Interaction of two sulfhydryl reagents with a cation recognition site on the neuronal dopamine carrier evidences small differences between [3H]GBR 12783 and [3H]cocaine binding sites

We have compared the effect of treating rat striatal cell membranes with ionic hydrophilic sulfhydryl reagents on the specific bindings of [³H]cocaine and of [³H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-[1-³H]propenyl)-piperazine) to the neuronal transporter of dopamine. Treatment with 1 mmol/1 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in similar time-and concentration-dependent reductions of the specific binding of both radioligands. None of the uptake blockers tested afforded any protection against 1 mmol/1 DTNB. Addition of (sub)millimolar concentrations of CaCl₂ or MgCl₂, or 250 mmol/1 KCl to a treatment medium containing 10 mmol/l Na + significantly increased the DTNB-induced reduction of the specific binding of both radioligands. Cations were likely to be responsible for this effect since ions in combination with DTNB induced similar reductions in binding when either 1 mmol/l CaCl₂ or 50–250 mmol/l NaCl were added. Effects of cations on the DTNB-induced inhibition of binding were generally more marked on [³H]GBR 12783 than on [³H]cocaine binding. When added to a medium containing 10 mmol/1 Na⁺ 1 mmol/1 DTNB induced a reduction in the B_max of the specific binding of both radioligands. Addition of 1 mmol/l Ca²⁺ maintained or increased this B_max reduction and elicited a decrease in affinity which was significant for [³H]GBR 12783 binding.Treatment of membranes with the sodium salt of p-hydroxymercurybenzenesulfonate (pHMBS) induced time-and concentration-dependent decreases in [³H]GBR 12783 binding which were significantly greater than decreases in [³H]cocaine binding. However, 50mol/lpHMBS produced a similar decrease in the B_max of the specific binding of both radioligands. The pHMBS-induced reduction of [³H]GBR 12783 binding was not reversed by drugs whose action is purely that of uptake inhibition or by substrates of the dopamine carrier. Some of these drugs (100 mol/l dopamine, 1 mol/l mazindol or 100 mol/l cocaine) protected the specific binding of [³H]cocaine against the effects of pHMBS, whereas 1 mmol/1 p-tyramine, 10 mol/l nomifensine and 10 nmol/l GBR 12783 were ineffective. Addition of 120 mmol/l Na⁺, 1 mmol/l Ca²⁺ or 10 mmol/l Mg²⁺ to a treatment medium containing 10 mmol/l Na⁺ significantly reduced the effects of pHMBS on the specific binding of both radioligands. When striatal cell membranes were treated in a medium containing 130 mmol/1 Na⁺, there was a general decrease in the effects of ions on the reductions of specific binding produced by DTNB or pHMBS. Cation concentrations which interfered with the actions of DTNB and pHMBS were approximately those which blocked the specific binding of [³H]GBR 12783 when they were present during association of the radioligand (K⁺, Ca ²⁺, Mg²⁺), or, in the case of Na⁺, which are effective in reducing this blockade (Bonnet et al. 1988).The present data are consistent with the existence of mutually exclusive binding sites for [³H]GBR and [³H]cocaine on the neuronal dopamine transporter. The hypothesis of a cation recognition site which could gate admission of uptake inhibitors or carrier substrates to their binding domain on the transporter is discussed.     

Characterization of binding of [3H]GBR 12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine) to membranes and to solubilized membrane extracts from terminal field regions of mesolimbic, mesocortical and nigrostriatal dopamine pathways

