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Biomedical subjects

R B Rothman

Publications and source records attributed to R B Rothman.

At least 145 records · Page 8Linked to original sources
PubMed1994

An electrophilic affinity ligand based on (+)-MK801 distinguishes PCP site 1 from PCP site 2.

The electrophilic affinity ligand, (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycl ohepten-5,10 - imine hydrochloride [(+)-MK801-NCS] was characterized for its ability to acrylate phencyclidine (PCP) and sigma binding sites in vivo. Initial studies, conducted with mouse brain membranes, characterized the binding sites labeled by [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP). The Kd values of [3H]TCP for PCP site 1 (MK801-sensitive) and PCP site 2 (MK801-insensitive) were 12 nM and 68 nM, with Bmax values of 1442 and 734 fmol/mg protein, respectively. Mice were sacrificed 18-24 hours following intracerebroventricular administration of the acylator. The administration of (+)-MK801-NCS increased [3H]TCP binding to site 2, but not to site 1. Although (+)-MK801-NCS decreased [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d; ccyclohepten-5,10-imine maleate ([3H](+)-MK801) binding to site 1, it had no effect on [3H]TCP binding to site 1. Viewed collectively with other published data, these data support the hypothesis that PCP sites 1 and 2 are distinct binding sites, and that [3H]TCP and [3H](+)-MK801 label different domains of the PCP binding site associated with the NMDA receptor.

Affinity Labels↗
PubMed1994

Lack of evidence for context-dependent cocaine-induced sensitization in humans: preliminary studies.

Cocaine-induced behavioral sensitization is the well-documented phenomenon where repeated doses of cocaine elicit increasingly greater effects on motoric activity in rats. Some observations suggest that behavioral sensitization may provide a model for understanding the mechanisms of drug-craving elicited by environmental triggers or cues. The process of fully validating such an animal model for its ability to detect effective anticraving medicines is a difficult and long-term undertaking. As a first step in that direction, we decided to determine if cocaine can produce conditioned behavioral sensitization in humans using a paradigm fairly similar to that used for rodents. Because humans do not react to cocaine with the pronounced motor activation observed in rodents, we measured a variety of end points, including blood pressure (BP), heart rate (HR), respiratory rate, pupil diameter, hormones (prolactin and cortisol), and subjective responses using the questionnaire for drug-related feelings (QDRF) and the EEG. To mimic the home and test cages used in rodent studies, two rooms were used: a small test chamber and a regular room with a window and furnishings. On day 1 each subject received a drug infusion (either saline or 40 mg cocaine IV) in both locations. On day 2, all subjects received an infusion (saline or 25 mg cocaine IV) in the test chamber. All drug infusions were conducted double blind. The paired group received cocaine on both days in the test chamber. The unpaired group received cocaine in regular room on day 1, and cocaine in the test chamber on day 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Effects of electroconvulsive shock on the retention of cocaine-induced conditioning.

The purpose of these studies was to determine if ECS is capable of preventing the retention of cocaine-induced conditioned increases in locomotor behavior. One group of rats (paired) was injected with 40 mg/kg of cocaine immediately before a 30 min exposure to a locomotor monitoring chamber while the other group (unpaired) was injected with saline prior to a similar exposure. One hour following return to their home cages, the paired rats were injected with saline while the unpaired animals were injected with 40 mg/kg of cocaine. On day 2, both groups were injected with 10 mg/kg of cocaine and returned to the test apparatus. The presence of conditioned cocaine effects are indicated by enhanced locomotor output in the paired group relative to the unpaired group on day 2. ECS delivered immediately following training on day 1 was effective in preventing the retention of conditioning. ECS delivered 1 h prior to training, 1 h after or 1 h before on day 2 were ineffective. Cocaine-induced conditioning appears to involve associative learning that can be disrupted by ECS delivered immediately following training.

Animals↗
PubMed1994

Low affinity of FMRFamide and four FaRPs (FMRFamide-related peptides), including the mammalian-derived FaRPs F-8-Famide (NPFF) and A-18-Famide, for opioid mu, delta, kappa 1, kappa 2a, or kappa 2b receptors.

