Structural basis for action by diverse antidepressants on biogenic amine transporters.

The biogenic amine transporters (BATs) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents including selective serotonin reuptake inhibitors (SSRIs), serotonin-noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCAs). Because eukaryotic BATs are recalcitrant to crystallographic analysis, our understanding of the mechanism of these inhibitors and antidepressants is limited. LeuT is a bacterial homologue of BATs and has proven to be a valuable paradigm for understanding relationships between their structure and function. However, because only approximately 25% of the amino acid sequence of LeuT is in common with that of BATs, and as LeuT is a promiscuous amino acid transporter, it does not recapitulate the pharmacological properties of BATs. Indeed, SSRIs and TCAs bind in the extracellular vestibule of LeuT and act as non-competitive inhibitors of transport. By contrast, multiple studies demonstrate that both TCAs and SSRIs are competitive inhibitors for eukaryotic BATs and bind to the primary binding pocket. Here we engineered LeuT to harbour human BAT-like pharmacology by mutating key residues around the primary binding pocket. The final LeuBAT mutant binds the SSRI sertraline with a binding constant of 18 nM and displays high-affinity binding to a range of SSRIs, SNRIs and a TCA. We determined 12 crystal structures of LeuBAT in complex with four classes of antidepressants. The chemically diverse inhibitors have a remarkably similar mode of binding in which they straddle transmembrane helix (TM) 3, wedge between TM3/TM8 and TM1/TM6, and lock the transporter in a sodium- and chloride-bound outward-facing open conformation. Together, these studies define common and simple principles for the action of SSRIs, SNRIs and TCAs on BATs.

Binding of mazindol and analogs to the human serotonin and dopamine transporters.

Mazindol has been explored as a possible agent in cocaine addiction pharmacotherapy. The tetracyclic compound inhibits both the dopamine transporter and the serotonin transporter, and simple chemical modifications considerably alter target selectivity. Mazindol, therefore, is an attractive scaffold for both understanding the molecular determinants of serotonin/dopamine transporter selectivity and for the development of novel drug abuse treatments. Using molecular modeling and pharmacologic profiling of rationally chosen serotonin and dopamine transporter mutants with respect to a series of mazindol analogs has allowed us to determine the orientation of mazindol within the central binding site. We find that mazindol binds in the central substrate binding site, and that the transporter selectivity can be modulated through mutations of a few residues in the binding pocket. Mazindol is most likely to bind as the R-enantiomer. Tyrosines 95 and 175 in the human serotonin transporter and the corresponding phenylalanines 75 and 155 in the human dopamine transporter are the primary determinants of mazindol selectivity. Manipulating the interaction of substituents on the 7-position with the human serotonin transporter Tyr175 versus dopamine transporter Phe155 is found to be a strong tool in tuning the selectivity of mazindol analogs and may be used in future drug design of cocaine abuse pharmacotherapies.

Altered levels of dopamine transporter in the frontal pole and the striatum in mood disorders: A postmortem study.

Dopamine dysregulation is known to play a major role in the pathophysiology of major depressive disorders (MDD) and bipolar disorders (BD). The dopamine transporter (DAT) plays a critical role in regulating dopamine concentration at the synaptic cleft and therefore could have an important role in the molecular pathology of MDD and BD. To test this hypothesis, we measured levels of [3H]mazindol binding to DAT in Brodmann's area (BA) 10, BA 17 as well as in the dorsal and ventral striatum from 15 controls, 15 patients with MDD and 15 patients with BD, obtained postmortem, using in situ radioligand binding with autoradiography. Compared to controls, levels of [3H]mazindol binding to DAT was significantly higher in BA10 from patients with MDD but not BD. There was no significant difference in [3H]mazindol binding to DAT in BA 17 or the dorsal and ventral striatum from patients with MDD or BD. In addition, levels of [3H]mazindol binding show no correlation with donor age, postmortem interval, tissue pH, sex or duration of illness. In conclusion, our data suggest that changes in levels of DAT may be selectively affecting dopamine homeostasis in BA 10 in patients with MDD.

