Adenosine A(1) receptor-dependent and -independent effects of the allosteric enhancer PD 81,723

The 2-amino-3-benzoylthiophene PD 81,723 has been shown to exhibit alloster ic enhancement of adenosine A(1) receptor binding and function. The aim of this study was to clarify the mechanism of this effect using membranes puri fied from rat brain and Chinese hamster ovary (CHO)-A(1) cells that stably express the rat adenosine A(1) receptor as well as intact CHO-A(1) and nont ransfected CHO cells. In membranes containing 100 mu M magnesium, (2-amino- 4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone (PD 81,723) si gnificantly increased the affinity of the adenosine A(1) receptor agonist, cyclopentyladenosine, for the low-affinity receptor without affecting high- affinity binding or B-max. In intact cells, PD 81,723 inhibited basal adeny lyl cyclase (AC) activity as well as forskofin-, cholera toxin-, and pertus sis toxin-stimulated AC activity in CHO-A(1) and CHO cells. Basal AC activi ty was inhibited 49% in CHO and 82% in CHO-A(1) cells by 30 mu M PD 81,723. In CHO-A(1) cells, half-maximal inhibition of forskolin-stimulated AC occu rred at 5 mu M PD 81,723 compared to 10 mu M in CHO cells. Cholera toxin-st imulated AC was reduced 90% in both CHO and CHO-A(1) cells by 30 mu M PD 81 ,723. At the same concentration of PD 81,723, pertussis toxin-stimulated AC activity was reduced 86% (CHO-A(1)) and 77% (CHO). [H-3]forskolin was disp laced from purified rat liver AC by PD 81,723 with an IC50 of 96 mu M. Thes e results demonstrate that two mechanisms appear to contribute to the obser ved effects of PD 81,723. One mechanism is allosteric enhancement of adenos ine A(1) receptor function. Results from transfected and nontransfected cel ls suggest that PD 81,723 also inhibits AC directly by binding to the catal ytic unit at or near the forskolin-binding site.