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.     

[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.     

[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     

Presynaptic opioid receptors modulating acetylcholine release in the hippocampus of the rabbit

Summary Inhibition of cardiae adenylate cyclase by adenosine receptor agonists was reinvestigated in a more homogeneous sarcolemmal vesicular preparation than used in a previous study. Microsomal particles obtained by differential centrifugation were further fractionated on a shallow density gradient of Percoll. Two populations of plasma membrane vesicles were partially resolved. Identical peaks were identified for adenylate cyclase activity and [³H]ouabain binding, whereas 5-nucleotidase activity and -adrenoceptor binding displayed an additonal peak at higher density, where angiotensin converting enzyme, a marker for endothelial plasma membranes, was at maximal activity. Significant inhibition by N⁶-cyclohexyladenosine (CHA), as measured in each fractionation step following homogenization, was observed only at the activity peak of adenylate cyclase. Moreover, analysis of the degree and rank order of potency of several adenosine analogs was indicative for interaction with A₁-adenosine receptors. Accordingly, the peak in adenosine receptor binding, using (-)[¹²⁵I]iodo-N⁶-hydroxyphenyl-isopropyladenosine as the radioligand, coincided with CHA-inhibitable adenylate cyclase activity. By contrast, adenylate cyclase was slightly stimulated by CHA in the higher density range, an action suggested to be mediated via A₂-adenosine receptors, which recently have been demonstrated to exist on guinea-pig coronary endothelium. It is concluded that the full extent of adenosine receptor-mediated adenylate cyclase inhibition in the heart is only to be demonstrated if contamination of the sarcolemmal preparation with endothelial membrane components is kept to a minimum.Abbreviations R-PIA (–)N⁶-(R-phenylisopropyl)-adenosine - NECA 5-(N-ethyl-carboxamido)-adenosine - ICYP (–)[¹²⁵I]iodo-cyanopindolol - dATP 2-deoxy-adenosine-5-triphosphate - S-PIA (+)N⁶-(S-phenylisopropyl)-adenosine - HPIA (–)N⁶-(4-hydroxy-phenylisopropyl)-adenosine - CHA N⁶-cyclohexyl-adenosine - Gpp(NH)p guanylyl imidodiphosphate - dAMP 2-deoxy-adenosine-5-monophosphate - HEPES 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid - EDTA (ethylenedinitrilo)-tetraacetic acid - [¹²⁵I]HPIA (–)N⁶-(3-[¹²⁵I]-iodo-4-hydroxyphenylisopropyl)-adenosine     

Glomeruli and microvessels of the rabbit kidney contain both A1- and A2-adenosine receptors

