title: 2-Chloro-N6-[3H]cyclopentyladenosine ([3H]CCPA)--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.
authors.affiliation: Pharmakologisches Institut, Universität Heidelberg, Federal Republic of Germany.
authors.forename: K N
authors.initials: KN
authors.lastname: Klotz
authors.forename: M J
authors.initials: MJ
authors.lastname: Lohse
authors.forename: U
authors.initials: U
authors.lastname: Schwabe
authors.forename: G
authors.initials: G
authors.lastname: Cristalli
authors.forename: S
authors.initials: S
authors.lastname: Vittori
authors.forename: M
authors.initials: M
authors.lastname: Grifantini
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism; D002384:Catalysis; D066298:In Vitro Techniques; D007553:Isotope Labeling; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D051381:Rats; D011983:Receptors, Purinergic / Q000187:drug effects*
chemical_list: D011983:Receptors, Purinergic; C058394:2-chloro-N(6)cyclopentyladenosine; D000241:Adenosine