Identification of Adenosine Receptor in Rat Pineal Gland: Evidence for A-2 Selectivity

We have examined the binding of the adenosine agonist radioligands [3H]cyclohexyladenosine [( 3H]CHA), R-N6-[3H]phenylisopropyladenosine [( 3H]R-PIA), and 5'-N-ethylcarboxamido[3H]adenosine [( 3H]NECA) to membranes prepared from rat pineal gland. The results showed that the A-1-selective ligands (CHA and R-PIA) had less than or equal to 10% specific binding. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 72% specific binding of the total binding. This specific binding was insensitive to cyclopentyladenosine (50 nM) or R-PIA (50 microM). To characterize this binding, we used the N-ethylmaleimide pretreatment method. Under these conditions, this binding was of high affinity with a KD of 51 +/- 10 nM and an apparent Bmax of 1,060 +/- 239 fmol/mg of protein. Specific binding was unaffected by the presence of MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM) (-25%), a result suggesting the involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. The rank of activity of adenosine analogues in displacing specific [3H]NECA binding was NECA greater than 2-chloroadenosine greater than S-adenosyl-L-homocysteine greater than CHA. Binding was also displaced by 3-isobutyl-1-methylxanthine (IC50 = 23.6 microM). These findings are consistent with the selective labeling by [3H]NECA of an A-2-type adenosine receptor in rat pineal membranes.     

Characterization of A-2 Receptors in Postmortem Human Pineal Gland

We have examined the binding of the adenosine agonist radioligands [3N]N6-cyclohexyladenosine ([3H]CHA) and [3H]5'-N-ethylcarboxamidoadenosine ([3H]NECA) to membranes prepared from postmortem human pineal glands. The results showed that the A-1-specific ligand CHA did not bind to membranes. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 68% specific binding of the total binding. This specific binding was nearly insensitive to the N-ethyl-maleimide pretreatment method. To characterize this binding, we used cyclopentyladenosine (50 nM). Under those conditions [3H]NECA binding at 30 degrees C was rapid and reversible; the KD determined from the kinetic studies was 141 nM. In postmortem human pineal gland, the rank order of potency of adenosine analogues and drugs competing with [3H]NECA showed the specificity for an A-2 receptor: NECA greater than 2-chloroadenosine greater than L-N6(2-phenylisopropyl)adenosine greater than 8-phenyltheophylline greater than 3-isobutyl-1-methylxanthine greater than caffeine. Guanylylimidodiphosphate (100 microM) induced a decrease in the affinity of [3H]NECA, a result suggesting the involvement of a G protein mechanism in the coupling of the adenosine receptor to other components of the receptor complex. Scatchard analysis revealed one class of binding sites for [3H]NECA with KD and Bmax ranging from 175 to 268 nM and 11.0 to 14.1 pmol/mg protein, respectively. The binding of [3H]NECA was not affected by age, sex, or postmortem delay. [3H]NECA should be a useful tool to assess brain A-2 receptor density in a variety of neuropsychiatric disorders.     

Dissociation between adenosine receptors and adenylate cyclase in the smooth muscle of guinea pig myometrium

We have previously demonstrated that adenosine causes contraction of guinea-pig myometrium in a fashion consistent with the presence of a purinergic receptor of the A1 subtype. Incubation of guinea-pig uterine smooth muscle membranes with the stable adenosine analogue [3H]cyclohexyladenosine [( 3H]CHA) resulted in rapid, reversible association of radioligand to saturable sites. The affinity (KD) of the receptor for [3H]CHA determined from kinetic experiments (3.14 nM) is in good agreement with that determined in saturation experiments (KD = 4.5 nM). Scatchard analysis of specific [3H]CHA binding (Bmax = 79 fmol/mg protein) is consistent with a single class of binding sites for [3H]CHA. Computer analysis of competition of [3H]CHA binding by the stereoisomers of phenylisopropyl adenosine, R-PIA (KI = 5.3 nM) and S-PIA (KI = 69 nM), as well as the 5'-substituted analogue, ethylcarboxamide adenosine (NECA; KI = 4.2 nM) suggest that [3H]CHA binding occurs to a single class of receptors of the AI subtype. Contractile studies employing these agents reveal that the relative order of potency, based on ED50 values, correlates well with the relative order of competition of agonist binding, based on equilibrium binding constants. Direct assay of myometrial adenylate cyclase failed to show that adenosine receptors in this smooth muscle are coupled to adenylate cyclase. We conclude here that a smooth muscle adenosine receptor is not coupled to adenylate cyclase, yet subserves muscle contraction. These data are important in light of recent attempts to classify adenosine receptors as dual regulators of adenylate cyclase.     