The binding affinities at opioid receptor subtypes in rat or guinea pig brain membranes were determined for the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2), the two mammalian-derived FMRFamide-related peptides F-8-Famide (NPFF; Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe- NH2) and A-18-Famide (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe -NH2), and the two other FMRFamide-related peptides Tyr-Phe-Met-Arg-Phe-NH2 (Tyr-FMRFamide) and Pro-Gln-Arg-Phe-NH2 (Pro-Gln-RFamide). The mu and delta sites were labeled in rat brain membranes using tritiated [D-Ala2, N-MePhe4,Gly-ol5] enkephalin ([3H]DAMGO) and [D-Ala2,D-Leu5]enkephalin ([3H]DADLE), respectively. The kappa sites were labeled in guinea pig brain using [3H]U-69,593 after treatment with BIT and FIT for kappa 1 and [3H]bremazocine after pretreatment with BIT and FIT for kappa 1 and [3H]bremazocine after pretreatment with BIT and FIT for kappa 2. The kappa 2a binding sites were assayed using [Leu5]enkephalin to block kappa 2b sites and the kappa 2b sites were assayed using (-)-(1S,2S)-U50,488 to block kappa 2a sites. Neither FMRFamide nor any of the FMRFamide-related peptides (up to 61.0 microM) displayed significant affinity at any of the subtypes of opioid receptor. Hence, the known ability of FMRFamide and FaRPs to interact with the opioid system does not appear to be related to direct binding to these opioid receptors.

Amino Acid Sequence↗
PubMed1994

Studies of the biogenic amine transporters. III. Demonstration of two binding sites for [3H]GBR12935 and [3H]BTCP in rat caudate membranes.

The present study addressed the hypothesis that there exist multiple sites/states associated with the dopamine (DA) transporter ligands. The authors used [3H](1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) (GBR12935) and [3H]-N-(1-(2-benzo[b]thiophenyl)cyclohexyl)piperidine ([3H]BTCP) to label binding sites present in striatal membranes and conducted experiments under nearly identical assay conditions, i.e., 18- to 24-hr incubations at 4 degrees C in 55.2 mM sodium phosphate buffer, pH 7.4, with a protease inhibitor and antioxidant cocktail. To obtain data suitable for quantitative curve fitting, it was necessary to repurify the [3H]ligands periodically by high-performance liquid chromatography. Under these conditions, greater than 90% specific binding was observed. The method of binding surface analysis was used to characterize the interaction of GBR12935, BTCP, mazindol and 2 beta-carbomethoxy-3 beta-(4-fluorophenyl) tropane with binding sites labeled by the [3H]ligands. Nonlinear least-squares curve fitting of the data to one- and two-site binding models demonstrated that, for both [3H]ligands, the two-site model fit the data far better than did the one-site model. The results indicated that [3H]GBR12935 labeled two binding sites, with higher (GBR site 1) and lower affinity (GBR site 2) for BTCP (Ki values of 5.84 nM and 1394 nM). [3H]BTCP labeled two sites with high affinity (Kd values of 9.3 nM and 6.3 nM) at which GBR12935 also had high affinity (Ki values of 8.9 nM and 0.98 nM). Intrastriatal 6-hydroxy-DA lesions decreased the density of both [3H]GBR12935 binding sites but affected site 1 much more than site 2, which indicated that a greater portion of GBR site 1 is localized on striatal nerve terminals. By contrast, the 6-hydroxy-DA lesions decreased BTCP site 2 without significantly decreasing BTCP site 1, which indicated that BTCP site 1 is not located on DA nerve terminals. The i.c.v. administration of 5,7,-dihydroxytryptamine did not decrease GBR site 1 or 2, which indicated that neither is located on serotonin striatal nerve terminals. Viewed collectively with other reports, these data support the hypothesis that DA transporter ligands label multiple binding sites in caudate membranes. The identification of selective agents for these sites may be valuable tools for identifying drugs that might modulate the effects of cocaine.

Animals↗
PubMed1994

GBR12909 attenuates cocaine-induced activation of mesolimbic dopamine neurons in the rat.

