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M E Reith

Publications and source records attributed to M E Reith.

At least 55 records · Page 3Linked to original sources
PubMed1997

Modeling of the interaction of Na+ and K+ with the binding of the cocaine analogue 3beta-(4-[125I]iodophenyl)tropane-2beta-carboxylic acid isopropyl ester to the dopamine transporter.

The present study examines the interaction of Na+ and K+ with the binding of the cocaine analogue 3beta-(4-[125I]iodophenyl)tropane-2beta-carboxylic acid isopropyl ester to dopamine transporters (DATs) in rat striatal synaptosomal membranes at 37 degrees C. The binding increases with [Na+] from 10 to 100 mM and decreases with higher [Na+]. The presence of K+ reduces the maximal stimulatory effect of Na+ and causes a nonlinear EC50 shift for Na+. K+ strongly inhibits the binding at low [Na+]. Increasing [Na+] produces a linear IC50 shift for K+. Saturation analysis indicates a single binding site changing its affinity for the radioligand depending on [K+]/[Na+] ratio in the assay buffer. A reduced Bmax was observed in the presence of 10 mM Na+ and 30 mM K+. Both high [Na+] and high [K+] accelerate the dissociation of the binding, and K+-induced acceleration was abolished by increasing [Na+]. Least squares model fitting of equilibrium data and kinetic analysis of dissociation rates reveal competitive interactions between Na+ and K+ at two sites allosterically linked on the DAT: One site mediates the stimulatory effect of Na+, and the other site involves the radioligand binding and the inhibitory effect of cations on the binding. Various uptake blockers and substrates, dopamine in particular, display reduced potency in inhibiting the binding at a higher [K+]/[Na+] ratio.

Animals↗
PubMed1997

Cations affect [3H]mazindol and [3H]WIN 35,428 binding to the human dopamine transporter in a similar fashion.

The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21 degrees C. Zn2+ (30-100 microM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1-100 microM) had no effect; Hg2+ at approximately 3 microM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0 degrees C, and at 30-100 microM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30-100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21 degrees C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21 degrees C and Hg2+ at 0 degrees C.

Animals↗
PubMed1997

WIN 35,428 and mazindol are mutually exclusive in binding to the cloned human dopamine transporter.

It has been suggested that cocaine and mazindol bind to separate sites on the dopamine transporter. In the present study, we address this issue by examining the inhibition by mazindol of the binding of [3H]WIN 35,428 ([3H]2beta-carbomethyoxy-3beta-(4-fluorophenyl)-tropane), a phenyltropane analog of cocaine, and the inhibition by WIN 35,428 of [3H]mazindol binding to the cloned human dopamine transporter expressed in C6 glioma cells. The design involved the construction of inhibition curves at six widely different radioligand levels, enabling the distinction between the nonlinear hyperbolic competition (i.e., negative allosteric) model and the competitive (i.e., mutually exclusive binding) model. Nonlinear computer curve-fitting analysis indicated no difference in the goodness of fit between the two models; the negative allosteric model indicated an extremely high allosteric constant of approximately > or = 100, which practically equates to the competitive model. The present results suggest that complex interactions reported between cocaine and mazindol in inhibiting dopamine transport are beyond the level of ligand recognition.

Binding, Competitive↗
PubMed1996

Regulation of the functional activity of the human dopamine transporter by the arachidonic acid pathway.

The role of arachidonic acid was examined in the regulation of dopamine transport in C6 glioma cells stably expressing the human dopamine transporter. Exogenously added arachidonic acid (20-160 microM) stimulated [3H]dopamine uptake when pre-incubated for short times (15-30 min); 160 microM arachidonic acid inhibited following longer pre-exposures (45-60 min). Under the same conditions, only decreases were observed in the binding of the cocaine analog [3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane ([3H]WIN 35,428). The reduction in dopamine transporter activity by arachidonic acid (at 160 microM for 60 min) was caused by a decrease in the Vmax (from 202 to 44 pmol/mg/min) opposed by a smaller reduction in K(m) (from 1.2 to 0.8 microM), whereas the effect of arachidonic acid (at 160 microM for 15 min) on [3H]WIN 35,428 binding was caused by a reduction in the Bmax (from 1.8 to 1.3 pmol/mg) without a change in Kd (7.2 nM). Upon 15-min exposure, melittin, an activator of phospholipase A2, and nordihydroguaiaretic acid, a lipooxygenase inhibitor, both expected to cause enhanced endogenous arachidonic acid, inhibited [3H]dopamine uptake and [3H]WIN 35,428 binding with an IC50 value close to 1 microM, whereas thimerosal, which raises arachidonic acid by inhibiting lipid reacylation, caused similar reductions at the sub-millimolar level. Co-presence of stauroporine (0.3-2 microM), an inhibitor of protein kinase C, had little or no effect on the melittin- or arachidonic acid-induced inhibition of [3H]dopamine uptake. Both the melittin- and arachidonic acid-, but not phorbol 12-myristate 13-acetate-induced inhibition of uptake were counteracted by bovine serum albumin (0.1 and 1 mg/ml) which binds arachidonic acid. The data taken together suggest that the inhibitory effects of arachidonic acid activators and those of protein kinase C activators on dopamine uptake are mediated by separate mechanisms.