Binding of the Adenosine A2 Receptor Ligand [3H]CGS 21680 to Human and Rat Brain: Evidence for Multiple Affinity Sites

A new radiolabeled adenosine receptor agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadeno sin e (CGS 21680), apparently specific for high-affinity binding sites of the A2 subtype in rat brain, was used to identify and pharmacologically characterize adenosine receptors in human brain. The binding of [3H]CGS 21680, as determined by standard radioligand binding technique in the presence of exogenously added adenosine deaminase, reached equilibrium after 40 min at 25 degrees C. In saturation studies, a single class of high-affinity binding sites with values for KD of 22 +/- 0.5 nM and Bmax of 444 +/- 63 fmol/mg of protein were observed. Similar binding characteristics were observed regardless of whether rapid filtration or centrifugation was used to separate bound versus free ligand. Of the 14 brain regions examined, [3H]CGS 21680 binding was highest in putamen, followed by globus pallidus and caudate nucleus. The level of [3H]CGS 21680 binding in these areas of basal ganglia was identical to 5'-N-[3H]ethylcarboxamidoadenosine ([3H]NECA) binding in the presence of 50 nM N6-cyclopentyladenosine (CPA). The rank order of agonist potencies as determined by a series of competition experiments was NECA greater than or equal to CGS 21680 greater than 2-chloroadenosine greater than N6-(R)-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than N6-(S)-phenylisopropyladenosine. This potency order was the same for the binding of [3H]CGS 21680 to rat, and of [3H]NECA in the presence of 50 nM CPA to rat and human, brain membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a low affinity binding site for N6-substituted adenosine derivatives in rat testis membranes

Abstract

The binding characteristics of radiolabeled N⁶-(cyclohexyl)adenosine ([³H]CHA), N⁶-(R-phenylisopropyl)adenosine ([³H]R-PIA), 5′-N-ethylcarboxamidoadenosine ([³H]NECA), and 2-[4-(2-carboxyethyl)phenyl]ethyl-amino-5′-N-ethylcarboxamidoadenosine ([³H]CGS 21680), to rat testis membranes were investigated. Specific binding of [³H]CGS 21680, a selective agonist for the A_2a adenosine receptor, was very modest whilst the nonselective agonist [³H]NECA bound to rat testis membranes showing high binding capacity. At least two types of binding sites for [³H]NECA could be identified in rat testis membranes: high affinity sites and high capacity sites. Selective agonists for the At adenosine receptor, [³H]CHA and [³H]R-PIA bound with high affinity to a single class of binding sites. This high affinity binding site showed the typical pharmacological specificity of the A₁ adenosine receptor with a potency order for agonists of CHA R-PIA > NECA > N⁶-(S-phenylisopropyl)adenosine (S-PIA). In order to detect the presence of the A₃ adenosine receptor in these membranes we selectively blocked the A₁ receptor with a large molar excess of a xanthine antagonist, either 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or xanthine amine congener (XAC). In the presence of an antagonist a low affinity binding site for [³H]CHA and [³H]R-PIA was detected. This low affinity binding site showed a different pharmacological specificity than the high affinity binding site. In fact the potency order for agonists was CHA NECA = R-PIA > S-PIA. This finding suggests that the low affinity binding site represents the A₃ adenosine receptor.     