Previous studies have shown that the dopamine (DA) reuptake inhibitor 1-(2-[bis(4-fluorophenyl)-[methoxy]ethyl)-4-(3-phenylpropyl) piperazine (GBR12909) antagonizes the increase in extracellular DA evoked by local perfusion of cocaine into the striatum. In the present work, in vivo microdialysis methods were used to examine the effects of i.v. cocaine, GBR12909 and combinations of the two drugs on DA overflow in the nucleus accumbens of awake rats. Both cocaine and GBR12909 (0.3, 1.0 and 3.0 mg/kg) caused dose-related elevations in extracellular DA when given alone. However, the temporal profile of DA overflow was different with each drug. Cocaine caused a rapid and short-lived increase in DA, whereas GBR12909 caused a slow and sustained elevation of transmitter. In drug combination studies, the rise in extracellular DA after a modest dose of cocaine (1.0 mg/kg) was significantly reduced from 250% to 175% of baseline by pretreatment with a subthreshold dose of GBR12909 (0.3 mg/kg). A high dose of cocaine (3.0 mg/kg) increased dialysate DA by 600%; this rise in DA was decreased to 450% and 325% of baseline by pretreatment with 0.3 and 1.0 mg/kg of GBR12909, respectively. The neurochemical effect of the combination of GBR12909 plus cocaine was clearly not additive. GBR12909 also blocked the DA-releasing action of amphetamine (1.0 mg/kg). Our findings show that GBR12909 antagonizes the rise in extracellular DA produced by systemic cocaine and these results provide further evidence that DA reuptake inhibitors may be useful pharmacological adjuncts in the treatment of cocaine addiction and withdrawal in human patients.

Amphetamine↗
PubMed1994

Studies of the biogenic amine transporters. IV. Demonstration of a multiplicity of binding sites in rat caudate membranes for the cocaine analog [125I]RTI-55.

The drug 3 beta-[4'-iodophenyl]tropan-2 beta-carboxylic acid methyl ester (RTI-55) is a cocaine congener with high affinity for the dopamine transporter (Kd < 1 nM). The present study characterized [125I]RTI-55 binding to membranes prepared from rat, monkey and human caudates and COS cells transiently expressing the cloned rat dopamine (DA) transporter. Using the method of binding surface analysis, two binding sites were resolved in rat caudate: a high-capacity binding site (site 1, Bmax = 11,900 fmol/mg of protein) and a low-capacity site (site 2, Bmax = 846 fmol/mg of protein). The Kd (or Ki) values of selected drugs at the two sites were as follows: (Ki for high-capacity site and Ki for low-capacity site, respectively): RTI-55 (0.76 and 0.21 nM), 1-[2-diphenyl-methoxy)ethyl]-4-(3-phenylpropyl)piperazine (0.79 and 358 nM), mazindol (37.6 and 631 nM), 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (45.0 and 540 nM) and cocaine (341 and 129 nM). Nisoxetine, a selective noradrenergic uptake blocker, had low affinity for both sites. Serotonergic uptake blockers had a high degree of selectivity and high affinity for the low-capacity binding site (Ki of citalopram = 0.38 nM; Ki of paroxetine = 0.033 nM). The i.c.v. administration of 5,7-dihydroxytryptamine to rats pretreated with nomifensine (to protect dopaminergic and noradrenergic nerve terminals) selectively decreased the Bmax of site 2, strongly supporting the idea that site 2 is a binding site on the serotonin (5-HT) transporter. This serotonergic lesion also increased the affinity of [125I]RTI-55 for the DA transporter by 10-fold. The ligand selectivity of the caudate 5-HT transporter was different from the [I125]RTI-55 binding site on the 5-HT transporter present in membranes prepared from whole rat brain minus caudate. The [125I]RTI-55 binding to the DA transporter was further resolved into two components, termed sites 1a and 1b, by using human and monkey (Macaca mulatta) caudate membranes but not the membranes prepared from rat caudate or COS cells that transiently expressed the cloned cocaine-sensitive DA transporter complementary DNA. Similar experiments also resolved two components of the caudate 5-HT transporter. Viewed collectively, these data provide evidence that [125I]RTI-55 labels multiple binding sites associated with the DA and 5-HT transporters.

Animals↗
PubMed1993

Effects of acute and chronic cocaine on the activity of tuberoinfundibular dopamine neurons in the rat.

We investigated the effects of acute and chronic cocaine on dopamine (DA) synthesis in tuberoinfundibular dopamine (TIDA) neurons by measuring the accumulation of 3,4-dihydroxyphenylalanine (DOPA) in the median eminence (ME). Cocaine-induced prolactin (PRL) responses were evaluated for comparison. Acute cocaine inhibited DA synthesis in the ME and decreased circulating PRL. Chronic cocaine did not alter basal DOPA levels in TIDA nerve terminals or PRL levels in plasma. After repeated cocaine injections, cocaine's inhibitory effect on DA synthesis was abolished in the ME while the PRL response to drug was unchanged. These results suggest that TIDA neurons become tolerant to cocaine-induced suppression of DA synthesis after chronic cocaine exposure, but the altered sensitivity of TIDA neurons is not revealed by plasma PRL measures.

