of [³H]guanylylimidodiphosphate ([³H]Gpp(NH)p) from human platelet membranes has been used to probe the interactions between alpha₂-adrenergic recpetors and N_i, the guanine nucleotide binding protein that couples those receptors to an inhibition of adenylate cyclase activity. We show here that ADP, which also acts through specific platelet receptors to inhibit adenylate cyclase activity, also promotes the release of [³H]Gpp(NH). The amount of [³H]Gpp(NH)-release elicited by epinephrine and by ADP together is equal to the sum of the amounts released by the two agents acting individually. Furthermore the maximal amounts of [³H]Gpp(NH)-release elicited by each of the two agents approximates the numbers of receptors for ADP and epinephrine present in the platelet membranes. These results suggest that the two receptor types interact with distinct portions of the pool of N_i molecules and that each receptor initiates guanine-nucleotide exchange on a single molecule of N_i.     

Pertussis vaccine reduces agonist binding to the rat heart muscarinic receptor and its guanine nucleotide modulation

The injection ofBordetella pertussis, inactivated by merthiolate, causes a 2-fold increase in the IC50 of carbamylcholine (carbachol) in displacing [³H];-L(–) quinuclidinyl benzilate binding ([³H]QNB) to the receptor. In control animals, 50 M Gpp(NH)p causes a 6-fold decrease in the affinity of carbachol binding, whereas after vaccination the reduction is only 1.6-fold. After pertussis treatment there is no alteration in the affinity and number of [³H]QNB binding sites of to the muscarinic receptor.     

Differential effect of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on [3H]SCH23390 and [3H]forskolin binding in rat striatum

The binding of [3H]forskolin to a homogeneous population of binding sites in rat striatum was enhanced by NaF, guanine nucleotides and MgCl2. These effects of NaF and guanylylimidodiphosphate (Gpp(NH)p) were synergistic with MgCl2, but NaF and Gpp(NH)p together elicited no greater enhancement of [3H]forskolin binding. These data suggest that [3H]forskolin may label a site which is modulated by the guanine nucleotide regulatory subunit which mediates the stimulation of adenylate cyclase (NS). The D1 dopamine receptor is known to stimulate adenylate cyclase via NS. In rat striatum, the Bmax of [3H]forskolin binding sites in the presence of MgCl2 and NaF was approximately two fold greater than the Bmax of [3H]SCH23390-labeled D1 dopamine receptors. Incubation of striatal homogenates with the protein modifying reagent EEDQ elicited a concentration-dependent decrease in the binding of both [3H]SCH23390 and [3H]forskolin, although EEDQ was approximately 14 fold more potent at inactivating the D1 dopamine receptor. Following in vivo administration of EEDQ there was no significant effect on [3H]forskolin binding sites using a dose of EEDQ that irreversibly inactivated greater than 90% of D1 dopamine receptors. These data suggest that EEDQ is a suitable tool for investigating changes in the stoichiometry of receptors and their second messenger systems.     

Differential inhibition of brain specific [3H]flunitrazepam binding by several types of dyes

Several dyes, representing different structural classes, inhibit [³H]flunitrazepam binding to brain specific receptors in the rat with 50% inhibition in the 1 to 100 M range. Crystal Violet and Methyl Violet 2B inhibited more potently in the forebrain than in the cerebellum. Congo Red yielded a Hill number near 2.3, probably reflecting positive cooperativity between interacting binding sites in benzodiazepine receptor complexes. Toluidine Blue 0 was the most potent of the dyes tested (IC₅₀=1 M in cerebellum) and inhibited more potently in cerebellum than in forebrain.     

Differential regulation of high-affinity agonist binding to muscarinic sites in the rat heart, cerebellum, and cerebral cortex

The muscarinic agonist [3H]cismethyldioxolane ([3H]CD) was used to characterize the effects of regulators upon high-affinity agonist binding sites of the rat heart, cerebral cortex and cerebellum. Comparative studies with sodium ions (Na+), magnesium ions (Mg++), N-ethylmaleimide (NEM) and the guanine nucleotide Gpp(NH)p revealed tissue-specific effects. Mg++ preferentially enhanced while Gpp(NH)p and NEM reduced high-affinity [3H]CD binding in the heart and cerebellum. By comparison NEM enhanced high-affinity agonist binding in the cerebral cortex while Gpp(NH)p and Mg++ had little or no effect. Kinetic studies support an allosteric mechanism for these effects and provide further evidence for muscarinic receptor subtypes in mammalian tissues.     