5'-N-ethylcarboxamide[3H]adenosine binding sites of mouse mastocytoma P815 cell membranes: characterization and solubilization

Mouse mastocytoma P815 cell membranes were found to possess adenosine binding sites as assessed by using the adenosine agonist [3H]5'-N-ethylcarboxamideadenosine (NECA). The Kd and Bmax for the [3H]NECA binding at 0 degrees C were 380 nM and 17 pmol/mg of protein, respectively. The rank order of potency for inhibition of [3H]NECA binding was NECA greater than 5'-N-cyclopropylcarboxamideadenosine greater than 2-chloroadenosine greater than 2',5'-dideoxyadenosine greater than isobutylmethylxanthine greater than theophylline greater than N6-[(R)-1-methyl-2-phenylethyl]adenosine = N6-[(S)-1-methyl-2- phenylethyl]adenosine. Thermodynamic analyses of the adenosine receptor agonist and antagonist binding showed that all such ligands displayed negative values of both enthalpy and entropy which suggested that the driving force for the binding was enthalpic. [3H]NECA binding sites of P815 cell membranes were solubilized with sodium cholate and retaining the same ligand-binding characteristics as those of the membrane-bound form. By gel filtration on a Sepharose CL-6B column, the adenosine binding site was estimated to have a Stokes radius of approximately 6.7 nm.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

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.     

Purification and characterization of bovine cerebral cortex A1 adenosine receptor

A1 adenosine receptors (A1AR) acting via the inhibitory guanine nucleotide binding protein inhibit adenylate cyclase activity in brain, cardiac, and adipose tissue. We now report the purification of the A1AR from bovine cerebral cortex. This A1AR is distinct from other A1ARs in that it displays an agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine greater than (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine (NECA) compared to the traditional potency series of R-PIA greater than NECA greater than S-PIA. The A1AR was solubilized in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and then purified by chromatography on an antagonist [xanthine amine congener (XAC)]-coupled Affi-Gel 10 followed by hydroxylapatite chromatography. Following purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of Mr 36,000 by silver staining, Na125I iodination with chloramine T and photoaffinity labeling with [125I]8-[4-[[[[2-(4-aminophenyl acetylamino) ethyl] carbonyl] methyl] oxy]-phenyl]-1,3- dipropylxanthine. This single protein displayed all the characteristics of the A1AR, including binding an antagonist radioligand [( 3H]XAC) with high affinity (Kd = 0.7 nM) and in a saturable manner (Bmax greater than 4500 pmol/mg). Agonist competition curves demonstrated the expected bovine brain A1AR pharmacology: R-PIA greater than S-PIA greater than NECA. The overall yield from soluble preparation was 7%. The glycoprotein nature of the purified A1AR was determined with endo- and exoglycosidases. Deglycosylation with endoglycosidase F increased the mobility of the A1AR from Mr 36,000 to Mr 32,000 in a single step. The A1AR was sensitive to neuraminidase but resistant to alpha-mannosidase, suggesting the single carbohydrate chain was of the complex type. This makes the bovine brain A1AR similar to rat brain and fat A1AR in terms of its carbohydrate chains yet the purified A1AR retains its unique agonist potency series observed in membranes.     

Adenosine binding sites of rat pheochromocytoma PC 12 cell membranes: partial characterization and solubilization

Rat pheochromocytoma PC 12 cell membranes were shown to possess A2-like adenosine binding sites as assessed by using 5'-N-ethylcarboxamide[3H]adenosine [( 3H]NECA). Specific [3H]NECA binding to PC 12 cell membrane at 0 degrees C was saturable and showed a monophasic saturation profile. In contrast, [3H]NECA binding to PC 12 cell membrane at 30 degrees C exhibited a biphasic profile suggesting the presence of two specific binding site. The rank order of potency for inhibition of [3H]NECA binding at 0 degrees C was NECA greater than 2-chloroadenosine greater than 2',5'-dideoxyadenosine greater than isobutylmethylxanthine much greater than phenylisopropyladenosine. These adenosine binding sites were solubilized with sodium cholate and the solubilized portion retained the same ligand binding characteristics as those of the membrane-bound form. Gel filtration experiments indicated an apparent Stokes radius of 6.7 nm for these adenosine binding sites/detergent complexes.     