Animals↗
PubMed1993

Identification of a GBR12935 homolog, LR1111, which is over 4,000-fold selective for the dopamine transporter, relative to serotonin and norepinephrine transporters.

The di-substituted piperazines, GBR12909 (1-[2-[bis(4-fluorophenyl)-methoxy]ethyl]-4-[3- phenylpropyl]piperazine) and GBR12935 (1-[2-(diphenyl-methoxy)-ethyl]-4-(3-phenylpropyl)piperazine), are potent and selective (20-to 100-fold) inhibitors of [3H]dopamine reuptake, relative to [3H]5-HT and [3H]norepinephrine uptake. The GBR12935 analog, 1-(2-(diphenylmethoxy)ethyl)-4-(3-phenylpropyl)homopiperazine (LR1111), was synthesized as part of a systematic structure-activity study of analogs of GBR12935 and GBR12909. LR1111 differs from GBR12935 by the addition of a methylene group into the piperazine ring to yield a compound with a seven-member homopiperazine ring. The IC50 values for LR1111 at the dopamine, norepinephrine, and serotonin transporters were 7.2 nM, 34,072 nM, and greater than 20,000 nM, respectively, whereas the IC50 values of GBR12935 were 3.7 nM, 289 nM, and 1261 nM for these same transporters. This demonstrates that the addition of a single methylene group in the piperazine ring results in a compound with similar affinity but significantly higher selectivity for the dopamine transporter. LR1111 increased motoric activity in rats after intravenous administration. These indicate that LR1111 is a potent and highly selective inhibitor of the dopamine transporter.

Animals↗
PubMed1993

Biosynthesis of dopamine and serotonin in the rat brain after repeated cocaine injections: a microdissection mapping study.

The purpose of the present study was to examine the effects of chronic cocaine on dopamine (DA) and serotonin (5-HT) synthesis in several rat brain regions implicated in drug reinforcement. Male rats were treated twice daily with cocaine (15 mg/kg, ip) or saline for 1 week. After 42 hr of abstinence, rats were challenged with either cocaine (15 mg/kg, ip) or saline, followed by the aromatic L-amino acid decarboxylase inhibitor 3-hydroxybenzylhydrazine (NSD-1015; 100 mg/kg, ip). Animals were decapitated 30 min after NSD-1015 and discrete brain regions were microdisected from 300 microns frozen sections. Postmortem tissue levels of 3,4-dihydroxyphenylalanine (DOPA) and 5-hyroxytryptophan (5-HTP) were quantified by HPLC with electrochemical detection and used to estimate biosynthesis of DA and 5-HT, respectively. In chronic saline-treated rats, cocaine dramatically suppressed DA and 5-HT synthesis in all forebrain regions examined, including: medial prefrontal cortex, nucleus accumbens, caudate nucleus, olfactory tubercle, and basolateral amygdala. The degree of inhibition ranged from 35-65% and was more pronounced in 5-HT neurons compared to DA neurons in the same tissue sample. In general, chronic cocaine did not significantly alter basal levels of DOPA or 5-HTP; a notable exception was lateral hypothalamus, where chronic cocaine reduced basal DA synthesis to 75% of control. After repeated cocaine injections, the synthesis-inhibiting effect of a challenge injection of cocaine was attenuated in many brain areas. These data suggest that whereas acute cocaine decreases DA and 5-HT synthesis in forebrain, chronic cocaine is not neurotoxic to DA and 5-HT neurons. In addition, the mechanism(s) mediating cocaine-induced suppression of monoamine synthesis may become desensitized by chronic exposure to the drug.

5-Hydroxytryptophan↗
PubMed1993

Stereochemical requirements for pseudoirreversible inhibition of opioid mu receptor binding by the 3-methylfentanyl congeners, RTI-46144 and its enantiomers: evidence for different binding domains.

