Differences in the GTP-regulation of membrane-bound and solubilized A1-adenosine receptors

Summary Using [³H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a ³H-labeled A₁-selective adenosine antagonist with high affinity and extremely low non-specific binding, it was possible to quantitatively evaluate the effect of GTP on agonist binding. Competition experiments on [³H]DPCPX binding to guinea-pig cerebral cortical membranes in the absence of GTP showed a high- and a low-affinity state for adenosine receptor agonists (82/18% for N⁶-cyclopentyladenosine). Addition of 1 mmol/l GTP only partially converted the high-affinity state of the A₁-adenosine receptor into a low-affinity state. This failure of complete conversion from high- to low-affinity state was also seen in membranes from rat testes under the same experimental conditions and, moreover, in guinea-pig brain membranes under different experimental conditions, such as in the presence of Na⁺ or when free Mg²⁺ has been reduced by EDTA. The only difference was that in the absence of Mg²⁺ the high-affinity state of the A₁-receptor was markedly smaller than in the presence of Mg²⁺ (36% vs. 82%). By contrast, in the solubilized state of the receptor total conversion of all receptors into the low-affinity state was obtained upon addition of 1 mmol/l GTP. Reduction of binding of the agonist radioligand [¹²⁵]iodo-N⁶-(4-hydroxyphenylisopropyl)-adenosine with increasing concentrations of GTP and Gpp(NH)p demonstrated that the guanine nucleotide affinity to the solubilized A₁-receptor was more than 100-fold higher than to the membrane-bound receptor. Hence, the incomplete transition of the high-affinity into the low-affinity state of the membrane-bound A₁-receptor upon addition of GTP may be attributable to the low affinity of the membrane-bound receptor-G-protein complex for GTP.Abbreviations CHAPS 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate - CPA N⁶-cyclopentyl-adenosine - dATP deoxy-ATP - DPCPX 8-cyclopentyl-1,3-dipropylxanthine - DPX 1,3-diethyl-8-phenylxanthine - Gpp(NH)p guanylylimidodiphosphate - HEPES 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid - [¹²⁵I]HPIA (–)-N⁶-3-([¹²⁵I]-iodo-4-hydroxyphenylisoprop-yl)-adenosine - NECA 5-(N-ethylcarboxamido)adenosine - PEI polyethylenimine - R-PIA (–)-N⁶-(R-phenylisopropyl)-adenosine - [³H]XAC [³H]xanthine amine congener Send offprint requests to Dr. Wolfgang Schütz at the above address     

[3H]5′-N-ethylcarboxamide adenosine binds to both Ra and Ri adenosine receptors in rat striatum

Summary Adenosine analogs such as 5-N-ethylcarboxamide adenosine and N⁶-cyclohexyladenosine stimulate or inhibit adenosine cyclase activity in preparations of rat striatum depending on the assay conditions. N⁶-cyclohexyladenosine inhibits but does not stimulate adenosine cyclase activity in preparations of hippocampus. These findings suggest that the striatum contains both R _a (stimulatory) and R _i (inhibitory) adenosine receptors while the hippocampus contains only R _i receptors. We have previously shown that [³H]N⁶-cyclohexyladenosine binds to R _i receptors in rat hippocampus (Yeung and Green 1983). Comparisons of the characteristics of [³H]5-N-ethylcarboxamide adenosine and [³H]N⁶-cyclohexyladenosine binding to hippocampus show that [³H]5-N-ethylcarboxamide adenosine also binds to R _i receptors with high affinity. [³H]5-N-ethylcarboxamide adenosine binds to R _i receptors in the striatum and to a second site that is present in striatum but not hippocampus. High affinity binding of both ligands to R _i receptors can be blocked by treatments with N-ethylmaleimide that do not markedly affect [³H]5-N-ethylcarboxamide adenosine binding to the second site in the striatum. The pharmacological characteristics of the second site indicate that it is the R _a adenosine receptor.The abbreviations used are NEM N-ethylmaleimide - Gpp(NH)p 5-guanylylimidodiphosphate - NECA 5-N-ethylcarboxamide adenosine - l-PIA N⁶-(l-phenylisopropyl)adenosine - d-PIA N⁶-(d-phenylisopropyl) adenosine - DPX 1,3-diethyl-8-phenylxanthine     

Autoradiographic evidence for G-protein coupled A2-receptors in rat neostriatum using [3H]-CGS 21680 as a ligand

