Dopamine transporter synthesis and degradation rate in rat striatum and nucleus accumbens using RTI-76

Intracerebroventricular injections of the irreversible dopamine transporter (DAT) inhibitor, RTI-76 [3beta-(3-p-chlorophenyl) tropan-2beta-carboxylic acid p-isothiocyanatophenylethyl ester hydrochloride], decreased DAT binding in both the striatum and nucleus accumbens as measured by both [3H]GBR12935 and by [3H]WIN35,428. This decrease was dose-related, with 100 nmol RTI-76 producing approximately a 50% decrease in both regions. The maximal inhibition of DAT binding was observed 24 h after RTI-76 injection, and binding was fully restored 7 days after injection. The DAT protein half-life determined under these conditions was about 2 days. [3H]Nisoxetine binding at norepinephrine transporters in the cortex was not altered by RTI-76 administration at any time point or dose examined.     

RTI-76, an isothiocyanate derivative of a phenyltropane cocaine analog, as a tool for irreversibly inactivating dopamine transporter function in vitro

Human dopamine transporters, stably expressed by human embryonic kidney-293 cells, were reacted with 3beta-(3p-chlorophenyl)tropan-2beta-carboxylic acid p-isothiocyanatophenylethyl ester (RTI-76) under varying conditions. Exposure to RTI-76 (1 microM) at 0 degrees C, followed by extensive wash-out, reduced subsequent binding of the cocaine analog [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl) tropane (WIN 35,428), which was caused by an increase in Kd in the absence of a Bmax change. Exposure to RTI-76 (50 nM(-1) microM) at 37 degrees C, however, caused concentration-dependent reductions in binding Bmax; increases in Kd were observed only at high levels of RTI-76 (0.5-1 microM). The reductions in Bmax are consonant with acylation of transporters, the increases in Kd with incomplete wash-out observed also for the amine precursor of RTI-76 which lacks the isothiocyanate group for irreversible binding and which did not lower Bmax at 37 degrees C. Reductions in binding Bmax generated by varying concentrations of RTI-76 up to 300 nM at 37 degrees C correlated with reductions in [3H]dopamine uptake Vmax on a one-to-one basis, with Km values showing a tendency towards a small reduction as a function of transporter inactivation, but the potency of WIN 35,428 in inhibiting uptake not showing a change. The results are discussed in the context of possible oligomeric assemblies of dopamine transporters carrying multiple recognition sites for cocaine analogs and dopamine.     

Isothiocyanate derivatives of cocaine: irreversible inhibition of ligand binding at the dopamine transporter

Isothiocyanate derivatives of (-)-cocaine were prepared and tested for inhibitory potency at the cocaine receptor in rat striatal membranes. Coincubation with m-isothiocyanatobenzoylecgonine methyl ester (m-ISOCOC), p-isothiocyanatobenzoylecgonine methyl ester (p-ISOCOC), and 3 beta-(4-isothiocyanatophenyl)tropane-2-carboxylic acid methyl ester (ISOWIN) resulted in inhibition of [3H]WIN 35,428 binding, but the compounds were about 10-fold weaker than (-)-cocaine. However, p-ISOCOC was approximately 3-fold more potent than metaphit, an isothiocyanate derivative of phencyclidine. p-ISOCOC was equipotent at the serotonin transporter but was much less potent at the norepinephrine transporter and was inactive at the D2 dopamine receptor at 1000 microM concentration. The IC50 value for m-ISOCOC and p-ISOCOC varied with tissue concentration, suggesting irreversible inhibition of binding. Preincubation with m-ISOCOC and p-ISOCOC resulted in inhibition of [3H]WIN 35,428 binding that could not be removed by washing of the membranes; in contrast, preincubation with (-)-cocaine caused inhibition that was readily removed by washing. Preincubation with 1 microM concentrations of p-ISOCOC resulted in a large reduction in Bmax of the high affinity binding site for [3H]WIN 35,428. Preincubation with 100 microM p-ISOCOC eliminated the high affinity site and apparently reduced the affinity at the low affinity site. Coincubation of 10 microM p-ISOCOC with 100 microM cocaine prevented the total loss of [3H]WIN 35,428 binding. The uptake of [3H]dopamine was inhibited by p-ISOCOC with an IC50 comparable to that of cocaine. Additionally, preincubation of rat striatal synaptosomes with 10 microM p-ISOCOC reduced the Vmax of [3H]dopamine uptake after washing. These data suggest that m-ISOCOC and p-ISOCOC are useful irreversible acylators of (-)-cocaine binding sites at the dopamine transporter.     

