Unaltered [3H]GBR-12935 binding after chronic treatment with dopamine active drugs

Rats were injected intraperitoneally with haloperidol 0.5 mg/kg, raclopride 1 mg/kg, bromocriptine 2.5 mg/kg,d-amphetamine 2.5 mg/kg, or cocaine 10 mg/kg twice daily for 21 days. The animals were sacrificed 72 h after last injection. Control rats were injected with saline, following the same schedule. The radioligand [³H]GBR-12935 was used as a presynaptic marker for dopamine neurites. There were no significant differences in [³H]GBR-12935 binding to striatum between drug-treated rats and controls.     

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     

Radiolabeling of dopamine uptake sites in mouse striatum: comparison of binding sites for cocaine,mazindol, and GBR 12935

Summary This study addressed the possibility of a unique binding interaction between cocaine and the dopamine transporter as compared with other blockers of dopamine uptake. Cocaine binding sites in a fresh P₂ fraction of mouse striatum were labeled with [³H]CFT, a phenyltropane analog of cocaine also known as WIN 35,428, and compared with sites labeled with [³H]mazindol or [³H]GBR 12935. Under the conditions used, homogeneous binding was observed that was inhibited monophasically by cocaine, CFT, and mazindol; the same potencies were observed with the three radioligands. Saturation analysis in the presence and in the absence of unlabeled inhibitor (CFT, mazindol, cocaine) indicated a change in the K_d but not the B_max, consonant with a competitive mechanism. Tris-HCl reduced the affinity of each radioligand and unlabeled inhibitor without changing the B_max. N-Ethylmaleimide reduced the binding of all radioligands equally and cocaine offered protection. The dissociation rate of [³H]CFT and [³H]mazindol binding was not affected by the presence of mazindol and CFT, respectively. The B_max of [³H]CFT and [³H]mazindol binding was the same; the relatively higher value for [³H]GBR 12935 binding in analyses involving varying tritiated GBR 12935 only, was due primarily to an underestimation of the specific activity of [³H]GBR 12935. All results are in agreement with a one-site model in which cocaine, CFT, mazindol, and GBR 12935 share a common binding site in mouse striatum.     

3H]cocaine binding and inhibition of [3H]dopamine uptake is similar in both the rat striatum and nucleus accumbens

Cocaine binds with high affinity to the dopamine transporter in both the striatum and the nucleus accumbens. We examined Na(+)-dependent [3H]cocaine binding, mazindol inhibition of [3H]cocaine binding and cocaine inhibition of [3H]dopamine uptake in both rat brain areas. The striatum and nucleus accumbens demonstrated Na(+)-dependent [3H]cocaine binding with similar densities. Mazindol inhibited [3H]cocaine binding with a similar IC50 in both the striatum and nucleus accumbens. Likewise, cocaine inhibited [3H]dopamine uptake in both brain regions with equivalent efficacy. From these data we conclude that the dopamine transporter is similar in both the striatum and nucleus accumbens.     

3H-dopamine uptake and 3H-haloperidol binding in striatum after administration of methyl mercury to rats

Methyl mercury inhibits dopamine (DA) and serotonin (5-HT) uptake by brain synaptosomes and decreases antagonist binding to striatal dopaminergic D₂ receptors in vitro. To assess the effects in vivo, adult rats were given methyl mercury, either as a single dose (10 mg/kg by gavage) or a cumulative total dose of 50 mg/kg in 2 weeks. The repeated dosing decreased body weight and caused neuromuscular dysfunction. In spite of this overt toxicity, neither ³H-DA uptake nor ³H-haloperidol binding changed in striatal synaptosomes. There were no significant alterations in ³H-5-HT uptake by hypothalamic synaptosomes or ³H-flunitrazepam binding in cerebellar synaptosomes. The results suggest that monoaminergic synapses and the benzodiazepine binding sites, associated with cerebellar GABA receptors, remain functionally normal at doses of methyl mercury that are otherwise toxic. The results also emphasize the importance of due care when extrapolating cellular or biochemical data to the level of the whole organism.     