Summary Recently [³H]-CGS 21680 (2-[p-(2-carbonylethyl)-phenylethylamino]-5-N-ethylcarboxamidoadeno-sine) has been identified as a selective adenosine A₂-receptor agonist. In this study the binding of [³H]-CGS 21680 to 10 m sections of rat neostriatum was investigated with quantitative autoradiography. Specific, saturable binding was detectable, and Scatchard analysis of saturation experiments gave estimates for K _D and B _max of 1.7 nM and 322 fmol/mg protein, respectively. The rank order of potency for inhibition of [³H]-CGS 21680 binding was 5-N-ethylcarboxamidoadenosine (1.9 nM) > 2-chloroadenosine (18 nM) > R-N⁶-phenylisoprop-yladenosine (59 nM) > S-N⁶-phenylisoprophyladeno sine (460 nM) > 1,3-dipropyl-8-cyclopentylxanthine (700 nM). The binding of [³H]-CGS 21680 was sensitive to GTP, since 1 M GTP reduced binding to 4.7% of control. These data support the identity of CGS 21680 as an agonist at high affinity adenosine A₂-receptors and indicate these receptors in rat striatum are coupled to guanine nucleotide binding proteins. Send offprint requests to F. E. Parkinson at the above address     

Separation of solubilized A2 adenosine receptors of human platelets from non-receptor [3H]NECA binding sites by gel filtration

Summary Human platelet membranes were solubilized with the zwitterionic detergent CHAPS (3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate) and the solubilized extract subjected to gel filtration. Binding of the adenosine receptor agonist [3H]NECA (5-N-ethylcarboxamidoadeno-sine) was measured to the eluted fractions. Two [³H]NECA binding peaks were eluted, the first of them with the void volume. This first peak represented between 10% and 25% of the [³H]NECA binding activity eluted from the column. It bound [³H]NECA in a reversible, saturable and GTP-dependent manner with an affinity of 46 nmol/1 and a binding capacity of 510 fmol/mg protein. Various adenosine receptor ligands competed for the binding of [³H]NECA to the first peak with a pharmacological profile characteristic for the A₂ adenosine receptor as determined from adenylate cyclase experiments. In contrast, most adenosine receptor ligands did not compete for [³H]NECA binding to the second, major peak. These results suggest that a solubilized A₂ receptor-GS protein complex of human platelets can be separated from other [³H]NECA binding sites by gel filtration. This allows reliable radioligand binding studies of the A₂ adenosine receptor of human platelets.Abbreviations CHAPS 3-[3-(cholamidopropyl)dimethylammoniol-l-propanesulfonate - CIA 2-chloroadenosine - CPA N⁶-cyclopentyladenosine - DPX 1,3-diethyl-8-phenylxanthine - NECA 5-N-ethylcarboxamidoadenosine - PAA 2-phenylaminoadenosine - PIA N⁶-phenyhsopropyladenosine - XAC 8-{4-[([{(2-aminoethyl)amino}carbonyl}methyl)oxy]phenyl]-1,3-dipropylxanthine Send offprint requests to M. J. Lohse     

[125I] N6-p-hydroxyphenylisopropyladenosine,a new ligand for Ri adenosine receptors

Summary N⁶-p-Hydroxyphenylisopropyladenosine (HPIA) has been labelled with carrier-free Na[¹²⁵I] to very high specific activity (2,175 Ci/mmol) and used as an agonist ligand to characterize R_i adenosine receptors in rat cerebral cortex membranes. The binding is saturable, reversible, stereospecific and dependent on protein concentration. The specific binding at 37°C was of high affinity with an equilibrium dissociation constant K_D of 0.48 nmol/l and was saturable with 0.23 pmol of [¹²⁵I]HPIA per mg of protein. The rate constant of association, k₁, was 3.25×10⁸ l mol^–1 min^–1 and that of dissociation, k₂, 0.0110 min^–1 yielding a t_1/2 of 63 min. In competition experiments the (–)isomer of N⁶-phenylisopropyladenosine (PIA) was 16-fold more potent than the (+)isomer in competing for the binding sites. Specific binding was most effectively displaced by N⁶-cyclohexyladenosine (CHA, k_i=0.26 nmol/l), (–)PIA (k_i=0.33 nmol/l) and HPIA (k_i=0.52 nmol/l), whereas 5-N-ethylcarboxamidoadenosine (NECA, k_i-1.42 nmol/l) was less effective. The methylxanthines 3-isobutyl-1-methylxanthine (IBMX), theophylline and caffeine which have been classified as adenosine antagonists had k_i values between 5–34 mol/l. Binding of [¹²⁵I]HPIA was regulated by guanine nucleotides and divalent cations. The results indicate that [¹²⁵I]HPIA labels R_i adenosine receptors in rat brain membranes.     