Cocaine receptors on dopamine transporters are related to self-administration of cocaine.

Although cocaine binds to several sites in the brain, the biochemical receptor mechanism or mechanisms associated with its dependence producing properties are unknown. It is shown here that the potencies of cocaine-like drugs in self-administration studies correlate with their potencies in inhibiting [3H]mazindol binding to the dopamine transporters in the rat striatum, but not with their potencies in binding to a large number of other presynaptic and postsynaptic binding sites. Thus, the cocaine receptor related to substance abuse is proposed to be the one associated with dopamine uptake inhibition.

Decrease of D2 receptor binding but increase in D2-stimulated G-protein activation, dopamine transporter binding and behavioural sensitization in brains of mice treated with a chronic escalating dose 'binge' cocaine administration paradigm.

Understanding the neurobiology of the transition from initial drug use to excessive drug use has been a challenge in drug addiction. We examined the effect of chronic 'binge' escalating dose cocaine administration, which mimics human compulsive drug use, on behavioural responses and the dopaminergic system of mice and compared it with a chronic steady dose (3 x 15 mg/kg/day) 'binge' cocaine administration paradigm. Male C57BL/6J mice were injected with saline or cocaine in an escalating dose paradigm for 14 days. Locomotor and stereotypy activity were measured and quantitative autoradiographic mapping of D(1) and D(2) receptors, dopamine transporters and D(2)-stimulated [(35)S]GTPgammaS binding was performed in the brains of mice treated with this escalating and steady dose paradigm. An initial sensitization to the locomotor effects of cocaine followed by a dose-dependent increase in the duration of the locomotor effect of cocaine was observed in the escalating but not the steady dose paradigm. Sensitization to the stereotypy effect of cocaine and an increase in cocaine-induced stereotypy score was observed from 3 x 20 to 3 x 25 mg/kg/day cocaine. There was a significant decrease in D(2) receptor density, but an increase in D(2)-stimulated G-protein activity and dopamine transporter density in the striatum of cocaine-treated mice, which was not observed in our steady dose paradigm. Our results document that chronic 'binge' escalating dose cocaine treatment triggers profound behavioural and neurochemical changes in the dopaminergic system, which might underlie the transition from drug use to compulsive drug use associated with addiction, which is a process of escalation.

Time-course of SKF-81297-induced increase in glutamic acid decarboxylase 65 and 67 mRNA levels in striatonigral neurons and decrease in GABA(A) receptor alpha1 subunit mRNA levels in the substantia nigra, pars reticulata, in adult rats with a unilateral 6-hydroxydopamine lesion.

Striatal projection neurons use GABA as their neurotransmitter and express the rate-limiting synthesizing enzyme glutamic acid decarboxylase (GAD) and the vesicular GABA transporter vGAT. The chronic systemic administration of an agonist of dopamine D1/D5-preferring receptors is known to alter GAD mRNA levels in striatonigral neurons in intact and dopamine-depleted rats. In the present study, the effects of a single or subchronic systemic administration of the dopamine D1/D5-preferring receptor agonist SKF-81297 on GAD65, GAD67, PPD and vGAT mRNA levels in the striatum and GABA(A) receptor alpha1 subunit mRNA levels in the substantia nigra, pars reticulata, were measured in rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion. After a single injection of SKF-81297, striatal GAD65 mRNA levels were significantly increased at 3 but not 72 h. In contrast, striatal GAD67 mRNA levels were increased and nigral alpha1 mRNA levels were decreased at 72 but not 3 h. Single cell analysis on double-labeled sections indicated that increased GAD or vGAT mRNA levels after acute SKF-81297 occurred in striatonigral neurons identified by their lack of preproenkephalin expression. Subchronic SKF-81297 induced significant increases in striatal GAD67, GAD65, preprodynorphin and vGAT mRNA levels and decreases in nigral alpha1 mRNA levels. In the striatum contralateral to the 6-OHDA lesion, subchronic but not acute SKF-81297 induced a significant increase in GAD65 mRNA levels. The other mRNA levels were not significantly altered. Finally, striatal GAD67 mRNA levels were negatively correlated with nigral alpha1 mRNA levels in the dopamine-depleted but not dopamine-intact side. The results suggest that different signaling pathways are involved in the modulation by dopamine D1/D5 receptors of GAD65 and GAD67 mRNA levels in striatonigral neurons. They also suggest that the down-regulation of nigral GABA(A) receptors is linked to the increase in striatal GAD67 mRNA levels in the dopamine-depleted striatum.