2 beta-substituted analogues of cocaine. Synthesis and inhibition of binding to the cocaine receptor.

The potencies of a series of 2 beta-substituted cocaine analogues to displace [3H]-3 beta-(p-fluorophenyl)tropane-2 beta-carboxylic acid methyl ester binding in rat striatal membranes demonstrate the requirement for a 2 beta-substituent with two hydrogen-bond acceptors. The insensitivity of the ester moiety to steric and electronic factors suggests its modification to provide site-specific irreversible ligands.     

N-modified analogues of cocaine: synthesis and inhibition of binding to the cocaine receptor.

Cocaine methiodide (2), N-norcocaine (1b), N-benzyl-N-norcocaine (1c), and N-nor-N-acetylcocaine (1d) were synthesized and evaluated for their ability to inhibit binding of [3H]-3 beta-(4-fluorophenyl)tropane-2 beta-carboxylic acid methyl ester (WIN 35,428) to the cocaine receptor. The study showed that removal of the N-methyl group to give 1b, or replacement with the larger N-benzyl group to give 1c, has a relatively small effect on binding potency. In contrast, replacement of the N-methyl group by the acetyl moiety to give 1d, or the addition of a methyl group to give 2, reduces affinity for the receptor by a large factor. In order to gain preliminary information concerning the importance of the nitrogen location on the tropane ring system, the receptor binding affinity of 8-methyl-8-azabicyclo[3.2.1]octan-3 beta-ol benzoate (5, beta-tropacocaine) was compared to that of the isomeric 6-methyl-6-azabicyclo[3.2.1]octan-3 beta-ol benzoate (4d). The fact that both compounds have similar binding affinities for the cocaine receptor suggests that 3 beta-(benzoyloxy)-6-methyl-6-azabicyclo[3.2.1] octane-2-carboxylic acid methyl ester, which is isomeric with cocaine, may possess binding potency similar to cocaine.     

New, potent cocaine analogs: ligand binding and transport studies in rat striatum

Two potent cocaine analogs have been developed that have the highest known affinities for the cocaine binding site in rat striatum. Both 3 beta-(4-chlorophenyl)- (RTI-COC-31) and 3 beta-(4-methylphenyl)-tropane-2-carboxylic acid methyl ester (RTI-COC-32) compete for [3H]WIN 35,428 and [3H]mazindol binding with a IC50 that is 100 times more potent than that of (-) cocaine. Additionally, these compounds inhibit [3H]dopamine uptake with a similar, high potency. These results may lead to the development of high affinity probes for the cocaine binding site.     

The self-administration of WIN 35,428 and cocaine: comparisons of satiety threshold and elimination half-life in rats

Rats that self-administered cocaine at unit doses between 0.75 and 12 micromol/kg with mean inter-injection intervals between approximately 2 and 18 min also reliably self-administered the cocaine analogue WIN 35,428 (beta-CFT; (-)-3 beta-(4-fluorophenyl)tropane-2 beta-carboxylic acid methyl ester) at unit doses between 0.1 and 1.6 micromol/kg with mean intervals between 10 and 116 min. The long inter-injection intervals of WIN 35,428 necessitated sessions of more than 12 h. The inter-injection intervals were regular and proportional to the unit dose, consistent with the satiety threshold model. Analysis of the mean intervals as a function of unit doses generated values for the mean satiety threshold of cocaine and WIN 35,428 of 6.10 and 0.87 micromol/kg, respectively. The mean t(1/2) for cocaine and WIN 35,428 were 11.1 and 69.4 min, respectively. The approximately 43-fold lower rate of consumption of WIN 35,428 relative to cocaine was a product of the seven-fold greater pharmacodynamic potency and the six-fold greater pharmacokinetic potency.     

