2-Chloro-N6-[3H]cyclopentyladenosine ([3HCCPA) ?a high affinity agonist radioligand for A1 adenosine receptors

Abstract

The tritiated analogue of 2-chloro-N6-cyclopentyladenosine (CCPA), an adenosine derivative with subnanomolar affinity and a 10,000-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 affinity KD-value of 0.2 nmol/l. In the presence of GTP a KD-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 KD-value of 0.4 nmol/l and a Bmax-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.

Development of Polar Adenosine A2A Receptor Agonists for Inflammatory Bowel Disease: Synergism with A2B Antagonists

“…20 The synthesized 2-substituted (ar)alkylthioadenosine derivatives were investigated in radioligand binding studies of human and rat A 2A AR using the agonist radioligand [ 3 H]CGS21680. 21 Selectivity versus other adenosine receptor subtypes was assessed by performing radioligand binding studies with human and rat A 1 AR using [ 3 H]CCPA, 22 human A 2B AR using [ 3 H]PSB-603, 16 and human A 3 AR using [ 3 H]PSB-11 23 and/or [ 3 H]NECA 24 as radioligands (see Table 1). …”

Transitory expression of the A2b adenosine receptor during implantation chamber development

“…Equilibrium binding was carried out for 3 hr at 25°C and terminated by rapid vacuum filtration through Whatmann 2.4 cm GF/C filters (Maidstone, England) presoaked in 0.3% (v/v) polyethylenimine (Bruns et al, 1983;Stiles et al, 1988). Specific binding was determined by subtracting from total cpm the cpm bound in the presence of 1 mM theophylline (Klotz et al, 1989). Data were analyzed by nonlinear curvefitting with the program R ENZFITTER (ElsevierBiosoft, Cambridge, England).…”

1-Substituted 4-[Chloropyrazolyl][1,2,4]triazolo[4,3-a]quinoxalines: Synthesis and Structure-Activity Relationships of a New Class of Benzodiazepine and Adenosine Receptor Ligands

“…Inhibition of binding of [ 3 H]2-chloro-N 6 -cyclopentyladenosine (CCPA) [10] to A1-adenosine receptors of rat brain cortical membranes and inhibition of [ 3 H]3-(3-hydroxypropyl)-8-(3-methoxystyryl)-7-methyl-1-propargylxanthine (MSX-2) [11] to adenosine receptors in rat brain striatal membranes were assayed in analogy to published procedures [16] . As buffer Tris-HCl, 50 mM, pH 7.4 (at room temp.)…”

“…The A 1 -selective agonist [ 3 H]2-chloro-N 6 -cyclopentyladenosine (CCPA) [10] was used as A 1 ligand and [11] as A 2A ligand, caffeine was chosen as a standard reference compound. The results thus obtained are shown in Tables 2 and 3.…”

“…Inhibition of the receptor-radioligand binding was determined by a range of 5 to 6 concentrations of the compounds. The Cheng-Prusoff equation [14] and KD of 0.2 nM for [ 3 H]CCPA [10] and 8 nM for [ 3 H]MSX-2 [11] were used to calculate the Ki values from the IC50 values, determined by the non-linear curve fitting program PRISM TM (GraphPad, San Diego, California, USA).…”