Dysregulation of Striatal Dopamine Receptor Binding in Suicide.

Inconsistent evidence implicates disruptions of striatal dopaminergic indices in suicide and major depression. To determine whether there are alterations in the striatal dopamine system in suicide, we conducted a quantitative autoradiographic survey of dopamine transporter (DAT; [3H]mazindol), D1 receptor ([3H]SCH23390), and D2 receptor ([3H]sulpiride) binding in the dorsal striatum postmortem from matched suicides and controls. Axis I and axis II psychiatric diagnosis, recent treatment history, and early life adversity (ELA) were determined by psychological autopsy. Mean DAT, D2, and D1 receptor binding did not differ in suicide. However, there was a positive correlation between D1 and D2 receptor binding in the dorsal striatum of control subjects (R2=0.31, p<0.05) that was not present in suicides (R2=0.00, p=0.97). In suicides and controls with reported ELA, there was no correlation between striatal DAT and D1 receptor binding (R2=0.07, p=0.33), although DAT and D1 receptor binding was positively correlated in subjects with no report of ELA (R2=0.32, p<0.05). After controlling for age, there were no significant ELA-related mean differences. Binding of D1 receptors and DAT throughout the striatum correlated negatively with age (D1 receptor: R2=0.12, p<0.05; DAT: R2=0.36, p<0.001). There appears to be an imbalance in dopaminergic receptor and transporter expression related to suicide that differs from that associated with ELA or age.

Systematic review and meta-analysis of the efficacy and safety of amfepramone and mazindol as a monotherapy for the treatment of obese or overweight patients.

The aim of this study was to evaluate efficacy and safety of amfepramone, fenproporex and mazindol as a monotherapy for the treatment of obese or overweight patients. A systematic review of primary studies was conducted, followed by a direct meta-analysis (random effect) and mixed treatment comparison. Medline and other databases were searched. Heterogeneity was explored through I2 associated with a p-value. Of 739 identified publications, 25 were included in the meta-analysis. The global evaluation of Cochrane resulted in 19 studies with a high level of bias and six with unclear risk. Due to the lack of information in primary studies, direct meta-analyses were conducted only for amfepramone and mazindol. Compared to placebo, amfepramone resulted in higher weight loss in the short-term (<180 days; mean difference (MD) -1.281 kg; p<0.05; I2: 0.0%; p=0.379) and long-term (≥180 days; MD -6.518 kg; p<0.05; I2: 0.0%; p=0.719). Only studies with long-term follow up reported efficacy in terms of abdominal circumference and 5-10% weight reduction. These results corroborated the finding that the efficacy of amfepramone is greater than that of placebo. Treatment with mazindol showed greater short-term weight loss than that with placebo (MD -1.721 kg; p<0.05; I2: 0.9%; p=0.388). However, metabolic outcomes were poorly described, preventing a meta-analysis. A mixed treatment comparison corroborated the direct meta-analysis. Considering the high level of risk of bias and the absence of important published outcomes for anti-obesity therapy assessments, this study found that the evaluated drugs showed poor evidence of efficacy in the treatment of overweight and obese patients. Robust safety data were not identified to suggest changes in their regulatory status.

Endothelin-1 inhibits the neuronal norepinephrine transporter in hearts of male rats.

