Dissociation of high-affinity cocaine analog binding and dopamine uptake inhibition at the dopamine transporter

Cocaine initiates its euphoric effects by binding to the dopamine transporter (DAT), blocking uptake of synaptic dopamine. It has been hypothesized that the DAT transmembrane aspartic acid residue D79 forms an ionic interaction with charged nitrogen atoms in both dopamine and cocaine. We examined the consequences of novel and previously studied mutations of the D79 residue on DAT uptake of [3H]dopamine, DAT binding of the cocaine analog [3H]WIN 35,428, and drug inhibition of each process, all under identical conditions. The rat D79E DAT mutation decreased dopamine uptake Vmax by 7-fold and decreased dopamine turnover by 4-fold. Wild-type DAT displayed near-perfect agreement in the uptake and binding inhibition potencies for substrates, but cocaine and other nonsubstrate inhibitor drugs were approximately 3-fold less potent in uptake than in binding assays. Apparent affinities for substrates were unaffected by the D79E mutation unless the catechol moiety was modified. Strikingly, potencies for nonsubstrate inhibitors in uptake and binding assays matched for D79E DAT, because of a 3-fold lowering of binding affinities relative to WT DAT. The present findings reveal a complex role for D79 in determining substrate specificity and high-affinity binding of DAT inhibitors. We propose that at least two discrete inhibitor-binding DAT conformations or populations exist and that the DAT conformation/population responsible for inhibitor high-affinity binding is less responsible for dopamine uptake. The findings may be extensible to other psychostimulants and antidepressants that display discrepancies between binding affinity and monoamine uptake inhibition potency and may be relevant to development of a long-sought "cocaine antagonist".     

Doubtful role for phencyclidine metabolites in PCP enhancement of QNB binding

In vivo administration of phencyclidine (PCP) has been shown to enhance the accumulation of intravenously administered 3H quinuclidinyl benzilate (QNB) in certain regions of mouse brain. Since this effect can be blocked by prior administration of atropine, it has been interpreted as an enhancement by PCP of the specific binding of QNB. The present studies replicated this earlier work and determined that two major hydroxy metabolites of PCP found in rodents 1-(1-phenylcyclohexyl)4-hydroxy piperidine (4-OH-pip PCP) and 1-(1-phenyl-4-hydroxycyclohexyl)piperidine (4-OH-cyclo PCP) probably do not contribute substantially to the PCP effect on QNB binding. Though QNB accumulation in brain was increased by injection of 4-OH-cyclo PCP, the doses necessary for the effect were substantially higher than those needed for PCP. Furthermore, pretreatment of the animals with beta-diethylaminoethyl diphenylpropylacetate (SKF525A), a compound known to block the metabolism of PCP in liver microsomes and thus the formation (at least in part) of these metabolites, did not attentuate the PCP effect on QNB binding.     

Discriminable effects of phencyclidine analogs evaluated by multiple drug (PCP versus other) discrimination training

This study tested structural analogs of phencyclidine (PCP) using drug discrimination procedures to determine which analogs produced discriminable effects similar to those of PCP. It also tested the utility of multiple-drug discrimination training (PCP versus other drugs or saline) as a method for increasing the specificity produced by training. All discrimination training took place in two-lever operant compartments using FR-10 reinforcement of presses on the correct lever. During training, rats were required to concurrently discriminate PCP from one or more other drug conditions. Rats in group 1 discriminated PCP (lever 1) versus saline (lever 2). Rats in group 2 discriminated PCP (lever 1) versus saline, fentanyl, phenobarbital, amphetamine, or mescaline (lever 2). In both groups 1 and 2, the required discriminations were rapidly learned. The percentage of PCP choices and the ED₅₀ doses obtained during tests for generalization did not differ significantly in groups 1 and 2. Drugs to which responding on the PCP lever generalized included 1-[1-(2-thienyl)cyclohexyl]piperidine, N-3thyl-1-phenylcyclohexylamine, 1-phenylcyclohexylamine, ketamine, 1-(1-phenylcyclohexyl)morpholine, 1-[1-(2-thienyl)cyclohexyl]morpholine, N,N-diethyl-1-phenylcyclohexylamine, N-(iso-propyl)-1-phenylcyclo-hexylamine, N-methyl-1-phenylcyclohexylamine, N-(n-propyl)-1-phenylcyclohexylamine, Dextrorphan, (dl)-N-allyl-N-normetazocine, N-N-dimethyl-1-phenylcyclo-hexylamine N-(n-butyl)-1-phenylcyclohexylamine, 1-[1-(2-thienyl)cyclohexyl]pyrrolidine, and N-(s-butyl)-1-phenylcyclohexylamine, in agreement with previous reports. Rats in group 3 discriminated PCP (lever 1) versus saline, cyclazocine, dextrorphan, phenobarbital, or mescaline (lever 2). this discrimination was learned slowly, and asymptotic accuracy was low, thus demonstrating one of the limitations of drug discrimination training paradigms that require the concurrent discrimination of several drugs.     

