On the high affinity binding site for [3H]-1,3-dipropyl-8-cyclopentylxanthine in frog brain membranes.

1. Radioligand binding properties of the adenosine receptor ligands, [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), and [3H]-R-phenylisopropyladenosine ([3H]-R-PIA) were investigated in frog brain membranes. 2. The specific binding of the adenosine antagonist, [3H]-DPCPX to frog brain membranes showed one binding site with Kd and Bmax values of 43.8 nM and 0.238 +/- 0.016 pmol mg-1 protein, respectively. Guanosine 5'-triphosphate (GTP, 100 microM) decreased to 72 +/- 7% and Mg2+ (8 mM) increased to 121 +/- 3% [3H]-DPCPX (40 nM) binding to frog brain membranes. 3. [3H]-DPCPX saturation binding experiments performed in the presence of Mg2+ (8 mM), or in the presence of GTP showed that Mg2+ ions decreased the Kd value of [3H]-DPCPX to 14 nM, and GTP increased this value to 65.6 nM. Bmax values were not significantly (P > 0.05) modified (0.261 +/- 0.018 pmol mg-1 protein, with Mg2+, and 0.266 +/- 0.026 pmol mg-1 protein, in presence of GTP) by the presence of Mg2+ or GTP. 4. The specific binding of [3H]-R-PIA (15 nM) was decreased to 37 +/- 6% by GTP (100 microM) and increased to 123 +/- 4% by Mg2+ (8 mM). [3H]-R-PIA saturation binding experiments performed in the presence of Mg2+ (8 mM) showed one binding site with Kd and Bmax values of 0.9 nM and 0.229 +/- 0.008 pmol mg-1 of protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apparent affinity of 1,3-dipropyl-8-cyclopentylxanthine for adenosine A1 and A2 receptors in isolated tissues from guinea-pigs

1. The classification of adenosine receptor subtypes (A1 and A2) in intact tissues has been based on the order of agonist potency. In this study the apparent affinity of 1,3-dipropyl-8-cyclopentylxanthine (CPX), an antagonist which has been reported to be A1 selective, and the non-selective antagonist 1,3-dimethyl-8-phenylxanthine (8PT) has been evaluated on isolated tissues from the guinea-pig. 2. The isolated tissues used were atria (bradycardic response, proposed A1 sub-type), aorta and trachea (relaxant response, proposed A2 sub-type). 3. Both the xanthines antagonized responses to adenosine in the three tissues but had little or no effect on responses to carbachol (atria), sodium nitrite (aorta) or isoprenaline (trachea). 4. pA2 values for 8PT were similar on the three tissues (6.3-6.7), however, the pA2 value for CPX on the atria (7.9-8.4) was greater than that on the aorta (6.6) or trachea (6.6). 5. These results support the suggestion that the adenosine receptors which mediate bradycardia in the atrium are of the A1 sub-type and that those which mediate relaxation in the aorta and trachea are of the A2 type.     

Labeling of A1 adenosine receptors in porcine atria with the antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine

We have used the antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine to label adenosine recognition sites in porcine atrial membranes. 8-Cyclopentyl-1,3-[3H]dipropylxanthine bound saturably, reversibly and with high affinity to an apparently homogeneous population of recognition sites with a Bmax of 32.0 +/- 0.9 fmol/mg protein and a KD of 0.394 +/- 0.049 nM. Prototypic adenosine receptor agonists inhibited the specific binding of 8-cyclopentyl-1,3-[3H]dipropylxanthine in a manner consistent with the labeling of an A1 adenosine receptor. 8-Cyclopentyl-1,3-[3H]dipropylxanthine appears to be a valuable antagonist radioligand for the characterization of cardiac adenosine receptors.     

