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     

Evaluation of the binding of the A-1 selective adenosine radioligand,cyclopentyladenosine (CPA), to rat brain tissue

Summary The binding of [³H]-Cyclopentyladenosine (CPA), an N⁶-substituted analog of adenosine, was examined in vitro. CPA bound with high affinity (K _d=0.48 nmol/l) to rat brain membranes. Specific binding, which represented 90–97% of the total counts bound at a ligand concentration of 1 nmol/l, was saturable, reversible and sensitive to protein denaturation. The pharmacology of binding was consistent with the labeling of an A-1 receptor, the R- and S-diastereomers of N⁶-phenylisopropyladenosine (PIA) showing a sixteenfold difference in their ability to displace CPA. The prototypic A-1 selective adenosine agonist, N⁶-cyclohexyladenosine (CHA) was twofold less active than CPA in displacing radiolabeled CPA. Comparison of the ability of cold CHA and CPA to displace [³H]-CPA gave rat dissociation constants of 1.88 and 1.80×10⁴ s^–1, respectively suggesting that both CHA and CPA bound to the same recognition site. In contrast however, comparison of the binding of [³H]-CPA with that of [³H]-CHA showed distinct differences. The K _d for CHA was approximately twice that of CPA while the apparent B _max was 60% greater. In comparing the pharmacology of CPA binding with that of CHA, it was found that CHA, S-PIA and the antagonist, PACPX were more active in displacing CHA than CPA. In general however, CPA has a binding profile very similar to that observed with CHA.Abbreviations 2-CADO 2 chloroadenosine - CHA N⁶cyclohexyladenosine - CPA N⁶cyclopentyladenosine - EHNA Erythro-9-(2-hydroxy-3-nonyl)adenine - IBMX Isobutylmethylxanthine - NECA 5 N-ethylcarboxamide adenosine - PACPX [1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine] - PIA N⁶-Phenylisopropyladenosine     

Ontogeny of adenosine receptors in the longitudinal muscle and muscularis mucosae of the rat distal colon

The development of adenosine A₁ and A_2B receptors on the longitudinal muscle and muscularis mucosae of the neonatal rat distal colon has been investigated using homogenate binding, quantitative autoradiography and functional studies. In homogenate binding studies 1,3-[³H]-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) bound with high affinity to A₁ receptors in the muscularis mucosae and intact colon from rats aged 10, 15, 20, 25 and 30 days. The affinity of [³H]DPCPX was similar to that in the adult at all ages, but the density of binding sites was higher in the neonatal tissues. Quantitative autoradiography showed a higher density of [³H]DPCPX binding sites in the longitudinal muscle than in the muscularis mucosae at all ages studied (day 10 to adult), and this binding was concentration-dependently displaced by N ⁶-cyclopentyladenosine (CPA). In functional studies the longitudinal muscle relaxed in response to 5’-N-ethylcarboxamidoadenosine (NECA) and CPA at all ages studied (15–30 days), NECA being more potent than CPA. The potency of NECA remained constant and it was antagonised by 1 μM DPCPX at all ages with pA ₂-values consistent with activation of A₂ receptors. The inactivity of 2-[p-(carboxyethyl)-phenylethylamino]-5’-N-ethylcarbox-amidoadenosine (CGS 21680) indicated that the A₂ receptors were of the A_2B subtype. The muscularis mucosae contracted in response to CPA at all ages studied (day 15 to adult) and the antagonism by DPCPX (10 nM) were consistent with activation of A₁ receptors. In conclusion, binding, autoradiographic and functional studies identified A₁ receptors on the rat colon muscularis mucosae at all ages studied. However, while binding and autoradiographic localisation showed the presence of A₁ receptors in the longitudinal muscle at all ages studied, functional data only revealed the presence of A_2B receptors. Received: 3 July 1998 / Accepted: 25 November 1998     

[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.     