3H]Imipramine binding and [3H]5HT uptake in human blood platelets: changes after one week chlorimipramine treatment

In platelets of normal volunteers taking chlorimipramine (50 mg/day) for one week, the saturable uptake of [3H]5HT was fully inhibited at day 8, but returned to control values at day 15. The Bmax of [3H]imipramine binding was decreased by 65% at day 8 and remained significantly below control values at day 15. If the present findings can be extrapolated to other antidepressants, the reported decreases in [3H]imipramine binding in depression may partly reflect residual treatment effects. It cannot be excluded that, in depression, the platelet [3H]imipramine receptor already is down-regulated maximally which would preclude a further down-regulation due to antidepressant drug therapy.     

Chronic phencyclidine,like amphetamine,produces a decrease in [3H]spiroperidol binding in rat striatum

Rats were treated with d-amphetamine sulfate (5 and 10 mg/kg i.p.) and phencyclidine (PCP) (5 mg/kg i.p.) twice per day. After 21 days, [³H]spiroperidol binding in striatum was reduced by all treatments; receptor number (B_max) and not affinity (K_D) was affected. These results suggest that the psychotic effect of PCP may, like those of amphetamine, involve changes in dopamine receptors.     

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.     

3H]mazindol binding associated with neuronal dopamine and norepinephrine uptake sites

[3H]Mazindol labels neuronal dopamine uptake sites in corpus striatum membranes (KD = 18 nM) and neuronal norepinephrine uptake sites in cerebral cortex and submaxillary/sublingual gland membranes (KD = 4 nM). The potencies of various inhibitors of biogenic amine uptake in reducing [3H]mazindol binding in striatal membranes correlate with their potencies for inhibition of neuronal [3H]dopamine accumulation, whereas their potencies in reducing [3H]mazindol binding to cortical and salivary gland membranes correlate with their potencies for inhibition of neuronal [3H]norepinephrine accumulation. Similar to the dopamine and norepinephrine uptake systems, [3H]mazindol binding in all three tissues is dependent upon sodium (with potassium, lithium, rubidium, and Tris being ineffective substitutes) and chloride (with sulfate and phosphate being ineffective substitutes). In membranes of the cerebral cortex and salivary gland, half-maximal stimulation is observed at 50-80 mM NaCl, whereas in membranes of the corpus striatum half-maximal stimulation occurs at 240 mM NaCl. In striatal membranes NaCl increases the affinity of [3H]mazindol binding with no effect on the maximal number of sites. The enhancement of affinity is due to a selective slowing of the dissociation of the ligand from its binding site. The association of [3H]mazindol binding sites with neuronal dopamine uptake sites in the corpus striatum is further supported by the reduction of [3H]mazindol binding sites in striatal membranes following destruction of dopaminergic neurons by 6-hydroxydopamine. Similarly, the association of [3H]mazindol binding sites with neuronal norepinephrine uptake sites in cerebral cortex is supported by the reduction of [3H]mazindol binding to cortical membranes following destruction of noradrenergic neurons by N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine.     

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     

Characterization of dopamine autoreceptor and [3H]spiperone binding sites in vitro with classical and novel dopamine receptor agonists

The specific D2 receptor agonist, LY 141865, but not the specific D1-receptor agonist, SK&F 38393, potently inhibited electrically evoked [3H]dopamine release from slices of the cat caudate. Similarly, LY 141865, but not SK&F 38393, inhibited [3H]spiperone binding to membranes of the cat caudate. The inhibition by dopamine receptor agonists of electrically evoked [3H]dopamine release was antagonized by the specific D2-receptor antagonist S-sulpiride. The inhibition of the electrically evoked release of [3H]dopamine by apomorphine was not, however, antagonized by the specific D1-receptor antagonist, bulbocapnine. Similarly, S-sulpiride but not bulbocapnine potently inhibited [3H]spiperone binding to membranes of the cat caudate. These results suggest that the dopamine autoreceptor modulating the depolarization-evoked release of [3H]dopamine, and the binding site of [3H]spiperone, are valid in vitro models for D2-dopamine receptors. Contrary to some previous reports, DPI was inactive in both in vitro dopamine receptor models. The IC50 values of a series of dopamine receptor agonists correlated very well in the two in vitro dopamine receptor models. One exception to this correlation was bromocriptine, which was more potent at [3H]spiperone binding sites than at the dopamine autoreceptor. With the exception of bromocriptine, all dopamine receptor agonists had one-hundred fold higher potency at the dopamine autoreceptor than at [3H]spiperone binding sites. [3H]Spiperone binding sites are localized primarily postsynaptic to dopamine terminals. Possible differences between the pharmacological properties of pre- and postsynaptic dopamine receptors should become apparent in the comparison of the two in vitro dopamine receptor models. However, the order of potency of dopamine receptor agonists with both in vitro models, dopamine autoreceptor and [3H]spiperone binding, was the same: N-n-propylnorapomorphine greater than TL-99 = 7-HAT greater than M-7 greater than Apomorphine greater than LY 141865.     