Labelling of Ri adenosine receptors in rat fat cell membranes with (−)-[125iodo]N6-hydroxyphenylisopropyladenosine

Summary A new adenosine analogue, (–)-iodo-N⁶-p-hydroxyphenylisopropyladenosine [(–)-IHPIA], has been developed for radioligand binding studies of R_i adenosine receptors. In addition, the effects of (–)IHPIA on adenosine-mediated responses of rat fat cells have been characterized. (–)IHPIA is slightly less potent at R_i adenosine receptors than (–)N⁶-phenylisopropyladenosine [(–)PIA] as assessed by adenylate cyclase and lipolysis studies. (–)IHPIA inhibited basal adenylate cyclase activity with an IC₅₀ of 60 nmol/l compared to an IC₅₀ of 16.3 nmol/l for (–)PIA. (–)PIA and (–)IHPIA inhibited adenosine deaminase-stimulated lipolysis of intact rat fat cells with an IC₅₀ of 0.55 and 3.6 nmol/l. The potency of (–)N⁶-p-hydroxyphenylisopropyladenosine [(–)HPIA] was intermediate. (–)HPIA has been labelled with carrier-free Na[¹²⁵I] to very high specific activity (2,175 Ci/mmol) and used as agonist radioligand in binding studies of R_i adenosine receptors. The binding of (–)[¹²⁵I]HPIA was saturable, reversible and stereospecific. Saturation analysis revealed two affinity states with dissociation constants (K _D) of 0.7 and 7.6 nmol/l and maximal number of binding sites (B _max) of 0.94 and 0.95 pmol/mg protein. The rate constant of association, k ₁, was 3.7×10⁸ l×mol^–1×min^–1. Binding was slowly reversible with a t_1/2 of 88 min. In competition experiments specific binding was most potently inhibited by (–)PIA, N⁶-cyclohexyladenosine (CHA), (–)HPIA and (–)IHPIA, followed by 5-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine. 1,3-Diethyl-8-phenylxanthine (DPX) and 8-phenyltheophylline were the most potent adenosine antagonists with K _i-values of 67 and 83 nmol/l, whereas the methylxanthines 3-isobutyl-1-methylxanthine, theophylline and caffeine had K _i-values between 1 and 21 mol/l. Binding is highly stereospecific, as indicated by an approximately 20-fold higher K _i-value of the (+)isomer of PIA in comparison to the (–)isomer. The pharmacological profile of (–)[¹²⁵I]HPIA binding sites is consistent with an interaction at R _i adenosine receptors. (–)[¹²⁵I]HPIA appears to be a suitable agonist for radioligand binding studies at R _i adenosine receptors.     

Interaction of barbiturates with adenosine receptors in rat brain

Summary The effects of barbiturates on radioligand binding to inhibitory R _i adenosine receptors of rat brain membranes were investigated. Binding of the adenosine receptor agonist (–)N⁶-phenylisopropyl[³H]adenosine and the antagonist 1,3-diethyl-8-[³H]phenylxanthine was inhibited by several barbiturates. This inhibition was concentration-dependent and occurred in the range of pharmacologically effective concentrations. Pentobarbital was the most potent of the barbiturates tested with a K _i of 92 mol/l. The (+)isomers of hexobarbital and mephobarbital were more potent than the respective (–)isomers. Barbituric acid itself did not displace either radioligand in concentrations up to 1 mmol/l. The inhibitory effect of pentobarbital was reversed by a single wash of membranes preincubated with the barbiturate. The presence of pentobarbital caused a decrease of the affinity of the receptor for the antagonist radioligand but did not alter the number of binding sites, suggesting a competitive antagonism. The effects of pentobarbital on radioligand binding to the receptor were not changed by the presence of picrotoxinin nor by the absence of chloride ions. This indicates that they are not mediated via the picrotoxinin binding site. The barbiturates could not be classified as either agonists or antagonists at the R _i adenosine receptor. The presence of GTP did not influence the inhibition of radioligand binding by pentobarbital; this is also observed for antagonists, whereas the affinity of agonists is markedly reduced by GTP. Binding of antagonists to the receptor is enthalpy-driven; the interaction of pentobarbital with the receptor was entropy-driven and the same was true for agonists. Thus, the interaction of pentobarbital with R _i adenosine receptors of rat brain membranes differs from that of both adenosine agonists and antagonists. Our data suggest that R _i adenosine receptors may be involved in the mediation of the effects of barbiturates.     