The binding characteristics of [3H]GBR 12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine), a selective dopmaine uptake inhibitor, were examined in intact membrane preparations and solubilized extracts of terminal field regions of dopamine pathways in the brain of the rats. There were many similarities in the properties of binding sites for [3H]GBR 12935 in the striatum, nucleus accumbens and olfactory tubercle. The binding of [3H]GBR 12935 was saturable and the affinity constants were not significantly different between regions of the brain. The binding of [3H]GBR 12935 was inhibited by amfonelic acid, GBR 12909, mazindol, methylphenidate and cocaine, with comparable affinities in each region of the brain and with the same order of potency in both preparations. Furthermore, the rank order of potencies for inhibiting the binding of [3H]GBR 12935 was the same as for inhibiting the uptake of [3H]dopamine in these regions of the brain. There did appear to be some degree of heterogeneity of binding sites for [3H]GBR 12935 in each of these regions of the brain, as both amfonelic acid and mazindol were best fitted by two-site models. Whether this apparent heterogeneity was due to the existence of two distinct binding sites or to two components of a single site is unclear. It did not, however, appear to be due to binding to uptake sites for norepinephrine or serotonin, as neither nisoxetine nor fluoxetine, selective inhibitors of the uptake of norepinephrine and serotonin, respectively, inhibited the binding of [3H]GBR 12935, at concentrations which inhibit the uptake of norepinephrine or serotonin.     

Evidence for mutually exclusive binding of cocaine, BTCP, GBR 12935, and dopamine to the dopamine transporter.

The present study addressed the possibility that there are distinct but allosterically interacting populations of binding sites for dopamine/cocaine and BTCP/GBR (N-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine/1-(2-diphenylmethox y) - ethyl]-4-(3-phenylpropyl)piperazine) (selective dopamine uptake blockers) on the dopamine transporter in the rat striatum. Dopamine uptake sites were labeled in vitro with the cocaine analog [3H]CFT (2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane), and the inhibition of binding by CFT or cocaine was measured. A graphic method was adopted for studying shifts in inhibitory potency resulting from the addition of a second compound. Under the conditions used, the co-presence of dopamine, GBR 12935, or BTCP decreased the inhibitory potency of CFT or cocaine to the extent predicted by a model in which all compounds bind to the same site or the binding of all compounds is mutually exclusive. No evidence for negative allosteric interactions between CFT and BTCP was found in experiments comparing inhibition of [3H]CFT binding by BTCP at a low and high concentration of [3H]CFT.     

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     

Spermine interacts with cocaine binding sites on dopamine transporters

These studies were designed to assess the potential interaction of the polyamine spermine with cocaine binding to dopamine and serotonin transporters. The results of the experiments presented here indicate that spermine inhibits binding of the cocaine congener [³H] CFT to striatal synaptosomal membranes. Further, although [³H] CFT is known to interact with both dopamine and serotonin transporters, our results indicate that the observed inhibition of [³H] CFT binding is likely to reflect a specific inhibition of binding to dopamine transporters. Spermine significantly inhibited the binding of both [³H] CFT and [³H] mazindol to dopamine transporters, while it had no apparent effects on the binding of the potent serotonin uptake inhibitor [³H] paroxetine. Finally, saturation experiments show that the inhibition of ligand binding to the cocaine binding site on dopamine transporters appears not to be due to a modification of ligand affinity for the transporter, but to a decrease in the apparent density of ligand binding sites. The results of these experiments indicate that endogenously produced polyamines can alter cocaine binding to the dopamine transporter. The results are discussed in terms of possible impact on novel approaches for pharmacologically manipulating cocaine reinforcement and craving in clinical treatments for cocaine addiction, as well as for emergency treatment of cocaine overdose.     

Interaction of [3H]GBR 12935 and GBR 12909 with the dopamine uptake complex in nucleus accumbens

Although some behavioral effects of cocaine are hypothesized to be due to blockade of dopamine uptake in nucleus accumbens, it has been reported that in nucleus accumbens there are no specific cocaine binding sites and that cocaine is a weak inhibitor of dopamine uptake. [3H]GBR 12935 and an unlabelled analog, GBR 12909, are ligands that bind with great affinity and specificity to a site on dopamine uptake complex in striatum. We therefore investigated the interaction of these GBR compounds with the dopamine uptake complex in nucleus accumbens. We found specific high affinity [3H]GBR 12935 binding and a significant correlation between displacement of [3H]GBR 12935 binding by a series of compounds in striatum and nucleus accumbens. GBR 12909 inhibited dopamine uptake with equal potency in nucleus accumbens and striatum. Thus, there appear to be some aspects of the dopamine uptake complex in nucleus accumbens and striatum that are similar.     