The role of calcium in the regulation of [H]hemicholinium-3 binding sites in rat brain

The role of calcium in the regulation of sodium-dependent high-affinity uptake of choline was assessed in vitro in slices of the rat brain, by measuring the specific binding of [^3H]hemicholinium-3 ([^3H]HCh-3) and the uptake of [^3H]choline. Depolarization with potassium of slices of hippocampus, cortex, or striatum significantly increased the specific binding of [^3H]HCh-3 when compared to control slices. However, the observed potentiation of specific binding of [^3H]HCh-3 was markedly inhibited by the removal of calcium from the incubation medium in cortex or hippocampus, but not in slices of striatum. Alterations in the uptake of [^3H]choline directly paralleled the observed changes in the specific binding of [^3H]HCh-3 in striatum of the rat and were unaffected by the reduction of calcium in the incubation medium. The inorganic calcium channel antagonists, cadmium and cobalt, but not magnesium, zinc, manganese or lanthanum, significantly inhibited the 40 mM potassium chloride-induced stimulation of the binding of [^3H]HCh-3 in the striatum. Finally, the calcium ionophore A23187 significantly increased the binding of [^3H]HCh-3 in slices of striatum, either in the presence or absence of calcium in the bathing medium. This study demonstrates regional differences in the role of extracellular calcium in the regulation of the uptake of choline and suggests the involvement of intracellular release of calcium in the in vitro regulation of the sodium-dependent high-affinity uptake of choline in the striatum     

Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

The interactions of dopaminergic agonists and antagonists with 3H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [3H]dopamine and [3H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed "D3" binding site. Antagonist/[3H]dopamine competition curves are of uniformly steep slope (nH = 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [3H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [3H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [3H]dopamine binding are also correlated with estimates of these agonists' affinities for the high affinity binding component of agonist/[3H]flupentixol competition curves. Both D3 specific [3H] dopamine binding and the high affinity agonist-binding component of dopamine/[3H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor.     

[123I]β-CIT SPECT imaging of dopamine transporter availability after mazindol administration in human cocaine addicts

The in vivo potency of mazindol for binding to striatal dopamine transporters (DAT) was assessed by [¹²³I]β-CIT ([¹²³I]2β-carbomethoxy-3β-(4-iodophenyl)tropane) single photon emission computed tomography (SPECT). Cocaine-dependent subjects (n=12) underwent three SPECT scans; one before, between, and after subchronic (1 week) administration of 2mg/day and 4mg/day mazindol. For each scan, subjects were injected with [¹²³I]β-CIT and imaged 24h later under equilibrium conditions. Results showed a statistically significant main effect of mazindol dose (df=2, F=10.30, P<0.001, repeated measures ANOVA) in reducing the specific to non-displaceable equilibrium partition coefficient, V₃″ (a measure proportional to DAT binding potential). Regression analysis of the logit transformed data enabled estimation of the 50% displacement dose of mazindol (ED₅₀=30mg/ day). These data suggest that low doses of mazindol (i.e., 2–4mg) occupy a small percentage (i.e., <25%) of DAT in human cocaine abusers and that much higher, potentially intolerable doses (i.e., ≥30mg/day) may be required to antagonize significantly cocaine binding in vivo. Received: 14 November 1996/Final version: 17 December 1997     

Comparison of [3H]YM-09151-2 with [3H]spiperone and [3H]raclopride for dopamine d-2 receptor binding to rat striatum

The Kd value of [3H]YM-09151-2, a potent and highly selective dopamine D-2 antagonist, for binding to rat striatum was about 20 pM (half of that for [3H]spiperone and one-fiftieth of that for [3H]raclopride). The Bmax of [3H]YM-09151-2 binding was about 30% higher than that of [3H]raclopride or [3]spiperone. The ratio (bout 3%) of non-specific to specific binding of [3H]YM-09151-2 was smaller than that of [3H]spiperone and [3H]raclopride. The Hill coefficient values of dopamine D-2 antagonists, SCH23390, mianserin and phentolamine for the inhibition of binding of [3H]YM-09151-2 were near 1.0, and their Ki values with [3H]YM-09151-2 were consistent with those for inhibiting [3H]raclopride and [3H]spiperone binding to D-2 receptors. Thus, [3H]YM-09151-2 may be the most suitable ligand for the labelling of dopamine D-2 receptors in the brain.     