The coexistence of adenosine A1 and A2 receptors in guinea-pig aorta

The effects of adenosine, 5'-(N-ethyl)carboxamidoadenosine (NECA), 2-chloroadenosine (2-CA), No-cyclohexyladenosine (CHA) and N6(R-2-phenylisopropyl)-adenosine (R-PIA) on the tone of phenylephrine-constricted guinea-pig isolated aorta have been examined. For aortic relaxation the analogues exhibited the following rank order of potency: NECA > adenosine > 2-CA > R-PIA > CHA. This is consistent with previous reports that relaxation of this tissue is mediated by the adenosine A₂ receptor. An unexpected finding was that R-PIA, 2-CA and CHA all induced contractions at concentrations lower than were required for relaxation, giving a biphasic dose-response curve. Neither NECA nor adenosine contracted the aorta. This is consistent with activation of vascular A₁ receptors. An A₁-selective concentration of the antagonist l,3-dipropyl-8-cyclopentyl xanthine abolished the contraction elicited by R-PIA in the guinea-pig aorta. This further suggests that the contraction is mediated by a₁ receptors.     

Effects of adenosine analogues on contractile response and CAMP content in guinea-pig isolated ventricular myocytes

Summary In the present study the effects of adenosine analogues were investigated on cAMP content and contractile response in guinea-pig ventricular myocytes. The adenosine analogues (–)-N⁶-phenylisopropyladenosine (R-PIA), 5-N-ethylcarboxamideadenosine (NECA) and (+)-N⁶-phenyl-isopropyladenosine (S-PIA) in the presence of 0.01 mol/l isoprenaline reduced contractile response concentration-dependently. R-PIA and NECA were about equipotent (IC₂₅: 0.01 mol/l and 0.039 mol/l respectively), while S-PIA was less potent (IC₂₅: 0.6 mol/l). Isoprenaline stimulated cAMP content was reduced by R-PIA (IC₂₅: 0.004 mol/l) and with lower potency by S-PIA (IC₂₅: 0.15 mol/l) but the extent of reduction of cAMP by R-PIA and S-PIA (to 55% and 64% respectively) was less than the reduction of contractile response (to 26% and 55% respectively). This suggests that the effects of R- and S-PIA on contractile response are only in part due to a reduction in cAMP content. In addition, NECA did not decrease cAMP content but decreased contractile response to the same extent as R-PIA. Similar results were obtained in the presence of the phosphodiesterase inhibitor Ro 20-1724. Time course studies revealed that the effects of R-PIA (1 mol/l) on cAMP content and contractile response coincided reaching steady state after 5 min and remained stable thereafter. The effects of NECA (1 µmol/l) on contractility also reached steady state within 5 min, whereas it did not change cAMP content. It is concluded that the reduction of contractility by adenosine analogues in the presence of isoprenaline can only in part be explained by a reduction of cAMP content. It is suggested that a cAMP-independent effect, possibly an activation of phosphatases, might be involved additionally.Abbreviations R-PIA (–)-N⁶-phenylisopropyladenosine - NECA 5-N-ethylcarboxamideadenosine - S-PIA (+)-N⁶-phenylisopropyl-adenosine - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone Send offprint requests to J. Neumann at the above address     

Pharmacological characterization and autoradiographic localization of dopamine receptors in the rat adrenal medulla

The pharmacological profile and the anatomical localization of dopamine D₁-like and D₂-like receptors were studied in sections of rat adrenal medulla, with radioligand binding and autoradiographic techniques, respectively. [³H]([R]-(+)-chloro-2,3,4,5-tetrahydro-5-phenyl-1H-3benzazepin-al hemimaleate) (SCH 23390) was used as a ligand for dopamine D₁-like receptors and [³H]spiperone was used as a ligand for dopamine D₂-like receptors. Radioligand binding and light microscope autoradiography did not show specific [³H]SCH 23390 binding in sections of rat adrenal medulla. This suggests that rat adrenal medulla does not express dopamine D₁-like receptors. [³H]Spiperone was specifically bound to sections of rat adrenal medulla. The binding was time-, temperature- and concentration-dependent, with a dissociation constant (K_d) of 1.05 nM and a maximum density of binding sites (B_max) of 100.2 ± 3.8 fmol/mg tissue. The pharmacological profile of [³H]spiperone binding to rat adrenal medulla was similar to that displayed by neostriatum, which is known to express dopamine D₂ receptors. Light microscope autoradiography showed the accumulation of specifically bound [³H]spiperone as silver grains within sections of adrenal medulla. Silver grains were found primarily over the cellular membrane of chromaffin cells. The above data indicate that chromaffin cells of the rat adrenal medulla express dopamine receptors belonging to the dopamine D₂ receptor subtype. These receptors are probably involved in the modulation of catecholamine release from chromaffin cells, as documented by functional studies.     

