Adenosine is a neuromodulator with potent influences upon epileptic processes. These actions are mediated by two subtypes of cell surface receptors, A1 and A2. The exact mechanisms mediating these actions remain undetermined. The long-term objectives of this research are to investigate the following two aspects of adenosine's mechanism of action: 1) Ligand-receptor interactions - In order to better define this step in adenosine action, we will investigate a class of ligands which are termed """"""""bivalent"""""""" because they consist of two adenosyl groups. Their binding ability should be determined, in part, by the distance separating the two adenosyl groups on the ligand, and on the distance between neighboring receptor molecules on the cell surface. Such agents have potential uses as probes of the distance between receptors. They will thus serve to probe the distance between receptors. Furthermore, any differences between the receptor subtypes in terms of their inter-receptor spacing should result in the discovery of compound which have specificity for each subtype. The finding of A2 specificity, in particular, would be of significance as few such agents are currently available. Specificity of this nature should allow better definition of the role of each receptor subtype in anticonvulsant activity. 2) Alteration of gene expression - We hypothesize that adenosinergic input may influence gene expression. Both adenosine levels and c-fos mRNA levels have been reported to rise in the period immediately following a seizure, suggesting that adenosine may be in part responsible for the induction of the c-fos gene. Results of our preliminary experiments show that adenosine analogs can induce c-fos in tissue culture cells. This finding will be extended both in tissue culture and using an in vivo seizure model. In pursuing these long term objective, the following specific aims are proposed: 1) To synthesize agents having two adenosyl groups separated by bridging groups which vary in length, composition, and rigidity. Later, we will extend these efforts to include some multivalent agents, with three or more adenosyl groups. 2) To characterize these compounds both in vitro and in vivo. For in vitro testing, radioligand binding assays of membrane-receptor preparations will be used to measure the affinity, subtype specificity, and agonist/antagonist qualities of these agents. For the vivo testing, the maximal electroshock and pentylenetetrazole seizure models will be used to assess anticonvulsant properties. 3) To examine the effects of adenosine analogs on gene expression in cultured cells. Preliminary results indicate that adenosine can modulate the expression of the proto-oncogene c-fos. Factors controlling this response, including receptor type and second messenger pathways, will be determined. 4) To investigate the effects of adenosine agonists and antagonists on the c-fos induction seen following metrazol-induced seizures. Seizures cause the release of adenosine and the induction of c-fos. The use of adenosine analogs will help to determine the contribution of the purinergic pathway to this alteration in gene expression.