Ra Adenosine receptors in human platelets

Summary Adenosine receptors in human platelet membranes have been characterized by radioligand binding and measurement of adenylate cyclase activity. Binding of 5-N-ethylcarboxamido[³H]adenosine ([³H]NECA) was rapid, reversible and dependent on protein concentration, pH and temperature. Due to a rapid rate of dissociation (t _1/2 approximately 20 s) binding was highest at 0° C. Adenosine deaminase and GTP alone did not influence [³H]NECA binding, whereas several divalent cations decreased binding. Saturation experiments revealed two different binding sites for [³H]NECA, with K _d values of 0.16 and 2.9 mol/l and B _max values of 8.4 and 33.4 pmol/mg of protein. In competition experiments NECA was the most potent adenosine agonist (IC₅₀ 0.5 mol/l), followed by 2-chloroadenosine (IC₅₀ 6.3 mol/l) and adenosine (IC₅₀ 12mol/l). A similar rank order of potencies was observed for the stimulatory effect of adenosine analogues on platelet adenylate cyclase. NECA stimulated adenylate cyclase activity with an EC₅₀ value of 0.5 mol/l and was approximately 4-fold more potent than (–)N⁶-phenylisopropyladenosine [(–)PIA]. However, (–)PIA and N⁶-cyclohexyladenosine did not significantly affect [³H]NECA binding, an observation not consistent with the stimulatory effect on adenylate cyclase. The adenosine antagonists 3-isobutyl-1-methylxanthine, theophylline and caffeine showed IC₅₀ values between 98 and 5,600 mol/l. [³H]PIA bound to platelet membranes with very low affinity and was not displaced by NECA. The [³H]NECA binding to human platelet membranes satisfies essential criteria for R _a adenosine receptors and, with some limitations, should be of value for the characterization of adenosine receptors in R _a subtype selective cells.     

Effects of N-ethylmaleimide on adenosine receptors of rat fat cells and human platelets

N-Ethylmaleimide (NEM) differentially modified Ri adenosine receptors in rat fat cells and Ra adenosine receptors in human platelets. Pretreatment of rat fat cell membranes with NEM inhibited the binding of the agonist (-)N6-phenylisopropyl[3H]adenosine [( 3H]PIA), but did not affect the binding of the antagonist 1,3-diethyl-8-[3H]phenylxanthine [( 3H]DPX). The IC50-value for inhibition of [3H]PIA binding was 0.067 mM. Saturation of [3H]PIA binding revealed that NEM converts the high affinity form of the Ri receptor into a low affinity form. NEM also decreased the potency of agonists to displace [3H]DPX binding, as shown by a 74-fold shift of the Ki-value for (-)PIA, whereas antagonist-induced displacement remained unchanged. In addition, low concentrations of NEM (0.01-0.1 mM) attenuated the (-)PIA-induced inhibition of adenylate cyclase activity of rat fat cells. At higher concentrations (0.1-1 mM) NEM reduced basal and stimulated adenylate cyclase activities in rat fat cells and human platelets, presumably by inactivation of the catalytic unit. Radioligand binding of 5'-N-ethylcarboxamido[3H]-adenosine [( 3H]NECA) to Ra adenosine receptors of human platelet membranes was not changed by NEM at low radioligand concentrations. Saturation analysis of [3H]-NECA binding showed that NEM led to an apparent increase of agonist affinity with a concomitant decrease in total [3H]NECA binding sites. These results suggest that NEM reduces the affinity of Ri adenosine receptors, probably by affecting the inhibitory guanine nucleotide binding protein (Ni), whereas [3H]NECA binding sites are inversely affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of A1 adenosine receptors in brain from different species by radioligand binding and photoaffinity labelling