OBJECTIVE: Endothelin-1 (ET-1) potentiates norepinephrine (NE)-induced contractile responses. An impairment of cardiac NE re-uptake by the neuronal NE transporter (NET) contributes to an increased NE net release in failing hearts. We hypothesized that both phenomena are caused by ET-1-mediated inhibition of NET. METHODS: [3H]-NE-uptake, electrical field stimulation-evoked NE overflow and left ventricular contractility (LV-dp/dt(max)) were measured in isolated perfused rat hearts. NET density on cardiac plasma membranes was determined by [3H]-mazindol binding. Experimental heart failure in rats was induced by transverse aortic constriction (TAC). RESULTS: ET-1 inhibited cardiac [3H]-NE-uptake in a concentration- and time-dependent manner. The endothelin A receptor (ET(A)) antagonist BQ123 but not the endothelin B receptor (ET(B)) antagonist BQ788 abolished ET-1-induced reduction of [3H]-NE-uptake. Likewise, ET-1, but not the ET(B) agonist sarafotoxin S6c, enhanced the stimulated overflow of endogenous NE. In contrast, ET-1 inhibited the stimulated NE overflow during NET blockade (exocytotic NE release) via activation of ET(B). In isovolumically contracting healthy hearts, ET-1 potentiated the NE- but not isoprenaline-induced increase in LV-dp/dt(max). Since isoprenaline is not a NET substrate, the enhanced LV-dp/dt(max) response to NE thus depends on NET. In TAC rats, ET(A) antagonism by darusentan improved both impairment of cardiac [3H]-NE-uptake and reduction of [3H]-mazindol binding sites. CONCLUSION: ET-1 inhibits cardiac NE re-uptake via ET(A) but attenuates exocytotic NE release via ET(B), resulting in opposite effects on cardiac NE net release. In healthy hearts, ET(A)-mediated inhibition of NE re-uptake exceeds ET(B)-mediated silencing of NE release and potentiates the NE-induced increase in left ventricular contractility. In TAC rats, endogenous ET-1 impairs NE re-uptake and promotes sympathetic overstimulation of failing hearts.

Neurochemical effects of chronic dietary and repeated high-level acute exposure to chlorpyrifos in rats.

Very little is known about the effects of chronic exposure to relatively low levels of anticholinesterase insecticides or how the effects of chronic exposure compare to those of higher, intermittent exposure. To that end, adult male rats were fed an anticholinesterase insecticide, chlorpyrifos (CPF), for 1 year at three levels of dietary exposure: 0, 1, or 5 mg/kg/day (0+oil, 1+oil, and 5+oil). In addition, half of each of these groups also received a bolus dosage of CPF in corn oil ("spiked" animals; 60 mg/kg initially and 45 mg/kg thereafter) every 2 months (0+CPF, 1+CPF, 5+CPF). Animals were analyzed after 6 or 12 months of dosing, and again 3 months after cessation of dosing (i.e., "recovery" animals-six experimental groups with n = 4-6/group/time point). Cholinesterase (ChE) activity was measured in retina, whole blood, plasma, red blood cells, diaphragm, and brain [pons, striatum, and the rest of the brain (referred to simply as "brain")]. Muscarinic receptor density was assessed in retina, pons, and brain, whereas dopamine transporter density and the levels of dopamine and its metabolites were assessed in striatum. Cholinesterase activity at 6 and 12 months was not different in any of the tissues, indicating that a steady state had been reached prior to 6 months. The 1+oil group animals showed ChE inhibition only in the blood, whereas the 5+oil group exhibited > or = 50% ChE inhibition in all tissues tested. One day after the bolus dose, all three groups (0+CPF, 1+CPF, 5+CPF) showed > or = 70% ChE inhibition in all tissues. Muscarinic receptor density decreased only in the brain of the 5+oil and 5+CPF groups, whereas dopamine transporter density increased only at 6 months in all three spiked groups. Striatal dopamine or dopamine metabolite levels did not change at any time. Three months after CPF dosing ended, all end points had returned to control levels. These data indicate that, although chronic feeding with or without intermittent spiked dosages with CPF produces substantial biochemical changes in a dose- and tissue-related manner, there are no persistent biochemical changes.

