While much information is available regarding the interactions of cocaine with the amine transporters, there is a gap in our knowledge about the effects of acute and chronic exposure to agents with longer actions than cocaine. We have previously characterize various tropane molecules which have higher affinities for and found a longer duration of activity at the dopamine transporter (DAT). One of the goals of this study is to examine how exposure to cocaine and long-acting tropane compounds induces various alterations in the dopamine transporter (DA), including functional activity, levels of protein, phosphorylation and intracellular localization. Each of these criteria are important indices and their modifications could result in altered dopaminergic neurotransmission. We propose to examine if these modification occur in both in vitro and in vivo models. The in vitro systems we will utilize are CHO cells transfected with the human DAT (hDAT) and mid-brain primary cultures containing intact dopamine (DA) cells. By utilizing these systems, we can compare the response of intact DA cells (possessing intrinsic dopaminergic characteristics such as auto-receptors and DA packaging) to those obtained from cells which do have these intrinsic dopaminergic qualities (CHO cells). The goal of these studies is to provide a comparison between 1) short-acting (cocaine) and long-acting (tropanes) compounds regarding their ability to alter the DAT, 2) exposures to these compounds both acutely and chronically and 3) the results obtained in vivo versus from in vitro experiments. Another goal of these studies is to develop (with Dr. Davies) and characterize novel tropanes that have high affinity for and interact with the DAT for prolonged periods of time, primarily by modifying the degree of lipophilicity. By utilizing the results from the above described experiments, we also propose to develop new compounds which will have not only a high affinity and duration over the other amine carrier proteins. Agents which have along duration of action at the DAT than cocaine could provide important insights into future pharmacotherapies for treating cocaine addiction. These studies will yield critical information concerning the biochemical adaptations induced by acute and repeated exposure to cocaine and tropane compounds involving DAT modifications, providing a better understanding of the mechanisms and adaptations which result from prolonged drug exposure.
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