Effects of Chronic Administration of Interferon α A/D on Serotonergic Receptors in Rat Brain

The effects of chronic administration of interferon (IFN; recombinant human IFN -A/D) on serotonergic binding sites in rat brain were investigated. IFN was injected daily for 2 weeks at a dose of 100000 I.U./kg, (i.p.) in male Wistar rats. IFN did not alter either [³H]ketanserin binding to 5-HT_2A receptors or [³H]paroxetine binding to 5-HT transporters. Scatchard analysis of [³H]8-hydroxy-dipropylaminotetraline (8-OH-DPAT) binding to 5-HT_1A receptors demonstrated the presence of high- and low-affinity binding sites in both treatment and control groups. IFN significantly increased both Kd and Bmax measures of [³H]8-OH-DPAT binding at low-affinity binding sites, but not at the high-affinity sites. These results suggest that IFN affects the low-affinity 5-HT_1A receptors sites and may be involved in the development of IFN-induced psychiatric disturbances.     

Characterization of adenosine receptors in Mullus surmuletus

The intent of the present study was to investigate adenosine receptor sites in brain membranes of the saltwater teleost fish, Mullus surmuletus, using the A₁ receptor selective agonist, [³H]CHA, and A_2a receptor selective agonist [³H]CGS 21680. The A₁ selective agonist, [³H]CHA, bound saturably, reversibly and with high affinity to a single-class of binding sites (K_d 1.47 nM; B_max 100–190 fmol/mg protein, dependent on fish length). The A_2a selective agonist, [³H]CGS 21680, also bound saturably, reversibly and with relative high affinity to a single-class of binding sites (K_d 44.2 nM; B_max 150–300 fmol/mg protein dependent on fish length). In equilibrium competition experiments, adenosine analogous, NECA, CGS 21680, CHA, CPA, S-PIA, R-PIA, CPCA, DPMA, and xanthine antagonists, DPCPX, XAC, and THEO all displaced [³H]CHA and [³H]CGS 21680 specifically bound to brain membranes from Mullus surmuletus. Specific binding of both [³H]CHA and [³H]CGS 21680 was inhibited by GDPβS. For [³H]CHA the IC₅₀ value was 2.5 ± 0.1 μM, while for [³H]CGS 21680 the IC₅₀ value was 7.7 ± 0.3 μM. Our results indicate that the high affinity binding sites for [³H]CHA have some pharmacological characteristics of mammalian A₁ adenosine receptors, while the binding sites for [³H]CGS 21680 appear to be virtually identical to the binding sites for [³H]CHA.     

Ontogenetic Profile of the Adenosine Uptake Sites in Rat Forebrain

The ontogenesis of rat forebrain adenosine uptake sites labelled by [3H]nitrobenzylthioinosine ([3H]NBI) was determined and compared to that of rat forebrain adenosine receptors labelled by N6-cyclohexyl[3H]adenosine ([3H]-CHA). [3H]NBI binding is highly invariant with similar levels of [3H]NBI binding sites from embryonic day 19 to day 30 postpartum. Scatchard and Hill analyses reveal the binding of [3H]NBI in 6-day-old tissue to be indistinguishable from such binding in 30-day-old tissue. In contrast, [3H]-CHA binding is highly variant. [3H]CHA binding develops slowly but steadily from about embryonic day 19, with adult binding levels being achieved at around 25 days postpartum. The ontogenetic profile of [3H]CHA appears to coincide with synaptogenesis whereas that of [3H]NBI does not.     

The amphiphilic peptide adenoregulin enhances agonist binding to A1-adenosine receptors and [35S]GTPγS to brain membranes

Summary 1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog,Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes.2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 µM) for A₁-adenosine receptors, 30% (100 µM) for A_2a-adenosine receptors, 20% (2 µM) for ₂-adrenergic receptors, and 30% (100 µM) for 5HT_1A receptors. High affinity agonist binding for A_1-, _2-, and 5HT_1A-receptors was virtually abolished by GTPS in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin.3. The effect of adenoregulin on binding of N⁶-cyclohexyladenosine ([³H]CHA) to A₁-receptors was relatively slow and was irreversible. Adenoregulin increased the B_max value for [³H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [³H]CHA dissociation. Binding of the A₁-selective antagonist, [³H]DPCPX, was maximally enhanced by only 13% at 2 µM adenoregulin. Basal and A₁-adenosine receptor-stimulated binding of [³⁵S]GTPS were maximally enhanced 45% and 23%, respectively, by 50 µM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [³H]CHA and [³H]DPCPX were enhanced by adenoregulin. Binding of [³H]CHA to membranes from DDT₁ MF-2 cells was maximally enhanced 17% at 20 µM adenoregulin. In intact DDT₁ MF-2 cells, 20 µM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediatedvia the adenosine A₁ receptor.4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein.