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     

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     

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     

Purification and characterization of an adenotin-like adenosine binding protein from human platelets

A low-affinity adenosine binding protein (adenotin) was purified from human platelet membranes by a four-step procedure. Purification was achieved after extraction from human platelet membranes with 0.30% 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Further purification included Sepharose CL 6 B gel filtration, DEAE-Sepharose CL 6 B, and hydroxylapatite chromatography. The protein was purified 884-fold to homogeneity with a 25% yield of binding activity from the membranes. 5-[8(n)-³H]-N-ethyl-carboxamidoadenosine ([³H]NECA) binds to the purified protein with a K_D of 155 (144–167) nmol/l and a B_max of 1.85±0.10 nmol/mg of protein. Sodium dodecylsulfate polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa. The 2-chloro-substituted adenosine analogs 2-chloro-5-N-methylcarb-oxamidoadenosine (CIMECA) and 2-chloro-5-N-ethyl-carboxamidoadenosine (CINECA) were identified as new high affinity ligands of the purified protein showing K_i values of 18 nmol/l and 28 nmol/l, respectively. The low-affinity adenosine binding protein showed a pharmacological profile as follows: CIMECA > 5-N-ethylcarbox-amidoadenosine (NECA) > 2-chloroadenosine (CIA) > 2-[4-(2-carboxyethyl)phenethylamino]-5-N-ethylcarbox amidoadenosine (CGS 21680) > R-N⁶-phenylisopropyl-adenosine (R-PIA).Amino-terminal sequence analysis revealed homologies to endoplasmin, glucose regulated protein (GRP 94), tumor rejection antigen precursor (GP96), and some stress related proteins.Abbreviations CGS 21,680 2-[4-(2-carboxyethyl)phenethylamino]-5N-ethylcarboxamidoadenosine - CIA 2-chloroadenosine - CIMECA 2-chloro-5-N-methylcarboxamidoadenosine - CINECA 2-chloro-5-Nethylcarboxamidoadenosine - CHAPS 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate - NECA 5-N-ethylcarboxamidoaden-osine - DPCPX 1,3-dipropyl-8-cyclopentylxanthine - MECA 5-N-meth-ylcarboxamidoadenosine - R-PIA R-N⁶-phenylisopropyladenosine - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - XAC xanthine amine congener, 8-{4-8[([{(2-aminoethyl)amino}carbonyl]-methyl)oxy]phenyl}-1,3-dipropyl-xanthine Correspondence to: T. Fein at the above adress     

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     

Functional evidence for the presence of adenosine A2-receptors in cultured coronary endothelial cells

Summary Adenosine and the adenosine receptor agonists, R- and S-N⁶-phenylisopropyladenosine (R- and S-PIA) and 5-N-ethylcarboxamidoadenosine (NECA), enhanced [³H]cAMP accumulation in [³H]adenine-labelled cultured endothelial cells isolated from the microvasculature of guinea pig hearts. As shown by their concentration-response curves, NECA was a more potent agonist than R-PIA or adenosine. Their respective concentrations at half-maximal stimulation of [³H]cAMP accumulation were 0.7 M, 10.5 M and 12.6 M, indicating a 15- to 18-fold potency difference between NECA and the other agonists. The increased [³H]cAMP accumulation elicited by 10^–5 M NECA was inhibited by the xanthine derivative 8-phenyltheophylline, 3-isobutyl-l-methylxanthine, theophylline or caffeine. These findings provide functional evidence for the presence of adenosine receptors of the A₂-type in microvascular coronary endothelial cells in culture. The functional significance of these receptors remains to be established, but they may be involved in the regulation of vascular permeability.Abbreviations NECA 5-N-ethylcarboxamidoadenosine - PIA N⁶-phenylisopropyladenosine - 8-PT 8-phenyltheophylline - IBMX 3-isobutyl-l-methylxanthine - IBMX shirlyDL,-4-(3-butoxy-4methoxybenzyl)-2-imidazolidinone Postdoctoral fellow of the Medical Research Council of Canada Send offprint requests to S. Nees at the above address     

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     

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.     