Synthesis and transporter binding properties of 3beta-[4'-(phenylalkyl, -phenylalkenyl,and -phenylalkynyl)phenyl]tropane-2beta-carboxylic acid methyl esters: evidence of a remote phenyl binding domain on the dopamine transporter

A series of 4'-substituted 3beta-phenyltropane-2beta-carboxylic acid methyl esters were synthesized and evaluated for binding at the dopamine transporter (DAT) in order to better define the pharmacophore for the cocaine binding site on the DAT. Results from the study of 3beta-[(4'-phenylalkyl)phenyl]tropane-2beta-carboxylic acid methyl esters (5a-c and 6a,b) revealed strong evidence of a previously unknown remote binding domain. The 3beta-[(4'-phenylethyl)phenyl]tropane-2beta-carboxylic acid methyl ester (5a), which has a two methylene linker between the 3beta-phenyl group and the remote phenyl group, has an IC(50) value of 5.14 nM at the DAT. The 3beta-[4'-(benzyl)phenyl] and 3beta-[4'-(phenylpropyl)phenyl] analogues 6b and 5b, respectively, are 102- and 68-fold less potent than 5a at the DAT. Compound 5a also has good affinity for the serotonin and norepinephrine transporters (K(i) = 21 and 6.5 nM, respectively) and is thus a nonselective monoamine uptake inhibitor. Electrostatic effects make a significant contribution to the DAT binding affinity of the 3beta-[(4'-phenylalkenyl)phenyl]tropane-2beta-carboxylic methyl esters (6c, 7a,b, and 8) and 3beta-[(4'-phenylalkynl)phenyl]tropane-2beta-carboxylic acid methyl esters (4a-e). However, the results from the DAT binding on these compounds suggest that there may be another binding domain even further remote from the 4'-position on the 3beta-phenyl group. In both cases, steric barriers have to be overcome before potent binding to the DAT is observed. 3beta-(4'(3-Phenyl-1-propynyl)phenyl)tropane-2beta-carboxylic acid methyl ester (4b), with an IC(50) value of 1.82 nM, was the most potent compound studied. This compound possessed K(i) values of 1.19 and 16.5 nM for the serotonin and norepinephrine transporter and is thus a nonselective monoamine uptake inhibitor.     

Synthesis and receptor binding of N-substituted tropane derivatives. High-affinity ligands for the cocaine receptor

The synthesis and pharmacological characterization of a series of N-substituted 3-(4-fluorophenyl)tropane derivatives is reported. The compounds displayed binding characteristics that paralleled those of cocaine, and several had substantially higher affinity at cocaine recognition sites. Conjugate addition of 4-fluorophenyl magnesium bromide to anhydroecgonine methyl ester gave 2 beta-(carbomethoxy)-3 beta-(4-fluorophenyl)tropane (4a, designated CFT, also known as WIN 35,428) after flash chromatography. N demethylation of 4a was effected by Zn/HOAc reduction of the corresponding 2,2,2-trichloroethyl carbamate to give 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)nortropane (5), which was alkylated with allyl bromide to afford the N-allyl analogue, 6. The N-propyl analogue, 7, was prepared by catalytic reduction (Pd/C) of 6. The most potent analogue, 4a, was tritiated at a specific activity of 81.3 Ci/mmol. [3H]4a bound rapidly and reversibly to caudate putamen membranes; the two-component binding curve typical of cocaine analogues was observed. Equilibrium was achieved within 2 h and was stable for at least 4 h. High- and low-affinity Kd values observed for [3H]4a (4.7 and 60 nM, respectively) were more than 4 times lower than those for [3H]cocaine, and the density of binding sites (Bmax = 50 pmol/g, high, and 290 pmol/g, low) for the two drugs were comparable. Nonspecific binding of [3H]4a was 5-10% of total binding.     