Summary Radioligand binding to A₁ adenosine receptors at brain membranes from seven species was investigated. The antagonist 8-cyclopentyl-1,3-[³H]dipropyl-xanthine ([³H]DPCPX) bound with affinities between 0.17 nM in sheep brain and 2.1 nM in guinea pig brain. Competition of several antagonists for [³H]DPCPX binding showed that the most potent compounds were DPCPX with K _i values of 0.05 nM in bovine brain and 1.1 nM in guinea pig brain and xanthine amine congener (XAC) with K _i values of 0.03 nM in bovine brain and 5.5 nM in guinea pig brain. The differences in affinity of the agonist radioligand 2-chloro-N ⁶-[³H]cyclopentyl-adenosine ([³H]CCPA) were less pronounced, ranging from a K _D value of 0.12 nM (hamster brain) to 0.42 nM (guinea pig brain). Agonist competition for [³H]DPCPX binding of photoaffinity labelling, however, exhibited marked species differences. N-Ethylcarboxamidoa-denosine (NECA) and S-N ⁶-phenylisopropyladenosine (S-PIA) showed 20 to 25-fold different K _D values in different species. NECA had a particularly high affinity in guinea pig brain and was only two-fold less potent than R-PIA. Thus, the difference from the classical A₁ receptor profile (R-PIA > -NECA > S-PIA) is not sufficient to speculate that A₁ receptor subtypes may exist that are coupled to different effector systems. Our data show that these difference can easily be explained by species differences.     

Demonstration of Ri-type adenosine receptors in bovine myocardium by radioligand binding

Summary Adenosine has been shown to have negative inotropic, chronotropic and dromotropic effects on the heart. The pharmacological profiles of these effects suggest that they are mediated via R_i (A₁) adenosine receptors, but a direct demonstration of these receptors is still missing. In the present study we report direct labelling of these receptors with (-)N⁶-[¹²⁵I]-p-hydroxyphenylisopropyladenosine ([¹²⁵I]HPIA). The radioligand bound in a saturable and reversible manner to a crude membrane preparation, the B _max-value was 30.5 fmol/mg protein and the K _D-value 1.1 nmol/l. A similar affinity of the ligand was obtained in kinetic and competition experiments. Competition experiments with a variety of adenosine analogues gave a pharmacological profile characteristic of R_i adenosine receptors with high affinities of N⁶-substituted derivatives and a marked stereospecificity for N⁶-phenylisopropyladenosine (PIA). Purification of the membrane preparation by density gradient centrifugation resulted in a 30-fold increase in the number of binding sites which was paralleled by a similar increase in the number of binding sites for [³H]ouabain. Guanine nucleotides decreased binding of [¹²⁵I]HPIA in a dose-dependent manner, but the IC₅₀-values were considerably higher than those reported in other tissues. Finally, binding of [¹²⁵I]HPIA appeared to be entropy-driven which has been shown to be characteristic of agonist binding to R_i adenosine receptors. These results suggest the presence of R_i adenosine receptors in ventricular myocardium which may be responsible for the mediation of the effects of adenosine and its analogues.Abbreviations [¹²⁵I]HPIA (-)N⁶-[¹²⁵I]-p-hydroxyphenylisopropyladenosine - (-)IHPIA (-)N⁶-iodo-p-hydroxyphenylisopropyladenosine - (+)/(-)PIA (+)/(-)N⁶-phenylisopropyladenosine - CHA N⁶-cyclohexyladenosine - NECA 5-N-ethylcarboxamidoadenosine - App(NH)p 5-adenylylimidodiphosphate - Gpp(NH)p 5-guanylylimidodiphosphate     

Guanine nucleotide and cation regulation of the binding of [3H]cyclohexyladenosine and [3H]diethylphenylxanthine to adenosine A1 receptors in brain membranes

Guanine nucleotides, divalent cations, and sodium differentially regulate agonist and antagonist binding to adenosine A1 receptors in brain membranes. Guanine nucleotides decrease the binding of the adenosine A1 receptor agonist [3H]N6-cyclohexyladenosine ([3H]CHA) to guinea pig and bovine brain membranes by about 50% at 1--3 microM, while not affecting binding of the antagonist [3H]1,3-diethyl-8-phenylxanthine ([3H]DPX) to A1 receptors in bovine brain. GTP decreases the potency of agonists competing for [3H]DPX binding by 3--6 times, without altering the potency of antagonists. This effect can be used to grade experimental substances along an adenosine agonist-antagonist continuum. The 66% inhibition of [3H]CHA binding by 1 mM EDTA, with no change in [3H]DPX binding, suggests that endogenous divalent cations may regulate adenosine receptor interactions. Removal of endogenous divalent cations by EDTA treatment greatly increases the enhancement of [3H]CHA binding by divalent cations. Specific binding of [3H]CHA to guinea pig brain is increased 150--170% by 0.3--1.0 mM Mn2+, Mg2+, and Ca2+ following EDTA preincubation, secondary to an increase in apparent affinity and receptor number. Sodium ions also selectively regulate the binding of [3H]CHA. Sodium decreases [3H]CHA binding 40%, whereas lithium and potassium are ineffective. Sodium does not affect [3H]DPX binding.     