[3H]5′-N-Ethylcarboxamidoadenosine selectively labels the low affinity adenosine binding protein,adenotin, on intact chinese hamster ovary cells

The ability of [³H]5′-N-ethylcarboxamidoadenosine (NECA) to specifically bind recognition sites on intact Chinese hamster ovary (CHO) cells was examined in the present study. Saturation experiments indicated that [³H]NECA bound with moderate affinity (Kd = 400 nM) and large capacity (apparent Bmax = 3.2 pmol/10⁵ cells) to intact CHO cells. No specific binding to these cells was observed with the A₁-selective agonist 20 nM [³H]cyclohexyladenosine or with the A₂-selective agonist 20 nM [³H]CGS 21680. Competition studies revealed that close structural analogs of NECA and the xanthine phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) inhibited 20 nM [³H]NECA binding with moderate affinity (IC₅₀s 0.5–15 μM). Adenosine also showed weak activity (IC₅₀ = 100 μM) for inhibiting [³H]NECA binding. However, a wide variety of prototypic adenosine receptor agonists and antagonists did not significantly interact with these [³H]NECA recognition sites on CHO cells. [³H]NECA binding to CHO cell membranes was not sensitive to guanine nucleotides and NECA did not stimulate cAMP formation. These results are consistent with the previously demonstrated ability of [³H]NECA to bind low affinity adenosine binding proteins (adenotin proteins), as well as, adenosine receptors in a variety of mammalian tissues. The present results further indicate that [³H]NECA selectively labels in adenotin-like recognition site on intact CHO cells in the absence of detectable binding to high affinity adenosine receptors. © 1993 Wiley-Liss, Inc.     

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.     

Pharmacological properties of adenosine receptors and adenosine binding proteins

Two major subtypes of adenosine receptors occur in different tissues which have been distinguished by pharmacological and biochemical criteria. The A₁ adenosine receptor has a high-affinity for adenosine and mediates inhibition of adenylyl cyclase, whereas the A₂ adenosine receptor usually has a lower affinity and mediates stimulation of the enzyme. Furthermore, evidence has been obtained that A₁ receptors increase the conductance of receptor-regulated potassium channels, induce inactivation of calcium channels, and modulate the breakdown of phosphoinositides by phospholipase C. Selective agonists and antagonists have been developed for both receptor subtypes. In addition, both adenosine receptors have extensively been characterized by radioligand binding studies. Suitable radioligands for the A₁ receptor are the agonist [³H]2-chloro-N⁶-cyclopentyladenoisine (CCPA) and the antagonist [³H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX)and for the A_2a receptor [³H]2-[p-(carboxyethyl)phenethylamino]- 5′-N-carboxamidoadenosine (CGS 21860). Furthermore, photoaffinity ligands were developed from adenosine derivatives, which can be covalently incorporated into the binding unit of both receptor subtypes. With this approach, it has been shown that the A₁ receptor has an apparent molecular weight of approximately 36 kDa and the A_2a receptor of 45 kDa. A second approach to elucidate the structure of adenosine receptors involves the purification of receptor protein by affinity chromatography. With this procedure, cerebral A₁ receptors have been purified to apparent homogeneity. More recently, the structure of receptor subtypes has been elucidated by cloning the receptors from a cDNA library. Furthermore, a novel adenosine binding with [³H] 5′ -N-ethylcarboxamidoadenosine ([³H]NECA). The pharmacological profile of this NECA-binding protein has been determined in competition experiments with adenosine receptor ligands. It can be distinguished from that of A_2a adenosine receptors and other adenosine binding proteins. We propose the name A_x for this unique adenosine binding protein. © 1993 Wiley-Liss, Inc.     

Purinoceptor-mediated modulation by endogenous and exogenous agonists of stimulation-evoked [3H]noradrenaline release on rat iris