The binding of [3H]4-diphenylacetoxy-N-methylpiperidine methiodide to longitudinal ileal smooth muscle muscarinic receptors

Muscarinic receptors present in longitudinal ileum were characterized using the non-selective radioligand [3H]N-methylscopolamine [( 3H]NMS) and the M3 selective radioligand [3H]4-diphenylacetoxy-N-methylpiperidine methiodide [( 3H]4DAMP). In saturation studies, [3H]4DAMP, but not [3H]NMS, identified two populations of binding sites with 17% of the sites (155 fmol/mg protein) displaying high affinity (Kd = 0.39 nM) for [3H]4DAMP and the remaining sites displaying low affinity for the radioligand (Kd = 4.43 nM). In competition studies gallamine and methoctramine, but not AF-DX 116, identified two populations of [3H]NMS binding sites. Affinity estimates for gallamine and methoctramine indicated that 80% of the [3H]NMS binding sites were of the M2 subtype. The minor population of [3H]NMS binding sites could not be readily characterized, due partly to the low selectivity of the competing ligands and also to the relatively low density of the sites. In studies using the M3 muscarinic receptor selective radioligand [3H]4DAMP, the minor population of sites could be preferentially labeled by using a low concentration (0.4 nM) of [3H]4DAMP. Under these conditions, [3H]4DAMP labeled approximately equal levels of the two muscarinic receptor binding sites present in the ileum. Competition studies with AF-DX 116, gallamine and methoctramine indicated that the two [3H]4DAMP binding sites displayed the pharmacology expected of the M2 and M3 receptors, respectively. These results provide additional evidence that longitudinal ileal smooth muscle membranes contain both M2 and M3 muscarinic receptors and indicate that [3H]4DAMP is a useful ligand for identifying heterogeneity of muscarinic receptor subtypes.     

Diuretic and saliuretic effects of 1,3-dipropyl-8-cyclopentylxanthine, a selective A1-adenosine receptor antagonist.

We have previously shown that 8-phenyltheophylline (8-PT), a non-selective antagonist at adenosine A1- and A2-receptors, has a diuretic effect. In this study, the diuretic and adenosine antagonist effects of the A1-receptor selective compound 1,3-dipropyl-8-cyclopentylxanthine (CPX) have been examined in the conscious rat. CPX (0.1 and 0.3 mg kg-1 i.v.) significantly attenuated bradycardic but not hypotensive responses evoked by adenosine. In contrast, 8-PT (3 mg kg-1 i.v.) significantly antagonized both adenosine-induced bradycardia and hypotension. CPX (0.1 and 0.3 mg kg-1 i.v.) evoked a dose-related diuretic and saliuretic response in the conscious rat. These results indicate that the diuretic effects of adenosine antagonists are associated with blockade of the A1-receptor sub-type.     

A1 Adenosine receptors can occur manifesting two kinetic components of 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX) binding

The results described in this paper show, for the first time, that At adenosine receptors can have two kinetic components for the binding of the antagonist [³H]DPCPX. At low ionic strength ( 42mmo1/l), dissociation of [³H]DPCPX bound to A₁ receptors fitted better to a two kinetic components model than to a one kinetic component model. The kinetic constants were consistent with comparable Kd values for the two components of the antagonist binding, and therefore these two components cannot be distinguished by saturation isotherm analysis. Correspondence to: E.I. Canela at the above address     

Characterization of adenosine binding sites in bovine testicular tissue using 8-cyclopentyl-1,3-[3H]dipropylxanthine

The existence of specific adenosine binding sites in bovine testicular tissue was evaluated using the novel antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX). Saturation analysis revealed specific binding that was saturable at approximately 1 nM. Scatchard analysis indicated a single class of binding sites with a KD = 0.26 nM and a Bmax = 0.37 pmol/mg protein. Affinity profiles suggest an A1 subtype recognition site that is different from the classical A1 adenosine receptor. The results presented should prove useful in subsequent studies concerning heterogeneity among adenosine receptors and also aid in discerning the role of adenosine in reproduction.     