Binding of uptake blockers to the neuronal dopamine transporter: further investigation about cationic and anionic requirements

Effects of ions on the binding of uptake blockers to the rat dopamine transporter (rDAT) labelled with [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)-[3H] tropane] and [3H]mazindol were studied at 20 degrees C. [3H]WIN 35,428 binding increased with Na+ concentrations of up to 10-60 mM and decreased at higher concentrations. At pH 7.4, incubation media containing NaCl and/or Na2HPO4/NaH2PO4 were less stimulant than an NaHCO3/NaH2PO4 medium and they shifted maximal binding values to higher ionic concentrations. In an NaHCO3/NaH2PO4-buffered medium, Na+ concentrations >10 mM decreased the binding of 0.2 nM [3H]WIN 35,428, but an increase of the radioligand concentration shifted this decrease to the right. [3H]Mazindol binding was stimulated by Na+ concentrations < or =10 mM and was rather unaffected at higher concentrations. The inhibition of [3H]WIN 35,428 binding produced by 130 mM Na+ was independent of the nature of the anion; in contrast, isothionate and H2PO4-/HCO3 produced a more pronounced inhibition of the [3H]mazindol binding than Cl- and Br-, whereas I- tended to be a stimulant. Ca2+ and Mg2+ more potently inhibited the [3H]WIN 35,428 binding than K+. All these cations recognize a site which is not mutually exclusive with that of the radioligand since they induced the dissociation of the [3H]WIN 35,428-rDAT complex, an effect which was reduced (K+) or modified (Ca2+) when the Na+ concentration was increased. This site is likely to be the Na+ site by which low Na+ concentrations allosterically stimulate the uptake blocker binding. However, the intensity of the cation-induced dissociations was moderate and the main component of the binding inhibition that these cations produced results from the occupancy of a cation site, mutually exclusive with that of the radioligand. Thus, the WIN 35,428 binding inhibition produced by Ca2+, K+ and Na+ was competitive, and Na+ reduced the inhibitory potency of Ca2+ and K+. This reduction was more intense for Ca2+ and Mg2+ than for K+, suggesting that occupancy of the cation site by a divalent cation activated a strong negative allosteric interaction between this site and the Na+ site. Decrease in the Na+ concentration from 10 mM to 5 mM, or replacement of 5 mM HCO3-/H2PO4- by an equimolar concentration of isethionate or Cl- did not modify [3H]WIN 35,428 binding dissociation. Level(s) at which anions stimulate and inhibit the binding of uptake blockers remain uncertain and could be specific for each radioligand.     

Synthesis and pharmacology of site-specific cocaine abuse treatment agents: restricted rotation analogues of methylphenidate

A series of threo-1-aza-3 or 4-substituted-5-phenyl[4.4.0]decanes (quinolizidines), which were envisioned as restricted rotational analogues (RRAs) of methylphenidate (MP), was synthesized and tested for inhibitory potency against [(3)H]WIN35,428, [3H]citalopram, and [3H]nisoxetine binding to the dopamine, serotonin, and norepinephrine transporters, respectively. Two different synthetic schemes were used; a Wittig reaction or acylation (followed by an intramolecular condensation) was a key feature of each scheme. The unsubstituted RRA, threo(trans)-1-aza-5-phenyl[4.4.0]decane (12a), was equipotent to unconstrained threo-MP against [(3)H]WIN35,428 binding. The extra ring in these RRAs (which reduces the conformational freedom) and the orientation and polarity of substituents at the 4-position on this extra ring are of critical importance to the biological activity. Generally, the RRAs paralleled the corresponding unconstrained MP derivatives in binding affinity to the three transporters. The results suggest that the conformation of MP in which the carbonyl group of the methyl ester is H-bonded to the piperidinyl N-H may be the bioactive form of the molecule.     

