Adenosine A1 Receptor-Dependent and -Independent Effects of the Allosteric Enhancer PD 81,723

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 288; no. 2; p. 446
• Main Authors: Musser, B, Mudumbi, R V, Liu, J, Olson, R D, Vestal, R E
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.02.1999
• Subjects: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases - drug effects; Adenylyl Cyclases - metabolism; Adjuvants, Immunologic - pharmacology; Animals; Brain - metabolism; Brain - ultrastructure; CHO Cells - metabolism; Cholera Toxin - pharmacology; Colforsin - pharmacology; Cricetinae; Cyclic AMP - metabolism; Drug Interactions; Enzyme Activation - drug effects; Enzyme Inhibitors - pharmacology; Kinetics; Magnesium - pharmacology; Membranes - metabolism; Pertussis Toxin; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Radioligand Assay; Rats; Receptors, Purinergic P1 - physiology; Stimulation, Chemical; Thiophenes - pharmacology; Transfection; Virulence Factors, Bordetella - pharmacology
• ISSN: 0022-3565; 1521-0103

The 2-amino-3-benzoylthiophene PD 81,723 has been shown to exhibit allosteric enhancement of adenosine A 1 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)-A 1 cells that stably express the rat adenosine A 1 receptor as well as intact CHO-A 1 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 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 adenylyl cyclase (AC) activity as well as forskolin-, cholera toxin-, and pertussis toxin-stimulated AC activity in CHO-A 1 and CHO cells. Basal AC activity was inhibited 49% in CHO and 82% in CHO-A 1 cells by 30 μM PD 81,723. In CHO-A 1 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-A 1 cells by 30 μM 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). [ 3 H]forskolin was displaced from purified rat liver AC by PD 81,723 with an IC 50 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 A 1 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.