Effects of phencyclidine (PCP)-like drugs on turning behavior, 3H-dopamine uptake, and 3H-PCP binding

In this study representatives from three chemical classes which are known to produce a phencyclidine (PCP)-like discriminative stimulus cue in rats were tested for their ability to inhibit synaptosomal uptake of 3H-dopamine (3H-DA) and to compete for a binding site labeled with 3H-PCP. Although there was a good correlation between these two in vitro activities within the arylcycloalkylamine class (PCP, N-ethyl-phenylcyclohexylamine (PCE), and ketamine) it did not hold when representatives from the benzomorphans, N-allynormetazocine (NANM), cyclazocine (CYCL), and ethylketocyclazocine (EKC), or a substituted dioxolane (etoxadrol) were included. At some dose each of these drugs with the exception of EKC also produced ipsilateral turning in rats with a unilateral 6-hydroxydopamine lesion of the substantia nigra. This effect was also not well correlated with inhibition of 3H-DA uptake. However, a significant correlation was found to exist between turning behavior and affinity for the putative PCP/sigma receptor. The possibility that a non-dopaminergic mechanism involving the PCP/sigma receptor underlies the ability of these agents to induce turning behavior is discussed.     

The effects of the phencyclidine analogs BTCP and TCP on nigrostriatal dopamine neuronal activity

Extracellular single unit recording techniques were used to evaluate the effects of two phencyclidine (PCP) derivatives. N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine (BTCP) and N-[1-(2-thiophenyl)cyclohexyl]piperidine (TCP) on the electrophysiological activity of antidromically identified nigrostriatal dopamine (DA) neurons in chloral hydrate-anesthetized rats. I.v. BTCP produced a dose-dependent decrease in the firing rate of identified nigrostriatal DA neurons whereas TCP elicited a dose-dependent biphasic effect which was characterized by an activation of cell firing at low doses followed by a reversal of the response with larger doses. A hemitransection of the brain anterior to the substantia nigra significantly reduced the inhibitory effect of BTCP while this surgical procedure did not affect the response to TCP. However, iontophoretic application of BTCP induced a current-dependent inhibition of the spontaneous activity of cells while local application of TCP had no effect on the firing rate of these cells. These data indicate that PCP analogs are able to interact with the nigrostriatal DAergic pathway through distinct and opposing mechanisms. The results are discussed in light of recent observations that BTCP is selective for the DA uptake site while TCP is selective for the high affinity PCP binding site.     

Inhibition by deprenyl of dopamine uptake in rat striatum: a possible correlation between dopamine uptake and acetylcholine release inhibition

Optical isomers of deprenyl inhibit the resting and ouabain induced release of acetylcholine (ACh) in isolated striatal slices of the rat and this effect correlates with the capability of deprenyl to inhibit the uptake of 3H-dopamine in striatal homogenate in a concentration of 10(-4)--10(-4) M. The release of ACh is significantly increased in striatal slices taken from rats pretreated with a single dose of 250 micrograms of 6-hydroxydopamine. 5 mg/kg of deprenyl given 30 min prior to 6-hydroxydopamine treatment prevented completely the chemical destruction of the dopaminergic neurons. In contrast to deprenyl, clorgyline potentiated the effect of 6-hydroxydopamine.     

1-methyl-4-phenylpyridinium (MPP+) analogs: in vivo neurotoxicity and inhibition of striatal synaptosomal dopamine uptake

The ability of various 1-methyl-4-phenylpyridinium (MPP+) analogs to inhibit the uptake of tritium labeled dopamine and MPP+ by synaptosomes prepared from neostriata of male C57 Black mice was measured and compared with their dopaminergic neurotoxic potential which was estimated by an in vivo intracerebral microdialysis technique. The correlation observed between these two properties suggests that nerve terminal uptake is an important step in the expression of the nigrostriatal toxicity of structural analogs of MPP+. The uptake inhibition and neurotoxic properties of this series of compounds appear to be highly structurally sensitive and suggest that few nitrogenous bases will be potent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-type neurotoxins.     