Summary To investigate whether endogenous purinoceptor agonists affect the sympathetic neurotransmission in the rat isolated iris, and to classify the purinoceptors modulating exocytotic [³H]-noradrenaline release, we have determined the effect of adenosine receptor antagonists on, and the relative potency of selected agonists in modulating, the field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow. In addition, the apparent affinity constants of 8-phenyltheophylline (8-PT) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in antagonizing the prejunctional effects of purinoceptor agonists were estimated.The relatively A₁-selective DPCPX 10 and 100 nmol/l increased the evoked [³H]-noradrenaline overflow by about 25%–35%a indicating a minor inhibition of evoked release by endogenous purinoceptor agonists probably via an A₁ adenosine receptor. Whereas the A₁/A₂-antagonist 8-PT failed to increase the evoked [³H]-noradrenaline overflow in the absence of exogenous agonists (without or with dipyridamole 1 pmol/l present), the relatively A₂-selective antagonist CP-66,713 (4-amino-8-chloro -1-phenyl(1,2,4)triazolo(4,3-a)quinoxaline) 100 nmol/l decreased it by 20%–30% in the absence and continuous presence of DPCPX. This may be compatible with a minor A₂-mediated facilitation by an endogenous purinoceptor agonist.All exogenous agonists tested (except UTP 100 mol/l) inhibited the evoked [³H]-noradrenaline overflow. The relative order of agonist potency (IC4o, concentration in mol/l for inhibition of evoked release by 40%) was CPA (N⁶-(cyclopentyl)adenosine, 0.004) > R-PIA (R(–)N6-(2phenylisopropyl)adenosine, 0.066) = CHA (N⁶-(cyclohexyl)adenosine, 0.082) > NECA (N⁵-(ethyl-carboxamido)adenosine 0.44) > ADO (adenosine, 4.1). ATP was n early equipotent with ADO. Maximum inhibition was 70%–80% and similar for all agonists. Adenosine deaminase 1 u/ml failed to affect the ATP-induced, but abolished the adenosine-induced prejunctional inhibition. The adenosine uptake inhibitor S-p-nitrobenzyl-6-thioguanosine (NBTG) failed to enhance the potency of ADO and ATP. The A₁-selective antagonist DPCPX 10 nmol/l did not reduce the ATP potency indicating an effect of ATP per se not mediated via an A₁ purinoceptor.Prejunctional affinity constants of 8-PT were 6.07 when tested against adenosine (in the presence of dipyridamole), and 6.60 against CHA. The apparent -log K_B of DPCPX tested against CPA was 9.71. The high DPCPX affinity is compatible with an A₁ adenosine receptor mediating inhibition of sympathetic neurotransmission in rat iris. This receptor may not be the only prejunctional purinoceptor on rat iris sympathetic nerves. The receptor by which ATP acts prejunctionally in this tissue remains to be determined.This study was supported by the Deutsche Forschungsgemeinschaft (Fu 163/2 and 163/3) Send offprint requests to H. Fuder at the above address     

Evidence for high-affinity binding sites for the adenosine A2A receptor agonist [3H] CGS 21680 in the rat hippocampus and cerebral cortex that are different from striatal A2A receptors

The binding of the adenosine A_2A receptor selective agonist 2-[4-(2-p-carboxyethyl) phenylamino]-5-N-ethylcarboxamidoadenosine (CGS 21680) to the rat hippocampal and cerebral cortical membranes was studied and compared with that to striatal membranes. [³H] CGS 21680, in the concentration range tested (0.2–200 nM), bound to a single site with a K _d of 58 nM and a B _max of 353 fmol/mg protein in the hippocampus, and with a K _d of 58 nM and a B _max of 264 fmol/mg protein in the cortex; in the striatum, the single high-affinity [³H] CGS 21680 binding site had a K _d of 17 nM and a B _max of 419 fmol/mg protein. Both guanylylimidodiphosphate (100 M) and Na⁺ (100 mM) reduced the affinity of [³H] CGS 21680 binding in the striatum by half and virtually abolished [³H] CGS 21680 binding in the hippocampus and cortex. The displacement curves of [³H] CGS 21680 binding with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), N ⁶-cyclohexyladenosine (CHA), 5-N-ethyl-carboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) were biphasic in the hippocampus and cortex as well as in the striatum. The predominant [³H]CGS 21680 binding site in the striatum (80%) had a pharmacological profile compatible with A_2A receptors and was also present in the hippocampus and cortex, representing 10–25% of [³H]CGS 21680 binding. The predominant [³H]CGS 21680 binding site in the hippocampus and cortex had a pharmacological profile distinct from A_2A receptors: the relative potency order of adenosine antagonists DPCPX, 1,3-dipropyl8-{4-[(2-aminoethyl)amino]carbonylmethyloxyphenyl} xanthine (XAC), 8-(3-chlorostyryl) caffeine (CSC), and (E)-1,3-dipropyl-8-(3,4-dimethoxystyryl)-methylxanthine (KF 17,837) as displacers of [³H] CGS 21680 (5 nM) binding in the hippocampus and cerebral cortex was DPCPX > XAC CSC KF 17,837, and the relative potency order of adenosine agonists CHA, NECA, CADO, 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5-N-ethylcar-boxamidoadenosine (APEC), and 2-phenylaminoadenosine (CV 1808) was CHA NECA CADO > APEC CV1808 > CGS 21680. In the presence of DPCPX (20 nM), [³H] CGS 21680 (0.2-200 nM) bound to a site (A_2A-like) with a K _d of 20 nM and a B _max of 56 fmol/mg protein in the hippocampus and with a K _d of 22 nM and a B _max of 63 fmol/mg protein in the cortex. In the presence of CSC (200 nM), [³H]CGS 21680 (0.2–200 nM) bound to a second high-affinity site with a K _d of 97 nM and a B _max of 255 fmol/mg protein in the hippocampus and with a K _d of 112 nM and a B _max of 221 fmol/mg protein in the cortex. Two pharmacologically distinct [³H]CGS 21680 binding sites were found in synaptosomal membranes of the hippocampus and cortex and in the striatum, one corresponding to A_2A receptors and the other to the second high-affinity [³H]CGS 21680 binding site. In contrast, the pharmacology of [³H]CHA binding was similar in synaptosomal membranes of the three brain areas. The present results establish the existence of at least two high-affinity [³H]CGS 21680 binding sites in the CNS and demonstrate that the [³H]CGS 21680 binding site predominant in the hippocampus and cerebral cortex has different binding characteristics from the classic A_2A adenosine receptor, which predominates in the striatum.     

