Characterization of the binding of a novel nonxanthine adenosine antagonist radioligand, [3H]CGS 15943, to multiple affinity states of the adenosine A1 receptor in the rat cortex

MF Jarvis, M Williams, UH Do and MA Sills

Research Department, CIBA-GEIGY Corp., Summit, New Jersey.

The triazoloquinazoline CGS 15943 is the first reported nonxanthine adenosine antagonist that has high affinity for brain adenosine receptors. In the present study, the binding of [3H] CGS 15943 to recognition sites in rat cortical membranes was characterized. Saturation experiments revealed that [3H]CGS 15943 labeled a single class of recognition sites with high affinity (Kd = 4 nM) and limited capacity (Bmax = 1.5 pmol/mg of protein). Competition studies revealed that the binding of [3H]CGS 15943 was consistent with the labeling of brain adenosine A1 receptors. Adenosine agonists inhibited 1 nM [3H]CGS 15943 binding with the following order of activity N6- cyclopentyladenosine (IC50 = 15 nM) greater than 2-chloroadenosine greater than (R)-N6-phenylisopropyladenosine greater than 5'-N6- ethylcarboxamidoadenosine greater than (S)N6-phenylisopropyladenosine greater than CGS 21680 greater than CV 1808 (IC50 greater than 10,000 nM). The potency order for adenosine antagonists was CGS 15943 (IC50 = 5 nM) greater than 8-phenyltheophylline greater than 1,3-dipropyl-8-(4- amino-2-chloro)phenylxanthine greater than 1,3-diethyl-8-phenylxanthine greater than theophylline = caffeine (IC50 greater than 10,000 nM). Antagonist inhibition curves were steep and best described by a one- site binding model. In contrast, adenosine A1 agonist competition curves were shallow, as indicated by Hill coefficients less than unity. Computer analysis revealed that these inhibition curves were best described by a two-site binding model. Agonist competition curves generated in the presence of 1 mM GTP resulted in a rightward shift and steepening of the inhibition-concentration curves, whereas antagonist binding was not altered in the presence of GTP. The complex binding interactions found with adenosine agonists indicate that [3H]CGS 15943 labels both high and low affinity components of the adenosine A1 receptor in the rat cortex. Additionally, the present data also provide some evidence that [3H]CGS 15943 may also recognize an additional low affinity binding component, which may represent a putative low affinity A2b receptor in this tissue.

Volume 39, Issue 1, pp. 49-54, 01/01/1991
Copyright © 1991 by American Society for Pharmacology and Experimental Therapeutics