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ORIGINAL ARTICLE

Evidence for the Functional Expression and Pharmacological Characterization of Adenine Receptors in Native Cells and Tissues

Pharmaceutical Institute, Pharmaceutical Chemistry Poppelsdorf, (S.G., C.E.M.) and Department of Pharmacology and Toxicology (I.v.K.), University of Bonn, Bonn, Germany; and Department of Chemical Sciences, University of Camerino, Camerino, Italy (S.V., R.V., G.C.)

Abstract

An orphan G protein-coupled receptor from rat has recently been discovered to be activated by the nucleobase adenine (Proc Natl Acad Sci USA 99:8573–8578, 2002). In the present study, we show for the first time that the adenine receptor is expressed in membrane preparations of native tissues and cell lines in high density, including rat brain cortex, rat brain striatum, and the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. Saturation analysis with [³H]adenine at rat brain cortical membranes exhibited a single high-affinity binding site with a K_D value of 27.2 nM, and a binding capacity of 2.28 pmol/mg of protein. Kinetic studies revealed unusual binding kinetics of [³H]adenine with rapid association and slow dissociation. A series of compounds were investigated in [³H]adenine competition experiments at rat brain cortex. Only minor substitution of the adenine structure was tolerated, the most potent compounds of the present series being 2-fluoroadenine (K_i value of 620 nM), 8-thioadenine (K_i value of 2.77 µM), N⁶-methyladenine (K_i value of 3.64 µM), and 7-methyladenine (K_i value of 4.13 µM), all of which were partial agonists (40–60% intrinsic activity). Adenine dose dependently inhibited forskolin-stimulated adenylate cyclase in membrane preparations of NG108-15 cells as well as in intact cells, showing that the receptor is functional in NG108-15 cells. Reverse transcriptase-polymerase chain reaction experiments followed by sequencing indicate that the NG108-15 cells express the murine ortholog of the adenine receptor. Moreover, preliminary radioligand binding studies with [³H]adenine at membranes of human astrocytoma 1321N1 cells suggest that a human ortholog of the rat adenine receptor exists.

Received for publication August 25, 2004.

Accepted for publication December 14, 2004.

Address correspondence to: Dr. Christa E. Müller; Pharmaceutical Institute Poppelsdorf, University of Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany. E-mail: christa.mueller{at}uni-bonn.de

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