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First published on December 16, 2004; DOI: 10.1124/mol.104.006601

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Received for publication August 26, 2004.
Revised November 29, 2004.
Accepted for publication December 14, 2004.

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

Simone Gorzalka 1, Sauro Vittori 2, Rosaria Volpini 2, Gloria Cristalli 2, Ivar von Kugelgen 1, Christa E Muller 1*

1 University of Bonn 2 University of Camerino

* Address correspondence to: E-mail: christa.mueller{at}uni-bonn.de

Abstract

An orphan G protein-coupled receptor from rat has recently been discovered to be activated by the nucleobase adenine (Bender et al. Proc. Natl. Acad. Sci. USA 2002, 99:8573-8578). 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 KD value of 27.2 nM, and a binding capacity of 2.28 pmol/mg protein. Kinetic studies revealed unusual binding kinetics of [3H] adenine with rapid association and slow dissociation. A series of compounds was 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 (Ki 620 nM), 8-thioadenine (Ki 2.77 µM), N6-methyladenine (Ki 3.64 µM) and 7-methyladenine (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 adenine receptor is functional in NG108-15 cells. RT-PCR 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.


Key words: Adenosine, Purinergic, cAMP, Receptor binding studies


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I. von Kugelgen, A. C. Schiedel, K. Hoffmann, B. B. A. Alsdorf, A. Abdelrahman, and C. E. Muller
Cloning and Functional Expression of a Novel Gi Protein-Coupled Receptor for Adenine from Mouse Brain
Mol. Pharmacol., February 1, 2008; 73(2): 469 - 477.
[Abstract] [Full Text] [PDF]


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