A new class of adenosine receptors in brain. Characterization by 2-chloro[3H]adenosine binding

Micromolar concentrations of adenosine and its analogs have profound depressant effects on neuronal firing and synaptic transmission in many brain areas. Using the adenosine agonist 2-chloro[3H]adenosine (Cl[3H]Ado), we have identified a distinct class of micromolar-affinity adenosine binding sites in rat forebrain membranes. Specific Cl[3H]Ado binding was reversible and saturable with an apparent KD of 9.1 microM and a Bmax of 61 pmoles/mg protein. The present studies were conducted using washed brain membrane fractions not treated with adenosine deaminase. Specific Cl[3H]Ado binding under these conditions was insensitive to (-)-N6-(R-phenylisopropyl)adenosine ((-)PIA) and treatment with 3 mM N-ethylmaleimide, unlike high-affinity A1 adenosine receptor binding. Treatment of membranes with adenosine deaminase revealed an additional population of binding sites sensitive to (-)PIA. Inhibition of Cl[3H]Ado binding by adenosine analogs exhibited an order of potency ClAdo greater than 5'-N-ethylcarboxamide adenosine (NECA) greater than (-)PIA which differs from that of both A1 and A2 adenosine receptors. The potent A1 and A2 receptor antagonist 8-phenyltheophylline had no significant effect on binding up to 10 microM. Specific binding, however, was inhibited by the adenosine antagonists 8(p-sulfophenyl)theophylline, isobutylmethylxanthine, theophylline, and caffeine. Micromolar Cl[3H]Ado binding was highly selective for adenosine agonists and antagonists. These results suggest that the micromolar-affinity Cl[3H]Ado binding sites may represent a novel central purinergic receptor, distinct from the A1 and A2 adenosine receptors involved in the regulation of adenylate cyclase.     

Interactions of spironolactone with (+)-[3H]-isradipine and (-)-[3H]-desmethoxyverapamil binding sites in vascular smooth muscle.

Spironolactone partially inhibited the specific binding of (+)-[3H]-isradipine and (-)-[3H]-desmethoxyverapamil to vascular smooth muscle membranes. It is suggested that spironolactone interacts at a binding site of the calcium channel complex and allosterically modulates ligand binding at receptor sites in the channel.     

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

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

Effects of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a highly selective adenosine receptor antagonist,on force of contraction in guinea-pig atrial and ventricular cardiac preparations

Summary The effects of the A₁ adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) on force of contraction were examined in isolated electrically driven auricles and papillary muscles from guinea-pigs in the absence and presence of (–)-N⁶-phenylisopropyladenosine (PIA) and 5-N-ethylcarboxamidadenosine (NECA).In auricles DPCPX (30–1000 nmol/l) alone increased force of contraction. DPCPX produced only a minor inhibition of phosphodiesterase I–III activity. PIA and NECA alone exerted concentration-dependent negative inotropic effects and the concentration-response curves for PIA and NECA were shifted competitively to the right by the adenosine receptor antagonist DPCPX with similar potency and efficacy. The pA₂-value for the inhibition of the effects of PIA and NECA were 9.1 and 8.8, respectively.In papillary muscles DPCPX alone had no inotropic effect. In the presence of isoprenaline PIA and NECA alone exerted concentration-dependent negative inotropic effects and again DPCPX shifted the concentration-response curves for PIA and NECA competitively to the right with similar potency and efficacy. The pA₂-value for the inhibition of the effects of PIA and NECA were 9.3 and 9.0, respectively.It is concluded that DPCPX is a potent competitive A₁ adenosine receptor antagonist in guinea-pig atrial and ventricular cardiac preparations. Since PIA and NECA were equally potent the cardiac adenosine receptor may constitute a subtype of A₁ adenosine receptors differing from the receptor in other tissues such as fat cells. Furthermore, DPCPX has a positive inotropic effect in atrial tissue which cannot be attributed to the A₁ receptor antagonism.Abbreviations cAMP cyclic adenosine-3,5-monophosphate - DPCPX 1,3-dipropyl-8-cyclopentylxanthine - IBMX 3-isobutyl-lmethylxanthine - NECA 5-N-ethylcarboxamidadenosine - PDE phosphodiesterase - PIA (–)-N⁶-phenylisopropyladenosine Send offprint requests to J. Scholz at the present addressParts of the results have been presented at the 54th Annual Meeting of the German Society for Heart and Circulation Research (Scholz et al. 1988)     

Interaction of 1,3-di(2-[5-3H]tolyl) guanidine with sigma 2 binding sites in rat heart membrane preparations.