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     

[3H]MDL 100,907: a novel selective 5-HT2A receptor ligand

In studies using standard radioligands, unlabeled MDL 100,907 (R-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol) has been shown to have a high degree of selectivity for the 5-HT_2A receptor. The present study was undertaken to investigate the receptor binding characteristics of [³H]MDL 100,907 in rat cortical homogenates. [³H]MDL 100,907 was found to reach equilibrium at 37°C after 15 min. Saturation experiments indicated binding to a single site with a K_D of 0.56 nM, Hill slope of 1.15, and a B_max of 512 fmol/mg protein. In parallel experiments with the standard 5-HT_2A receptor radioligand, [³H]ketanserin, with prazosin added to block ₁ receptors, a similar Hill slope and B_max was noted but a two-fold higher K_D was found. In competition binding studies using 0.5 nM [³H]MDL 100,907, some 19 standard ligands to various receptors including the 5HT_1A, D₂, ₁, and receptors resulted in estimated K_I values that were consistent with [³H]MDL 100,907 selectively binding to the 5-HT_2A receptor. A comparison of the K_I values for 17 standard 5-HT_2A receptor agonists and antagonists displacing [³H]MDL 100,907 versus [³H]ketanserin resulted in a highly significant linear correlation (R² = 0.96, P<0.001). Taken together these results suggest that [³H]MDL 100,907 is binding to the 5-HT_2A receptor with a sub-nanomolar affinity without the use of secondary blocking agents.     

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     

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 selective A1 and A2 adenosine receptor agonists on cardiovascular tissues

Summary We investigated the negative chronotropic and vasodilating properties of new selective A₁ and A₂ adenosine agonists such as 2-chloro-N⁶-cyclopentyladenosine (CCPA) and 2-hexynyl-5-N-ethyl-carboxamidoadenosine (2-hexynyl-NECA) as compared with reference adenosine analogues. The potency of these compounds on heart rate was assessed in the rat atrial preparation and their activity on the vascular tone was determined in both rat aorta and bovine coronary artery. CCPA was found to be the most potent At agonist of those currently available in producing negative chronotropic effects (EC₅₀ = 8.2 nM). The A1 antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX) blocked CCPA activity in a dose-dependent manner. There was also a significant correlation between its biological effect and the affinity for A₁ receptors as measured in the rat brain by [³H]-N⁶-cyclohexyladenosine (³[H]-CHA) binding. The A₂ selective agonist 2-hexynyl-NECA showed vasodilating properties comparable with those observed with the reference compounds, CGS 21680 and NECA. EC₅₀ values were 596 and 569 nM in rat aorta and bovine coronary artery, respectively. Moreover, the rank order of potency was similar in the two vascular districts examined, suggesting that the rat aorta is a useful model for studying the effects of adenosine derivatives on vascular tone. In addition, the potency of the compounds in inducing vasodilation was found to be correlated with their affinity for A₂ receptors as measured in the rat striatum by ³[H]-CGS 21680 binding.These data further support that A₁ receptors are involved in depressing cardiac activity and A₂ receptors in inducing vasorelaxation.Correspondence to A. Conti at the above address     

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)]     

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