Summary Rabbit renal cortices were fractionated by collagenase dispersion and glomeruli, microvessels and tubuli purified on a discontinuous sucrose gradient. Binding experiments with (–)[¹²⁵I]N⁶-(4-hydroxyphenylisopropyl)-adenosine ([¹²⁵I]HPIA) provided evidence for the presence of A₁-adenosine receptors in the glomerular and microvascular fraction. With glomeruli, saturation isotherms for specific [¹²⁵I]HPIA binding were mono-phasic with a K _D of 1.3 nmol/l and a B _maxof 7.7 fmol/mg protein. In kinetic experiments, an association rate constant of 4.9 × 105 (mol/ 1)^–1 s^–1 and a dissociation rate constant of 4.3 × 10^–4 s^–1 were obtained, yielding a K _D of 0.9 nmol/l. Adenosine analogs displaced [¹²⁵I]HPIA binding with a rank order of potency typical of A₁-adenosine receptors; furthermore, binding was inhibited by methylxanthines and modulated by GTP. Saturation experiments with the microvessels revealed a K _D of 1.9 nmol/l and a B _max of 13.4 fmol/mg protein. However, no inhibition of glomerular and microvascular adenylate cyclase activity could be demonstrated, but instead both 5-N-ethylcarboxamido-adenosine (NECA) and N⁶-(R-phenylisopropyl)-adenosine (R-PIA) stimulated enzyme activity, with EC₅₀ values of 0.14 mol/l and 1.5 mol/l, respectively. The concentration-response curve for NECA was shifted to the right (factor 9) by 10 mol/l 8-phenyltheophylline. On the other hand, computer simulation of biphasic curves (adenylate cyclase inhibition in the presence of activation via a stimulatory receptor) indicates that the failure to observe an A₁-adenosine receptor-mediated inhibition of adenylate cyclase activity in the presence of stimulatory adenosine receptors may be attributable to methodological constraints. The results demonstrate that both A₁- and A₂-adenosine receptors are present in rabbit glomeruli and microvessels. It is suggested that both receptors are involved in the control of renin secretion.Abbreviations R-PIA (–)N⁶(R-phenylisopropyl)-adenosine - NECA 5-(N-ethyl-carboxamido)-adenosine - S-PIA (+)N⁶-(S-phenylisopropyl)-adenosine - I-HPIA (–)N⁶-(3-iodo-4-hydroxy-phenylisopropyl)-adenosine - HPIA (–)N⁶-(4-hydroxyphenylisopropyl)-adenosine - [¹²⁵I]HPIA (–)N6-(3-[¹²⁵I]iodo-4-hydroxy-phenylisopropyl)-adenosine - ATP adenosine-5-triphosphate - cAMP cyclic 3,5-adenosine-monophosphate - GTP guanosine-5-triphosphate - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - EDTA (ethylenedinitrilo)-tetraacetic acid Send offprint requests to W. Schütz at the above addressThis study was supported by the Fonds zur Förderung der wissenachiftlichen Forschung in Österreich (Project 5712)     

Apparent heterogeneity of cardiac A 1 adenosine receptors as revealed by radioligand binding experiments on N-ethylmaleimide-treated membranes

Summary While G protein-coupled receptors are often studied by analyzing antagonist radioligand: cold agonist inhibition curves using an independent site model, it is now clear that K_L and K_H values determined in these analyses are not reliable estimates of the affinities of the agonists for free and G protein-coupled forms of the receptor. Thus, such experiments cannot be used to contrast the characteristics of a given type of receptor in different tissues, i.e., to probe for the existence of receptor subtypes. Since treatment with N-ethylmaleimide treatment blocks receptor: G_i/G₀ protein interactions, such analyses on N-ethylmaleimide-pretreated membranes should allow direct assessment of the affinities of competing ligands for the free receptor or for multiple receptor subtypes.As A₁ adenosine receptors couple to G_i, and perhaps to G_o, we have performed A₁ adenosine receptor radio-ligand competition studies first on control, then on N-ethylmaleimide-pretreated bovine cardiac and cerebral cortical membranes. Results of experiments with the antagonist radioligand [³H]xanthine amine congener appeared to be confounded by ligand binding to A₂ adenosine receptors present in the cardiac membrane preparations. Further experiments utilized the A₁-specific radioligand [³H] 1,3-dipropyl-8-cyclopentylxanthine. These experiments confirmed once more that the K_L values determined by computer analysis of competition curves performed on control membranes are not reliable estimates of the affinities of the competing ligand for free receptors. Furthermore the results supported the hypothesis that similar analyses on NEM-treated membranes provide reliable estimates of the affinity(s) of competing ligands for free receptors. Lastly, the results suggest that cardiac membranes contain two subtypes of A₁ adenosine receptors that are differentiated by 5-modified but not N⁶-modified adenosine analogs. One of these receptor subtypes appears to be the same as the A₁ receptor detected in cortical membranes.Abbreviations [¹²⁵I]ABA [1251](N⁶-p-aminobenzyl)adenosine - [³H]CPX [³H]8-cyclopentyl-1,3-dipropylxanthine - [³H]R-PIA [³H]N⁶-R-phenylisopropyladenosine - [⁶H]XAC [⁶H]xanthine amine congener - CHA N⁶-cyclohexyladenosine - EDTA ethylenediaminetetraacetic acid - [¹²⁵I]BW-A844U [¹²⁵I]3-(4-amino)phenethyl-l-propyl-8-cyclopentylxanthine - NCCA 5-N-cyclopropylcarboxamide adenosine - NECA 5-N-ethylcarboxamide adenosine - PMSF phenylmethylsulphonyl fluoride - NEM N-ethylmaleimide Recipient of a Postdoctoral Fellowship from the Chicago Heart Association     

