MEASUREMENT OF GUANINE-NUCLEOTIDE-BINDING PROTEIN-ACTIVATION BY A1 ADENOSINE RECEPTOR AGONISTS IN BOVINE BRAIN MEMBRANES - STIMULATION OF GUANOSINE-5'-O-(3-[S-35]THIO)TRIPHOSPHATE BINDING

Signal transduction by A1 adenosine receptors was investigated by meas uring the modulation by adenosine agonists of guanosine-5'-O-(3-[S-35] thio)triphosphate ([S-35]GTP[S]) binding to guanine nucleotide-binding proteins (G proteins) The extent of stimulation of [S-35]GTP[S] bindi ng was dependent on the presence of high concentrations of Mg2+ (1-10 mm), GDP (10 muM), and NaCl (100 mm). Under optimal conditions, the ag onist (R)-N6-phenylisopropyladenosine [(R)-PIA] stimulated binding of [S-35]GTP[S] to G proteins approximately 2.3-fold. All adenosine recep tor agonists tested stimulated the binding of [S-35]GTP[S] with a rank order of potency typical of A1 adenosine receptors in bovine tissues, (R)-PIA > 2-chloro-N6-cyclopentyladenosine > (S)-PIA > 5'-N-ethylcarb oxamidoadenosine > yl)phenethylamino]-5'-N-ethylcarboxamidoadenosine. The EC50 values for G protein activation correlated with the K(i) valu e of agonists for inhibition of radioligand binding to the high affini ty state of the A1 adenosine receptor. The inclusion of 100 mm NaCl as well as increasing GDP concentrations led to a parallel increase of K (i) values and EC50 values. In addition, both compounds induced a shif t of A1 adenosine receptors from the high affinity state for agonists to the low affinity state.