Animals↗
PubMed1996

Binding domains for blockers and substrates on the cloned human dopamine transporter studied by protection against N-ethylmaleimide-induced reduction of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane ([3H]WIN 35,428) binding.

Binding sites for 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane ([3H]WIN 35,428) on the human dopamine transporter expressed in C6 glioma cells were alkylated with N-ethylmaleimide (NEM), and the protective potency of the blockers cocaine, N[1-(2-benzo[b]thiophenyl) cyclohexyl]piperidine (BTCP), and benztropine, and of the substrates dopamine, d-amphetamine, and norepinephrine was measured. In general, the protective potency was lower (at least 4-5 times) than the potency in inhibiting [3H]WIN 35,428 binding with the compounds present under the same experimental conditions used for the NEM alkylation. However, the disparity was substantially greater for all substrates tested (23- to 44-fold) than for the blockers (4- to 11-fold), especially cocaine (5-fold) and BTCP (4-fold). Benztropine took an intermediate place (11-fold) between cocaine (5-fold) and BTCP (4-fold), on the one hand, and dopamine (23-fold), on the other hand. [3H]WIN 35,428 binding was best described by a one-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.

Animals↗
PubMed1996

Extracellular dopamine in the rat ventral tegmental area and nucleus accumbens following ventral tegmental infusion of cocaine.

Rats were implanted with dual dialysis probes, one in the ventral tegmental area, and another one ipsilateral in the nucleus accumbens. Infusion of cocaine (10, 100, 1000 mM) into the ventral tegmental area gradually increased extracellular dopamine to 164, 329 and 991% of baseline in the ventral tegmental area, but reduced dopamine to 76, 47 and 38% of baseline in the nucleus acumbens. These results are consistent with cocaine-induced feedback regulation of dopamine cell activity involving somatodendritic impulse regulating dopamine D2 autoreceptors.

Animals↗
PubMed1996

Focal ethanol elevates extracellular dopamine and serotonin concentrations in the rat ventral tegmental area.

The present study describes the effects of local perfusion with ethanol on extracellular dopamine and serotonin (5-hydroxytryptamine, 5-HT) concentrations in the ventral tegmental area. Various concentrations of ethanol in artificial cerebrospinal fluid (ACSF) (0.1-10%, v/v) were administered through a microdialysis probe into the ventral tegmental area of freely moving Sprague-Dawley rats. A significant and concentration-dependent increment in dialysate output of both dopamine and serotonin was observed after local infusion of ethanol. Perfusion with Ca(2+)-free medium or tetrodotoxin (1 microM in ACSF) produced a significant reduction in basal extracellular dopamine and serotonin concentration but failed to block dopamine or serotonin release produced by infusion of 10% ethanol. Perfusion with 100 mM K+ before and after infusion of 10% ethanol revealed that the second perfusion with high K+ solution still produced an increase in dopamine and serotonin concentration, similar in magnitude to the first response, indicating that perfusion with 10% ethanol did not cause irreversible damage to either dopamine cell bodies or serotonin terminals in the ventral tegmental area. These results suggest that dopamine and serotonin release from the ventral tegmental area produced by focal application of 10% ethanol is mediated, at least in part, by a non-exocytotic mechanism. Direct stimulation of the ventral tegmental area dopamine neurons by ethanol might be involved in the reinforcing properties of the drug.

Animals↗
PubMed1996

[3H]WIN 35,428 [2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane] binding to rat brain membranes. Comparing dopamine cell body areas with nerve terminal regions.

