Identification of specific [3H]adenine-binding sites in rat brain membranes.
We recently reported that adenine acts as a neurotrophic factor independent of adenosine or P2 receptors in cultured Purkinje cells [Watanabe S. et al. (2003) J. Neurosci. Res. 74, 754-759], suggesting the presence of specific receptors for adenine in the brain. In this study, the characterization of adenine-binding activity in the rat brain was performed to further characterize the receptor-like adenine-binding sites. Specific binding sites for [(3)H]adenine were detected in membrane fractions prepared from rat brains. The kinetics of [(3)H]adenine binding to membranes was described by the association and dissociation rate constants, 8.6 x 10(5) M(-1) min(-1) and 0.118 +/- 0.045 min(-1), respectively. A single binding site for [(3)H]adenine with a K (D) of 157.1 +/- 20.8 nM and a B (max) of 16.3 +/- 1.1 pmol/mg protein (n = 6) was demonstrated in saturation experiments. A displacement study involving various related compounds showed that the [(3)H]adenine binding was highly specific for adenine. It was also found that [(3)H]adenine-binding activity was inhibited by adenosine, although other adenosine receptor ligands were ineffective as to [(3)H]adenine binding. The brain, especially the cerebellum and spinal cord, showed the highest [(3)H]adenine-binding activity of the tissues examined. These results are consistent with the presence of a novel adenine receptor in rat brain membranes.
Watanabe
Shun
S
Department Molecular Cell Signaling, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526.
Ikekita
Masahiko
M
Nakata
Hiroyasu
H
D000225:Adenine / Q000378:metabolism*; D000241:Adenosine / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D011687:Purines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D011983:Receptors, Purinergic / Q000032:analysis*; D013329:Structure-Activity Relationship; D014018:Tissue Distribution; D014316:Tritium
D011687:Purines; D011983:Receptors, Purinergic; D014316:Tritium; D000225:Adenine; D000241:Adenosine