Fentanyl and its congeners are of interest not only because of their clinical applications, but also because certain members of this series of opioid analgesics exhibit unique properties, such as acting as pseudoirreversible inhibitors of mu receptor binding, both in vitro and in vivo. Previous studies showed that pretreatment of membranes with (+)-cis-3-methylfentanyl resulted in a lower affinity interaction of [3H]ohmefentanyl with the mu binding site, as well as an increased dissociation rate. The present study was undertaken to determine the stereochemical requirements for pseudoirreversible inhibition of mu receptor binding using the methylfentanyl congeners, (+-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide HCl (RTI-4614-4) and its four resolved enantiomers. AR configuration of the 2-hydroxy group was essential for high affinity binding and pseudoirreversible inhibition. The two enantiomers with this configuration, 1b((2R,3R,4S)-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide oxolate) and 1c 1c ((2R,3S,4R)-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide HCl), acted as pseudoirreversible inhibitors of the mu receptor as labeled with [3H][D-Ala2-MePhe4,Gly-ol5]enkaphalin, [3H]fentanyl or [3H]etorphine. RTI-4614-4, 1b, and 1c decreased the Bmax of [3H][D-Ala2-MaePhe4,Gly-ol5]enkepalin binding sites without altering the dissociation rate. These drugs had a lesser effect on steady-state [3H]fentanyl and [3H]etorphine binding but did produce statistically significant changes in the parameters of the two-component dissociation model, which accurately described the dissociation of these [3H]ligands. Viewed collectively, these data indicate that the mechanism of the pseudoirreversible inhibition appears to depend on the radioligand used to label the mu receptor. To explain these data, a pseudoallosteric model is proposed that postulates that certain mu ligands bind to different domains of the drug recognition site of the mu receptor and that the prebinding of pseudoirreversible inhibitors to the recognition site changes the domains available to a radioligand, leading to alterations in steady-state binding levels and dissociation kinetics.

Animals↗
PubMed1993

Studies of the biogenic amine transporters. II. A brief study on the use of [3H]DA-uptake-inhibition to transporter-binding-inhibition ratios for the in vitro evaluation of putative cocaine antagonists.

The cocaine receptor on the dopamine transporter is a logical target binding site for the design and synthesis of novel agents for evaluation as possible cocaine antagonists. Although there is no widely accepted and validated assay for detecting a cocaine antagonist, one commonly accepted strategy is to compare the IC50 value of a test agent for inhibition of [3H]dopamine uptake and its IC50 value for inhibition of the binding of a transporter ligand such as [125I]RTI-55. The goal of such a comparison is to guide the synthesis of agents which have high "uptake-to-binding ratios", i.e. agents which are much more potent in the binding assay than they are in the uptake assay. In the present study we tested the hypothesis that ratios different from unity can result from the fact that the two assays are conducted under markedly different conditions. The results showed that conducting the uptake and binding assays under identical conditions reduced the GBR12935 uptake-to-binding ratio of 6.20 (under standard assay conditions) to 0.36. These data indicate that uptake-to-binding ratios must be interpreted with caution, and emphasizes the need for simpler and less expensive methods than cocaine self-administration paradigms to screen compounds as modulators of cocaine reinforcement.

Animals↗
PubMed1992

Probes for narcotic receptor mediated phenomena. 18. Epimeric 6 alpha- and 6 beta-iodo-3,14-dihydroxy-17-(cyclopropylmethyl)-4,5 alpha-epoxymorphinans as potential ligands for opioid receptor single photon emission computed tomography: synthesis, evaluation, and radiochemistry of [125I]-6 beta-iodo-3,14-dihydroxy-17-(cyclopropylmethyl)-4,5 alpha-epoxymorphinan.

The epimeric 6 beta- and 6 alpha-iodo-3,14-dihydroxy-17-(cyclopropylmethyl)-4,5 alpha-epoxymorphinans (1, ioxy) and (2, epioxy), respectively, were each synthesized in five steps starting with naltrexone. The configuration of the 6-iodo group of 1 was unequivocally determined to be beta-based on single crystal X-ray analysis of its precursor 3-acetoxy-6 beta-iodo-14-hydroxy-17-(cyclopropylmethyl)-4,5 alpha-epoxymorphinan (10). Both 1 and 2 as well as their corresponding 3-O-acetates 10 and 11 were found to readily cross the blood-brain barrier and completely reverse the analgesic effects of a 10 mg/kg intraperitoneal dose of morphine sulfate as determined by the paw withdrawal latency test. Compounds 1 and 2 were found to bind with high affinity to mu, delta, and kappa receptors in vitro. In general, 1 and 2 exhibited higher affinity for mu and kappa receptors than naltrexone while the 6 beta-iodo epimer 1 (ioxy) was more potent than its epimer 2. In a comparison of the 6 beta-halogen substituent on binding affinity across opioid receptor subtypes, it was generally found that I greater than Br greater than F. On the basis of the results of in vitro and in vivo testing, 1 was selected as a target for radioiodination and evaluation as a potential single photon emission computed tomography imaging agent for opioid receptors. Carrier-free [125I]-1 was synthesized in near quantitative yield by the sequence of reaction of excess 3-acetoxy-6 alpha-[[(trifluoromethyl)sulfonyl]oxy]-14-hydroxy-17- (cyclopropylmethyl)-4,5 alpha-epoxymorphinan (8) with anhydrous Na125I in dry acetonitrile for 90 min at 76 degrees C followed by deacetylation of the product with 1:1 aqueous ammonia/acetonitrile at 25 degrees C. The potential of [125I]-1 as an in vivo imaging agent for opioid receptors is evaluated and discussed.

