Psychostimulants increase extracellular concentration of the neurotransmitter, dopamine, in the nucleus accumbens and other brain regions comprising the prefrontal-cortico-striatal loop. Dysregulation of this system has been implicated in the transition from casual to compulsive drug use and to the preservative behavior that characterizes addiction. Psychostimulants increase dopamine concentrations by binding to and inhibiting the dopamine transporter, a membrane bound protein that clears dopamine released into the synaptic cleft. This action mediates the abuse liability of these agents as well as their psychomotor stimulant effects. We and others have shown that D2 and D3 receptors, members of the D2 family of G-protein coupled receptors, regulate extracellular dopamine concentrations in the nucleus accumbens and striatum. Extracellular dopamine concentrations are decreased in response to D2/D3 receptor agonists and increased in response to receptor antagonists. Our in-vivo and in-vitro studies provided evidence that D2/D3 receptors regulate dopamine transmission presynaptically by two distinct mechanisms. They affect transporter mediated dopamine re-uptake and they affect release. Due, however, to the lack of ligands selective for these receptor sub-types and the presence of both D2 and D3 receptors in the brain regions examined, the role of each in the regulation of dopamine dynamics could not be delineated.? ? Using a recently developed live-cell imaging technique, our studies have provided the first direct demonstration that D2 receptors regulate the activity of the dopamine transporter. For these studies, human embryonic kidney (HEK) cells and a neuronally-derived cell line were co-transfected with the human D2 receptor and the human dopamine transporter. Dopamine transporter function was monitored in real time using the high affinity, fluorescent dopamine transporter substrate, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (ASP+). Addition of the D2/D3 agonist, quinpirole to cells co-expressing these proteins induced a rapid and concentration-dependent increase in ASP+ accumulation. The structurally dissimilar D2/D3 agonist, PD128907 also increased ASP+ accumulation. Increases in accumulation was pertussis toxin-sensitive and only apparent in transfected cells. D2 receptor stimulation induced phosphorylation of extracellular related kinase (ERK1/2) and Akt, a major target of phosphoinositide 3-kinase (PI3K), confirming that the D2 receptor signals through these kinase cascades. Pretreatment of cells with the ERK 1/2 inhibitor, PD 98059, prevented the quinpirole-evoked increase in ASP+ accumulation. PI-3 kinase inhibition was without effect indicating that D2 receptor stimulation upregulates DAT function in mammalian cell lines via an ERK1/2-dependent and PI3K-independent mechanism. Co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) techniques revealed that the dopamine transporter and D2 receptor are in close proximity providing a cellular basis for the interaction of these two proteins. ? ? In subsequent studies we have used the ASP+ technique to determine whether dopamine transporter function is regulated by D3 receptors. In cells co-expressing D3 receptors and the dopamine transporter, we observed that D3 receptor activation rapidly increased ASP+ uptake. Similar effects were observed using a conventional radioligand binding assay. D3 receptor stimulation, like D2 receptor stimulation, activated ERK 1/2 and PI3-K. In contrast, however, to D2 receptor stimulation, the inhibition of either kinase prevented the increase in uptake evoked by D3 receptor activation. Using biotinylation techniques to quantify the intracellular and membrane expression of the dopamine transporter, we have shown that D3 receptor activation differentially affected transporter subcellular distribution depending on the duration of agonist exposure. The rapid increase in transporter function is associated with increased transporter cell surface expression and decreased intracellular transporter. In contrast, following prolonged D3 receptor activation, dopamine transporter function is down-regulated and transporter cell surface expression is reduced. Amphetamine and other transporter substrates promote redistribution of the dopamine transporter from the plasma membrane to the cytosol where they are unable to regulate uptake. Therefore, increased intracellular transporter accumulation would result in an elevation of extracellular dopamine concentrations. This effect appears paradoxical since decreased cell surface transporter expression decreases dopamine clearance from the extracellular fluid and high extracellular DA concentrations are neurotoxic. Our results suggest that if dopamine is available to transiently stimulate D2 or D3 receptors, transporter internalization will decrease in the vicinity of these receptors, resulting in rapid clearance of extracellular dopamine and prevention of prolonged receptor activation. On-going studies are determining the role of D2/D3 regulation of the dopamine transporter in mediating alterations in dopamine transmission produced by amphetamine and other transporter substrates. ? ? Kappa opioid receptor agonists decrease extracellular dopamine concentrations in the nucleus accumbens and dorsal striatum. Our recent studies have shown that acute administration of the potent kappa opioid receptor agonist, salvinorin A, an alkaloid found in the mint leaf and used by Indian tribes for its psychotomimetic actions, produces similar effects. Since kappa opioid receptors are apposed to dopamine transporters in dopamine nerve terminals in the nucleus accumbens we have initiated studies to determine whether this G-protein coupled receptor regulates dopamine transmission by affecting the activity of the dopamine transporter. Our studies in native tissue and heterologous expression systems have revealed that salvinorin, dynorphin, the endogenous kappa opioid receptor ligand, as well as synthetic agonists modulate extracellular dopamine concentrations, at least in part, by facilitating dopamine uptake. Furthermore, the interaction of kappa opioid receptors with the dopamine transporter, like that of D2 and D3 receptors is ERK 1/2- dependent. These findings suggest that the previously documented cocaine-antagonist-like effects of kappa opioid receptor agonists may result, at least, in part, from the ability of kappa opioid receptors to regulate the dopamine transporter. ? ? Cocaine binds with high affinity to the dopamine transporter as well as other monoamine transporters. Studies, currently in progress, are examining the role of kappa opioid receptors in regulating the activity of these monoamine transporters. The contribution of kappa- and other opioid receptor types to the regulation of amino acid transporter function will also be assessed in view of our recent findings indicating the involvement of endogenous opioid system in regulating in glutamate and GABA homeostasis in-vivo.
