The objective is to gain a better understanding of the role that acidic peptides play in millisecond excitatory signaling in the central nervous system. We propose to carry out a detailed structure-activity relationship study on the endogenous brain dipeptides N-acetylaspartylglutamate and aspartylglutamate. Flexible and conformationally restricted analogues of these two peptides will be synthesized to help define the required spatial disposition of the three carbosyl moieties. Attempts will also be made to develop specific antagonists of Ac-Asp-Glu and Asp-Glu. These attempts will include replacing the carboxyl groups with other isofunctional moieties (e.g. tetrazole, sulfonate, and phosphonate) and developing affinity labeling agents in which either the acetyl moiety is replaced with acyl moieties possessing reactive groups or the chloromethyl ketone function is substituted for one of the carboxyl moieties. A potential metabolically stable analogue of Ac-Asp-Glu in which the Asp-Glu amide bond is replaced with the olefinic function will also be synthesized. The proposed compounds will be evaluated for electrophysiological activity using two in vitro slice preparations from rat brain, namely olfactory cortical slices and hippocampal slices. Both extracellular and intracellular methods will be employed. Radioligand binding studies will be conducted using preparations of synaptosomes and synaptic plasma membranes, and will measure the potency of the analogues of the acidic peptides to displace the specific binding of [3H]-glutamate, [3H]-Ac-Asp-Glu, and [3H]-2-amino-4-phosphonobutanoic acid. We will also attempt to develop an effective radioligand for Ac-Asp-Glu receptors using the metabolically stable analogue of Ac-Asp-Glu that will be synthesized. IT is hoped that through this study, new and novel pharmacological probes can be developed that can be used to delineate in more detail the number and kinds of receptors and neurotransmitters that are involved in millisecond excitatory neurotransmission in the CNS and the roles that such receptors and neurotransmitters play in normal and abnormal neurologic function.
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