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.     

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.     

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     

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.     

2-Chloro-N6-cyclopentyladenosine: a highly selective agonist at A1 adenosine receptors

Summary 2-Chloro-N⁶-cyclopentyladenosine (CCPA) was synthesized as a potential high affinity ligand for A₁ adenosine receptors. Binding of [³H]PIA to A₁ receptors of rat brain membranes was inhibited by CCPA with a K _i-value of 0.4 nM, compared to a K _i-value of 0.8 nM for the parent compound N⁶-cyclopentyladenosine (CPA). Binding of [³H]NECA to A₂ receptors of rat striatal membranes was inhibited with a K _i-value of 3900 nM, demonstrating an almost 10,000-fold A₁-selectivity of CCPA.CCPA inhibited the activity of rat fat cell membrane adenylate cyclase, a model for the A₁ receptor, with an IC₅₀-value of 33 nM, and it stimulated the adenylate cyclase activity of human platelet membranes with an EC₅₀-value of 3500 nM. The more than 100-fold A₁-selectivity compares favourably with a 38-fold selectivity of CPA. Thus, CCPA is an agonist at A₁ adenosine receptors with a 4-fold higher selectivity and 2-fold higher affinity than CPA, and a considerably higher selectivity than the standard A₁ receptor agonist R-N⁶-phenylisopropyladenosine (R-PIA). CCPA represents the agonist with the highest selectivity for A₁ receptors reported so far.Abbreviations CCPA 2-choro-N⁶-cyclopentyladenosine - CPA N⁶-cyclopentyladenosine - NECA 5-N-ethylcarboxamidoadenosine - PIA N⁶-phenylisopropyladenosine Send offprint requests to M. J. Lohse at the above address     

Pharmacological characterization of benzodiazepine receptors in the brain

Receptors in rat brain membranes which specifically bind ³H-diazepam were characterized pharmacologically using reference substances representing several pharmacological classes of drugs. Of 28 benzodiazepines tested, several “classical” ones (diazepam, clonazepam, lorazepam, oxazepam, nitrazepam, flurazepam, bromazepam and chlorazepate) with known clinical efficacy, as well as three newer “triazolo” benzodiazepines (estazolam, U 35,005, U 31,957), one new “imidazolo” benzodiazepine, U 31,219, and one new 2-carbamoylmethylene-benzodiazepine, displaced ³H-diazepam binding at low concentrations (K_i = 1–60 nM). For these benzodiazepines there was a statistically significant correlation between K_i values for displacement and ED₅₀ (or MED) values in several pharmacological tests predictive of anxiolytic activity in man. More than 100 nonbenzodiazepines, representing 22 distinct pharmacological classes as well as 14 presumed neurotransmitters in the CNS, including 4 peptides, were much weaker as ³H-diazepam displacers (K_i > 0.1 nM). These results that in vitro ³H-diazepam binding represents the physiologically relevant binding to hitherto unknown receptors in the CNS.     

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.     

