Molecular Pharmacology, Vol 19, 228-235, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Adenosine Receptors in Fat Cells

Identification by (-)-N⁶-[³H]Phenylisopropyladenosine Binding

¹ Institut für Pharmakologie und Toxikologie, Medizinische Hochschule Hannover, 3000 Hannover 61, and Institut für Pharmakologie und Toxikologie, Universität Bonn, 5300 Bonn 1, Federal Republic of Germany

The binding of (-)-N⁶-[³H]phenylisopropyladenosine to its receptor sites was investigated in plasma membranes of rat fat cells. The binding was rapid, reversible, stereospecific, saturable, and dependent on protein concentration, pH, and temperature. Pretreatment of fat cell membranes with adenosine deaminase resulted in a 3 to 4-fold increase in specific binding. The specific binding was of high affinity with an equilibrium dissociation constant (K_D) of 6.1 nM and was saturable with 1.9 pmoles of (-)-N⁶-[³H]phenylisopropyladenosine per milligram of protein. Rate constants of association and dissociation were k₁ 1.95 x 10⁷ M^-1 min^-1 and k₂ = 0.040 min^-1, respectively. In competition experiments the (-)-isomer of N⁶-phenylisopropyladenosine was 11-fold more potent than the (+)-isomer in competing for the binding sites. Specific binding was most effectively displaced by the following purine-modifled adenosine derivatives which have been classified as R site adenosine agonists: (-)-N⁶-phenylisopropyladenosine monophosphate, N⁶-isobutyladenosine, N⁶-cyclohexyladenosine, 2-chloroadenosine, 2-fluoroadenosine, N⁶-phenyladenosine, and N⁶-benzyladenosine, with IC₅₀ values ranging from 17 to 139 nM. The ribose-modified adenosine derivatives 3'-deoxyadenosine, 2',5'-dideoxyadenosine, and 2'-deoxyadenosine, which have been classified as P site adenosine agonists, showed IC₅₀ values ranging from 9.7 to 78 µM. Similar IC₅₀ values were obtained by several 5'-phosphorylated adenosine derivatives. Pharmacologically inactive compounds such as inosine, hypoxanthine, and adenine did not substantially inhibit binding at concentrations up to 100 µM. The methylxanthines isobutylmethylxanthine (IC₅₀ 55 µM), theophylline (IC₅₀ 61 µM), and caffeine (IC₅₀ 150 µM), which have been classified as antagonists for R site adenosine receptors, competed for the binding sites of (-)-N⁶-[³H]phenylisopropyladenosine in a manner which parallels their known pharmacological activity in fat cells. Specific binding was not displaced by other phosphodiesterase inhibitors (papaverine or Ro 20-1724), and not by dipyridamole or nicotinic acid. Our results indicate that rat fat cells have adenosine receptors which are labeled by (-)-N⁶-[³H]phenylisopropyladenosine. On the basis of the order of affinity of agonists and antagonists they appear identical with the R site adenosine receptors which mediate the inhibitory effects of adenosine on cyclic AMP formation and lipolysis in fat cells.

Note:
ACKNOWLEDGMENTS The excellent technical assistance of Miss Helga Hannemann and Mrs. Anna-Carmen Bauer is gratefully acknowledged.

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