Perinatal heptachlor exposure increases expression of presynaptic dopaminergic markers in mouse striatum.

Although banned in the 1970s, significant levels of the organochlorine pesticide heptachlor are still present in the environment raising concern over potential human exposure. In particular, organochlorine pesticides have been linked to an increased risk of Parkinson's disease. Studies from our laboratory and others have demonstrated that exposure of laboratory animals to heptachlor alters the levels and function of the dopamine transporter (DAT), an integral component of dopaminergic neurotransmission and a gateway for the dopaminergic neurotoxin MPTP. In this study, we examined the effects of developmental exposure to heptachlor on DAT, and other key components of the dopaminergic system, including the vesicular monoamine transporter 2 (VMAT2), tyrosine hydroxylase (TH), and aromatic amino acid decarboxylase (AADC). Female C57BL/6J mice received 0 or 3mg/kg heptachlor in peanut butter every 3 days for 2 weeks prior to breeding and throughout gestation and lactation until the offspring were weaned on postnatal day (PND) 21. On postnatal day 28, DAT, VMAT2, and TH levels were increased by 100, 70, and 30%, respectively, with no change in AADC levels or total dopamine levels. The ratio of DAT:VMAT2 was increased 29%. Since an increase in the DAT:VMAT2 ratio appears to predict susceptibility of brain regions to Parkinson's disease (PD) and results in increased toxicity of MPTP, these results suggest that alterations of the dopaminergic system by developmental heptachlor exposure may increase the susceptibility of dopamine neurons to toxic insult.

Long-term effects of pergolide and (-)-deprenyl on 3H-mazindol and 3H-spiperone binding in rat brain.

The effects of long-term administration of the putative neuroprotective agents pergolide and (-)-deprenyl was assessed by studying 3H-mazindol and 3H-spiperone binding at 12 and 20 months in the major dopamine brain regions. Male Wistar rats were treated from 3 to 20 months, together with their respective untreated and saline injected control groups. The main findings were: 1) there was a decrease in both 3H-mazindol and 3H-spiperone binding with age between 12 and 20 months; 2) there were no differences at 20 months between the pergolide or the (-)-deprenyl treated groups and their controls, thus providing no evidence for long-term neuroprotection; and 3) there was a marked decrease in 3H-mazindol binding in the injected controls compared with the untreated controls at both 12 and 20 months. This raises the possibility that mild chronic stress may accelerate the aging of the dopamine system.

Intrastriatal grafts derived from fetal striatal primordia: II. Reconstitution of cholinergic and dopaminergic systems.

Reconstitution of striatal cholinergic and dopaminergic systems was studied in intrastriatal grafts derived from embryonic day 15 rat striatal primordia and implanted into adult host rats in which unilateral ibotenic acid lesions had previously been made in the striatum. Histochemical, immunohistochemical, and ligand binding autoradiographic techniques were applied to analyze different constituents of these two systems and to study their locations relative to each other in grafts allowed to grow for 9-17 months following transplantation. For the cholinergic system, a modular organization was found in the striatal grafts with stains for choline acetyltransferase and acetylcholinesterase, respectively the synthetic and degradative enzymes for cholinergic neurons; by autoradiographic [3H]hemicholinium binding, specific for high affinity choline uptake sites associated with cholinergic terminals; and by autoradiographic [3H]pirenzepine binding, selective for M1 receptors. For the dopaminergic system, a comparable modular organization was found in the grafts by immunostaining for tyrosine hydroxylase, the catecholamine synthetic enzyme; by autoradiographic [3H]mazindol binding for dopamine uptake sites; and by [3H]SCH23390 binding for dopamine D1 receptors and [3H]sulpiride binding for dopamine D2 receptors. The results indicate that the distributions of the cholinergic and dopaminergic markers in striatal grafts are in close anatomical register. These markers for intracellular and membrane-associated components of the cholinergic and dopaminergic systems were preferentially localized in the acetylcholesterase-rich patches of the grafts in which cortical and thalamic fibers have also been found in striatal grafts, and in which output neurons projecting to the pallidum are located. This anatomical correlation suggests that the substrates for cholinergic-dopaminergic interactions typical of the normal striatum may be reinstated in the grafts both in relation to efferent neurons establishing connections with the host brain that are typical of normal striatofugal connections, and in relation to major afferent fiber systems from the host brain originating in regions known to project densely to the normal striatum. Accordingly, the cholinergic and dopaminergic systems in such grafts may regulate the functional influence of the grafts on the behavior of host animals.