Bovine Brain Coated Vesicles Contain Adenosine A1 Receptors. Presence of Adenylate Cyclase Coupled to the Receptor

Clathrin-coated vesicles purified from bovine brain express adenosine A1 receptor binding activity. N6-Cyclohexyl[3H]adenosine [( 3H]CHA), an agonist for the A1 receptor, binds specifically to coated vesicles. High and low agonist affinity states of the receptor for the radioligand [3H]CHA with KD values of 0.18 and 4.4 nM, respectively, were detected. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Binding competition experiments using agonists (N6CHA, N-cyclopentyladenosine, 5'-(N-ethylcarboxamido)adenosine, and N6-[(R)- and N6-[(S)-phenylisopropyl]adenosine) and antagonists (theophylline, 3-isobutyl-1-methylxanthine, and caffeine) confirmed the typical adenosine A1 nature of the binding site. This binding site presents stereospecificity for N6-phenylisopropyladenosine, showing 33 times more affinity for N6-[(R)- than for N6-[(S)-phenylisopropyl]adenosine. The specific binding of [3H]CHA in coated vesicles is regulated by guanine nucleotides. [3H]CHA specific binding was decreased by 70% in the presence of the hydrolysis-resistant GTP analogue guanyl-5-yl-imidodiphosphate. Bovine brain coated vesicles present adenylate cyclase activity. This activity was modulated by forskolin and CHA. The results of this study support the evidence that adenosine A1 receptors present in coated vesicles are coupled to adenylate cyclase activity through a Gi protein.     

The Adenosine Receptors Present on the Plasma Membrane of Chromaffin Cells Are of the A2b Subtype

The adenosine receptors in the plasma membrane of chromaffin cells from bovine adrenal medulla were characterized. The presence of A1 receptors was discounted owing to the absence of R-[3H]phenylisopropyladenosine (R-PIA) and [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) binding. The binding of the specific A2a ligand CGS-21680 was low. In contrast, the binding of 5'-(N-[3H]-ethylcarboxamidoadenosine ([3H]NECA) was relatively high (1.7 pmol/mg of protein at a ligand concentration up to 90 nM). This binding did not correspond to non-adenosine receptor NECA binding sites because the specific [3H]-NECA binding was similar when unlabeled adenosine, NECA, or R-PIA was used to measure the nonspecific binding. The rank order of potency of different ligands for the displacement of specific [3H]NECA binding was DPCPX greater than NECA greater than chloroadenosine greater than R-PIA greater than theophylline = CGS-21680. These results indicate that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A2b subtype.     

