[Assessment of the in vitro and in vivo toxicity of 3,4-dihydroxyphenylacetaldehyde (DOPAL)]. / Recherche d'une toxicité du 3,4-dihydroxyphénylacétaldéhyde (DOPAL) in vitro et in vivo.

This work was carried out in order to evaluate the in vitro and in vivo toxicity of 3,4-dihydroxyphenylacetaldehyde (DOPAL). This aldehyde is formed from dopamine (DA) by monoamine oxidases (MAO) and is mainly oxidised to 3,4-dihydroxyphenylacetic acid by brain aldehyde dehydrogenases (ALDH), or eventually reduced to 3,4-dihydroxyphenylethanol by aldose/aldehyde reductases. In vitro, catecholaminergic SH-SY5Y cells were incubated with DA and disulfiram (DSF), an irreversible inhibitor of ALDH. As evidenced by MTT assays, a 24-h treatment with 10(-4) M DA and/or 10(-6) M DSF followed by a 24-h incubation in a drug-free medium evidenced that the toxicity of each of these drugs was potentiated by the second drug. HPLC measurements demonstrated that this drug association induced an early DOPAL production that could result in a delayed cell toxicity. For in vivo studies, male Sprague-Dawley rats were treated with L-DOPA-benserazide, which increases the production of DOPAL by MAO, and DSF. An acute injection of DSF (100mg/kg i.p.) and L-DOPA/benserazide (100mg/kg+25mg/kg, 24h later) significantly increased the DOPAL striatal level. However, a 30-day treatment with DSF (100mg/kg i.p., once every two days) and L-DOPA/benserazide (100mg/kg+25mg/kg, twice a day) did not affect both indexes used to assess the integrity of the nigro-striatal dopaminergic terminals (i.e. the striatal content in DA and the binding to the vesicular monoamine transporter on striatal membranes). These results do not support the hypothesis of a DOPAL toxicity and argue against the toxicity of L-DOPA therapy.

Gas chromatography/mass spectrometric identification of dopaminergic metabolites in striata of rats treated with L-DOPA.

The ex vivo identification of dopaminergic metabolites present in rat striata after (L-DOPA + benserazide) treatment is reported. The different metabolites have been identified as the trimethylsilyl derivatives in the striatal extracts by gas chromatography/mass spectrometry.

Involvement of the NH2 terminal domain of catecholamine transporters in the Na(2+) and Cl(-)-dependence of a [3H]-dopamine uptake.

The ionic dependence of the [3H]-dopamine uptake was studied in transfected cells expressing the human neuronal transporter for dopamine (hDAT) or noradrenaline (hNET), and chimeric transporters resulting from the symmetrical exchange of the region from the NH2 terminal through the first two transmembrane domains (cassette I). Chimera A is formed by hDAT comprising cassette I from hNET, whereas chimera B corresponds to the reverse construct. The appearance or the intensity of a Cl(-)-independent component of transport was linked to the presence of the COOH terminal part of hNET in both monoclonal and polyclonal Ltk(-) cells (Cl(-) substituted by isethionate and NO3(-), respectively), and in transiently transfected COS-7 cells. Cassette I was also involved in the Cl(-)-dependence because the transport activity of polyclonal Ltk(-) cells expressing A was partly Cl(-)-independent and because Ltk(-) cells expressing transporters containing cassette I of hDAT displayed higher K(mCl)- values than cells expressing the reverse constructs. In monoclonal Ltk(-) cell lines, K(mNa)+ values and biphasic vs monophasic dependence upon Na(+) concentrations differentiate transporters containing cassette I of hNET from those containing cassette I of hDAT. In COS-7 cells, the exchange of cassette I produced a significant change in Hill number values. In Na(+)-dependence studies, exchange of the COOH terminal part significantly modified Hill number values in both Ltk(-) and COS-7 cells. Hill number values close to two were found for hNET and hDAT when sucrose was used as substitute for NaCl. The NH2 terminal part of the transporters bears some of the differences in the Na(+) and Cl(-)-dependence of the uptake that are observed between hDAT and hNET. Present results also support a role of the COOH terminal part in the ionic dependence.

Differential sensitivity to NaCl for inhibitors and substrates that recognize mutually exclusive binding sites on the neuronal transporter of dopamine in rat striatal membranes.