Sodium dependent [3H]cocaine binding associated with dopamine uptake sites in the rat striatum and human putamen decrease after dopaminergic denervation and in Parkinsons disease

Summary The binding of radiolabelled cocaine, an inhibitor of dopamine uptake, to the post-mortem human putamen was studied and compared to that in the rat striatum. Saturation analysis of [³H]cocaine binding to the human putamen revealed the presence of a high affinity component of binding with a K _d of 0.21 mol/l and a B _max of 1.47 pmol/mg protein. In addition a low affinity component (K _d=26.4 mol/l) was demonstrated, having a B _max of 42.2 pmol/mg protein. Also in the rat striatum [³H]cocaine binding was both of high affinity (K _d=0.36 mol/l, B _max=5.56 pmol/mg protein) and low affinity (K _d=25.9 mol/l, B _max=35.6 pmol/mg protein). A pharmacological characterisation of high affinity [³H]cocaine binding to rat striatal membranes clearly indicates an association with the neuronal dopamine transporter. The IC₅₀ values of 8 selected drugs for inhibition of [³H]cocaine binding in the rat striatum were highly significantly correlated with their potency to inhibit [³H]dopamine uptake into slices of the rat striatum. [³H]Cocaine binding was stereospecifically inhibited by (+)nomifensine and (+)diclofensine which were 50–80-fold more active than their respective (-)isomers. Drugs with dopamine releasing activity were more potent at inhibiting [³H]dopamine uptake than at competing for the high affinity site of [³H]cocaine binding. A highly significant correlation was found between IC₅₀ values for [³H]cocaine binding in the rat striatum and the human putamen. Further evidence in support of an association of [³H]cocaine binding in the rat striatum with the dopamine transporter was obtained from lesion studies. Thus, intranigral 6-hydroxydopamine administration produced a marked (67%) decrease in striatal [³H]cocaine binding. Also in the human putamen high affinity [³H]cocaine binding sites appear localized on dopaminergic nerve terminals as evidenced by a prominent decrease in binding in the putamen obtained from subjects with Parkinsons disease. It is concluded that [³H]cocaine may be a useful ligand to examine the dopamine transporter in the rat striatum and the human putamen. Therefore it offers a new and valuable approach in the study of drug effects and neuropsychiatric diseases.Preliminary results on some parts of this study have appeared previously in abstract form (Langer et al. 1984a) or as a rapid communication (Pimoule et al. 1983)     

3H]GBR-12935 binding sites in human striatal membranes: binding characteristics and changes in parkinsonians and schizophrenics

The binding of the diphenyl-substituted piperazine derivative, [3H]GBR-12935, a selective dopamine uptake inhibitor, to the post-mortem human putamen was studied. Inhibition curves by dopamine uptake inhibitors suggested the existence of two populations of [3H]GBR-12935 binding sites: one is potently inhibited by mazindol and/or nomifensine, and the second binding site is benztropine- and/or GBR 12909-sensitive. In the human putamen, [3H]GBR-12935 labeled both these two distinct binding sites. The [3H]GBR-12935 binding displaced by mazindol was enriched in the mouse and rat striatum, but not in the cultured mouse neuroblastoma cell N1E-115. The mazindol-sensitive [3H]GBR-12935 binding site increased in the presence of sodium and markedly decreased in the putamen from parkinsonians (45% of controls). On the other hand, the [3H]GBR-12935 binding displaced by benztropine showed no sodium-dependent increase and was not decreased in the putamen from parkinsonians. In the putamen from schizophrenics, the [3H]GBR-12935 binding did not significantly change in the density, while that displaced by mazindol tended to increase. It is concluded that in the human putamen, [3H]GBR-12935 binds to two distinct sites. One site is partially sodium-dependent and appears to be associated with a high-affinity dopamine uptake system on dopaminergic nerve terminals. The second binding site shows no sodium-dependency and may be associated with a nondopaminergic and/or extraneuronal DA uptake system.     