Characteristics of an adenosine A1 binding site in human placental membranes

Binding sites were solubilized from human placental membrane using 1.5% sodium cholate and were assayed using polyethylene glycol precipitation. These soluble binding sites had properties of an adenosine A1 binding site. 2-[3H]Chloroadenosine and N-[3H]-ethylcarboxamidoadenosine (NECA) binding were time dependent and reversible. Scatchard plots indicate two classes of binding sites with Kd values of 6 and 357 nM for 2-chloro[8-3H]adenosine and 0.1 and 26 nM with [3H]NECA. The specificity of [3H]NECA binding was assessed by the ability of adenosine analogs to complete for binding sites. Using this approach the estimated IC50 values were 60 nM for (R-PIA), 160 nM for S-PIA, 80 nM for NECA, and 20 nM for 2-chloroadenosine. Binding of [3H]NECA to the soluble sites is inhibited to 48% of the control value by 100 microM guanylyl-5'-imidodiphosphate (Gpp(NH)p). The IC50 value for NECA binding to the soluble binding site was increased from 80 nM to 1500 by Gpp(NH)p. There was a shift of binding affinity from a mixture of high and low affinity to only low affinity with 100 microM Gpp(NH)p. Despite these alterations a NECA prelabeled molecular species of 150 kDa did not decrease in molecular weight upon the addition of 100 microM Gpp(NH)p during high-performance liquid chromatography on a Superose 12 column. Other evidence to support the concept of preferential solubilization and assay of a small population of A1 binding sites was obtained. Following solubilization adenosine A2-like binding sites could be detected only in reconstituted vesicles. The existence of small amounts of A1 binding sites in intact human placental membranes was directly demonstrated using the A1 agonist ligand N6-[3H]cyclohexyladenosine and the A1 antagonist ligand 8-[3H]cyclopentyl-1,3-dipropylxanthine. JAR choriocarcinoma cells have "A2-like" membrane binding sites. In contrast to placental membranes, only A2-like binding sites could be solubilized from JAR choriocarcinoma cells. These observations indicate that human placental membranes contain adenosine A1 binding sites in addition to A2-like binding sites. These sites are guanine nucleotide sensitive, but do not shift to a lower molecular weight form upon assumption of a low affinity state.     

Effect of 5''-(N-Ethylcarboxamido)adenosine on Adenosine Transport in Cultured Chromaffin Cells

Extracellular adenosine is transported into chromaffin cells by a high-affinity transport system. The action of adenosine receptor ligands was studied in this cellular model. 5'-(N-Ethylcarboxamido)adenosine (NECA), an agonist of A2 receptors, activated adenosine transport. Km values for adenosine were 4.6 +/- 1.0 (n = 5) and 10.2 +/- 3.0 microM (n = 5) for controls and 100 nM NECA, respectively. The Vmax values were 66.7 +/- 23.5 and 170.2 +/- 30 pmol/10(6) cells/min for controls and 100 nM NECA, respectively. The A1 agonist N6-cyclohexyladenosine, the A1 antagonist 8-cyclopentyl-1, 3-dipropylxanthine, and the A1-A2 antagonist 1,3-dipropyl-8-(4-[(2-aminoethyl)amino]-carbonylmethyloxyphenyl)- xanthine did not significantly modify the adenosine transport in this system. Binding studies done with [3H]dipyridamole, a nucleoside transporter ligand, did not show changes in either the number or affinity of transporter sites after NECA treatment. This ligand can enter cells and quantifies the total number of transporters. The binding studies with [3H]-nitrobenzylthioinosine, which quantifies the plasma membrane transporters, showed a Bmax of 19,200 +/- 800 and 23,200 +/- 700 transporters/cell for controls and 100 nM NECA, respectively. No changes in the KD were obtained. The effects of NECA were not mediated through adenylate cyclase activation, because its action was not imitated by forskolin.     

Evidence for the presence of A1 and A2 adenosine receptors in the ventral aorta of the dogfish shark,Squalus acanthias