Xanthine derivatives as antagonists at A1 and A2 adenosine receptors

Summary A variety of alkylxanthines has been comparatively examined as antagonists of A₁ adenosine receptors in rat fat cells, rat and bovine cerebral cortex and of A₂ adenosine receptors in human platelets. With few exceptions all xanthine derivatives with 7-position substituents such as diprophylline, proxyfylline, pentoxifylline and etofylline were less potent antagonists than xanthine itself which hadK _i-values of 170 mol/l (A₁) and 93 mol/l (A₂). Theophylline, caffeine and 3-isobutyl-1-methylxanthine were more potent than xanthine but nearly equipotent antagonists at both receptor subtypes. 8-Phenyl substituents considerably increased the antagonist potency at A₁ and A₂ receptors. 1,3-Diethyl-8-phenylxanthine was the most potent A₂ antagonist (K _i 0.2 mol/l) in human platelets. At A₁ receptors 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) was the most potent antagonist in all three tissues withK _i-values from 0.3 to 8.6 nmol/l. Several 8-phenylxanthine derivatives were remarkably selective antagonists at A₁ receptors. 8-Phenyltheophylline was approximately 700 times more potent as antagonist at A₁ receptors (bovine brain) than at A₂ receptors (human platelets), and PACPX was even 1,600 times more potent as A₁ adenosine receptor antagonist. These compounds offer a possibility for a subtype-selective blockade of adenosine receptors.     

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.     

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.     

Involvement of adenosine A1 receptors in the discriminative-stimulus effects of caffeine in rats

Rationale Caffeine is a non-selective adenosine receptor antagonist in vitro, but involvement of different adenosine receptor subtypes, particularly adenosine A₁ and A_2A receptors, in the central effects of caffeine remains a matter of debate.Objective Investigate the role of adenosine A₁ and A_2A receptors in the discriminative-stimulus effects of caffeine.Methods Rats were trained to discriminate an injection of 30 mg/kg (i.p.) caffeine from saline. The selective A₁ receptor antagonist CPT, the selective A_2A receptor antagonist MSX-3 and the non-selective adenosine receptor antagonist DMPX were assessed for their ability to produce caffeine-like discriminative effects. The ability of CPT, MSX-3, the A₁ receptor agonist CPA and the A_2A receptor agonist CGS21680 to reduce the discriminative effects of caffeine was also tested. Radioligand binding experiments with membrane preparations from rat striatum and transfected mammalian cell lines were performed to characterize binding affinity profiles of the different adenosine antagonists used in the present study (caffeine, DMPX, CPT and MSX-3) in relation to all known adenosine receptors (A₁, A_2A, A_2B, A₃).Results DMPX and CPT, but not MSX-3, produced significant caffeine-like discriminative effects. MSX-3, but not CPT, markedly reduced the discriminative effects of caffeine and the caffeine-like discriminative effects of CPT. Furthermore, the A₁ receptor agonist CPA, but not the A_2A agonist CGS21680, reduced caffeines discriminative effects.Conclusions Adenosine A₁ receptor blockade is involved in the discriminative-stimulus effects of behaviorally relevant doses of caffeine; A_2A receptor blockade does not play a central role in caffeines discriminative effects and counteracts the A₁ receptor-mediated discriminative-stimulus effects of caffeine.     

Chronic intrathecal morphine treatment does not cause down-regulation of spinal adenosine A1 receptors in rats

We have shown previously that systemic chronic morphine treatment causes down-regulation of spinal adenosine A₁ receptors in rats. Recently, we have found that chronic supraspinal morphine treatment also causes this effect. In the present study, we investigated whether chronic spinal morphine treatment has the same effect of down-regulation of spinal adenosine A₁ receptors. Adult male Sprague-Dawley rats were rendered tolerant to morphine either by multiple intrathecal (i.t.) injections or continuous Lt. infusion by osmotic pump administration for 2 or 4 days. Spinal A₁-adenosine receptor binding activity was measured by using the selective A₁ adenosine agonist [³H]cyclohexyladenosine. No significant decrease in [³H]cyclohexyladenosine binding was found in the spinal cord after 2 or 4 days of multiple Lt. injections of morphine. There was also no significant change in the amount of spinal [³H]cyclohexyladenosine bound after 4 days of continuous Lt. infusion of morphine by osmotic pump. From these and our previous results, it is concluded that only supraspinal chronic morphine treatment down regulates the spinal A₁ adenosine receptor and this may play a role in the mechanism of supraspinal morphine tolerance but not spinal morphine tolerance.