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.     

N‐n‐alkylnicotinium and N‐n‐alkylpyridinium analogs inhibit the dopamine transporter: Selectivity as nicotinic receptor antagonists

N‐n‐Alkylnicotinium and N‐n‐alkylpyridinium analogs act as antagonists at nicotinic acetylcholine receptors (nAChRs) mediating nicotine‐evoked [³H]dopamine (DA) overflow from superfused rat striatal slices in the presence of a DA transporter (DAT) inhibitor. However, the potential interaction of these nAChR antagonists with DAT has not been evaluated. In the present study, analog inhibition of [³H]DA uptake into striatal synaptosomes and inhibition of [³H]GBR 12935 binding to striatal membranes was determined. N‐n‐Alkylnicotinium analogs with n‐alkyl chains of C_6–12 and N‐n‐alkylpyridinium analogs with n‐alkyl chains of C_7–20 inhibited [³H]DA uptake with a wide affinity range. With the exception of the C₂₀ N‐n‐alkylpyridinium, a linear relationship between chain length and inhibition of [³H]DA uptake was found in both analog series. Similarly, these analogs inhibited [³H]GBR 12935 binding (K_i=5.7–250 μM), and a linear relationship with chain length was observed, with the exception of the C₈ N‐n‐alkylnicotinium analog. Kinetic analyses of inhibition of [³H]DA uptake and [³H]GBR 12935 binding using representative C₁₂ analogs from each series revealed decreases in maximal [³H]DA transport velocity and maximal [³H]GBR 12935 binding without alterations in affinity, indicating noncompetitive interactions with DAT. In comparison, classical nAChR antagonists (mecamylamine, dihydro‐β‐erythroidine and methyllycaconitine) did not inhibit [³H]DA uptake or [³H]GBR 12935 binding. Moreover, inhibition of DAT function occurred at analog concentrations 10–120‐fold higher than those inhibiting nAChR function. Taken together with the inability of these analogs to inhibit field‐stimulation‐evoked [³H]DA overflow, the results indicate that these analogs act selectively as antagonists at nAChRs mediating nicotine‐evoked [³H]DA overflow. Drug Dev. Res. 60:270–284, 2003. © 2003 Wiley‐Liss, Inc.     

Pharmacological characterisation of the GlyT-1 glycine transporter using two novel radioligands