Isolated, endothelium-free rings of vascular smooth muscle (VSM) from the ventral aorta of the dogfish shark, Squalus acanthias, were used to examine the vasoactive effects of various adenosine agonists. Cumulative addition of 2-chloroadenosine (2 Cl-ADO) over the concentration range 10 nM-1 mM resulted in a biphasic response, with a significant increase in tension at 1 microM and a more significant decline in tension at 100 microM and 1 mM, suggesting that this tissue may possess both A1 and A2 adenosine receptors. N6-Cyclopentyladenosine (N-6 CPA) and N6-(2-phenylisopropyl)adenosine, R(-)isomer (R-PIA), generally considered to be more A1 specific, also produced slight, but significant increases in tension, but only at relatively high concentrations. The more specific A1 agonist, N6-(25)-[2-endo-norbonyl] adenosine [(S)-ENBA] produced a significant increase in tension at 1 pM, reaching 28% above control at 10 nM. The response to (S)-ENBA was also biphasic, with a fall in tension at 10 microM. The relatively non-specific agonist 5'-N-ethylcarboxamidoadenosine (NECA) produced a small, but significant, increase in tension at 1 microM, with no subsequent decline in tension at higher concentrations. These results allow us to assign a tentative structure-activity relationship (SAR) for an increase in tension of (S)-ENBA much much greater than R-PIA greater than or equal to 2-Cl ADO = N-6 CPA = NECA; for the decrease, the SAR is (S)-ENBA greater than 2-Cl ADO greater than R-PIA greater than N-6 CPA = NECA.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Interaction of Adenosine and Guanine Derivatives in the Rat Hippocampus: Effects on Cyclic AMP Levels and on the Binding of Adenosine Analogues and GMP

Guanine nucleotides (GN) have been implicated in many intracellular mechanisms. Extracellular actions, probably as glutamate receptor antagonists, have also been recently attributed to these compounds. GN may have a neuroprotective role by inhibiting excitotoxic events evoked by glutamate. Effects of extracellular GN on adenosine-evoked cellular responses have also been reported. However, the exact mechanism of such interaction is not known. In the present study, we showed that GN potentiated adenosine-induced cAMP accumulation in slices of hippocampus from young rats. However, neither GMP nor the metabotropic glutamate receptor agonist, 1S,3R-ACPD, inhibited the binding of the adenosine receptor agonist [³H]NECA (when binding to adenosine A2 receptors), or the binding of the adenosine A2a receptor agonist [³H]CGS 21680 in hippocampal membrane preparations. GppNHp, probably by interacting with G-proteins, decreased [³H]CGS 21680 binding. [³H]GMP binding was assayed in order to evaluate the GN sites which are not G-proteins. [³H]GMP binding was inhibited by GMP and GppNHp, but not by 1S,3R-ACPD. The interaction of endogenous adenosine with the GMP-binding sites was determined by incubating membranes in the presence or absence of adenosine deaminase (ADA). NECA, CADO, CGS 21680 and CPA (only at the highest concentration used) increased GMP binding in the presence of ADA. However, in the absence of ADA, the control levels of GMP binding were as high as in the presence of added ADA plus adenosine agonists, indicating that endogenous adenosine modulates the binding of GMP. If this site has a neuroprotective role, adenosine may be increasing its neuromodulator and proposed protective action.     

Discrete Distributions of Adenosine Receptors in Mammalian Retina

Binding sites for both the adenosine A1 receptor agonists [3H]phenylisopropyladenosine and [3H]cyclohexyladenosine and the mixed A1-A2 agonist N-[3H]ethylcarboxamidoadenosine [( 3H]NECA) were localized in rabbit and mouse retinas using autoradiographic techniques. These two classes of agonists bound to very different regions of mammalian retinas. A1 agonist binding was localized to the inner retina, particularly over the inner plexiform layer. The binding of [3H]NECA was observed primarily over the retinal pigmented epithelium and the outer and inner segments of photoreceptors. [3H]NECA labeling was not affected either by including a low concentration of unlabeled A1 agonist or by pretreating tissue with N-ethylmaleimide to inhibit ligand binding at A1 sites. While virtually all of the [3H]NECA binding was displaced by an excess of unlabeled NECA, displacement with antagonist or a large excess of cyclohexyladenosine revealed that approximately 30% of the [3H]NECA binding was at non-A1,A2 sites. The majority of the binding in the outer retina thus labeled A2 receptor sites. The unique localizations of the two classes of adenosine receptors suggest different functions in visual processing.     

Adenosine-receptor-mediated stimulation of low-Km GTPase in guinea-pig cerebral cortex.