Addition of NaCl (90--290 mM) to a 10 mM Na(+) medium did not significantly modify B(max) and K(d) values for [3H]mazindol binding to the dopamine neuronal transporter (DAT) studied on rat striatal membranes at 20 degrees C. Addition of NaCl differentially affected the ability of other uptake inhibitors and substrates to block the [3H]mazindol binding. Ratios of 50% inhibiting concentrations calculated for 290 and 90 mM NaCl allowed to distinguish three groups of agents: substrates which were more potent in the presence of 290 mM NaCl (group 1; ratio < 1) and two groups of uptake inhibitors displaying ratio values either ranging around two (group 2: WIN 35,428, cocaine, methylphenidate, pyrovalerone) or close to unity (group 3: BTCP, mazindol, benztropine, nomifensine). However, agents from these three groups recognize mutually exclusive binding sites since in interaction studies the presence of WIN 35,428 (group 2) or mazindol (group 3) increased the 50% inhibiting concentrations of D-amphetamine (group 1) and WIN 35,428 on the [3H]mazindol binding to theoretical values expected for a competition of all of these compounds for the same binding domain on the DAT.

Structural domains of chimeric dopamine-noradrenaline human transporters involved in the Na(+)- and Cl(-)-dependence of dopamine transport.

Catecholamine transporters constitute the biological targets for several important drugs, including antidepressants, cocaine, and related compounds. Some information exists about discrete domains of these transporters that are involved in substrate translocation and uptake blockade, but delineation of domains mediating the ionic dependence of the transport remains to be defined. In the present study, human neuronal transporters for dopamine and noradrenaline (hDAT and hNET) and a series of six functional chimeras were transiently expressed in LLC-PK1 cells. Substitution of Cl(-) by isethionate reveals that cassette IV (i.e., the region of the transporter encompassing transmembrane domain 9 through the COOH terminal) plays an important role in the Cl(-)- dependence of the uptake. Substitutions of Na(+) and NaCl by Tris(+) and sucrose, respectively, demonstrate that three different segments scattered across the transporter are involved in the Na(+)- dependence of the transport activity: cassette I (i.e., the region from the amino terminus through the first two transmembrane domains), cassette IV, and junction between transmembrane domains 3 to 5 and 6 to 8. Results of the present work also suggest that the use of Tris(+) as a substitute for Na(+) results in a biased estimate of the Hill number value for hDAT. This study provides useful clues for identifying specific residues involved in the uptake function of the catecholamine transporters.

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.

Recovery of dopamine neuronal transporter but lack of change of its mRNA in substantia nigra after inactivation by a new irreversible inhibitor characterized in vitro and ex vivo in the rat.

1. In vitro, the ability of DEEP-NCS {1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-isothiocyanatophenyl)ethyl]- piperazine} to inhibit [3H]-dopamine uptake by rat striatal synaptosomes was concentration-dependent and inversely related to the protein concentration. This inhibition was irreversible and resulted from changes in Vmax and KM. DEEP-NCS was less potent on noradrenaline, serotonin and choline transport. 2. One day after intrastriatal injections of DEEP-NCS (100 and 1000 pmol) in 20% dimethylsulphoxide, moderate decreases in the ex vivo dopamine uptake were observed in synaptosomes obtained from striatum injected with DEEP-NCS or solvent, and the contralateral uninjected striatum. 3. In similar conditions, 300 pmol DEEP-NCS in 45% 2 hydroxypropyl-gamma-cyclodextrin - 0.5% dimethylsulphoxide solution sub-totally reduced ex vivo dopamine uptake and mazindol binding, and moderately decreased choline and serotonin transport. These reductions were specific to DEEP-NCS-injected striata. A clomipramine pretreatment (16 mg kg-1 i.p. 1 h before) was performed in following experiments, since it reduced the DEEP-NCS-elicited decrease in serotonin uptake without affecting other indices. 4. One day after intrastriatal injection, DEEP-NCS elicited similar dose-dependent decreases in ex vivo dopamine uptake and mazindol binding (ID50=6.9-8 ng striatum-1). Changes in KM and Vmax for ex vivo dopamine transport produced by DEEP-NCS disappeared according to similar time-courses. 5. The t(1/2) for transporter recovery was 6. 1 days. This value should correspond to its actual turnover rate in vivo, since no change in transporter mRNA level was observed in substantia nigra ipsilateral to 300 pmol DEEP-NCS-injected striatum. 6. The results indicate that DEEP-NCS behaves as a potent, quite selective, irreversible inhibitor of the DAT, in vitro and in vivo. Its use in vivo suggests that the physiological half-life of the rat striatal DAT is close to 6 days.