Inhibitors of the glycine transporter GlyT-1 are being developed as potential treatments for schizophrenia. Here we report on the use of two novel radioligands, [³H]-SB-733993 and [³H]-GSK931145, for the characterisation of GlyT-1 in both cells and native tissue. Binding was evaluated in membranes either from HEK293 cells expressing recombinant human GlyT-1 (hGlyT-1) or from rat cerebral cortex. Specific binding of both [³H]-SB-733993 and [³H]-GSK931145 to hGlyT-1 HEK293 cell membranes and rat cerebral cortex membranes was saturable and comprised >90% of total binding. K_d and B_max values for the two radioligands were fairly similar, with K_d values of 1-2 nM and B_max values of around 7000 fmol/mg protein in hGlyT-1 membranes and 3000 fmol/mg protein in rat cortex membranes. Association of [³H]-SB-733993 was faster, with binding reaching equilibrium within 30 min compared with 90 min for [³H]-GSK931145. Dissociation was also much slower for [³H]-GSK931145 than for [³H]-SB-733993, with 50% of specific binding being dissociated by approximately 40 min and 5 min, respectively. Autoradiography studies with [³H]-GSK931145 showed widespread distribution of binding in rat brain, with generally higher binding in caudal compared with rostral areas. Initial studies in human frontal cortex membranes showed clear specific binding of [³H]-GSK931145, though with much lower density (B_max 570 fmol/mg protein) and slightly lower affinity (K_d 4.5 nM) compared with rat cortex. A human brain autoradiography study showed higher specific binding in cerebellum compared with frontal cortex. All GlyT-1 inhibitors tested, as well as glycine itself, competed fully for the binding of both [³H]-SB-733993 and [³H]-GSK931145 in both hGlyT-1 and rat cortex membranes. Studies on the effect of varying NaCl concentration showed that [³H]-SB-733993 binding was reduced by >90% in the absence of added Na⁺ ions, whilst [³H]-GSK931145 binding was unaffected. Glycine produced concentration-dependent decreases in binding affinity of both radioligands without major changes in B_max values, suggesting that both [³H]-SB-733993 and [³H]-GSK931145 bind to sites on GlyT-1 that are orthosteric to the site at which glycine itself binds. Overall, these results show that both [³H]-SB-733993 and [³H]-GSK931145 are useful radioligands for studies on GlyT-1 in both cell lines and native tissues, with [³H]-GSK931145 being the radioligand of choice for further studies on GlyT-1 expression and pharmacology.     

3H]GBR 12935 binding to dopamine uptake sites: subcellular localization and reduction in Parkinson's disease and progressive supranuclear palsy

[3H]GBR 12935 bound with high affinity to dopamine uptake sites in rat striatum where a close parallelism was observed between the subcellular localization profiles for [3H]dopamine uptake and [3H]GBR 12935 specific binding. Using the same ligand, we characterized the dopamine uptake sites in human striatum: the mean KD value was 3.2 nM and the specific binding was inhibited by several dopamine uptake blockers but with slightly lower affinities than those observed in the rat. The subcellular localization profile revealed a synaptosomal enrichment of the specific binding in human striatum. [3H]GBR 12935 binding was decreased in the putamen and caudate nucleus of subjects with Parkinson's disease (33 and 46% of control values, respectively) and progressive supranuclear palsy (38 and 57% of control values, respectively). It is very unlikely that the remaining binding sites in both diseases correspond to piperazine acceptor sites that are not involved in dopamine uptake. However, we cannot exclude the possibility that some of these remaining dopamine transporter sites are not functional, since the reduction in [3H]GBR 12935 specific binding was less marked than the decrease in the dopamine content of the same areas.     

Binding of [<Superscript>3</Superscript>H]mazindol to cardiac norepinephrine transporters: kinetic and equilibrium studies

The norepinephrine transporter (NET) is the carrier that drives the neuronal norepinephrine uptake mechanism (uptake₁) in mammalian hearts. The radioligand [³H]mazindol binds with high affinity to NET. In this study, the kinetics of [³H]mazindol binding to NET were measured using a rat heart membrane preparation. Results from these studies were used to set up saturation binding assays designed to measure cardiac NET densities (B_max) and competitive inhibition assays designed to measure inhibitor binding affinities (K_I) for NET. Saturation binding assays measured NET densities in rat, rabbit, and canine hearts. Assay reproducibility was assessed and the effect of NaCl concentration on [³H]mazindol binding to NET was studied using membranes from rat and canine hearts. Specificity of [³H]mazindol binding to NET was determined in experiments in which the neurotoxin 6-hydroxydopamine (6-OHDA) was used to selectively destroy cardiac sympathetic nerve terminals in rats. Competitive inhibition studies measured K_I values for several NET inhibitors and substrates. In kinetic studies using rat heart membranes, [³H]mazindol exhibited a dissociation rate constant k_off=0.0123±0.0007 min^–1 and an association rate constant k_on=0.0249±0.0019 nM^–1min^–1. In saturation binding assays, [³H]mazindol binding was monophasic and saturable in all cases. Increasing the concentration of NaCl in the assay buffer increased binding affinity significantly, while only modestly increasing B_max. Injections of 6-OHDA in rats decreased measured cardiac NET B_max values in a dose-dependent manner, verifying that [³H]mazindol binds specifically to NET from sympathetic nerve terminals. Competitive inhibition studies provided NET inhibitor and substrate K_I values consistent with previously reported values. These studies demonstrate the high selectivity of [³H]mazindol binding for the norepinephrine transporter in membrane preparations from mammalian hearts.     