Chronic phencyclidine treatment decreases phencyclidine and dopamine receptors in rat brain

Chronic phencyclidine treatment (10 mg/kg/day, SC for 14 days) significantly decreased the number of [³H]phencyclidine and [³H]spiperone binding sites in rat brain. [³H]Dihydromorphine binding was not affected by the same treatment. An acute treatment with phencyclidine (10 mg/kg, SC) did not modify any of the binding sites under study. These results suggest that a chronic phencyclidine treatment induces a down-regulation of phencyclidine and dopamine receptors without affecting opiate receptors. These reductions in the number of phencyclidine and dopamine binding sites might be related to the development of tolerance and/or dependence to phencyclidine.     

A multisubstrate mechanism of striatal dopamine uptake and its inhibition by cocaine.

A study of Na+ and Cl- as co-substrates in dopamine uptake into striatal suspensions and inhibition of dopamine uptake by cocaine was made by monitoring the initial velocity of the uptake of exogenously added non-radioactively labeled dopamine using a rotating disk electroanalytical technique with 50 msec resolution. Dopamine, in the concentration range of 0.025 to 4.00 microM, was found to be taken up rapidly into the tissue phase of striatal suspensions following the apparent zero order rate law for the first 25 sec. The observed, dopamine concentration-dependent, initial velocity data were first analyzed graphically using the Eadie-Hofstee transformation of the Michaelis-Menten kinetic equation and, subsequently, using all of the velocity data and the results of the graphical analyses, by non-linear curve fitting. Dopamine uptake was found to be first order in dopamine with a Vmax of 582 pmol/sec/g wet weight and a Km of 1.2 microM. The results of experiments in which choline and isethionate were substituted for Na+ and Cl-, respectively, suggested that the uptake process is second order in Na+ and first order in Cl-. Multisubstrate analyses of the initial velocities of uptake over the concentration range of 0.025 to 1.5 microM dopamine suggested that the mechanism of binding of dopamine to the uptake carrier is a partially random, sequential mechanism where dopamine or Na+ binds first with the uptake carrier and Cl- binds last. Cocaine was found to uncompetitively inhibit dopamine uptake and competitively inhibit both Na+ and Cl- binding (apparent Km values: 131 and 51 mM, respectively), suggesting that the mechanism of cocaine inhibition may be to bind to the dopamine occupied uptake carrier complex at the Na+ binding site.     

Acute amphetamine and/or phencyclidine effects on the dopamine receptor specific binding in the rat brain

Psychotic-like behaviour was induced in rats with a single i.p. injection of AMPH (20 mg/kg b.w.) and/or PCP (10 mg/kg b.w.). The D₁ and D₂ dopamine receptor (DAR) specific binding of [³H]SCH 23390 and [³H]spiperone, respectively, during the 120 min period upon the treatment was examined on cryosections using computerized scanning and image analysis. AMPH, alone or in combination with PCP, induced a transient decrease of the D₁ receptor specific binding in the striatum (30 min; AMPH, −18%; AMPH+PCP, −31%) and nucleus accumbens (30 min; AMPH, −30%; AMPH+PCP, −40%), which was completely abolished at the 120 min point. Only AMPH persistently elevated nigral D₁ receptor specific binding. PCP-induced striatal and accumbal D₁ receptor down-regulation was intensive throughout the 120 min period, while in the s. nigra it was non-significant. A significant increase of the D₂ receptor specific binding was observed only 30 min after the treatment in striatum (AMPH, 15%; PCP, 16%; AMPH+PCP, 13%) and n. accumbens (AMPH, 16%). These alterations of DAR specific binding may reflect a regulation of the DAR and the changes in nigrostriatal and mesolimbic DA-ergic neurotransmission during an intensive drug-induced psychotic-like behavioral expression.     

The psychotomimetic drug phencyclidine labels two high affinity binding sites in guinea pig brain: evidence for N-methyl-D-aspartate-coupled and dopamine reuptake carrier-associated phencyclidine binding sites