Inhibition of receptor-coupled adenylate cyclase by hormones is proposed to be associated with GTP hydrolysis. Since adenosine inhibits cerebral-cortical adenylate cyclase via A1 adenosine receptors, the present study attempts to verify this mechanism for A1-selective adenosine derivatives. In guinea-pig cortical membranes N6-(phenylisopropyl)adenosine (PIA) increased the Vmax. of the low-Km GTPase, with an EC50 (concentration causing 50% of maximal stimulation) of about 0.1 microM, and the stimulatory effect was competitively antagonized by 5 microM-8-phenyltheophylline. The rank order of potency of the stereoisomers of PIA and of 5-(N-ethylcarboxamido)adenosine (NECA) to stimulate GTPase correlated with their ability to inhibit adenylate cyclase activity (R-PIA greater than NECA greater than S-PIA). Competition binding studies with (-)-N6- ([125I]iodo-4-hydroxyphenylisopropyl)adenosine suggest that adenylyl imidodiphosphate (p[NH]ppA), an essential component of the GTPase assay system, is a more potent A1-receptor agonist than ATP, with an IC50 (concentration giving half-maximal displacement of radioligand binding) of 7.9 microM. On the basis of the p[NH]ppA concentration used in the GTPase assay (1.25 mM), enzyme stimulation by adenosine seems to be highly underestimated. Nevertheless, adenosine-induced GTP hydrolysis reflects an increased turnover of guanine nucleotides at the Ni regulatory site and appears to be a crucial step in the sequence of events processing the inhibitory signal to adenylate cyclase.     

Magnesium-Dependent Enhancement of Endogenous Agonist Binding to A1 Adenosine Receptors: A Complicating Factor in Quantitative Autoradiography

Quantitative autoradiography was used to investigate the effects of Mg2+ on agonist and antagonist binding to A1 receptors in rat striatum. A1 receptors were labelled with the selective agonist N6-[3H]cyclohexyladenosine ([3H]CHA) or the selective antagonist 1,3-[3H]dipropyl-8-cyclopentylxanthine ([3H]DPCPX). Mg2+ had no significant effect on equilibrium binding constants for [3H]CHA [control: KD (95% confidence interval) of 0.34 (0.15-0.80) nM and Bmax of 267 +/- 8 fmol/mg of gray matter; with 10 mM Mg2+: KD of 0.8 (0.13-4.9) nM and Bmax of 313 +/- 8.9 fmol/mg of gray matter] or [3H]DPCPX [control: KD of 0.54 (0.30-0.99) nM and Bmax of 256 +/- 2.3 fmol/mg of gray matter; with 10 mM Mg2+: KD of 1.54 (0.2-11.0) nM and Bmax of 269 +/- 35.7 fmol/mg of gray matter]. In contrast, Mg2+ slowed the apparent association rate for both ligands; this was observed as a shift from a one-component to a two-component model for [3H]DPCPX. Mg2+ also affected the dissociation rates of both ligands; for [3H]CHA, dissociation in the presence of Mg2+ was not detected. Mg2+ produced a concentration-dependent inhibition of [3H]CHA binding only prior to equilibrium. HPLC was performed on untreated sections, sections preincubated with adenosine deaminase (ADA), and sections preincubated with ADA and incubated with ADA in the absence or presence of Mg2+. Adenosine was found in measurable quantities under all conditions, and the concentration was not influenced by Mg2+ or by the inclusion of GTP in the preincubation medium. From these data, we conclude the following: (a) adenosine is present and may be produced continuously in brain sections; (b) ADA is not capable of completely eliminating the produced adenosine; (c) Mg2+ apparently does not influence adenosine production or elimination; (d) A1 receptor-guanine nucleotide binding protein coupling is maximal in this preparation; and (e) Mg2+ decreases the dissociation rate of bound endogenous adenosine from A1 receptors, thus limiting the access of [3H]CHA and [3H]DPCPX to the receptors. Thus, enhancement of endogenous adenosine binding to A1 receptors by Mg2+ is a complicating factor in receptor autoradiography and may be so in other preparations as well.