Animals↗
PubMed1992

A review of the role of anti-opioid peptides in morphine tolerance and dependence.

Studies on the mechanisms of tolerance and dependence have mostly focused on changes at the receptor level. These experiments, conducted with model systems ranging from clonal cell lines to whole animals, have identified a number of important adaptive mechanisms which occur at the receptor level. However, none of these adaptive mechanisms can completely account for the phenomena which serve to define the state of morphine tolerance and dependence, especially the observation that as an animal becomes more tolerant to morphine, less naloxone is required to trigger withdrawal. The data reviewed in this paper provide strong support for the hypothesis that the brain synthesizes and secretes neuropeptides which act as part of a homeostatic system to attenuate the effects of morphine and endogenous opioid peptides. According to this model, administration of morphine releases anti-opioid peptides (AOP), which then attenuate the effects of morphine. As more morphine is given, more AOP are released, thereby producing tolerance to the effects of morphine. Cessation of morphine administration, or administration of naloxone, produces a relative excess of anti-opioid, which is in part responsible for the withdrawal syndrome. Since endogenous and exogenous antagonists might together produce synergistic effects, less naloxone might be required to trigger withdrawal in the presence of higher levels of AOPs. Although the study of AOP is in its infancy, a deeper understanding of the central nervous system (CNS) anti-opioid systems may lead to new treatments for chronic pain, substance abuse, and psychiatric disorders.

Animals↗
PubMed1992

MPTP lesions of the nigrostriatal dopaminergic projection decrease [3H]1-[1-(2-thienyl)cyclohexyl]piperidine binding to PCP site 2: further evidence that PCP site 2 is associated with the biogenic amine reuptake complex.

Our previous studies have demonstrated that, using membranes of guinea pig brain, [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) labels not only the phencyclidine binding site associated with the NMDA receptor (PCP site 1), but also a second high affinity binding site which is associated with the biogenic amine reuptake carrier (termed PCP site 2). To test this hypothesis, the binding of [3H]GBR12935 to the dopamine transporter, and [3H]TCP binding to PCP sites 1 and 2 were measured in caudates harvested from control, MPTP-treated and reserpine-treated dogs. MPTP treatment decreased dopamine levels by over 99%, decreased [3H]GBR12935 binding by over 90%, decreased [3H]TCP binding to PCP site 2 by about 50%, and had no significant effect on [3H]TCP binding to PCP site 1. These data are consistent with the hypothesis that a portion of PCP site 2 is associated with dopaminergic nerve terminals in dog caudate.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1992

[3H] cocaine labels a binding site associated with the serotonin transporter in guinea pig brain: allosteric modulation by paroxetine.

We studied the characteristics of [3H]cocaine binding to membranes prepared from whole guinea pig brain. Cocaine binding was specific and saturable. A one-site binding model fit the data adequately: the Kd value of [3H]cocaine was 44 nM with a Bmax value of 280 fmol/mg protein. The rank order of potency for the [3H]cocaine binding site was paroxetine > clomipramine > (-)-cocaine > fluoxetine > mazindol > desipramine > GBR12909 > phencyclidine > benztropine > GBR12935 > (+)-cocaine. The IC50 values of these drugs for inhibition of [3H]cocaine binding were highly correlated with their IC50 values for inhibition of [3H]5-HT uptake into synaptosomes prepared from whole guinea pig brain. High affinity 5-HT uptake inhibitors produced dose-dependent wash-resistant (pseudoirreversible) inhibition of [3H]cocaine binding. The wash-resistant inhibition produced by paroxetine was due to an increase in the Kd of [3H]cocaine binding sites, and was accompanied by an increase in the dissociation rate, consistent with an allosteric mechanism. These studies suggest that, using membranes prepared from whole guinea pig brain, [3H]cocaine labels a binding site associated with serotonin transporter and that paroxetine and cocaine bind to different sites on the serotonin transporter.

Allosteric Regulation↗