Recently it was hypothesized by others that the D2dopamine receptor can regulate the uptake of dopamine. However, the evidence in support of this hypothesis, although compelling, was not based on observations related to direct measures of the kinetic activity of the transporter itself. Here kinetic evidence in support of this hypothesis is shown. The apparent time‐resolved initial velocity of the transport of 1.0 μM dopamine into striatal suspensions, measured using rotating disk electrode voltammetry, was found to increase in the presence of the D2 receptor agonist, quinpirole, at 100 μM. This effect was reversed by sulpiride. In separate studies it was shown that acute and chronic treatments with haloperidol at 0.5 mg/kg, i.p., reduced the reuptake transport of dopamine in vivo following intrastriatal stimulation of its release by K+. Thus, it appears that D2 receptors may influence the functioning of the striatal transporter for dopamine. These results are consistent with a model in which presynaptically released dopamine may feed back onto the function of its transporter to increase the velocity of the clearance of synaptic dopamine following an action potential, suggesting the existence of a mechanism, in addition to release and synthesis modulation, for fine‐tuning dopaminergic chemical signaling.

Experiments were conducted to determine how (−)‐cocaine and S(+)‐amphetamine binding sites relate to each other and to the catechol substrate site on the striatal dopamine transporter (sDAT). In controls, m‐tyramine and S(+)‐amphetamine caused release of dopamine from intracellular stores at concentrations ≥12‐fold those observed to inhibit inwardly directed sDAT activity for dopamine. In preparations from animals pretreated with reserpine, m‐tyramine and S(+)‐amphetamine caused release of preloaded dopamine at concentrations similar to those that inhibit inwardly directed sDAT activity. S(+)‐Amphetamine and m‐tyramine inhibited sDAT activity for dopamine by competing for a common binding site with dopamine and each other, suggesting that phenethylamines are substrate analogues at the plasmalemmal sDAT. (−)‐Cocaine inhibited sDAT at a site separate from that for substrate analogues. This site is mutually interactive with the substrate site (Kint = 583 nM). Mazindol competitively inhibited sDAT at the substrate analogue binding site. The results with (−)‐cocaine suggest that the (−)‐cocaine binding site on sDAT is distinct from that of hydroxyphenethylamine substrates, reinforcing the notion that an antagonist for (−)‐cocaine binding may be developed to block (−)‐cocaine binding with minimal effects on dopamine transporter activity. However, a strategy of how to antagonize drugs of abuse acting as substrate analogues is still elusive.

The inhibition by cocaine of the apparent initial rate of the transport of striatal dopamine was compared with inhibitions produced by cocaethylene, benztropine, GBR-12909, mazindol, and nomifensine . Rotating disk electrode voltammetry was used to measure the kinetically resolved, inwardly directed transport of dopamine in striatal suspensions . Evidence is presented that the primary site of action of cocaine may be at the external face of the transporter . Experiments to determine whether or not the other inhibitors bind to the same site as cocaine were conducted by comparing the inhibitions observed for each of the inhibitors alone with that observed when paired with cocaine . The resulting changes in the velocity of the transport of dopamine induced by the inhibitors were then fit to one of the previously developed models of inhibition by pairs of inhibitors affecting the kinetics of actively transporting systems : a single-site model, a twosite model in which the two binding sites for the inhibitors interact, and a two-site model in which the two binding sites for the two inhibitors act independently . Cocaine inhibited the transport of dopamine competitively with its structural analogues, cocaethylene and benztropine . The structurally dissimilar inhibitor, GBR-12909, was found also to be competitive with cocaine . In contrast, mazindol and nomifensine were found to bind to separate interactive sites when individually paired with cocaine . These results suggest that mazindol and nomifensine may interact with the kinetically active transporter for dopamine in a manner different from that of cocaine . Mazindol was tested and found to inhibit competitively the inward transport of dopamine into striatal suspensions . In contrast, our previous published findings show cocaine to be an uncompetitive inhibitor of the transport of striatal dopamine . These results suggest that cocaine inhibits inward transport of dopamine by reducing the intramembrane turnover of the transporter, whereas mazindol alters the kinetics of the recognition of dopamine by the transporter . Finally, the potential effects of these binding modes of inhibitors on synaptic chemical communication in dopaminergic systems were analyzed . The results of these analyses suggest that different effects on the extracellular concentrations of dopamine can result from the different patterns of inhibition, suggesting that different modula-1683 tory influences on pre-and postsynaptic receptor occupation can result from inhibition of the transport of dopamine .

The apparent second‐order association rate constant of dopamine binding to the striatal transporter (∼1 ± 106M−1 s−1) as well as the transporter turnover number (∼1.5 s−1) was estimated using rotating disk electrode voltammetry to monitor apparent zero trans entry of dopamine into striatal suspensions. The substrate specificity of the transporter was also assessed using catechol derivatives. Dopamine and norepinephrine were transported, whereas epinephrine and the acidic metabolites of dopamine were not transported. The metabolite, 3‐meth‐oxytyramine, was transported with a Km seven times greater than and a Vmax close to that for dopamine. 4‐Methoxytyramine was transported more facilely than the 3‐methoxy derivative. N‐Alkylation of the amine side chain of dopamine reduced transport dramatically. 4‐Ethylcatechol and 3,4‐dihydroxybenzylamine were transported with velocities 79 and 91 % less than that for dopamine, respectively. The rigid analogue 6,7‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene was transported with a greater velocity than the 5,7‐dihydroxy derivative. Finally, the apparent Kmvalues for 4‐ethylcatechol, 1‐amino‐2‐phenylethane, tyramine, and m‐tyramine as cosubstrates with dopamine were 1.1, 11, 17, and 2.6 μM, respectively. Pretreatments of striatal suspensions with chloroethylnorapomorphine, N‐ethylmaleimide, Hg2+, 4,5‐dihydroxy‐4,5‐dioxo‐1H‐pyrrolo[2,3‐f]quinoline‐2,7,9‐tricarboxylic acid (a redox modulator of receptors in neuronal as well as other tissues), and neuraminidase reduced the velocity of transport of dopamine, whereas N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline had no effect. Thus, the dopamine transporter requires an intact catechol with a primary ethylamine side chain for optimal activity relative to shorter side chain derivatives (side chains longer than two carbons were not tested), the 3‐hydroxyl group of dopamine is the more critical hydroxyl group, and the β rotamer of the extended conformation of dopamine is transported preferentially. The catechol appears to mediate the recognition of the substrate, whereas the amine side chain apparently facilitates the conformational change of the transporter that results in movement of dopamine into or across the membrane. The transporter distinguishes between agents known to block dopamine recognition sites on dopamine receptors? appears to possess a reduction/oxidation modulatory site, and requires sulfhydryl groups and external glycosylation for optimal function.

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