Turnover of specific [3H]spiperone and [3H]N,n-propylnorapomorphine binding sites in rat striatum following phenoxybenzamine administration

Inclusion of phenoxybenzamine into incubates containing rat striatal preparations equipotently displaced specific striatal [3H]spiperone and [3H]NPA binding. Pre-incubation of striatal membranes with phenoxybenzamine followed by extensive washing equipotently inhibited the subsequent specific [3H]spiperone or [3H]NPA binding. In both displacement and pre-incubation experiments phenoxybenzamine caused complete inhibition of specific [3H]spiperone binding to rat striatal membranes, but only partially inhibited specific [3H]NPA binding. Following parenteral administration to rats, phenoxybenzamine caused a marked inhibition of ex vivo specific [3H]spiperone binding in striatal tissue preparations from these animals which lasted approximately 24 hr following in vivo drug administration. In contrast, administration of phenoxybenzamine caused only a transient change in ex vivo specific [3H]NPA binding. Phenoxybenzamine causes irreversible inhibition of [3H]spiperone and [3H]NPA binding in vitro. In vivo administration of phenoxybenzamine discriminates between [3H]spiperone and [3H]NPA in ex vivo studies suggesting that these binding sites have different turnover rates.     

Unaltered 5-HT- and desipramine-sensitive [3H]imipramine binding and [3H]5-HT uptake in rat brain after chronic imipramine and norzimeldine treatment

Several reports have shown heterogeneity of [³H]imipramine binding to brain membranes. Recently, a high affinity and 5-HT sensitive [³H]imipramine binding site of protein nature, that was suggested to be identical to the substrate recognition site for 5-HT uptake, was demonstrated. Since most studies on the regulation of the [³H]imipramine binding sites by antidepressants have used desipramine displaceable binding, which is heterogenous in nature and contains binding not related to 5-HT uptake sites, the present report studies the possible effects of chronic (3 weeks) administration of imipramine or norzimeldine (10 mg/kg intraperitoneally twice daily) on 5-HT sensitive [³H]imipramine binding sites. For comparison, desipramine sensitive binding was also studied, as well as the physiological correlate 5-HT uptake. There were no changes in either [³H]imipramine binding or 5-HT uptake after the antidepressant treatment.Supported by the Swedish Medical Research Council Offprint requests to: J. Marcusson at Dept. of Geriatric Medicine     

Central administration of dopamine D3 receptor antisense to rat: effects on locomotion, dopamine release and [3H]spiperone binding

A 15-mer, all-phosphorothioate-modified antisense oligodeoxynucleotide (ASO) targeted against rat dopamine D3 receptor mRNA (4 μM, 5 days) significantly reduced (28%) the amount of binding sites labelled with [³H]spiperone in monolayer cultured Chinese hamster ovary (CHO) cells transfected with the complementary desoxyribonucleic acid (cDNA) for the rat D3 receptor. In contrast, D3-ASO treatment did not reduce the amount of bound [³H]spiperone in CHO cells transfected with D2 receptor cDNA. Intracerebroventricular infusion of D3-ASO (osmotic minipump, 10 μg/μl/h, 7 days) influenced dopamine receptor density in the limbic forebrain such that the upper part of the dopamine/[³H]spiperone displacement curve – tentatively representing the D3 receptor – was altered significantly. Spontaneous locomotor activity of non-habituated rats was increased significantly in D3-ASO-treated animals; in addition, in vivo microdialysis revealed a moderate increase in dopamine release in the nucleus accumbens in these animals. In all experiments, an oligodeoxynucleotide comprising the same nucleotides as the antisense sequence, but in random order, was used as control. It is concluded that the antisense strategy is useful for investigating the functional role of dopamine D3 receptors and that the dopamine D3 receptor is involved in rat locomotor behaviour. Received: 23 June 1997 / Accepted: 19 May 1998