8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) — a selective high affinity antagonist radioligand for A1 adenosine receptors

Summary The properties of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as an antagonist ligand for A₁ adenosine receptors were examined and compared with other radioligands for this receptor. DPCPX competitively antagonized both the inhibition of adenylate cyclase activity via A₁ adenosine receptors and the stimulation via A₂ adenosine receptors. The K ₁-values of this antagonism were 0.45 nM at the A₁ receptor of rat fat cells, and 330 nM at the A₂ receptor of human platelets, giving a more than 700-fold A₁-selectivity. A similar Al-selectivity was determined in radioligand binding studies. Even at high concentrations, DPCPX did not significantly inhibit the soluble cAMP-phosphodiesterase activity of human platelets. [³H]DPCPX (105 Ci/mmol) bound in a saturable manner with high affinity to A₁ receptors in membranes of bovine brain and heart, and rat brain and fat cells (K _D-values 50–190 pM). Its nonspecific binding was about 1 % of total at KD, except in bovine myocardial membranes (about 10%). Binding studies with bovine myocardial membranes allowed the analysis of both the high and low agonist affinity states of this receptor in a tissue with low receptor density. The binding properties of [³H]DPCPX appear superior to those of other agonist and antagonist radioligands for the A₁ receptor.Abbreviations CHA N⁶-cyclohexyladenosine - DPX 1,3-diethyl-8-phenylxanthine - HPIA N⁶-p-hydroxyphenylisopropyladenosine - NECA, -N-ethylcarboxamidoadenosine - PIA N⁶-phenylisopro-pyladenosine - XAC (xanthine amine congener) 8-{4-[([{(2aminoethyl)amino}carbonyl]methyl)oxy]phenyl-1,3-dipropyl-xanthine. 8-Cyclopentyl-1,3-dipropylxanthine is abbreviated DPCPX (from 1,3-dipropyl-8-cyclopentylxanthine) Send offprint requests to M. J. Lohse at the above address     

Guanine nucleotide and cation regulation of radioligand binding to R i adenosine receptors of rat fat cells

Summary The modulation of radioligand binding at R _i adenosine receptors of rat fat cells by guanine nucleotides and cations was investigated. Guanine nucleotides (in the order of potency: GTP=GDP>Gpp(NH)p>5-GMP) decreased the binding of the R _i receptor agonist (–)N⁶-phenylisopropyl[³H]adenosine ([³H]PIA), but did not affect binding of the antagonist 1,3-diethyl-8[³H]phenylxanthine ([³H]DPX). Saturation of [³H]PIA binding revealed that GTP (100 mol/l) converts the high affinity form of the R _i receptor into a low affinity form. This effect was confirmed in kinetic experiments. GTP decreased the potency of agonists in competing for [³H]DPX binding, as shown by a 50-fold shift of the K _i-value for (–)PIA, whereas antagonist-induced inhibition of binding remained unchanged. The divalent cations Mg²⁺ and Ca²⁺ produced a slight increase in [³H]PIA binding but did not affect [³H]DPX binding. Mn²⁺ markedly decreased both agonist and antagonist binding at R _i adenosine receptors. Divalent cations reversed the guanine nucleotide-induced decrease of affinity of the R _i receptor. Na⁺ did not significantly affect agonist or antagonist binding but abolished the stimulatory effect of Mg²⁺ on agonist binding in the presence of GTP. Our data indicate that guanine nucleotides convert the R _i adenosine receptor of rat fat cells from a high to a low agonist affinity state and that the modulation of radioligand binding by mono-and divalent cations differs from that of R _i receptors of other tissues.     

