Vol. 288, Issue 2, 446-454, February 1999

Adenosine A₁ Receptor-Dependent and -Independent Effects of the Allosteric Enhancer PD 81,723¹

Beth Musser, Ramagopal V. Mudumbi , Jingwen Liu² , Richard D. Olson and Robert E. Vestal

Clinical Pharmacology and Gerontology Research Unit, Department of Veterans Affairs Medical Center, Boise, Idaho (B.M., R.V.M., J.L., R.D.O., R.E.V.); Mountain States Medical Research Institute, Boise, Idaho (R.V.M., J.L., R.D.O., R.E.V.); and Department of Medicine and Pharmacology, University of Washington School of Medicine, Seattle, Washington (R.D.O., R.E.V.)

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 or B_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.