Localization of dopaminergic markers in the human subthalamic nucleus.

The potential role for dopamine in the subthalamic nucleus was investigated in human postmortem tissue sections by examining; (1) immunostaining for tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis; (2) binding of [(3)H]-SCH23390 (D1-like), [(3)H]-YM-09151-2 (D2-like), and [(3)H]-mazindol (dopamine uptake); and (3) expression of dopamine D1 and D2 receptor mRNAs. Immunostaining for tyrosine hydroxylase was visualized in Bouin's-fixed tissue by using a monoclonal antibody and the avidin-biotin-complex method. The cellular localization of the dopamine D1 and D2 receptor mRNAs was visualized by using a cocktail of human specific oligonucleotide probes radiolabeled with (35)S-dATP. Inspection of immunostained tissue revealed a fine network of tyrosine hydroxylase-immunostained fibers traversing the nucleus; no immunopositive cells were detected. Examination of emulsion-coated tissue sections processed for D1 and D2 receptor mRNA revealed, as expected, an abundance of D1 and D2 mRNA-positive cells in the caudate nucleus and putamen. However, no D1 or D2 receptor mRNA-expressing cells were detected in the subthalamic nucleus. Further, semiquantitative analysis of D1-like, D2-like and dopamine uptake ligand binding similarly revealed an enrichment of specific binding in the caudate nucleus and putamen but not within the subthalamic nucleus. However, a weak, albeit specific, signal for [(3)H]-SCH23390 and [(3)H]-mazindol was detected in the subthalamic nucleus, suggesting that the human subthalamic nucleus may receive a weak dopaminergic input. As weak D1-like binding is detected in the subthalamic nucleus, and subthalamic neurons do not express dopamine D1 or D2 receptor mRNAs, together these data suggest that the effects of dopaminergic agents on the activity of human subthalamic neurons may be indirect and mediated via interaction with dopamine D1-like receptors.

Effect of nicotine and amphetamine on the neurotoxicity of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice.

The present results show the potentiating effect of amphetamine on the ability of MPTP to destroy dopaminergic neurons in striatum of the mouse. A single injection of MPTP (8 mg/kg, retro-orbital) reduced the binding of [3H]mazindol, a marker for dopamine terminals, by 24%. When D-amphetamine (10 mg/kg, s.c.) was given 20 min prior to MPTP, the binding of [3H]mazindol, measured 3-5 days later, was reduced by 58%. It is proposed that the mechanism of this potentiation primarily involves an increased release of dopamine by D-amphetamine, and free radical-mediated processes. Although nicotine also releases dopamine from the striatum, no effect was observed when it was administered prior to MPTP. The lack of effect is probably related to short duration of action of nicotine and the modest effect on release of dopamine as compared to that of amphetamine.

Catecholamine-uptake inhibitors prevent the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mouse brain.

Male albino mice were injected subcutaneously with 50 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Seven days after administration, the striatal levels of dopamine(DA) and its metabolites were markedly reduced, as was the cortical level of noradrenaline (NA). Pretreatment with the selective inhibitor of DA uptake, GBR 13098, selectively and totally prevented the reduction of DA and its metabolites, whereas the selective inhibitor of NA uptake, maprotiline, selectively protected against NA depletion. The unselective inhibitors of DA and NA uptake, mazindol and nomifensine, prevented the MPTP-induced depletion of both DA and NA.

Protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity by the antioxidant ascorbic acid.