Potent substituted-3 beta-phenyltropane analogs of cocaine have cocaine-like discriminative stimulus effects.

Two novel phenyltropane analogs of cocaine, 3 beta-(4-chlorophenyl) tropane-2 beta-carboxylic acid methyl ester (RTI-COC-31) and 3 beta-(4-methylphenyl) tropane-2 beta-carboxylic acid methyl ester (RTI-COC-32), were evaluated for cocaine-like discriminative stimulus effects in rats. Two groups of rats were trained to discriminate 10 mg/kg cocaine from saline using a standard 2-lever discrimination procedure with correct-lever responding reinforced under a fixed-ratio 20 schedule of food reinforcement. One group of rats was used to evaluate RTI-COC-31 and the unsubstituted-3 beta-phenyltropane reference compound WIN 35,065-2 in substitution tests. Another group of rats was used to evaluate RTI-COC-32. For purposes of comparison, a cocaine dose-effect curve was also determined in each group. Both RTI-COC-31 and RTI-COC-32, as well as WIN 35,065-2, substituted completely for cocaine. RTI-COC-31 was 26.8-fold and RTI-COC-32 was 6-fold more potent than cocaine for producing cocaine-lever responding. The potent cocaine-like effects of the RTI analogs in vivo correspond with their high affinities for a cocaine recognition site on the dopamine transporter, providing further evidence that this site may mediate behavioral effects of cocaine relevant to its abuse.     

Binding domains for blockers and substrates on the dopamine transporter in rat striatal membranes studied by protection against N-ethylmaleimide-induced reduction of [3H]WIN 35,428 binding

Binding sites for 2β-carbomethoxy-3β-(4-fluorophenyl)[³H]tropane ([³H]WIN 35,428) on rat striatal membranes were alkylated with N-ethylmaleimide (NEM), and the protective potency was measured of the blockers cocaine, N[1-(2-benzo[b]thiophenyl) cyclohexyl]piperidine (BTCP), benztropine, WIN 35,428, and nomifensine, and of the substrates dopamine, norepinephrine, S(+)-amphetamine, tyramine, and metaraminol. In general, the protective potency was lower (at least 3 times) than the potency in inhibiting [³H]WIN 35,428 binding with the compounds present under the same experimental conditions used for the NEM-induced alkylation. However, the disparity was substantially greater for all substrates tested (10- to 93-fold) than for the blockers (2- to 6-fold), especially cocaine and BTCP (3-fold). [³H]WIN 35,428 binding was best described by a 1-site model under the present conditions. The results are discussed in terms of models involving blocker-induced conformational changes and overlapping nonidentical binding domains for blockers and substrates. Received: 14 May 1996 / Accepted: 11 September 1996     

Novel tropane-based irreversible ligands for the dopamine transporter.