Acute interactions between L-DOPA and the neurotoxic effects of 1-methyl-4-phenylpyridinium or 6-hydroxydopamine in mice.

We have compared the effects of an i.p. pretreatment with L-DOPA (200 mg/kg) associated with benserazide (25 mg/kg) on neurotoxic effects of either 6-hydroxydopamine (6-OHDA) (50 microg, 10 microl per mouse) or 1-methyl-4-phenylpyridinium (MPP+) (17.5 microg, 10 microl per mouse). The striatal dopamine (DA) content, the vesicular monoamine transporter (VMAT2) density, as well as the hypothalamic norepinephrine (NE) content were measured 8 days after treatments. The L-DOPA-benserazide pretreatment worsened by 65% the 6-OHDA-induced depletion in striatal DA. On the contrary, it reduced by 42% the MPP+-induced depletion in striatal DA and by 54% the MPP+-induced decrease in VMAT2 density. It was noticed that the L-DOPA-benserazide pretreatment did not modify the marked decrease in hypothalamic NE content induced by 6-OHDA.

Evidence for the sequential formation of two complexes between an uptake inhibitor, GBR 12783 [1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine], and the neuronal transporter of dopamine.

Incubation of a crude synaptosomal fraction from rat striatum with GBR 12783 at 37 degrees C produced an inhibition of the specific uptake of [3H]dopamine that increased with time. The inhibition increased when GBR 12783 was present during preincubation and incubation (IC50 = 1.85+/-0.1 nM) instead of incubation alone (IC50 = 25+/-3.5 nM). Time-course studies of uptake inhibition demonstrated that a first collision transporter-inhibitor complex (TI) was formed immediately after addition of GBR 12783 so that the initial uptake velocity (V0) decreased for increasing concentrations of inhibitor (Ki > or = 20 nM). TI slowly isomerized to a more stable complex TI* (Ki* < or = 5 nM) with a value of t1/2 = 20-270 s. Fits of data to model 2 in which the steady-state uptake (VS) is set to zero were generally preferred, suggesting that formation of TI* could tend to irreversibility, as a consequence of a very low reverse isomerization. As expected, k, V0, and VS tended to steady-state values in an asymptotic manner for high concentrations of GBR 12783. GBR 12783 at 2.5 nM produced a mixed inhibition of the uptake, with an increase in KM and a decrease in Vmax; these effects were improved for 10 nM GBR 12783 and at 20 degrees C. These results are discussed in relation to previous data concerning [3H]GBR 12783 binding. The present work gives the first experimental demonstration that dopamine uptake blockers can act according to a two-step mechanism of inhibition; this is of great interest, because these inhibitors can oppose the effects of cocaine or amphetamine on the transporter according to a reaction that is partly nondependent on the concentration of the abused agent.

Effect of low concentrations of K+ and Cl- on the Na(+)-dependent neuronal uptake of [3H] dopamine.

The specific uptake of [3H] dopamine (DA) was studied using a crude synaptosomal fraction obtained from rat striatum. In a medium containing a 10 mM NaHCO3/NaH2PO4 buffer and no added K+ ions, addition of NaCl elicited an increase in DA uptake for Na+ concentrations from 10 to 60 mM, and then a decrease of uptake for Na+ concentrations up to 130 mM. These data confirm that rather low NaCl concentrations produce a maximal DA uptake. This biphasic curve of uptake resulted from significant changes in the Vmax of the DA uptake. Except for 10 mM Na+, this curve was not significantly modified when 9 mM NaHCO3/NaH2PO4 were replaced by 9 mM NaCl. This result indicates that the Cl- dependence of the DA uptake is mainly secondary to the Na+ dependence. Addition of KCl up to 3 mM did not modify the ascending part of the NaCl-dependent uptake curve. In contrast, the reduction in uptake produced by high Na+ concentrations was prevented in a concentration-dependent manner by KCl; this effect resulted from a decrease in the Km and an increase in the Vmax for the uptake. Measurements of membrane potential, with the help of the fluorescent probe 3, 3'-diethylthiadicarbocyanine iodide [DiSC2(5)] and purified synaptosomes prepared from rat striatum and cerebral cortex, revealed that addition of 3 mM KCl to a medium containing a high Na+ concentration and no K+ ions produced a marked and stable decrease in the fluorescence level. This decrease which corresponds to an increase in membrane polarization was blocked by 0.1 mM ouabain. These data suggest that low K+ concentrations are likely to prevent the decrease in uptake elicited by high Na+ concentrations by restoration, via a Na+/K+ ATPase-mediated mechanism, of the membrane potential and/or a transmembrane electrochemical Na+ gradient more favourable to DA uptake.

