A 7-transmembrane receptor responding to adenine has recently been identified in the rat, distinct from the P1 (adenosine) and P2 receptor families (Bender et al., 2002). mRNA coding for this receptor was most prominent in a subset of primary afferent neurones, but was also found to relatively high density in the brain. This receptor expressed heterologously bound [³H]-adenine with a K_d value of 24 nM. I have, therefore, investigated whether [³H]-adenine binding in the brain might label this adenine receptor.

Rats of the Wistar strain (male, 175-250 g) were used throughout. Membrane preparations were generated from whole brain using repeated homogenisation and centrifugation at 30 000 g for 15 minutes. [³H]-Adenine binding was conducted at room temperature at a radioligand concentration of 15-25 nM, in a total volume of 500 µL. The assay buffer consisted of 50 mM Tris, 10 mM MgCl₂, 1 mM EDTA, pH 7.4 with a membrane content equivalent to 0.2-20 µg original wet weight. Non-specific binding was estimated in the presence of 100 µM adenine. Bound radioligand was separated by filtration through Whatman GF/B filters. Untransformed data from 4 separate preparations were analysed for statistical significance using the Student’s unpaired t-test.

At room temperature, binding of [³H]-adenine was ‘complete’ at 5 minutes and was unchanged after 90 minutes incubation (107 ± 1 % of 5’ values, n=3). Homologous displacement of [³H]-adenine binding with adenine was monophasic (Hill slope of -0.90 ± 0.02) and allowed calculation of an apparent pK_i value of 7.24 ± 0.06. An apparent B_amx value could also be calculated (281 ± 54 pmol/mg protein). At a concentration of 10 µM, xanthine was without significant effect (91 ± 6 % control), while hypoxanthine almost completely displaced [³H]-adenine binding (6 ± 0 %, P<0.001). In contrast, 10 µM allopurinol evoked a significant enhancement of [³H]-adenine binding (116 ± 3 %, P<0.05). Uridine also evoked a concentration-dependent enhancement of [³H]-adenine binding to rat brain membranes, with a maximal enhancement of 166 ± 16 % control and a calculated pEC₅₀ value of 6.86 ± 0.23 (Hill slope 1.02 ± 0.18). Binding of [³H]-adenine was unaffected in the presence of 100 mM NaCl (91 ± 3 % control, n=4) or 10 µM GTPγS (96 ± 2 %, n=4). Omission of Mg²⁺ ions from the buffer, however, completely eradicated [³H]-adenine binding (2 ± 0 %, P<0.001).

Taken together, these observations are inconsistent with [³H]-adenine binding to an adenine 7TM receptor. The enhancing effects of allopurinol and uridine might be taken to indicate allosteric activation of the binding site or reduced metabolism of the [³H]-adenine, although the maintained level of binding over 90 minutes is not consistent with this hypothesis. The data are consistent with binding to a Mg²⁺-dependent membrane-bound protein (as yet unidentified) expressed to very high density in the brain.

Bender, E. et al. (2002). Proc. Natl. Acad. Sci. U. S. A., 99, 8573 - 8578.