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Although adenosine or ATP is known to control various physiological functions in the brain, including synaptic transmission, neuronal cell death, and neurite outgrowth via P1 or P2 purinergic receptors in the nervous system, little is known about the functions of many other purine derivatives. We examined the effects of various purines on survival in the cerebellar cortex of Purkinje cells with large cell bodies and highly branched dendrites, and it was found that some purine and pyrimidine derivatives influence Purkinje cell survival. Treatment with adenine, guanine, guanosine, guanine nucleotides, and uracil nucleotides protected Purkinje cells from cell death in the cerebellar primary cultures. Among the effective compounds, adenine had the most potent survival activities on Purkinje cells. Other adenine-based purines such as adenosine, AMP, ADP, and ATP did not promote Purkinje cell survival. Furthermore, metabolic inhibitors of adenine had no effect on the protective ability of adenine for Purkinje cells, suggesting that adenine itself, not adenine metabolites, maintains Purkinje cell survival. These results suggest that adenine is involved in the control of Purkinje cell survival in cerebellar primary cultures via a novel adenine-dependent mechanism., (Copyright 2003 Wiley-Liss, Inc.)

We examined the influence of nucleobases, nucleosides, nucleotides, and their analogs on rat cerebellar Purkinje cells in primary culture and found that the number of cultured Purkinje cells was greatly increased by the nucleobase adenine. Purkinje cells were cultured for 13 days in vitro in the presence of various reagents, and the resulting cell numbers were counted. As a result, the nucleobase adenine was most effective at increasing the number of Purkinje cells among the reagents tested. In the cultures supplemented with adenine in millimolar concentrations (1-2 mM), the number of Purkinje cells was increased by up to 30 times the number of Purkinje cells in the control. Adenine had no affect on the number of granule cells, and it reduced the number of astrocytes, both of which were cocultured in cerebellar primary cultures. Stimulation of purinoceptors by adenosine and adenosine 5'-triphosphate (ATP) did not result in an increase in the number of Purkinje cells. Furthermore, the adenine effect on Purkinje cells was not related to PKA, as determined with the use of a PKA inhibitor. Our findings suggest that adenine exerts neurotrophic effects that have not been described to date; in particular, the present study demonstrated that adenine increases the number of Purkinje cells by an unknown mechanism.

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 [3H]adenine were detected in membrane fractions prepared from rat brains. The kinetics of [3H]adenine binding to membranes was described by the association and dissociation rate constants, 8.6 × 105 M–1 min–1 and 0.118 ± 0.045 min–1, respectively. A single binding site for [3H]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 [3H]adenine binding was highly specific for adenine. It was also found that [3H]adenine-binding activity was inhibited by adenosine, although other adenosine receptor ligands were ineffective as to [3H]adenine binding. The brain, especially the cerebellum and spinal cord, showed the highest [3H]adenine-binding activity of the tissues examined. These results are consistent with the presence of a novel adenine receptor in rat brain membranes. [ABSTRACT FROM PUBLISHER]