3alpha-(diphenylmethoxy)tropane (benztropine) and its analogues are tropane ring-containing dopamine uptake inhibitors that display binding and behavioral profiles that are distinct from cocaine. We previously prepared a benztropine-based photoaffinity label [125I]-(N-[4-(4'-azido-3'-iodophenyl)butyl]-3alpha-[bis(4'-fluorophenyl)methoxy]tropane, [125I]1, that covalently attached to the 1-2 transmembrane spanning region of the dopamine transporter (DAT). This was in contrast to the 4-7 transmembrane spanning region labeled by a cocaine-based photoaffinity label, [125I] 2 (RTI 82). To characterize further these different binding domains, photoaffinity ligands that had the 4'-azido-3'-iodophenyl substituent extended from the same position on the tropane ring were desirable. Thus, identification of the optimal alkyl linker between this substituent and the tropane nitrogen in the benztropine series was investigated to ultimately prepare the identical N-substituted analogue of 2. In this pursuit, the N-[4-(4'-azido-3'-iodophenyl)propyl] analogue of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (9a) was synthesized as well as two isothiocyanate analogues that do not require photoactivation (10a,b) for irreversible binding. The synthesis of these target compounds was achieved using a modification of the strategy developed for 1. Evaluation of these compounds for displacing [3H]WIN 35 428 binding at DAT in rat caudate putamen revealed that the 4'-azido-3'-iodophenylbutyl substituent, found in 1, provided optimal binding affinity and was chosen to replace the N-CH3 group on 2. Both the 4'-azido-3'-iodophenyl- and the 4'-isothiocyanatophenylbutyl analogues of 2 (25 and 26, respectively) were synthesized. Both products bound to DAT with comparable potency (IC(50) = 30 nM) to RTI 82 (2). In addition, compound 26 demonstrated wash-resistant displacement of [3H]WIN 35 428 in HEK 293 cells stably transfected with hDAT. These ligands will provide important tools for further characterizing the binding domains for tropane-based dopamine uptake inhibitors at the DAT.     

Synthesis and ligand binding of cocaine isomers at the cocaine receptor

The cocaine binding site at the dopamine transporter has been found to be stereoselective. Thus, the seven possible stereoisomers of (-)-cocaine have been synthesized and found to inhibit [3H]-2 beta-carbomethoxy-3 beta-(4-fluoro-phenyl)tropane [( 3H]WIN 35,428) with potencies ranging from 1/60 to 1/600 of that of (-)-cocaine. The synthesis and characterization of all new compounds is presented.     

Translocation of dopamine and binding of WIN 35,428 measured under identical conditions in cells expressing the cloned human dopamine transporter

Translocation of [³H]dopamine and binding of [³H]WIN 35,428 were measured in intact C6 glioma cells expressing the cloned human dopamine transporter (hDAT) under identical conditions of assay buffer (phosphate-Krebs) and temperature (25°C) with uptake at initial velocity and binding at equilibrium. In the intact cells, [³H]dopamine uptake was a one-component process; in contrast, [³H]WIN 35,428 binding included both a high-affinity component, inhibitable by micromolar concentrations of dopamine, and a low-affinity component only partially inhibited by millimolar concentrations of dopamine. Binding (high-affinity) over uptake Ki ratios were on the average 2.3 for the inhibitors WIN 35,428, cocaine, GBR 12909, and BTCP. The potency of dopamine in inhibiting its own translocation was close to that in inhibiting [³H]WIN 35,428 binding consonant with a more rapid reorientation step of the DAT in the C6-hDAT system than in rat striatal synaptosomes. The similarity in turnover values of the DAT estimated in the current experiments with the C6-hDAT system and in our previous study on rat striatal synaptosomes, performed under comparable conditions, suggest that all DAT's inserted into the C6 cell membrane are functionally active.     

Synthesis and pharmacology of site-specific cocaine abuse treatment agents: 2-(aminomethyl)-3-phenylbicyclo[2.2.2]- and -[2.2.1]alkane dopamine uptake inhibitors

