Differential A1-adenosine receptor reserve for inhibition of cyclic AMP accumulation and G-protein activation in DDT1 MF-2 cells

1 The A(1)-adenosine receptor (A(1)AdoR) reserve for N-6-cyclopentyladenosine (CPA) mediated inhibition of (-)isoprenaline stimulated cyclic AMP accumulation and stimulation of [S-35]-guanosine-5'-O-(thiotriphosphate) (GTP gamma S) binding, a measure of guanine nucleotide binding protein (C-protein) activation, was determined in DDT1 MF-2 cells. 2 Inactivation of the A(1)AdoRs with the chemoreactive ligand 8-cyclopentyl-3-[3 -[[4-(fluorosulphonyl)benzoyl]oxy]propyl]-1-propylxanthine (FSCPX) caused a progressive rightward shift of the concentration-response curves for CPA to inhibit cyclic AMP accumulation, with a maximum of 10 fold increase in the EC50 value. In contrast, inactivation of A(1)AdoR's caused only a 1.7 fold rightward shift in the CPA concentration-response for stimulation of [S-35]-GTP gamma S binding. 3 The A(1)AdoR occupancy-response relationship for CPA inhibition of cyclic AMP accumulation was hyperbolic with 43% receptor occupancy required to elicit the maximal response, i.e. a 57% A(1)AdoR reserve. In contrast, the A(1)AdoR occupancy-response relationship for CPA mediated stimulation of [S-35]-GTP gamma S binding was linear indicating little or no receptor reserve for G-protein activation. The relationship between CPA stimulation of [S-35]-GTP gamma S binding and cyclic AMP inhibition was also hyperbolic with 44% G-protein activation sufficient to cause maximal inhibition. 4 The data suggest that the A(1)AdoR reserve for CPA mediated inhibition of cyclic AMP accumulation occurs at the level of G-protein interaction with adenylyl cyclase. However, each A(1)AdoR appears to activate a constant fraction of the total G-protein population suggesting signal amplification at the receptor-G-protein level which may also contribute to the receptor reserve for CPA.