Potential differences between somatodendritic acid and axonal dopamine transporters were examined by comparing the binding constants of [3H]WIN 35, 428 [2 beta-carbomethoxy- 3 beta-(4-fluorophenyl)tropane] binding to membranes prepared from the rat ventral mesencephalon, containing A9 and A10 dopamine cell bodies, and from the nucleus accumbens. Saturation analysis of [3H]WIN 35,428 binding, in the presence of compounds to occlude norepinephrine and serotonin transporters, was performed by both the "unlabeled" method (varying unlabeled ligand) and "labeled" method (varying radioligand). The density of binding was substantially lower in the ventral mesencephalon than in the nucleus accumbens, but the binding affinity was only slightly different. Likewise, the differences between the two regions in the inhibitory potency of cocaine and GBR 12909 [1-(2-di(4-fluorophenyl)-methoxy-ethyl)4-(3-phenylpropyl)piperazine] were not substantial. The results suggest that somatodendritic and axonal dopamine transporters in the ventral mesencephalon and nucleus accumbens are not very different as far as their binding domains for uptake blockers such as cocaine and GBR 12909 are concerned.

Animals↗
PubMed1996

Structure-activity relationship studies of novel 4-[2-[bis(4-fluorophenyl)methoxy]ethyl]-1-(3-phenylpropyl)piperidine analogs: synthesis and biological evaluation at the dopamine and serotonin transporter sites.

Several analogs of the potent dopamine (DA) transporter ligand 4-[2-[bis(4-fluorophenyl)-methoxy]ethyl]-1-(3-phenylpropyl)piperidine, 1b, were made and biologically evaluated for their binding at the DA and serotonin (5HT) transporters in rat striatal membranes. Different alkyl chain lengths and substitutions were introduced in these molecules to generate an optimum activity and selectivity for the DA transporter. In general, unsubstituted and fluoro-substituted compounds were the most active and selective for the DA transporter. The compound 4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine, 9a, showed high potency and was the most selective for the DA transporter (5HT/DA = 49) in this series of compounds. Some of these novel analogs were found to be more selective in binding at the DA transporter than the original GBR 12909 molecule, 1-[2-]bis(4-fluorophenyl)methoxy]ethyl]-4-(3- phenylpropyl)piperidine.

Animals↗
PubMed1996

Elongation factor 1 alpha genes of the red alga Porphyra purpurea include a novel, developmentally specialized variant.

The life cycle of the red alga Porphyra purpurea alternates between two morphologically distinct phases: a shell-boring, filamentous sporophyte and a free-living, foliose gametophyte. From a subtracted cDNA library enriched for sporophyte-specific sequences, we isolated a cDNA encoding an unusual elongation factor 1 alpha (EF-1 alpha) that is expressed only in the sporophyte. A second EF-1 alpha gene that is expressed equally in the sporophyte and the gametophyte was isolated from a genomic library. These are the only EF-1 alpha genes detectable in P. purpurea. The constitutively expressed gene encodes an EF-1 alpha very similar to those of most eukaryotes. However, the sporophyte-specific EF-1 alpha is one of the most divergent yet described, with nine insertions or deletions ranging in size from 1 to 26 amino acids. This is the first report of a developmental stage-specific EF-1 alpha outside of the animal kingdom and suggests a fundamental role for EF-1 alpha in the developmental process.

Amino Acid Sequence↗
PubMed1996

The role of serotonin in the actions of psychostimulants: molecular and pharmacological analyses.

Cocaine is a highly abused psychostimulant which is a local anesthetic and inhibitor of the reuptake of dopamine (DA), serotonin (5-HT) and norepinephrine (NE). This manuscript details a brief summary and the primary conclusions of several presentations geared to present recent pharmacological analyses of the interaction of cocaine with 5-HT systems. These data illustrate the complexity of actions for cocaine in the brain and emphasize that, to fully understand the mechanisms which underlie its potent behavioural effects, the impact of this drug on 5-HT function as well as the interactions between 5-HT and the function of DA mesolimbic pathways must be considered.

Animals↗
PubMed1996

Extracellular dopamine, norepinephrine, and serotonin in the nucleus accumbens of freely moving rats during intracerebral dialysis with cocaine and other monoamine uptake blockers.

Monoamine-uptake blockers were applied focally (0.1-1,000 microM) through a dialysis probe in the nucleus accumbens of freely moving rats, and the extracellular concentrations of dopamine, norepinephrine, and serotonin were measured. The selective dopamine-uptake blocker GBR 12935 increased dopamine preferentially with only a small effect on norepinephrine, whereas the selective serotonin-uptake blocker fluoxetine increased serotonin output preferentially. In contrast, the selective norepinephrine-uptake blockers desipramine and nisoxetine enhanced not only norepinephrine, but also serotonin and dopamine appreciably. Cocaine increased all three amines with the greatest effects on dopamine and serotonin. As in our previous study on the ventral tegmental area, there was a positive association between dopamine and norepinephrine output when all blocker data were taken together. The present results suggest a contribution of the increase in norepinephrine, but not serotonin, to the enhancement of dopamine after cocaine applied focally in the nucleus accumbens.