As part of a program to develop site-specific medications for cocaine abuse, a series of 2-(aminomethyl)-3-phenylbicyclo[2.2.2]- and -[2.2.1]alkane derivatives was synthesized and tested for inhibitory potency in [3H]WIN 35,428 binding and [3H]dopamine uptake assays using rat striatal tissue. Selected compounds were tested for their ability to substitute for cocaine in rat drug discrimination tests. Synthesis was accomplished by a series of Diels-Alder reactions, using cis- and trans-cinnamic acid derivatives (nitrile, acid, acid chloride) with cyclohexadiene and cyclopentadiene. Standard manipulations produced the aminomethyl side chain. Many of the compounds bound with high affinity (median IC50 = 223 nM) to the cocaine binding site as marked by [3H]WIN 35,428. Potency in the binding assay was strongly enhanced by chlorine atoms in the 3- and/or 4-position on the aromatic ring and was little affected by corresponding methoxy groups. In the [2.2.2] series there was little difference in potency between cis and trans compounds or between N, N-dimethylamines and primary amines. In the [2.2.1] series the trans exo compounds tended to be least potent against binding, whereas the cis exo compounds were the most potent (4-Cl cis exo: IC50 = 7.7 nM, 27-fold more potent than 4-Cl trans-exo). Although the potencies of the bicyclic derivatives in the binding and uptake assays were highly correlated, some of the compounds were 5-7-fold less potent at inhibiting [3H]dopamine uptake than [3H]WIN 35,428 binding (for comparison, cocaine has a lower discrimination ratio (DR) of 2.5). The DR values were higher for almost all primary amines and for the trans-[2.2.2] series as compared to the cis-[2.2.2]. Most of the compounds had Hill coefficients approaching unity, except for the [2. 2.2] 3,4-dichloro derivatives, which all had nH values of about 2.0. Two of the compounds were shown to fully substitute for cocaine in drug discrimination tests in rats, and one had a very long duration of action.     

5-HT3 receptor antagonists block cocaine-induced locomotion via a PCPA-sensitive mechanism.

We report results in rats pretreated with (+/-)-zacopride (0.03 mg/kg, IP), ICS 205-930 (0.1 mg/kg, IP), and MDL 72222 (1.0 mg/kg, IP) 15 min before challenge with (-)-cocaine (10.0 mg/kg, IP). At a dose of 10 micrograms/kg, zacopride significantly inhibited (approximately 50%) cocaine-induced locomotion. We also investigated whether or not 5-hydroxytryptamine3 (5-HT3) antagonists block the cocaine binding site on the dopamine transporter and/or affect the ability of dopamine to regulate this binding site. In well-washed striatal membranes, neither zacopride nor ICS 205-930 (10(-9)-10(-5) M) inhibited [3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane ([3H]WIN 35,428) (0.3 nM) binding. Furthermore, neither of these compounds affected the ability of dopamine to block WIN 35,428 binding. To determine if 5-HT is required for the 5-HT3 antagonist effect, we examined the interaction between cocaine and zacopride in rats pretreated with p-chlorophenylalanine (PCPA) (3 days x 100 mg/kg/day). PCPA pretreatment shifted the cocaine dose-response curve to the right and blocked the ability of zacopride to reverse cocaine-induced activity.     

Chronic administration of the selective dopamine uptake inhibitor GBR 12909, but not cocaine, produces marked decreases in dopamine transporter density

The chronic continuous infusion of cocaine produces partial behavioral tolerance to cocaine and tolerance to the inhibition of dopamine uptake by cocaine, without changing dopamine transporter binding. In order to examine more closely the dopaminergic contribution to this effect, the selective dopamine uptake inhibitor GBR 12909 (30mg/kg/day), cocaine (50mg/kg/day), or vehicle, were continuously infused via osmotic minipump, and their effects on the dopamine transporter examined. Drug and vehicle pumps were implanted into male Sprague-Dawley rats and removed after seven days. [³H]WIN 35,428 binding and [³H]dopamine uptake were measured in caudate putamen and nucleus accumbens at varying intervals after pump removal. The B _max for [³H]WIN 35,428 binding was decreased by approximately 75% in the caudate putamen and by 40% in the nucleus accumbens of GBR 12909-treated rats both 1 and 4 days after pump removal, and was still significantly decreased after 10 days, but had returned to normal by 20 days post-treatment. In contrast, cocaine did not significantly alter [³H]WIN 35,428 binding. GBR 12909 produced both tolerance to the inhibition of [³H]dopamine uptake by cocaine, and a decrease in total uptake of dopamine, in the caudate putamen, with no change in the nucleus accumbens. The persistent reduction of [³H]WIN 35,428 binding following continuous GBR 12909 does not appear to result from residual drug binding. These findings suggest that GBR 12909 and cocaine may bind to and regulate the dopamine transporter in different ways.