Complex ionic control of [3H]GBR 12783 binding to the dopamine neuronal carrier.

At 20 degrees C, [3H]GBR 12783, {1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-([1-3H]propenyl)-pip era zine} dissociated from the dopamine neuronal carrier present in rat striatal membranes with a t1/2 value of 27 min. At this temperature, KCI, CaCl2 and MgCl2 increased the binding dissociation, revealing that they recognize a binding site which is not mutually exclusive with that of [3H]GBR 12783. The comparison of the ability of KCl to increase the binding dissociation (by 160% at 30 mM KCl) with its potency as a binding inhibitor (Ki-2.6 +/- 0.3 mM) suggests an involvement of two recognition sites for K+ in binding inhibition, a not mutually exclusive site and another, mutually exclusive, site. Divalent cations mainly inhibited the binding via a mutually exclusive site since 3 mM Ca2+ and 10 mM Mg2+ increased the binding dissociation by 90% at 20 degrees C whereas their Ki values were 0.049 +/- 0.006 and 0.141 +/- 0.035 mM, respectively. Involvement of this mutually exclusive site was also supported by the persistence of the binding inhibition elicited by Ca2+ and Mg2+ at 0 degree C, a temperature at which they reduced the binding dissociation. At 20 degrees C, 100 mM NaCl did not modify [3H]GBR 12783 binding but it antagonized the binding dissociation elicited by inhibitory cations. Ca2+ reduced the off-rate of [3H]GBR 12783 binding at 0 degree C and in the presence of 100 mM Na+. Finally, [3H]GBR 12783-binding dissociation was increased by high 'cytosolic' K+ while 'synaptic' concentrations of Na+, K+, Ca2+, Mg2+ and Cl- were ineffective. A reduction of H2PO4-/HCO3- from 10 to 5 mM and a substitution of 5 mM H2PO4-/HCO3- by 5 mM Cl- increased the binding dissociation, suggesting that an anion-binding site could also regulate the binding.

In vivo striatal binding of the D1 antagonist SCH 23390 is not modified by changes in dopaminergic transmission.

The in vivo striatal binding of [3H]SCH 23390, an antagonist of the D1 dopamine receptors, was investigated in mice submitted to pretreatment to either decrease (gammabutyrolactone 750 mg/kg, i.p.) or, increase (3,4-dihydroxyphenylalanine (L-DOPA) 200 mg/kg i.p. plus dexamphetamine 4 mg/kg, s.c.) dopaminergic transmission. Such conditions failed to modify [3H]SCH 23390 binding. However, we observed that dopamine (at concentrations > or = 1 microM), reduced the in vitro binding of [3H]SCH 23390 in membrane fractions. These results suggest that modifications in dopamine neurotransmission do not alter the in vivo quantification of D1 receptors with [3H]SCH 23390, for example, in studies that use positron emission tomography.

Pharmacological modifications of dopamine transmission do not influence the striatal in vivo binding of [3H]mazindol or [3H]cocaine in mice.

