[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     

Quantitative [3H] dipyridamole autoradiography: evidence for adenosine transporter heterogeneity in guinea pig brain

Summary [³H] Dipyridamole binding in guinea pig brain slices has been characterized. Binding of [³H] dipyridamole to guinea pig forebrian slices was found to be rapid, reversible and saturable. Saturation experiments revealed a class of high affinity binding sites with a B _max value of 592 ± 118 fmol/mg protein and K _d value of 10.8 nM ± 2.1 nM in the analysed concentration range. In competition experiments, the adenosine transport inhibitors hexobendine and dipyridamole itself were the most potent displacers (inhibition constants of 4.6 nM ± 1 nM and 11.5 nM ± 3 nM) with pseudo-Hill coefficients close to 1. Competition curves with nitrobenzylthioinosine, another adenosine transport inhibitor, however, showed a biphasic profile with a pseudo-Hill coefficient of 0.33 ± 0.04. Just 42% ± 4% of [³H] dipyridamole binding were inhibited by nanomolar concentrations of nitrobenzylthioinosine and only micromolar concentrations displaced the remainder. Subsequent quantitative autoradiography demonstrated regional differences in the inhibition of [³H] dipyridamole binding by submicromolar concentrations of nitrobenzylthioinosine. While in cortical areas of cerebrum and cerebellum 500 nM nitrobenzylthioinosine displaced binding of [³H] dipyridamole to only about one-third of its sites (in the Purkinje cell layer less than 10%), it showed similar potency as dipyridamole in various areas of the brainstem and hypothalamus. This biphasic and regionally heterogenous interaction of nitrobenzylthioinosine with [³H] dipyridamole binding sites in guinea pig brain slices strongly suggests heterogeneity of adenosine transporters.     

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

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

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

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

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

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

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     

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

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

[3H]-Spiroperidol labels dopamine receptors in membranes from rabbit mesenteric artery

Summary The potent dopamine receptor antagonist [³H]-spiroperidol was used to label binding sites in a membrane fraction derived from rabbit mesenteric artery which had characteristics expected for dopamine receptors. The binding was of high affinity with an equilibrium dissociation constant (K_D) of 13.1 nM; it was saturable with 110 fmol of [³H]-spiroperidol bound/mg protein at maximal occupancy of the sites. Binding at 37° C was rapid and readily reversible with rate constants of 0.0154 nM^–1 min^–1 and 0.114 min^–1 for forward and reverse reaction, respectively. Dopamine receptor antagonists were about 100–200 times more potent than -adrenolytic drugs in competing for the [³H]-spiroperidol binding sites and dopamine was much more potent than (–)-noradrenaline, adrenaline, (–)-isoprenaline, clonidine or serotonin. It is concluded that in a membrane fraction of the rabbit mesenteric artery there exist binding sites for [³H]-spiroperidol indistinguishable from dopamine receptors. Thus the present results support the view that in vascular smooth muscle there exist specific dopamine receptors.This work was supported by the Deutsche Forschungsgemeinschaft     

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     

[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.     

[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.     

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.     

Characterization of two distinct alpha-adrenoceptor binding sites in smooth muscle cell membranes from rat and bovine aorta

Summary In order to characterize postjunctional alpha adrenoceptor binding sites of aortic smooth muscle, the specific binding of (³H)prazosin and (³H)yohimbine to membranes prepared from the medial layers of rat and bovine thoracic aorta was investigated. Binding of (¹²⁵I)-BE 2254 (2-[B-(4-hydroxyphenyl)-ethylaminomethyl] tetralone) and (³H)RX 781094 (idazoxan) was also examined in bovine membranes. Each of the ligands displayed saturable, specific binding to a single population of sites; the K _D values of the respective ligands were similar in the two animal species. The number of (³H)prazison and (¹²⁵I)BE 2254 binding sites (160–190 fmol · mg protein^–1 in the two species) was higher than the number of (³H)yohimbine and (³H)RX 781094 binding sites (110–120 fmol · mg protein^–1 in the bovine and 50 fmol · mg protein^–1 in the rat). Alpha-adrenoceptor ligands inhibited binding of the ligands with the following orders of potency: prazosin > BE 2254 > yohimbine > RX 781094 > clonidine in the case of (³H)-prazosin, and yohimbine > RX 781094 > clonidine > prazosin in the case of (³H)yohimbine. Methoxamine, in concentrations up to 10 M, was without effect on the binding of either ligand. The absence or presence of Na⁺, K⁺ or Ca²⁺ added at physiological concentrations did not change the order of potency of displacing ligands whereas Ca²⁺ reduced by 50% the numbers of (³H)prazosin and (³H)-yohimbine sites and Na⁺ increased by 3-fold the affinity of (³H)yohimbine.It is concluded that post-junctional membranes from rat and bovine aortic smooth muscle contain two distinct ₁- and ₂-adrenoceptor binding sites, the number of the latter being less than the number of the former.     