Effect of bay K 8644, a calcium channel agonist,on dog cardiac muscarinic receptors

To investigate further whether the effects of the dihydropyridine (DHP) drugs on calcium channels are related to those of these drugs on muscarinic receptors, the binding characteristics of the DHP calcium channel agonist, Bay K 8644, on muscarinic receptors and calcium channels were compared to those of the DHP calcium channel antagonists, nicardipine and nimodipine in the dog cardiac sarcolemma. Bay K 8644, nicardipine and nimodipine inhibited the specific [³H]QNB binding with K _i values of 16.7μM, 3.5μM and 15.5μM respectively. Saturation data of [³H]QNB binding in the presence of these DHP drugs showed this inhibition to be competitive. Bay K 8644, like nicardipine and nimodipine, blocked the binding of [³H]nitrendipine to the high affinity DHP binding sites, but atropine did not, indicating that the muscarinic receptors and the DHP binding sites on calcium channels are distinct. The K _i value of Bay K 8644 for the DHP binding sites was 4 nM. Nicardipine and nimodipine (K _i :0.1–0.2 nM) were at least 20 times more potent than Bay K 8644 in inhibiting [³H]nitrendipine binding. Thus, the muscarinic receptors were about 4000 times less sensitive than these high affinity DHP binding sites to Bay K 8644. These results suggest that the DHP calcium agonist Bay K 8644 binds directly to the muscarinic receptors but its interaction with the muscarinic receptors is not related to its binding to the DHP binding sites on calcium channels.     

Adenosine receptor agonists: Binding and adenylate cyclase stimulation in rat liver plasma membranes

Summary N⁶-Cyclohexyl[³H]adenosine([³H]CHA),[³H]adenosine, and 5N-ethylcarboxamide[³H]adenosine ([³H]NECA), potent agonists in adenosine-responsive cellular systems, have been used to identify adenosine binding sites in rat liver plasma membranes. Endogenous ligands were removed by prior dialysis of the membranes. Specific binding of the ligands tested was characterized by rapid forward and reverse kinetics and heterogeneity as indicated by curvilinear Scatchard plots. The K _D in the high affinity range was 80 nM for [³H]adenosine, 84 nM for [³H]NECA, and 168 nM for [³H]CHA; the respective binding capacities of 1.19, 1.03, and 1.05 pmol/mg protein were of virtually the same magnitude, suggesting labeling of identical sites. However, all ligands also displayed binding to large numbers of low affinity sites. This high level of apparently non-receptor binding markedly influenced the adenosine structure-activity profile of [³H]CHA displacement, which differs with pharmacological findings. — NECA and CHA stimulated hepatic adenylate cyclase with an apparent ED₅₀ of 60 and 580 nM, respectively; adenosine was stimulatory at a concentration range from 0.1–2.0 M, but inhibitory at higher concentrations. Hence, estimation of the true ED₅₀ was not possible. Because the K _D of high affinity binding and the ED₅₀ of the biological effect of NECA and CHA are in the same range, it may be reasonable to assume that the high affinity sites represent adenosine receptors, recently classified as R_a-site receptors.Preliminary reports of this study have been presented at the 22nd Spring Meeting of the Deutsche Pharmakologische Gesellschaft (Naunyn-Schmiedeberg's Arch Pharmacol 316, R10, 1981) and at the 8th International Congress of Pharmacology [Tokyo 1981 (P 1459)]     

Binding of [H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes — a new, selective antagonist radioligand for A2A adenosine receptors

The present study describes the preparation and binding properties of a new, potent, and selective A_2A adenosine receptor (AR) antagonist radioligand, [³H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine ([³H]MSX-2). [³H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [³H]MSX-2 labeled a single class of binding sites with high affinity (K_d=8.0 nM) and limited capacity (B_max=1.16 fmol·mg⁻¹ of protein). The presence of 100 μM GTP, or 10 mM magnesium chloride, respectively, had no effect on [³H]MSX-2 binding. AR agonists competed with the binding of 1 nM [³H]MSX-2 with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680)>2-chloroadenosine (2-CADO)>N⁶-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K_i values for antagonists were in accordance with data from binding studies with the agonist radioligand [³H]CGS21680, while agonist affinities were 3–7-fold lower. [³H]MSX-2 is a highly selective A_2A AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20–30%, at 1 nM.     