Numerous studies have now demonstrated that a binding site for the psychotomimetic drug phencyclidine (PCP) exists within the receptor channel complex for the excitatory amino acid neurotransmitter glutamate, specifically the glutamate receptor selectively activated by N-methyl-D-aspartate (NMDA). Several lines of evidence support the hypothesis that all PCP receptors in rat brain are associated with the NMDA receptor complex. In the present study, we reexamine this hypothesis. We report that the PCP analog [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) labels two high affinity binding sites in membranes prepared from guinea pig brain site 1 (Kd = 14.1 nM, Bmax = 631 fmol/mg of protein) and site 2 (Kd = 46.5 nM, Bmax = 829 fmol/mg of protein). (+)-5-Methyl-10 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate bound to site 1 with high affinity (Kl = 3.2 nM) and to site 2 with low affinity (Kl = 5208 nM). The order of potency of drugs for inhibiting [3H]TCP binding to site 1 correlated with their ED50 values for inhibition of NMDA-mediated responses reported in the literature, whereas the order of potency of drugs for inhibiting [3H]TCP binding to site 2 correlated with their ED50 values for inhibition of [3H]dopamine reuptake reported in the literature. Kinetic experiments demonstrated that glutamate, 2-amino-7-phosphonoheptanoic acid, and Mg2+ modulated [3H]TCP binding to site 1 but not site 2. Preincubation of guinea pig striatal membranes with varying concentrations of the high affinity dopamine reuptake inhibitors N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3- phenylpropyl]piperazine caused a wash-resistant inhibition of [3H]TCP binding to site 2 but not site 1. Taken collectively, these data demonstrate the existence of a high affinity PCP binding site associated with the dopamine reuptake carrier and raise the possibility that the therapeutic and psychotomimetic effects of PCP in humans are separable and mediated via different binding sites.     

Disinhibitory effect of phencyclidine in the hippocampus in vitro: PCP receptors implicated

The effects of phencyclidine (PCP) and two dioxolane stereoisomers, dexoxadrol and levoxadrol, on hippocampal inhibition were compared. Field potentials were recorded in the CA1 pyramidal cell layer in the rat hippocampal slices in vitro. Recurrent inhibition of the population spikes evoked orthodromically by stimulation of the Schaffer collaterals was induced by antidromic conditioning stimulation at appropriate time intervals before the orthodromic stimulation. The drugs were applied by micropressure ejection in concentrations which did not affect the unconditioned population spike. After PCP or dexoxadrol administration, the orthodromically evoked population spike was much less reduced by the antidromic conditioning stimulation than before, suggesting that the recurrent inhibition was diminished. Levoxadrol had only negligible effect. Since dexoxadrol has many PCP-like pharmacological properties but levoxadrol does not, we concluded that PCP attenuates hippocampal recurrent inhibition by activating the PCP receptors. It is suggested that this action results in depression of excitatory synaptic transmission from axon collaterals to the inhibitory interneuron with possible involvement of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor.     

Artefactual inhibition of dopamine uptake by psychotropic drugs on striatal synaptosomal preparation

On striatal synaptosomal preparations, using a double labelling test, numerous antidepressant drugs demonstrated an inhibitory effect on [³H]DA uptake at the same high concentrations producing a [¹⁴C]DA release. This releasing effect was also shared by non-antidepressant agents and was observed on synaptosomes preloaded with [³H]5HT. The imipramine-induced release of DA was not modified by increasing concentration of K⁺, by decreasing concentration of Na⁺, by deleting Ca²⁺ from the incubation medium, or by blocking the catecholamine uptake systems with nomifensine or cocaine. The imipramine effect was reversible and was possibly initiated by a transient physico-chemical modification of the synaptosomal membrane. It was concluded that the DA uptake carrier is probably not involved in the effect of these drugs.     

Role of basolateral amygdala dopamine in modulating prepulse inhibition and latent inhibition in the rat

Rationale The dopamine (DA) projection to the basolateral amygdala (BLA) modulates nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) DA transmission. Given the involvement of the BLA, and of NAc and mPFC DA, in select forms of information processing, we sought to determine the role of BLA DA in modulating prepulse inhibition (PPI) and latent inhibition (LI).Objective The effects of BLA D₁ (SCH 23390) and D₂/D₃ (raclopride) receptor blockade on PPI and LI were examined.Methods Separate groups of male Long–Evans rats received bilateral intra-BLA infusions of SCH 23390 (3.2 or 6.4 g/0.5 l per side), raclopride (2.5 or 5.0 g/0.5 l per side) or saline prior to testing. In two experiments, the effects of BLA DA receptor antagonism on PPI of the acoustic startle response (ASR) and LI of conditioned taste aversion were determined. A control group received bilateral intra-striatal infusions of SCH 23390 or raclopride prior to PPI testing.Results Intra-BLA SCH 23390 or raclopride had no effect on the ASR. Intra-BLA SCH 23390 enhanced and raclopride disrupted PPI, both in a dose-related manner. Intra-striatal SCH 23390 or raclopride had no effect on PPI or ASR magnitude. Finally, BLA DA receptor blockade had no effect on LI.Conclusions These results indicate that PPI is modulated by BLA DA and suggest that this modulation occurs independently of changes in NAc and/or mPFC DA transmission. They also suggest that BLA DA is not involved in modulating LI and add to evidence indicating that PPI and LI are mediated by different neural substrates.     