Characterization of nicotinic acetylcholine receptors on cultured bovine adrenal chromaffin cells using modified l-[3H]nicotine binding assay

Summary To characterize the properties of nicotinic acetylcholine receptors (nAChRs) in autonomic ganglia, we examined l-[³H]nicotine binding to membrane fraction prepared from cultured bovine adrenal chromaffin cells, using a modified filtration method. Binding of l-[³H]nicotine to non-treated glass fiber filters interfered with the detection of specific binding to the membrane fraction. Presoaking glass fiber filters in 3% or higher concentrations of polyethyleneimine (PEI) solution (sixty times higher than earlier used concentration) for at least 5 h could reduce the binding of l-[³H]nicotine to the filters to the background level. Specific l-[³H]nicotine binding to the membrane fraction was detected only when the membrane fraction was prepared in Ca²⁺- and Mg²⁺ (EDTA, EGTA and protease inhibitors were added)-free buffer. Specific binding of l-[³H]nicotine was saturable and reversible. Both computer program and Scatchard analysis revealed a single class of high affinity binding sites with an average K_d of 8.9 nM and a B_max of 42.5 fmol/mg protein. The Hill coefficient was 0.98. In inhibition studies, both cholinergic agonists (carbachol and l-nicotine) and ganglionic agonists (lobeline and 1,1-dimethyl-4-phenylpiperazinium iodide) were much effective in inhibiting l-[³H]nicotine binding, whereas both neuromuscular blocking (-bungarotoxin and d-tubocurarine) and ganglionic blocking agents were less effective. These results suggest that high affinity nicotinic binding sites on adrenal chromaffin cells are nAChRs of the ganglion-type, which have properties different from nAChRs on the neuromuscular junction but similar to nAChRs in the brain. Send offprint requests to K. Lee at his present address     

Inhibition of 5-HT3 receptor function by imidazolines in mouse neuroblastoma cells: potential involvement of σ2 binding sites

The influence of several imidazolines and -site ligands on cation influx through the 5-HT₃ receptor channel in NIE-115 mouse neuroblastoma cells was studied by measuring the 2-min influx of the organic cation [¹⁴C] guanidinium induced by 1 M 5-HT (in the presence of 10 M substance P in all experiments). In addition, we determined specific binding of [³H]DTG (1,3-di(2-tolyl)-guanidine), a selective -site radioligand, and [³H] GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone), a selective 5-HT₃ receptor antagonist, to membranes prepared from NIE-115 cells.The 5-HT-induced [¹⁴C]guanidinium influx was inhibited by the imidazolines, ondansetron, antazoline, idazoxan, BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), cirazoline, naphazoline, clonidine and by the guanidine agmatine, but not by the catecholamine adrenaline. The inhibitory effect of the imidazolines on cation influx through the 5-HT₃ receptor channel was mimicked by the -site ligands, (±)-ifenprodil, (+)-3-PPP ((R)-3-(3-hydroxyphenyl)-N-propylpiperidine), DTG (1,3-di-tolyl-guanidine), haloperidol, dizocilpine, and ketamine as well as by the polyamines, arcane and spermidine. — Ondansetron inhibited [³H]GR65630 binding with high affinity, whereas inhibition of binding of this radioligand to the 5-HT₃ receptor by antazoline, BDF 6143, idazoxan, cirazoline, (±)-ifenprodil, (+)-3-PPP, DTG and haloperidol occurred in the high micromolar range. In the competition experiments with [³H]DTG, (±)-ifenprodil, haloperidol, unlabelled DTG, BDF 6143 and (+)-3-PPP inhibited binding of the radioligand at moderate affinity (K_i values in the range of 1 M or lower), whereas ondansetron, amazoline, idazoxan, cirazoline, naphazoline, clonidine, tolazoline, efaroxan, RX821002 (2-[2-(2-methoxy-1,4-benzodioxanyl)]imidazoline), ketamine and spermidine exhibited affinity in the high micromolar or millimolar range only. Comparison of the potencies of the ligands (pIC_50% values) in inhibiting 5-HT-induced [¹⁴C]guanidinium influx with their affinities (pK_i values) at the 5-HT recognition sites of the 5-HT₃ receptor and at the ₂-sites of the N1E-115 cells by means of multiple regression analysis revealed a significant correlation with the affinities at both sites.In conclusion, our data suggest that imidazolines and -ligands, which as a rule possess low affinity for the 5-HT recognition site of the 5-HT₃ receptor, may be assumed to exert their inhibitory effect on cation influx through the 5-HT₃ receptor channels, at least in part, by interacting with ₂-binding sites.     