2-Chloro-N6-[3H]cyclopentyladenosine ([3HCCPA) —a high affinity agonist radioligand for A1 adenosine receptors

Summary The tritiated analogue of 2-chloro-N⁶-cyclopentyladenosine (CCPA), an adenosine derivative with subnanomolar affinity and a 10000-fold selectivity for A₁ adenosine receptors, has been examined as a new agonist radioligand. [³H]CCPA was prepared with a specific radioactivity of 1.58 TBq/mmol (43 Ci/mmol) and bound in a reversible manner to A₁ receptors from rat brain membranes with a high affinityK _D-value of 0.2 nmol/l. In the presence of GTP aK _D-value of 13 nmol/l was determined for the low affinity state for agonist binding. Competition of several adenosine receptor agonists and antagonists for [³H]CCPA binding to rat brain membranes confirmed binding to an A₁ receptor. Solubilized A1 receptors bound [³H]CCPA with similar affinity for the high affinity state. At solubilized receptors a reduced association rate was observed in the presence of MgCl₂, as has been shown for the agonist [³H]N⁶-phenylisopropyladenosine ([³H]PIA). [³H]CCPA was also used for detection of A₁ receptors in rat cardio myocyte membranes, a tissue with a very low receptor density. A K_D-value of 0.4 nmol/l and aB _max-value of 16 fmol/ mg protein was determined in these membranes. In human platelet membranes no specific binding of [³H]CCPA was measured at concentrations up to 400 nmol/l, indicating that A2 receptors did not bind [³H]CCPA. Based on the subnanomolar affinity and the high selectivity for A₁ receptors [³H]CCPA proved to be a useful agonist radioligand for characterization of A₁ adenosine receptors also in tissues with very low receptor density.Abbreviations CHA N⁶-cyclopenyadenosine - CPA N⁶-cy-clopentyladen,osine - CCPA 2-chloro-N⁶-cyclopentyladenosine - CCCPA 2-chloro-5-chloro-5-deoxy-N⁶-cyclopentyladenosine; - CHAPS 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate - DPCPX 8-cyclopentyl-1,3-dipropylxanthine - NECA N-ethylcarboxamidoadenosine - PEI polyethylenimine - PIA N⁶-phenylisopropyladenosine Send offprint requests to K.-N. Klotz at the above address     

Characteristics of the [3H]-yohimbine binding on rat brain α2

Summary The labelling of rat cerebral cortex ₂-adrenoceptors with [³H]-yohimbine ([³H]-YOH) was investigated. At 25° C, binding equilibrium was reached in about 10 min and dissociation occurred with a half time of about 1 min. Saturation experiments gave an equilibrium K _D value of 10.13±1.95 nM and a maximum number of sites of 254±22 fmol/mg protein. The [³H]-YOH binding sites exhibited ₂-adrenergic receptor specificity; the order of potency for the antagonists was rauwolscine > yohimbine prazosin > corynanthine. For the agonists, the order was: oxymetazoline > clonidine > (–)-adrenaline > (–)-noradrenaline (–)-phenylephrine. Agonists exhibited shallow curves in inhibiting [³H]-YOH binding, with pseudo-Hill coefficients (n_H) of less than 1.0. These curves were shifted to lower overall affinity and steepened in the presence of 100 M GTP. Antagonist competition curves were also shallow but GTP had no significant effect.Divalent cations at millimolar concentrations decreased the [³H]-YOH binding: IC₅₀ values were about 6.0, 6.8 and 0.3 mM for Ca²⁺, Mg²⁺ and Mn²⁺ respectively.The maximal number of [³H]-YOH binding sites in the cortex was close to that labelled by the agonist [³H]-paraaminoclonidine ([³H]-PAC). The regional distribution of these sites in the brain, examined at a single concentration of [³H]-YOH and [³H]-PAC, showed a similar pattern except in the striatum. Taken together, the results indicate that like [³H]-PAC, [³H]-YOH labels ₂-adrenoceptors in rat brain cortex. They also show that [³H]-YOH is a useful tool for the study of the high and low affinity sites.     