Stereoselective behavioral effects of Lu 19-005 in monkeys: relation to binding at cocaine recognition sites

The effects of the monoamine uptake inhibitor Lu 19-005 ((±)-trans-3-(3,4-dichlorophenyl)-N-methyl-1-indanamine) and its (+) and (–) enantiomers, Lu 20-042 and Lu 20-043, were compared with those of cocaine and the selective dopamine uptake inhibitor GBR 12909 (1-{2-[bis-(4-fluorophenyl)methoxyl]ethyl}-4-(3-phenylpropyl)piperazin e) in behavioral and radioligand binding experiments. Behavioral experiments were conducted in groups of squirrel monkeys trained under fixed-interval schedules of reinforcement in which responding was maintained either by presentation of food or by termination of a visual stimulus associated with mild electric shock. Radioligand binding studies were conducted using [³H]CFT and [³H]GBR 12935 to label elements of the dopamine uptake system in caudate-putamen membranes of cynomolgus monkeys. All drugs produced dose-related increases in response rate under the fixed-interval schedules. Lu 19-005, Lu 20-042, and Lu 20-043 had relatively slow onsets (approximately 2 h) and relatively long durations of action, with effects persisting for two or more days following administration. Stereoselectivity was evident in the behavioral effects of the enantiomers of Lu 19-005, with Lu 20-042 being approximately 14 times more potent than Lu 20-043. In radioligand binding experiments, Lu 19-005 and its enantiomers were potent inhibitors of specifically bound [³H]CFT and [³H]GBR 12935. As in behavioral experiments, Lu 20-042 was more potent than Lu 20-043. The degree of stereoselectivity, however, varied with the temperature of the assay medium. At 37° C, Lu 20-043 was approximately 36 times more potent than Lu 20-043 in inhibiting the binding of [³H]CFT, whereas at 0–4° C, the difference in potency was only about 2-fold. The increased stereoselectivity at 37° C was due to a reduced potency for Lu 20-043. The results support the view that the behavioral effects of Lu 19-005 and its enantiomers are mediated at cocaine recognition sites associated with the dopamine uptake system. The results also show that incubation temperature can be a relevant factor in determining the relative potencies of drugs at [³H]CFT binding sites in vitro.Animals used in this study were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and of the Guide for Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, Department of Health, Education and Welfare, Publication No. (NIH)85-23, revised 1985. This research was supported by USPHS grants DA03774, DA06303, DA00499, and by Research Scientist Development Award DA00088 to RDS. Facilities and services were provided by the New England Regional Primate Research Center (USPHS Division of Research Resources Grant RR00168).     

Cocaine receptors labeled by [3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane

The potent cocaine analog 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane (CFT, also designated WIN 35,428) was tritiated and evaluated as a molecular probe for cocaine receptors in caudate putamen membranes of cynomolgus monkeys. Kinetic, saturation, and competition experiments indicated that [3H]CFT, like [3H]cocaine, bound to at least two components. Association and dissociation of the radioligand at 0-4 degrees occurred in two phases; the t 1/2 for dissociation of the fast and slow components was 2.5 and 23 min, respectively. Saturation analysis revealed high and low affinity binding components with affinities (Kd) of 4.7 +/- 1.2 and 60 +/- 12 nM (means +/- SE) and densities (Bmax) of 50 +/- 18 and 290 +/- 20 pmol/g of tissue, respectively. [3H]CFT was displaced stereoselectively by the enantiomers of cocaine and by the diastereoisomers of the phenyltropane analog of cocaine. Most congeners displaced [3H]CFT fully, with shallow competition curves (nH, 0.69-0.81). In contrast, several monoamine uptake inhibitors structurally unrelated to cocaine (GBR 12909, Lu 19-005, and mazindol) displaced a maximum of about 90% specifically bound [3H]CFT, with steeper competition curves (nH, 0.89-1.3), suggesting that these drugs bind to a subpopulation of [3H]CFT-labeled sites. The rank order of potency observed in the present study is identical to the rank order of potency at binding sites labeled by [3H]cocaine: Lu 19-005 greater than mazindol greater than CFT greater than GBR 12909 greater than (-)-cocaine greater than bupropion greater than WIN 35,140 greater than (+)-cocaine. Moreover, there is a high positive correlation (r, 0.99, p less than 0.001) between the affinities of drugs at sites labeled by [3H]CFT and [3H]cocaine. The results show that [3H]CFT and [3H]cocaine bind to a similar spectrum of sites in monkey caudate putamen. Because of its higher affinity and slower dissociation rate, [3H]CFT appears to be a superior radioligand probe for these sites.     

Further in vitro and in vivo studies with the putative presynaptic dopamine agonist N,N-dipropyl-7-hydroxy-2-aminotetralin

Summary The in vitro binding of the putative dopamine autoreceptor agonist [³H]DP-7-ATN to rat striatal membrane homogenates was investigated. The maximum number of binding sites B _max was 497.5 ± 50.2 fmol/mg protein and the affinity constant K _D was 8.3 ± 1.5 nM using 10 M (+) butaclamol to define non-specific binding. Lesion of the left medium forebrain bundle by 6-hydroxydopamine resulted in an almost complete loss of dopamine in the striatum but did not affect the binding of [³H]DP-7-ATN. The binding of [³H]DP-7-ATN to the homogenates of the dopaminergic cell bodies in the substantia nigra revealed a B _max of 542.4 ± 40.1 fmol/mg protein and a K _D of 11.1 ± 1.3 nM. The pharmacological profile of the binding was characterized as being to D-2 receptors. No direct in vitro evidence could be found for a selective binding to DA autoreceptors. The dopamine uptake inhibitor GBR 12909 interacted in a noncompetitive manner with the in vitro binding of [³H]DP-7-ATN and the latter compounds uptake into isolated synaptosomes was not through the specific dopamine uptake system but rather through diffusion. GBR 12909 failed to reveal any agonistic or antagonistic activity in the GBL model but was able to antagonize the hypomotility in rats induced by 0.25 mg/kg DP-7-ATN. The inhibitory effect of DP-7-ATN on DA release was also demonstrated using in vivo brain dialysis in conscious rats. Based on the above results, the possibility is discussed that the release regulating DA autoreceptors, which might be coupled to the reuptake complex, and the DA biosynthesis regulating autoreceptors, are different entities. Send offprint requests to A. S. Horn     

Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced in depression.     

Metaphit prevents locomotor activation induced by various psychostimulants and interferes with the dopaminergic system in mice

Metaphit, an isothiocyanate analog of phencyclidine and a proposed phencyclidine receptor acylator, inactivated the carrier involved in the neuronal uptake of dopamine in in vitro experiments with preparations of the striatum in the mouse. In ex vivo experiments 2 and 24 hr after the intravenous administration of metaphit, no changes were observed either in the binding of [3H]cocaine to striatal membranes or in the uptake of [3H]dopamine into synaptosomes or slices. In in vivo experiments 24 hr after pretreatment with metaphit, selective labelling of uptake sites for dopamine in the striatum of the mouse with [3H]GBR 12935 was unaffected. In these in vivo experiments, however, metaphit antagonized the locomotor stimulation induced by blockers of the uptake of dopamine (methylphenidate, mazindol, cocaine, GBR 12909) but not that induced by drugs that affect locomotion by other mechanisms (amphetamine, phencyclidine). Twenty-four hours after treatment with metaphit there was an increase in homovanillic acid in all regions of the brain studied (striatum, olfactory tubercle, cerebral cortex). There was no effect of metaphit on the disappearance rate of 3,4-dihydroxyphenylacetic acid and homovanillic acid from the striatum during the inhibition of monoamine oxidase with pargyline. If the increase in homovanillic acid reflected a greater rate of dopamine catabolism in metaphit-treated mice, it could explain the lack of locomotor stimulation of blockers uptake of the dopamine in these animals, resulting from a rapid breakdown of extracellularly accumulated dopamine.     