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 2 X 8 mg/kg retro-orbital) to BALB/cBy mice reduced [3H]mazindol binding to striatal membranes by 50%. Reactive oxygen derivatives have been suggested to be involved in MPTP neurotoxicity; therefore we examined the effects of ascorbic acid (an antioxidant). Ascorbic acid (100 mg/kg) given 20 min prior to MPTP administration appreciably prevented the reduction of [3H]mazindol binding. The involvement of oxidative processes in the mechanism of MPTP neurotoxicity may suggest a relationship to the etiology of Parkinson's disease, and the possible benefit of treatment with ascorbic acid.

Phencyclidine binding to striatal cocaine receptors.

PCP and related compounds inhibit 3H-mazindol binding to the cocaine receptor on dopamine transporters. The relative potencies of these compounds are such that some of the behavioral effects of PCP could be related to its action at the cocaine receptor; however, the affinity of PCP at the cocaine site (Ki = 1.59 microM) is less than its affinity at its own receptor (Ki about 0.12 microM). More data will be needed to conclusively implicate the cocaine receptor in the action of PCP.

Differences in the temperature dependence of drug interaction with the noradrenaline and serotonin transporters.

High affinity, sodium-dependent binding of [3H]mazindol is associated with the noradrenergic transporter while the binding of [3H]imipramine and [3H]paroxetine are associated with the serotonin transporter, e.g. in human platelets. In general radioligand binding studies to the monoamine transporters are performed at a temperature different from the physiological temperature (37 degrees C) at which uptake occurs. Previously reported data show a temperature dependence for tricyclic but not for nontricyclic inhibitors of the uptake of serotonin in their interaction with radioligand binding to the serotonin transporter. In the present study, both tricyclic and nontricyclic inhibitors of the uptake of noradrenaline were shown to have equal affinity for the binding of [3H]mazindol to the noradrenergic transporter at 0 degrees and 25 degrees C. Moreover, whereas serotonin transporter substrates inhibit the binding of [3H]imipramine/paroxetine in human platelets in an essentially temperature-independent manner (0-37 degrees C), noradrenaline transporter substrates are of higher affinity in inhibiting the binding of [3H]mazindol at 0 degrees C than at 25 degrees C. At 0 degrees C, substrate affinity for inhibition of the binding of [3H]mazindol to the noradrenaline transporter in the salivary gland of the rat approximates to the substrate affinity for transport by the noradrenaline carrier. The present data point to significant differences in the interaction of both substrates and inhibitors with the noradrenergic and serotonergic transporters, as studied using the radioligand binding. Thus, the pharmacological profile of the serotonin transporter, in terms of substrate and inhibitor affinity, is best studied using radioligand binding at 37 degrees C, whereas radioligand binding to the noradrenaline transporter is most representative at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemistry and biology of the 2 beta-alkyl-3 beta-phenyl analogues of cocaine: subnanomolar affinity ligands that suggest a new pharmacophore model at the C-2 position.

A series of 2 beta-alkyl-3 beta-phenyltropanes (i.e., the 2 beta-alkyl analogues of the WIN series) were prepared as analogues of cocaine and tested for their ability to displace [3H]mazindol binding and to inhibit high-affinity dopamine uptake into striatal nerve endings (synaptosomes). These 2 beta-alkyl analogues were readily prepared in optically pure form starting from cocaine by proceeding through the 2 beta-phenyl-bearing aldehyde 6 as a key intermediate. Wittig reaction of 6 with the appropriate phosphorane and hydrogenation delivered the final products. All new compounds with the exception of 8e were found to exhibit nanomolar or subnanomolar affinity for the cocaine binding site in the rat striatum. These results are in apparent opposition to the binding model previously proposed which suggests a hydrogen bond donor-acceptor interaction to be present in the vicinity of the C-2 substituent. Taken together with our previous reports and recent findings from other laboratories, we suggest a new pharmacophore model in which 2 beta-substituents lacking H-bond acceptors enhance affinity to the binding site through hydrophobic interactions. The new SAR data contained herein may be relevant to the design of possible cocaine antagonists.