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     

Agonist radioligand interactions with the solubilized porcine atrial A1 adenosine receptor

Porcine atrial adenosine receptors have been solubilized using a detergent system consisting of digitonin and sodium cholate and characterized with the agonist radioligand N6[125I]hydroxyphenylisopropyl) adenosine [125I]HPIA. 125IHPIA labeled an apparently homogeneous population of solubilized recognition sites with a Bmax of 88 +/- 4 fmol/mg of protein and a KD of 1.4 +/- 0.1 nM. Solubilization resulted in a 2.5-fold enrichment of adenosine receptor specific activity and an enhanced signal to noise ratio over that observed for porcine atrial membrane preparations. Solubilized cardiac adenosine receptors were relatively stable and exhibited many of the properties of membrane-bound receptors. The rank order potency of adenosine receptor agonists inhibiting the binding of [125I]HPIA was consistent with the labeling of a solubilized A1 adenosine receptor. Association rate experiments suggested that the interaction of [125I]HPIA with solubilized cardiac adenosine receptors was consistent with that of a simple bimolecular reaction. The dissociation constant calculated from kinetic data (0.73 nM) was in good agreement with that determined by equilibrium binding measurements (1.4 nM). The interaction of cardiac A1 adenosine receptors and guanine nucleotide binding protein(s) G protein(s) was retained in this detergent system. Addition of guanosine-5'-O-(3-thio)triphosphate to an equilibrated mixture of solubilized cardiac adenosine receptors and [125I]HPIA resulted in a rapid and complete dissociation of [125I]HPIA. This dissociation was resolved into two kinetic phases, which appear to arise from two populations of independent, noninterconvertible receptor-G protein complexes that display differing sensitivities to guanine nucleotides. The A1 adenosine receptor-G protein complex solubilized in digitonin/cholate appears to provide an excellent system by which agonist radioligand-receptor-G protein interactions can be further studied.     

Characterization of the K+-channel-coupled adenosine receptor in guinea pig atria

Summary In the present work we studied the pharmacological profile of adenosine receptors in guinea pig atria by investigating the effect of different adenosine analogues on⁸⁶Rb⁺-efflux from isolated left atria and on binding of the antagonist radioligand 8-cyclopentyl-1,3-[³H]dipropylxanthine ([³H]DPCPX) to atrial membrane preparations. The rate of⁸⁶Rb⁺-efflux was increased twofold by the maximally effective concentrations of adenosine receptor agonists. The EC₅₀-values for 2-chloro-N⁶-cyclopentyladenosine (CCPA), R-N⁶-phenylisopropyladenosine (R-PIA), 5-N-ethylcarboxamidoadenosine (NECA), and S-N⁶-phenylisopropyladenosine (S-PIA) were 0.10, 0.14, 0.24 and 12.9 M, respectively. DPCPX shifted the R-PIA concentration-response curve to the right in a concentration-dependent manner with a K_B-value of 8.1 nM, indicating competitive antagonism. [³H]DPCPX showed a saturable binding to atrial membranes with a B_max-value of 227 fmol/mg protein and a K_D-value of 1.3 nM. Competition experiments showed a similar potency for the three agonists CCPA, R-PIA and NECA. S-PIA is 200 times less potent than R-PIA. Our results suggest that the K⁺ channel-coupled adenosine receptor in guinea pig atria is of an A₁ subtype.Abbreviations CCPA 2-chloro-N⁶-cyclopentyladenosine - DPCPX 8-cyclopentyl-1,3-dipropylxanthine - NECA 5-N-ethylcarboxami-doadenosine - PIA N⁶-phenylisopropyladenosine Send offprint requests to H. Tawfik-Schlieper at the above address     

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.     

Characterization of agonist radioligand interactions with porcine atrial A1 adenosine receptors