Chirospecific and subtype selective dopamine receptor binding of heterocyclic methoxynaphthamide analogs

Employing the D(3) and D(4) selective methoxynaphthalines nafadotride and FAUC 182, respectively, as lead compounds, the pyrazolo[1,5-a]pyridine-3-carboxamides of type 1a and 2a as well as their 2-substituted regioisomers 1b and 2b were synthesized when following an ex-chiral pool approach. Dopamine receptor binding studies involving the target compounds (1a,b, 2a,b) and the respective optical antipodes ent-1a,b and ent-2a,b revealed the heterocyclic carboxamide 2a as a strong and selective D(4) ligand (K(i) = 8.6 nM). According to a mitogenesis assay, 2a shows D(4) partial agonist effects (29%, EC(50) = 6.7 nM) and, thus, might be of interest for the treatment of sexual dysfunction.     

Nitric oxide scavenger carboxy-PTIO potentiates the inhibition of dopamine uptake by nitric oxide donors

2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) has been increasingly used as nitric oxide (NO) scavenger. Carboxy-PTIO reacts with NO to form nitric dioxide and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (carboxy-PTI). In rat C6 glioma cells expressing human dopamine transporter, carboxy-PTIO paradoxically potentiated the inhibition of [(3)H]dopamine uptake by two NO donors, diethylamine/NO and (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)-amino]/NO. Further examinations revealed that carboxy-PTI concentration-dependently reduced dopamine uptake, indicating that the formation of carboxy-PTI may account for the failure of carboxy-PTIO to abolish NO elicited effects. These results suggest that caution should be taken in interpreting data obtained using carboxy-PTIO and probably other NO scavengers.     

Halothane anesthesia enhances the effect of dopamine uptake inhibition on interstitial levels of striatal dopamine

The anesthetic, isoflurane, has been shown to potentiate the ability of the dopamine (DA)-uptake inhibitor, nomifensine, to increase the brain interstitial dopamine level ([DA]_e). Since the effect of the more commenly used anesthetic, halothane, on this system is unknown, we determined [DA]_e by microdialysis in the striatum of rats, conscious or anesthetized with halothane, in the presence of the more selective DA uptake inhibitor, vanoxerine (GBR 12909), or the DA releaser, d-amphetamine. Basal [DA]_e was not changed by halothane. However, in halothane-anesthetized rats, the vanoxerine (3 mg/kg i.v.)-induced DA response increased severalfold compared to the response in conscious rats. The initial peak response to d-amphetamine (1 mg/kg i.v.) did not change, but the late response (1–3 h after injection) was augmented in anesthetized rats. Halothane is believed to increase firing of DA neurons in the substantia nigra and, hence, to release striatal DA. We hypothesize that [DA]_e, is maintained at a normal level during the increased firing by equally increased activity of the DA transporter. However, when the DA transporter is blocked by vanoxerine, the increased DA release is unimpaired and [DA]_e rises. Correspondence to: A. Fink-Jensen at the above address     

Comparison of characteristics of dopamine uptake and mazindol binding in mouse striatum

Summary Biochemical and pharmacological studies suggest that the binding of [³H]mazindol is functionally related to the dopamine uptake carrier complex in rodent striatum. In order to study further the relationship between the substrate recognition site for dopamine uptake and the high-affinity binding site for mazindol the uptake of [³H]dopamine and the binding of [³H]mazindol was studied in BALB/cBy mouse striatum in various buffers (Tris, HEPES, bicarbonate-phosphate). Kinetic analysis showed that theK _d, of the binding of [³H]mazindol and theK _m of the uptake of [³Mdopamine was changed by different sodium concentrations and/or by the presence of Tris, while theB _max, and theV _max remained essentially the same. However, the shape of the Na⁺ dependency curves was not the same for mazindol binding and dopamine uptake in the various buffers. The inhibitory effect of other cations such as K⁺ and Tris was also different on binding and uptake under similar experimental circumstances. Dopamine did not slow down the dissociation of mazindol from its site and this effect was not sodium-sensitive. These complexities can be accomodated by a model that involves overlapping sites for mazindol and dopamine on the dopamine uptake carrier complex, and translocation -reorientation steps.Abbreviations K _d dissociation rate constant - HEPES N-2hydroxyethyl-piperazine-N-2-ethanesulfonic acid - Km half-saturating concentration for uptake - B _max maximal binding capacity - V _max maximal initial uptake rate - k _–1 dissociation rate constant - IC₅₀ concentration of inhibitor causing 50% inhibition - DA 3,4dihydroxyphenylethylamine (dopamine) On leave from the Institute of Experimental Medicine, H-1450, Budapest, P.O.B. 67, Hungary. Send offprint request to I. Zimányi at her present address