Animals↗
PubMed1996

Amygdala kindling of forebrain seizures and the occurrence of brainstem seizures in genetically epilepsy-prone rats.

Forebrain seizures were kindled in rats by daily electrical stimulation of the amygdala. Genetically epilepsy-prone rats scoring 9 (GEPR-9s) on the seizure severity scale during audiogenic seizure (AGS) screening ("brainstem seizure-experienced") required fewer stimulations to achieve fully kindled seizures (forelimb clonus with rearing and falling) than control rats. AGS-naive GEPR-9s required an intermediate number of stimulations, indicating a role for both genetic predisposition and previous acoustically evoked brainstem seizure experience. Other forebrain kindling indices such as afterdischarge threshold/duration and seizure latency/duration also involved genetic as well as phenotypic (previous seizure experience) factors. In most GEPR-9s in both groups, severe brainstem seizures occurred after forebrain stimulation. The occurrence of brainstem seizures had a random nature and was not related to the sequence of kindling-dependent forebrain seizure progression. The lack of a difference in the occurrence of brainstem seizures between seizure-experienced and AGS-naive GEPR-9s suggest that genetic predisposition is the major factor in forebrain seizure-induced activation of brainstem seizure circuitry. This brainstem seizure activity appears to model pertinent aspects of secondary generalization observed in human partial seizures.

Acoustic Stimulation↗
PubMed1996

Modeling of the pH dependence of the binding of WIN 35,428 to the dopamine transporter in rat striatal membranes: is the bioactive form positively charged or neutral?

Cocaine and its phenyltropane analogs, including 2beta-carbomethoxy-3beta-(4-fluorophenyl) tropane can exist in either the cationic or the neutral form, with the proportion of each form determined by the pKA and ambient pH. It is unknown which form is the active form for binding to the dopamine transporter. Our study examines the following models for the pH dependency of [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl) tropane binding to the dopamine transporter in rat striatal membranes: 1) pH does not affect the binding affinity of the ligand for the dopamine transporter, and the observed binding is a function only of the concentration of the active form; and 2) pH affects the binding affinity of the ligand for the transporter, and changes in this affinity as well as in the concentration of the active form of the ligand in the medium contribute to the observed binding changes upon varying pH. For each model, predictions are formulated for the case that either the cation, neutral base, or both are the active form, and these predictions are compared with the experimental data obtained over a pH range of 6 to 9.5. Observations for 2beta-carbomethoxy-3beta-(4-fluorophenyl) tropane, which can be both in the cationic and neutral form, are compared with those for cocaine methiodide, which is permanently cationic, and benzocaine, which is permanently neutral. Consonant with all data is a simple model in which the cationic and neutral form have similar binding activity and binding changes as a function of pH are caused by changes at the level of the transporter rather than the ligand.

Animals↗
PubMed1995

Translocation of dopamine and binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl) tropane (WIN 35,428) measured under identical conditions in rat striatal synaptosomal preparations. Inhibition by various blockers.

Translocation of [3H]dopamine and binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]-tropane ([3H]WIN 35,428) were measured in crude synaptosomal preparations from rat striatum under identical conditions of assay buffer (phosphate-Krebs) and temperature (25 degrees). [3H]Dopamine uptake as a function of time was close to linear for at least 8 min, whereas [3H]WIN 35,428 binding had reached equilibrium within 1 min and remained at its plateau value for at least 20 min. The following inhibitors were tested in uptake and binding assays run in parallel with the same synaptosomal preparation: cocaine, WIN 35,428, benztropine, nomifensine, mazindol, methylphenidate, N-[1-(2-benzo[b]-thiophenyl)cyclohexyl]piperidine (BTCP), Lu 19-005 (Indatraline), 1-(2-(di(4-fluorophenyl)-methoxy)-ethyl)-4-(3-phenyl-2-propyl)piperazine (GBR 12909), 1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenyl-2-propyl)piperazine (GBR 12935) and 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo [1,2-a]quinoxaline (CGS 12066B). When present together with [3H]dopamine or [3H]WIN 35,428 for 8 min, the observed binding IC50 values were generally higher (average 1.4-fold) than the uptake IC50 values, with a significant y-axis intercept in linear regression analysis of binding on uptake IC50. For slowly equilibrating inhibitors, estimates of uptake IC50 values were overestimates, and relatively lower values were obtained by monitoring [3H]dopamine uptake for 1 min only during the last minute of the 8-min presence of inhibitor; under these conditions, binding over uptake IC50 ratios were on the average 2.3. Kinetic calculations, taking into account both radioligand and inhibitor equilibration kinetics, indicated that the latter comparison between binding and uptake measurements was most relevant, and suggested the involvement of complexities beyond simple competitive inhibition of dopamine transport, such as different binding domains for substrate and blocker recognition, or spare receptors for blockers. The present data indicate that binding over uptake IC50 ratios should be interpreted with caution, depending on the experimental conditions used to measure these ratios.