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.     

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     

The modulation of [3H]noradrenaline and [3H]serotonin release from rat brain synaptosomes is not mediated by the α2B-adrenoceptor subtype

Summary The present study aimed at relating the presynaptic ₂-adrenoceptors, known to modulate noradrenaline and serotonin release, with the recently described _2A- and _2B-adrenoceptor subtypes. The effects of the agonist oxymetazoline (selective for _2A subtype) and of three adrenoceptor antagonists (idazoxan, 1-(2-pyrimidinyl)piperazine (PmP) and prazosin, the last one known to be _2B selective) were evaluated on [³H]noradrenaline and [^H]serotonin release in superfused synaptosomes from rat brain cortex. These drugs were also tested in [³H]yohimbine binding to human platelet membranes (containing only _2A receptors) and to neonatal rat lung membranes (containing only _2B receptors).The affinity pattern of these compounds at _2A-adrenoceptors in binding studies was oxymetazoline > = idazoxan > PmP > prazosin; at _2B-adrenoceptors it was idazoxan > = prazosin > PmP = oxymetazoline. Oxymetazoline inhibited with high and similar potencies the K⁺-evoked [³H]noradrenaline and [³H]serotonin release, IC₅₀ 18 and 7 nM, respectively; in the same conditions, the IC₅₀ values of noradrenaline were 42 and 168 nM, respectively. The antagonist affinity pattern (antagonism against noradrenaline) was idazoxan > PmP > prazosin, either on [³H]serotonin release.These results indicate that presynaptic ₂ auto- or heteroreceptors do not belong to the _2B subtype and suggest that the modulation of noradrenaline and serotonin release may be mediated by the _2A-adrenoceptor subtype. Send offprint requests to M. Gobbi at the above address     

Characterization of adenosine receptors in rat brain by (−)[3H]N6-phenylisopropyladenosine

Summary (–)N⁶-Phenylisopropyladenosine, a potent agonist in adenosine-responsive cellular systems, has been labeled with tritium to high specific activity (26 Ci/mmol) and used to identify adenosine binding sites in rat brain membranes. (–)[H³]N⁶-Phenylisopropyladenosine binding was studied by a vacuum filtration technique. The binding was rapid, rapidly reversible, dependent on pH and temperature and stereospecific since the (–)isomer of N⁶-phenylisopropyladenosine was 40-fold more potent than the (+)isomer in competition experiments. The stereospecific binding sites were saturable and bound 0.8 pmol of (–)N⁶-phenylisopropyladenosine per mg of membrane protein. The dissociation constant (K_D) of (–)N⁶-phenylisopropyladenosine for these sites was 5–12 nM as determined independently by saturation and kinetic binding studies. Endogeneous ligands seem to occupy the binding sites since pretreatment with adenosine deaminase increased the specific binding.Adenosine and several adenosine derivatives were studied for their ability to compete with (–)[³H]N⁶-phenylisopropyladenosine binding. (–)N⁶-Phenylisopropyladenosine-5-monophosphate, N⁶-phenyladenosine, N⁶-benzyladenosine, 2-chloroadenosine and adenosine were most potent in displacing the radioligand from its binding sites and the IC₅₀-values ranged from 0.3–7 M. Physiologically inactive compounds such as inosine, hypoxanthine, adenine and the ribose-modified analogues 2-deoxyadenosine and 2,5-dideoxyadenosine did not substantially inhibit binding at concentrations up to 100 M. The adenosine antagonists isobutylmethylxanthine (IC₅₀ 3.2 M), theophylline (IC₅₀ 7.6 M) and caffeine (IC₅₀ 99 M) competed for the binding sites of (–)[³H]N⁶-phenylisopropyladenosine in a manner which parallels their known pharmacological activity whereas other phosphodiesterase inhibitors were ineffective.The (–)[³H]N⁶-phenylisopropyladenosine binding sites in rat brain membranes appear to be equivalent to adenosine receptor sites on the cell surface which have recently been classified as R-site adenosine receptors.This work is dedicated in memory of Erik Westermann (1923–1978) and Klaus Stock (1931–1978) who first described the potent pharmacological effects of N⁶-phenylisopropyladenosine and stimulated much of the recent interest in adenosine receptor research