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Indirect effect of guanine nucleotides on antagonist binding to A1 adenosine receptors: occupation of cryptic binding sites by endogenous vesicular adenosine.

Guanine nucleotides such as guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) have been found to increase the binding of antagonists to adenosine A1 receptors. This response can be attributed either to a direct effect of GTP on receptors to increase antagonist affinity or to an indirect effect to decrease the affinity of receptors for a pool of endogenous adenosine that cannot be readily removed from membranes. In this study, adenosine content was measured in preparations of membranes and 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS)-solubilized receptors by a sensitive radioimmunoassay. In both preparations, pools of adenosine (2.5-10 pmol/mg of protein) were detected that were resistant to deamination by added adenosine deaminase (0.5-3 units/ml) unless membrane lipids were first dissolved in acetone. Electron microscopic examination of crude CHAPS-solubilized receptors revealed the existence of small vesicles (< 1 microns in diameter). Furthermore, most "solubilized" receptors were retained by a 0.1-microns filter. The effects of GTP gamma S were evaluated on the binding of an antagonist, 3-(4-amino-3-125I-phenethyl)-1-propyl-8-cyclopentylxanthine (125I-BW-A844U), to A1 receptors of bovine brain membranes, receptors solubilized in CHAPS (crude solubilized), or receptors partially co-purified with G proteins by agonist affinity chromatography (partially purified). GTP gamma S (10 microM) increased antagonist binding to membranes (20-50%) and crude CHAPS-solubilized receptors (> 200%) but increased binding to partially purified receptors by only 10-15%. GTP gamma S decreased agonist (125I-N6-aminobenzyladenosine) binding and increased antagonist Bmax, but did not significantly decrease (5%) the dissociation rate of the antagonist. Omission of Mg2+ mimicked the effects of GTP gamma S on agonist and antagonist binding and increased both the association and dissociation rates of 125I-BW-A844U. These data suggest that a Mg(2+)-dependent GTP gamma S-induced increase in antagonist binding to membranes and solubilized receptors is primarily due to unmasking of cryptic binding sites occupied by contaminating vesicular adenosine. These findings are consistent with the observation that adenosine receptor antagonists have been found to have little or no inverse agonist physiological effects in well oxygenated tissues.