The agonist radioligand (-)-N6-[125I]-p-hydroxyphenylisopropyl-adenosine ([ 125I]HPIA) was used to characterize adenosine recognition sites in porcine atrial membranes. [125I]HPIA showed saturable binding to an apparently homogeneous population of sites with a maximum binding capacity of 35 +/- 3 fmol/mg of protein and an equilibrium dissociation constant of 2.5 +/- 0.4 nM. Kinetic experiments were performed to address the molecular mechanism of [125I]HPIA binding in porcine atrial membranes. [125I]HPIA apparently interacts with the cardiac adenosine receptor in a simple bimolecular reaction. A kinetically derived [125I] HPIA dissociation constant (2.4 nM) was in good agreement with that parameter measured at equilibrium. Guanyl nucleotides negatively modulated [125I]HPIA binding by increasing its rate of dissociation. This finding is consonant with the formation of a ternary complex in porcine atrial membranes, consisting of ligand, receptor, and guanyl nucleotide-binding protein. Prototypic adenosine receptor agonists and antagonists inhibited specific binding in a manner consistent with the labeling of an A1 adenosine receptor. The results of these experiments suggest that the adenosine receptor present in porcine atrial membranes, as labeled by [125I]HPIA, is of the A1 subtype.     

Purification and characterization of (-)[125I]hydroxyphenylisopropyladenosine, an adenosine R-site agonist radioligand and theoretical analysis of mixed stereoisomer radioligand binding

(-)-N6-(R-4-Hydroxyphenylisopropyl)adenosine (HPIA) was iodinated with NaI and trace 125I. Mono- and diiodinated reaction products and the starting material were separated by high pressure liquid chromatography and the structures of the reaction products were verified by NMR. (-)-N6-(R-Phenylisopropyl)adenosine (PIA), IHPIA, and I2HPIA decreased rat atrial contractility with ED50 values of 24, 28, and 33 nM, respectively. The contractile effects of these compounds were competitively blocked by theophylline (KI = 7.9 microM), but were not affected by adenosine deaminase. IHPIA also inhibited (-)isoproterenol-stimulated cyclic AMP accumulation in adipocytes with an ED50 (10 nM) and to an extent (83%) nearly identical to PIA. [125I]HPIA prepared using carrier-free 125I bound to adenosine receptors on membranes from rat cerebral cortex, adipocyte ghosts, and heart ventricles. Binding was inhibited stereospecifically by PIA and by other adenosine analogues and alkylxanthines. The KD of [125I]HPIA determined kinetically using brain membranes at 21 degrees was 0.94 nM (K1 = 2.55 X 10(7) M-1 min-1; K-1 = 0.024 min-1) in good agreement with the equilibrium determination of 1.94 nM. The density of adenosine receptors in brain membranes was found to be 871 fmol/mg of protein. When normalized to protein, the density of receptors in heart membranes and adipocyte ghosts, respectively, was found to be 39- and 2.3-fold less than in brain membranes. We conclude that [125I]HPIA can be rapidly synthesized and purified, binds to adenosine R-sites and is an agonist radioligand resistant to adenosine deaminase. Computer modeling of the equilibrium binding resulting from the use of mixed stereoisomers of a radioligand indicates that the combined use of (-)[125I]HPIA and (+)[125I]HPIA would result in the generation of nonlinear Scatchard plots.     

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.     

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.     

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     

Identification of a novel high affinity adenosine binding protein from bovine striatum

Summary In solubilized extracts from bovine striatal membranes three different binding sites for 5-N-ethylcarboxamidoadenosine ([³H]NECA) were observed after separation of the extract by gel filtration on Sepharose CL-6B. The first peak was eluted in the void volume and contained the AZ adenosine receptor. In the second peak, [³H]NECA binding sites were eluted with a pharmacological profile characteristic of adenotin, a low affinity non-receptor adenosine binding protein. The third peak represented approximately 50% of the [³H]NECA binding activity. This site bound [³H]NECA in a reversible and saturable manner with K _D of 17 nmol/l and a binding capacity of 11.3 pmol/mg protein. In competition experiments, adenosine, NECA, NAD, nnosine, 5-AMP and S-adenosyl-L-homocysteine were the most potent ligands. In contrast to adenosine receptors, this site did neither bind adenosine receptor antagonists nor the A₂ selective agonist CGS 21,680 (2-[p-(2-carboxyethyl)phenethylamino]5-N-ethylcarboxamidoadenosine). These results suggest the existence of a novel high affinity binding site for adenosine of unknown function in bovine striatum.Abbreviations AMPPCP ,-methyleneadenosine-5-triphosphonate - CCPA 2-chloro-N⁶-cyclopentyladenosine - CHAPS 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate - CGS 21,680 2-[p-(2-carboxyethyl)phenethylamino]-5-N-ethylcarbox-amidoadenosine - CPA N⁶-cyclopentyladenosine - DPCPX 1,3-dipropyl-8-cyclopentylxanthine - GppNHp guanosine-5-[,-imido]triphosphate - GTP[S] guanosine-5-O-(3-thiotriphosphate) - NBTI S-4-nitrobenzyl-6-thioinosine - NECA 5-N-ethylcarbox-amidoadenosine - NECG 5-N-ethylcarboxamidoguanosine - PIA N⁶-phenylisopropyladenosine - SAH S-adenosyl-L-homocysteine - XAC 8-{4-[([{(2-aminoethyl)-amino}carbonyl]-methyl)oxy]-phenyl}-1,3-dipropylxanthine Send offprint requests to A. Lorenzen at the above address     