We have considered the in vivo striatal binding of two ligands of the neuronal dopamine uptake complex: [3H]cocaine and [3H]mazindol. The [3H]cocaine tracer dose labelled the dopamine uptake complex in striatum but not the noradrenaline complex in cerebellum. On the contrary, the [3H]mazindol tracer dose induced a marked labelling of the noradrenaline uptake complex in cerebellum; its prevention by desipramine (5 mg/kg) increased simultaneously the cerebral bioavailability and thereby the striatal labelling of the dopamine transporter. In mice submitted to treatments modifying dopaminergic transmission either to decrease it (gammabutyrolactone, 750 mg/kg, i.p.) or to increase it (L-DOPA, 200 mg/kg, i.p., dexamphetamine, 4 mg/kg, s.c., or their combination), only dexamphetamine pretreatment significantly reduced [3H]cocaine and [3H]mazindol binding. Thus it appears that the level of dopamine transmission would not interfere with the in vivo quantification of striatal dopamine uptake sites assessed with either ligands.

Acute L-DOPA pretreatment potentiates 6-hydroxydopamine-induced toxic effects on nigro-striatal dopamine neurons in mice.

We have studied the effect of various agents on the decreases in striatal levels of dopamine (DA) and its metabolites which were observed 14 days after an intracerebroventricular (i.c.v.) administration of 50 micrograms 6-hydroxydopamine (6-OHDA) to mice. A pretreatment of mice with either a tyrosine hydroxylase inhibitor (alpha-methyl-p-tyrosine), a D2 receptor agonist (bromocriptine) or antagonist (haloperidol), or a vesicular uptake inhibitor (tetrabenazine) did not modify the 6-OHDA-induced decreases in DA and metabolites, indicating that DA synthesis, vesicular storage and neuronal firing rates are not mainly involved in the 6-OHDA-induced toxicity on the DA neurons. Conversely, a pretreatment with L-DOPA + benserazide potentiated the 6-OHDA-induced decreases in striatal levels of DA, homovanillic acid and 3-methoxy-tyramine. This effect was not due to an increased 6-OHDA uptake via the neuronal carrier since a pretreatment with L-DOPA + benserazide, performed 1-1.5 h before sacrifice, decreased the apparent affinity of the uptake, an effect which disappeared when considering the total DA concentration present in incubation medium ([3H]DA and cold released DA). In conclusion, potentiation of the 6-OHDA neurotoxicity by L-DOPA rises again the important problem of the safety of the latter drug in therapeutics.

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)-piperaz ine) to the neuronal transporter of dopamine. Treatment with 1 mmol/l 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/l DTNB. Addition of (sub)millimolar concentrations of CaCl2 or MgCl2, or 250 mmol/l 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/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/l Na+ 1 mmol/l DTNB induced a reduction in the Bmax of the specific binding of both radioligands. Addition of 1 mmol/l Ca2+ maintained or increased this Bmax 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, 50 mumol/l pHMBS produced a similar decrease in the Bmax 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 mumol/l dopamine, 1 mumol/l mazindol or 100 mumol/l cocaine) protected the specific binding of [3H]cocaine against the effects of pHMBS, whereas 1 mmol/l p-tyramine, 10 mumol/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/l Na+, there was a general decrease in the effects of ions on the reductions of specific binding produced by DTNB or pHMBS.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that pure uptake inhibitors including cocaine interact slowly with the dopamine neuronal carrier.

We have studied the ability of various uptake blockers to protect the dopamine neuronal carrier labeled with [3H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-(propenyl)-piperazine) against N-ethylmaleimide-induced alkylation, using membrane preparations obtained from rat striatum. Pure uptake inhibitors such as mazindol, pyrovalerone, nomifensine and methylphenidate, and substrates (dopamine, d-amphetamine, m-tyramine) protected the [3H]GBR 12783 binding site in a concentration-dependent manner. Preincubation of the membranes with these agents prior to N-ethylmaleimide treatment did not modify the protecting ability of substrates, whereas it significantly improved that of pure uptake inhibitors including cocaine. When the preincubation was omitted, the concentration dependence of the protection observed with pure uptake inhibitors decreased and a maximal 40% protection was observed for 10 microM to 1 mM cocaine concentrations. Effective protecting concentrations of blockers are correlated with their Ki determined in standard binding studies. These results reveal that all pure uptake inhibitors bind slowly to the dopamine neuronal carrier whereas substrates interact with it rapidly.

Thermodynamics of the binding of BTCP (GK 13) and related derivatives on the dopamine neuronal carrier.

