Abstract

The 2-amino-3-benzoylthiophene PD 81,723 has been shown to exhibit allosteric enhancement of adenosine A₁ receptor binding and function. The aim of this study was to clarify the mechanism of this effect using membranes purified from rat brain and Chinese hamster ovary (CHO)-A₁ cells that stably express the rat adenosine A₁ receptor as well as intact CHO-A₁ and nontransfected CHO cells. In membranes containing 100 μM magnesium, (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone (PD 81,723) significantly increased the affinity of the adenosine A₁ receptor agonist, cyclopentyladenosine, for the low-affinity receptor without affecting high-affinity binding orB_max. In intact cells, PD 81,723 inhibited basal adenylyl cyclase (AC) activity as well as forskolin-, cholera toxin-, and pertussis toxin-stimulated AC activity in CHO-A₁ and CHO cells. Basal AC activity was inhibited 49% in CHO and 82% in CHO-A₁ cells by 30 μM PD 81,723. In CHO-A₁ cells, half-maximal inhibition of forskolin-stimulated AC occurred at 5 μM PD 81,723 compared to 10 μM in CHO cells. Cholera toxin-stimulated AC was reduced 90% in both CHO and CHO-A₁ cells by 30 μM PD 81,723. At the same concentration of PD 81,723, pertussis toxin-stimulated AC activity was reduced 86% (CHO-A₁) and 77% (CHO). [³H]forskolin was displaced from purified rat liver AC by PD 81,723 with an IC₅₀ of 96 μM. These results demonstrate that two mechanisms appear to contribute to the observed effects of PD 81,723. One mechanism is allosteric enhancement of adenosine A₁ receptor function. Results from transfected and nontransfected cells suggest that PD 81,723 also inhibits AC directly by binding to the catalytic unit at or near the forskolin-binding site.