Affinities of barbiturates for the GABA-receptor complex and A1 adenosine receptors: a possible explanation of their excitatory effects

Summary The effects of barbiturates on the GABA-receptor complex and the A₁ adenosine receptor were studied. At the GABA-receptor complex the barbiturates inhibited the binding of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPT) and enhanced the binding of [³H]diazepam. Kinetic and saturation experiments showed that both effects were allosteric. Whereas all barbiturates caused complete inhibition of [³⁵S]TBPT binding, they showed varying degrees of maximal enhancement of [3H]diazepam binding; (±)methohexital was identified as the most efficacious compound for this enhancement. At the A₁ adenosine receptor all barbiturates inhibited the binding of [³H]N⁶-phenylisopropyladenosine ([³H]PIA) in a competitive manner. The comparison of the effects on [³H]diazepam and [³H]PIA binding showed that excitatory barbiturates interact preferentially with the A₁ adenosine receptor, and sedative/anaesthetic barbiturates with the GABA-receptor complex. It is speculated that the interaction with these two receptors might be the basis of the excitatory versus sedative/anaesthetic properties of barbiturates.Abbreviations GABA -aminobutyric acid - TBPT t-butylbicyclophosphorothionate 1073 - DMBB 5-(1,3-dimethyl)butyl-5-ethylbarbituric acid - MCB N-methyl-5-(1-cyclohexen-1-yl)-5-ethylbarbituric acid - MPPB N-methyl-5-phenyl-5-propylbarbituric acid - PIA N⁶-phenylisopropyladenosine Send offprint requests to M. J. Lohse at the above address     

Binding of adenosine receptor ligands to brain of adenosine receptor knock-out mice: evidence that CGS 21680 binds to A1 receptors in hippocampus

The adenosine receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5-N-ethylcarboxamidoadenosine (CGS 21680) is generally considered to be a selective adenosine A_2A receptor ligand. However, the compound has previously been shown to exhibit binding characteristics that are not compatible with adenosine A_2A receptor binding, at least in brain regions other than the striatum. We have examined binding of [³H]CGS 21680 and of antagonist radioligands with high selectivity for adenosine A₁ or A_2A receptors to hippocampus and striatum of mice lacking either adenosine A₁ (A1R^(–/–)) or A_2A (A2AR^(–/–)) receptors. Both receptor autoradiography and membrane binding techniques were used for this purpose and gave similar results. There were no significant changes in the binding of the A₁ receptor antagonist [³H]DPCPX in mice lacking A_2A receptors, or in the binding of the A_2A receptor antagonists [³H]SCH 58261 and [³H]ZM 241385 in mice lacking A₁ receptors. Furthermore, [³H]CGS 21680 binding in striatum was abolished in the A2AR^(–/–), and essentially unaffected in striatum from mice lacking A₁ receptors. In hippocampus, however, binding of [³H]CGS 21680 remained in the A2AR^(–/–), whereas binding was virtually abolished in the A1R^(–/–). There were no adaptive alterations in A_2A receptor expression in this region in A1R^(–/–) mice. Thus, most of the [³H]CGS 21680 binding in hippocampus is dependent on the presence of adenosine A₁ receptors, but not on A_2A receptors, indicating a novel binding site or novel binding mode.