We have studied the thermodynamic properties of the binding of a coherent series of uptake inhibitors derived from BTCP (GK 13 = N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine) to the dopamine neuronal carrier labelled with [3H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-propenyl)-piperazine). GK 13 (30 nM) and its 2-naphthyl derivative GK 189 (15 nM) competitively inhibited the specific binding of [3H]GBR 12783 to sites present in rat striatal membranes. Hill numbers calculated for the inhibition of the specific binding of [3H]GBR 12783 by BTCP derivatives were close to 1 (range 0.79-1.18). Increasing the temperature from 0 degrees to 30 degrees C induced a decrease in the affinity of [3H]GBR 12783 and GK derivatives which was generally less pronounced than that obtained when temperature was raised from 30 degrees C to 37 degrees C. Increasing the incubation temperature led to a decrease in both enthalpy (delta H degrees) and entropy (delta S degrees). We observed at 37 degrees C a large negative enthalpy change (range -48, -79 kJ/mol) and a negative, binding unfavorable, change in entropy. This indicates that the GK derivatives binding is enthalpy-driven. Furthermore, data obtained in the present study show that changes in thermodynamic parameters are not a function of the inhibitor's affinity for the dopamine neuronal carrier and this suggests that bonds involved in the inhibitor-carrier interaction are more likely related to the carrier configuration than to the chemical structure of the inhibitor.

Cocaine and GBR 12783 recognize nonidentical, overlapping binding domains on the dopamine neuronal carrier.

In incubation medium containing Na+ as the only cation, the specific binding of [3H]cocaine to a membrane preparation obtained from rat striatum reached a maximal level for 10 mM Na+, whereas higher concentrations decreased its affinity. The specific binding of [3H]cocaine was inhibited monophasically by GBR 12783, mazindol, nomifensine and substrates of the transporter; in saturation experiments, GBR 12783 competitively blocked the [3H]cocaine specific binding and vice versa. Treatment of the striatal membranes with N-ethylmaleimide resulted in a concentration-dependent reduction of the specific binding of [3H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-[1-3H]propenyl)-piperaz ine) which was significantly more marked than that of the specific binding of [3H]cocaine, the nonspecific binding of [3H]cocaine being measured with either cocaine or dopamine. Addition of substrates or pure uptake inhibitors to the treatment medium afforded protection against the N-ethylmaleimide-induced reduction in both bindings. In particular, cocaine offered protection for [3H]GBR 12783 binding and vice versa. All results are consistent with a model in which pure uptake blockers and substrates recognize nonidentical but overlapping binding domains on the neuronal carrier of dopamine.

Effect of CH3HgCl and several transition metals on the dopamine neuronal carrier; peculiar behaviour of Zn2+.

CH3Hg+ and metal ions inhibited the specific binding of (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-3H]propenyl) piperazine) ([3H]GBR 12783) to the dopamine neuronal carrier present in membranes from rat striatum with a general rank order of potency CH3Hg+ > Cu2+ > Cd2+ > Zn2+ > Ni2+ = Mn2+ = Co2+, suggesting that -SH groups are chiefly involved in this inhibition. Five millimolar dithiothreitol reversed the rather stable block of the specific binding produced by Cd2+ or Zn2+. An increase in the concentration of Na+, or addition of either K+ or Ca2+ reduced the inhibitory effects of metal cations, except Cu2+. Zn2+ (3 microM) reduced the inhibitory potency of Cd2+ on the binding but was ineffective against CH3Hg+ and Cu2+. Zn2+ at 0.3 to 10 microM significantly enhanced the specific binding of [3H]GBR 12783 and [3H]cocaine by 42 to 146%. Zn2+ (3 microM) increased the affinity of all pure uptake inhibitors tested and of the majority of the substrates for the [3H]GBR 12783 binding site. Dissociation experiments revealed that Zn2+ both inhibited and enhanced the [3H]GBR 12783 binding by recognizing amino acids located close to or in the radioligand binding site. Micromolar concentrations of Zn2+ noncompetitively blocked the [3H]dopamine uptake but they did not modify the block of the transport provoked by pure uptake inhibitors. These findings suggest that Na+, K+, Ca2+ and metal ions could recognize some -SH groups located in the [3H]GBR 12783 binding site; low concentrations of Zn2+ could allow a protection of these -SH groups.