Membrane preparations of rat hearts displayed specific binding activity for the prototypic sigma (sigma) receptor ligand, 1,3-di(2-[5-3H]tolyl) guanidine [( 3H]DTG), but not for the phencyclidine (PCP) receptor ligand, [3H]MK-801. Scatchard plot analysis of [3H]DTG binding revealed the presence of one high affinity saturable binding site with a KD of 8.7 nM and a Bmax of 100 pmol/g protein. The drug specificity profile of the receptor correlated with that of the sigma receptor with the following order of potency: DTG greater than haloperidol greater than (-)-pentazocine greater than (-)-butaclamol greater than (+)-butaclamol greater than (-) SKF-10047 greater than (+)pentazocine greater than PCP greater than TCP greater than MK-801 greater than (+)SKF-10047. [3H]DTG binding was sensitive to the Ca2+ channel blocker, verapamil (Ki 202 nM) but not to the K+ channel blocker, 4-aminopyridine. The reverse stereoselectivity of [3H]DTG binding for (-)-SKF-10047 and (-)-pentazocine (Ki of 1289 and 140 nM as compared with 17,582 and 2190 nM for (+)-SKF-10047 and (+)-pentazocine, respectively) indicated that the heart contains sigma receptors with characteristics of the sigma 2 subtype.     

Effects of some organophosphorus compounds on the binding of a radioligand (8-cyclopentyl 1, 3-[3H]dipropylxanthine) to adenosine receptors in ovine cardiac membranes.

8-Cyclopentyl-1,3-[3H] dipropylxanthine [( 3H]CPX) is a potent radioligand that specifically binds to the A1 adenosine receptors. Its high specificity makes it a suitable ligand for the characterization of A1 adenosine receptors in tissues with low receptor densities. We have demonstrated that the organophosphorus compounds soman, tabun and sarin, at relatively high concentrations, all bind to the A1 adenosine receptors in ovine cardiac membranes with Ki values of 36.7, 328 and 175 microM, respectively. The binding of soman to the receptor site was found to be totally reversible. We suggested that these organophosphorus compounds affect the mechanical responses of the heart through interaction with a potassium channel that does not seem to be closely linked to the A1 adenosine receptors.     

Nicotinic acetylcholine receptors in the rat stomach: I. (-)-[3H]nicotine binding

The nicotinic acetylcholine receptors in the rat stomach were characterized by means of a radioligand binding assay with (-)-[3H]nicotine as ligand. Saturation binding studies on the gastric fundus membranes revealed the presence of two binding sites with dissociation constant (KD) values of 3.1 and 289 nM, and maximum binding capacity (Bmax) values of 3.6 and 76 fmol/mg protein, respectively. The Bmax of the high affinity binding site was greatest in the cardia, followed by fundal mucosa, fundal muscle, and, finally antrum. The IC50 values of cholinergic drugs to inhibit (-)-[3H]nicotine binding in fundus membranes were as follows: (-)nicotine, 0.12 nM; cytosine, 9.3 nM; acetylcholine, 17.7 nM; carbachol, 700 nM; hexamethonium, 2270 nM. The IC50 values of alpha-bungarotoxin, d-tubocurarine and atropine were greater than 100 microM. The muscarinic acetylcholine receptors were also characterized with [3H]quinuclidinyl benzilate and the choline acetyltransferase activity was measured. These results suggest that nicotinic acetylcholine receptors as well as muscarinic acetylcholine receptors are present in the rat stomach and that the regional distribution of these receptors is uneven.