High-affinity [3H]GBR 12783 binding to a specific site associated with the neuronal dopamine uptake complex in the central nervous system

We labelled the neuronal dopamine uptake system by using the potent dopamine uptake inhibitor GBR 12783 in its tritiated form (18.3 Ci/mmol). The binding of [3H]GBR 12783 to rat striatal membranes was saturable and specific with a Kd of 1.6 nM and a Bmax of 10.3 pmol X mg protein-1 as determined by Scatchard analysis. [3H]GBR 12783 binding to rat striatal membranes was inhibited by dopamine uptake inhibitors with IC50 highly correlated with their IC50 for inhibiting [3H]dopamine uptake by a rat striatal synaptosomal preparation. The rank order of potency was the following: GBR 12783 greater than amfonelic acid greater than mazindol greater than pyrovalerone greater than nomifensine greater than benztropine greater than amineptine greater than methylphenidate greater than cocaine. Substrates of dopamine uptake competed with [3H]GBR 12783 binding at concentrations higher than those at which they inhibited [3H]dopamine uptake. In rats with a unilateral section of the medial forebrain bundle, the decrease in [3H]GBR 12783 binding to membranes prepared from the ipsilateral striatum was equal to the decrease in [3H]dopamine uptake by a synaptosomal preparation obtained from the same striatum. [3H]GBR 12783 bound in a sodium-dependent manner to membranes prepared from striatum, nucleus accumbens and tuberculum olfactorium. GBR 12783 displayed an approximately 150-fold lower affinity for the cortical norepinephrine uptake system labelled with [3H]desipramine than for the dopamine transport complex labelled with [3H]GBR 12783. [3H]GBR 12783 appears an attractive tool for the selective characterization of the dopamine uptake system in vitro.     

GBR12909 antagonizes the ability of cocaine to elevate extracellular levels of dopamine.

Rats were administered various IP doses of the high-affinity dopamine (DA) reuptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine (GBR12909). The caudate nuclei were removed 60 min after drug administration and stored at -70 degrees C. Striatal membranes were prepared later. The results demonstrated that GBR12909 produced a dose-dependent decrease in the binding of [3H]cocaine or [3H]GBR12935 to the DA transporter (ED50 about 10 mg/kg). Saturation binding studies with [3H]GBR12935 showed that this was due to both an increase in the Kd, due to residual drug, and to a decrease in the Bmax. At a dose of 25 mg/kg IP, GBR12909 produced a 50% decrease in the Bmax, and a 3.4-fold increase in the Kd. In the in vivo microdialysis studies, GBR12909 (25 mg/kg IP) produced a modest, long-lasting and stable elevation of extracellular DA. Administration of cocaine through the microdialysis probe to rats pretreated with either saline or GBR12909 (25 mg/kg IP) produced a dose-dependent increase in extracellular DA in both groups. GBR12909 inhibited cocaine-induced increases in extracellular DA by about 50% at all doses. These data collectively indicate that at a dose sufficient to decrease by 50% the Bmax of [3H]GBR12935 binding sites, GBR12909 antagonizes the ability of cocaine to elevate extracellular DA by 50%. Further studies will be needed to evaluate a possible role for GBR12909 in the medical treatment of cocaine addiction.