In the present study, we have characterized the effects of guanine nucleotides on agonist and antagonist binding to A1 adenosine receptors, which mediate inhibition of adenylate cyclase via the inhibitory G protein (Gi) in adipocytes. Our data indicate that guanosine-triphosphate (GTP) and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) enhance the binding of 8-(4-[(([(2-amino-ethyl)amino]carbonyl) methyl)oxyl]phenyl)-1,3-dipropylxanthine ([3H]XAC) to adipocyte membranes in a dose-dependent manner, with EC50 values being 1.8 and 2.2 microM, respectively. The stimulatory effect of GTP was abolished in pertussis toxin-intoxicated membranes, implying a role of a pertussis toxin-sensitive G protein in mediating this effect. Furthermore, the ranked order of efficacy for a series of guanine nucleotides to enhance [3H]XAC binding was GTP = Gpp(NH)p greater than GDP greater than GDP beta S = cGMP, which paralleled their ability to inhibit forskolin-stimulated adenylate cyclase activity. Saturation isotherms performed in the absence and presence of GTP and Gpp(NH)p indicate that the guanine nucleotide decreased the equilibrium dissociation constant (KD) but had no effect on the maximal binding (Bmax) of [3H]XAC. In contrast, Gpp(NH)p decreased agonist binding as manifested by a decrease in the percentage of A1 adenosine receptors in the agonist high affinity state (from 81% to 27%) without changing the high (KH) and low (KL) affinity constants. Kinetic experiments conducted to assess the effect of guanine nucleotide on [3H]XAC binding parameters demonstrate that Gpp(NH)p enhanced the observed rate of association (Kobs) of the radioligand with the receptor by 2-fold but had no effect on the rate of dissociation (K-1) of the radioligand-receptor complex.(ABSTRACT TRUNCATED AT 250 WORDS)