Animals↗
PubMed1995

Monoamine interactions measured by microdialysis in the ventral tegmental area of rats treated systemically with (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin.

The effect of (+/-)-8-hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT), a selective serotonin 5-HT1A agonist, on levels of extracellular norepinephrine (NE), dopamine (DA), and 5-HT (measured simultaneously) was investigated by microdialysis in the ventral tegmental area (VTA) of freely moving rats, and their behavioral activity was monitored. At 50 micrograms/kg s.c., 8-OH-DPAT reduced 5-HT levels but enhanced NE and DA levels in VTA dialysate. These effects were not altered by pretreatment with systemic idazoxan (5 mg/kg i.p.), a selective alpha 2 antagonist, or local sulpiride (10 microM), a selective D2/D3 antagonist. At 500 micrograms/kg s.c., 8-OH-DPAT further enhanced or more persistently reduced dialysate NE or 5-HT content but had little effect on dialysate DA content. Its DA level-increasing effect could be seen dramatically with local infusion of cocaine (30 microM) and, to a lesser extent, sulpiride (10 microM). Depletion of endogenous 5-HT with p-chlorophenylalanine attenuated both the 5-HT level-reducing and DA level-enhancing effects of 8-OH-DPAT without affecting its maximal NE effect and the locomotor-stimulatory effect. Partial depletion of endogenous NE with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine failed to change the monoamine response but diminished the locomotion induced by 8-OH-DPAT. These results suggested that (a) the low dose of 8-OH-DPAT may act at presynaptic 5-HT1A receptors to modulate 5-HT and DA release, while acting at postsynaptic 5-HT1A receptors to modulate NE release; (b) the high dose of 8-OH-DPAT may activate D2 receptors to offset its DA level-increasing effect; and (c) the locomotor-stimulatory effect of 8-OH-DPAT might be mediated primarily by postsynaptic 5-HT1A receptors and the NE system.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1994

O-526, a piperidine analog of GBR 12909, retains high affinity for the dopamine transporter in monkey caudate-putamen.

1,4-Disubstituted piperazines such as GBR 12909 (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine) and GBR 12935 (1-[2-(bis(phenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine) are among the most potent and selective ligands for the dopamine transporter in brain. However, they also bind to a widely distributed "piperazine acceptor site". In order to assess whether the piperazine moiety of GBR 12909 is critical for binding to the dopamine transporter, two piperidine analogs of GBR 12909, one with the nitrogen proximal to the diphenylmethyloxy moiety (O-549, (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperidine), the other distal to this moiety (O-526, 4-[2-[bis(4-fluorophenyl)methoxy]ethyl]-1- (3-phenylpropyl)piperidine) were synthesized. The affinities of O-526, O-549 and GBR 12909 for the dopamine and serotonin transporters in monkey caudate-putamen (Macaca fascicularis) were evaluated using [3H]WIN 35,428 ([3H]CFT, 2 beta-carbomethoxy-3 beta-4-(fluorophenyl)tropane) or [3H]GBR 12935 and [3H]citalopram to label the dopamine and serotonin transporters, respectively. O-526 inhibited [3H]WIN 35,428 binding with potencies that were similar to GBR 12909 (IC50: 24.9 +/- 3.23 vs. 22.0 +/- 2.2 nM, respectively) whereas O-549 was 24 times less potent than O-526 (IC50: 595 +/- 148 nM). The selectivity of GBR 12909 for the dopamine over the serotonin transporter was retained by O-526 but not by O-549 which was relatively non-selective. These results indicate that only one of the two nitrogens on the basic GBR structure is needed for high affinity binding to the dopamine transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