[<Superscript>3</Superscript>H]LY334370, a novel radioligand for the 5-HT<Subscript>1F</Subscript> receptor. I. In vitro characterization of binding properties

[³H]LY334370 was developed as a radioligand to study the characteristics of this compounds interaction with the 5-HT_1F receptor. Monovalent or divalent cations did not enhance the binding of [³H]LY334370 to the cloned human 5-HT_1F receptor. In the presence of MgCl₂, the time to reach equilibrium was approximately 2 h, while in its absence equilibrium was reached in less than 1 h. [³H]LY334370 had high affinity for the cloned human 5-HT_1F receptor (K_d=0.446 nM) and the 5-HT_1F receptor in rat brain (K_d=0.388 nM). The expression density of 5-HT_1F receptors, as determined by binding to homogenates of cortical regions from rat, was low (B_max=79.1 fmol/mg protein). There was a statistically significant correlation between the apparent pK_i for inhibition of [³H]LY334370 binding and the pEC₅₀ for stimulation of [³⁵S]GTPS binding to homogenates of cells expressing the cloned human 5-HT_1F receptor. In addition, there was a statistically significant correlation between the apparent pK_i for inhibition of [³H]LY334370 binding to the cloned human 5-HT_1F receptor and the pID₅₀ for inhibition of trigeminal nerve stimulated dural plasma protein extravasation in the guinea pig. The conclusion from these studies is that [³H]LY334370 is a high affinity radioligand which can be used for the study of the 5-HT_1F receptor in rat brain or in cells transformed with the human 5-HT_1F receptor.     

Effects of several 5′-carboxamide derivatives of adenosine on adenosine receptors of human platelets and rat fat cells

Summary The effects of several 5-carboxamide derivatives of adenosine on stimulatory (R _a) adenosine receptors of human platelets and inhibitory (R _i) adenosine receptors of rat fat cells have been compared. 5-N-Cyclopropylcarboxamidoadenosine (CPCA) and 5-N-ethylcarboxamidoadenosine (NECA) most potently inhibited ADP-induced aggregation of human platelets as shown by IC₅₀-values of 0.24 and 0.34 mol/l. 5-N-Methylcarboxamidoadenosine (MECA; IC₅₀ 0.81 mol/l) and 5-N-carboxamidoadenosine (NCA; IC₅₀ 2.1 mol/l) were less potent, whereas adenosine, 2-chloroadenosine and (-)N⁶-phenylisopropyladenosine [(-)PIA] exhibit IC₅₀-values of about 1.5 mol/l. Nearly the same rank order of potency was obtained for stimulation of adenylate cyclase activity of platelet membranes and for inhibition of [³H]NECA binding to human platelets. In order to examine the effects of the carboxamide analogues on R _i adenosine receptors of rat fat cells inhibition of lipolysis and adenylate cyclase were studied. (-)PIA was the most potent inhibitor of lipolysis as shown by an IC₅₀ of 0.5 nmol/l, followed by CPCA (IC₅₀ 1.1 nmol/l) and NECA (IC₅₀ 1.3 nmol/l), whereas MECA (IC₅₀ 17.9 nmol/l) and NCA (IC₅₀ 20.1 nmol/l) were much less potent than NECA in inhibiting lipolysis. Similar results were obtained for inhibition of adenylate cyclase activity of fat cell membranes and for competition with [³H]PIA binding to fat cell membranes. The relative potencies of the adenosine analogues at both receptor subclasses were calculated from the ratio of the IC₅₀-values for inhibition of platelet aggregation and of lipolysis. (-)PIA showed the highest selectivity for R _i receptors as indicated by a 2,900-fold lower IC₅₀ for the antilipolytic than for the antiaggregatory effect. The R _a/R _i activity ratio for NECA was about 260, for CPCA 220, for NCA 105 and for MECA 45. These results indicate that all 5-carboxamide adenosine derivatives are more potent agonists at R _i receptors than at R _a receptors. Since MECA has a higher selectivity for R _a receptors than NECA, it may be useful for the characterization of stimulatory adenosine receptors in different tissues.     

Guanine nucleoside diphosphate and triphosphate modulate [3H]CGS 21680 binding to A2 adenosine receptor in rat striatal membranes

In striatum and several other tissue, a guanine nucleotide binding protein (G_s) couples A₂ adenosine receptor to activation of adenylyl cyclase. We have examined the effect of guanine nucleoside diphosphate and triphosphate on [³H]CGS 21680 binding to A_2A adenosine receptors of rat striatum. Both GDP and GTP inhibited specific [³H]CGS 21680 binding to rat striatal membranes by 50% at about 0.1 mM. GMP was inhibitory only at higher concentrations, and the estimated IC₅₀ value was greater than 1mM. The nonhydrolyzable analog of GTP, GPP (NH)p, was as potent as GTP with an IC₅₀ value of approximately 86 μM. These results suggest that the regulation of A_2a adenosine receptor binding properties by guanine nucleotides is independent of G_s activation, since inhibition of agonist binding is achieved by addition of agonist binding is achieved by addition of both guanine nucleoside diphosphate and triphosphate © 1993 Wiley-Liss, Inc.