Novel drug interactions at D(2) dopamine receptors: modulation of [3H]quinpirole binding by monoamine oxidase inhibitors

D(2) dopamine receptors are the principal target of drugs used to treat schizophrenia and Parkinson's disease. Recent findings suggest novel drug interactions at D(2) receptors, specifically interactions of monoamine oxidase inhibitors (MAOIs) at a novel binding site that modulates the binding of [3H]quinpirole to the D(2) receptor. That MAOIs inhibit [3H]quinpirole binding challenges the traditional understanding of ligand interactions at dopamine receptors and may shed light on the mechanism of behavioral sensitization to psychostimulants and the pharmacology and toxicity of MAOIs.     

Photoaffinity labelling of dopamine D2 receptors by [3H]azidomethylspiperone

We have characterized the dopamine D2 receptor photoaffinity probe, [3H]azido-N-methylspiperone ([3H]AMS). In the absence of light, [3H]AMS bound reversibly and with high affinity (Kd 70 pM) to sites in canine striatal membranes and was competitively inhibited by dopaminergic agonists and antagonists with an appropriate D2 receptor specificity. Upon photolysis, [3H]AMS covalently incorporated into a peptide of Mr 92,000 as assessed by fluorography following SDS-polyacrylamide gel electrophoresis. Labelling of this peptide was specifically and stereoselectively blocked by D2 antagonists and agonists. Minor specifically labelled peptides of Mr 70,000-55,000 were observed under some conditions and were the result of proteolytic degradation of the peptide at Mr 92,000.     

Modulation of [3H]dopamine release from rabbit retina

The calcium-dependent release of [3H]dopamine ([3H]DA) elicited by field stimulation or potassium is modulated through activation of stereoselective inhibitory DA autoreceptors of the D-2 subtype that are pharmacologically different from the D-1 DA receptor subtype linked to the stimulation of adenylate cyclase (EC 4.6.1.1). The D-2 DA autoreceptors appear to be endogenously activated by DA because DA receptor antagonists such as S-sulpiride increased the stimulation-evoked release of [3H]DA. Nanomolar concentrations of norepinephrine (NE) and epinephrine (E) inhibited in a concentration-dependent manner the electrical stimulation-evoked release of [3H]DA. The inhibitory effect of these catecholamines was not modified by S-sulpiride, which, on the contrary, selectively antagonized the inhibition of [3H]DA release elicited by exogenous DA. Phentolamine or (+/-)-propranolol did not affect the release of [3H]DA from rabbit retina. The alpha antagonist phentolamine competitively antagonized the inhibitory effect of both NE and E, which suggests that these catecholamines activate alpha receptors in retina. The decrease by catecholamines of the calcium-dependent release of [3H]DA appears not to involve beta adrenoceptors because their inhibitory effect was not modified by propranolol. Under identical experimental conditions (i.e., nomifensine, 30 microM), serotonin did not modify the stimulated release of [3H]DA. In conclusion, in the rabbit retina, DA autoreceptors of the D-2 subtype appear to modulate endogenously released DA whereas inhibitory presynaptic alpha receptors might be of pharmacological importance as sites of action for retinal or blood-borne catecholamines.     

Specific [3H]raclopride binding to neostriatal dopamine D2 receptors: Role of disulfide and sulfhydryl groups

Receptor binding studies were performed in rabbit neostriatum (caudate-putamen) using the dopamine D2 antagonist [³H]raclopride. Treatment of the membrane preparations with the reducing agent L-dithiothreitol (L-DTT) as well as with the alkylating compoundN-ethylmaleimide (NEM), produced dose-dependent decreases of specific [³H]raclopride binding; the IC₅₀ values were of 3.1 and 1.2 mM, respectively. Saturation experiments showed that the reduction of disulfide (-S-S-) bonds by L-DTT (1 mM) decreased the number of binding sites, with only a slight increase in the affinity. On the other hand, alkylation of sulfhydryl (-SH) groups by NEM (1mM) decreased both receptor number and affinity. The properties of the remaining binding sites were examined in competition curves with the physiological substrate dopamine and the dopaminergic antagonist (+)butaclamol. The IC₅₀ values for (+)butaclamol in control and in L-DTT and NEM treated membranes were between 3.4 and 4.8 nM, with Hill coefficients (n_H) of 1, indicating that the remaining binding sites conserved a high affinity for antagonist binding. In the case of dopamine, the curves were shallow (n_H 0.45–0.64) and both compounds increased the IC₅₀ from 0.7 M (control) to 8 M and 11 M, for L-DTT and NEM respectively. Iterative analysis revealed that L-DTT produced a very important (>60%) decrease in the number of high-affinity (R_H) binding. After NEM, there was a decrease in both the number of (R_H) and the affinity (K_H) of the high-affinity binding sites, and in the affinity (K_L) of the low-affinity sites. These results demonstrate the participation of-S-S- and-SH groups in the agonist conformation of theprimary ligand recognition site of the dopamine D2 receptor. Alternatively,-S-S-and-SH groups could be related to the coupling of the primary ligand recognition protein with adenylate cyclase by means of an inhibitory type ofG protein.     

High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.     

Regulation of agonist and antagonist binding to striatal D-1 dopamine receptors: studies using the selective D-1 antagonist [3H]SK&F R-83566

The potent and D-1 versus D-2 selective dopamine receptor antagonist, SK&F R-83566, was radiolabelled with tritium and was used as a radioligand for examination of D-1 receptors in rat striatum. Binding of the radioligand was stereoselective, saturable and reversible. In homogenates of rat striatum, nonspecific binding of the radioligand was less than 5% of total binding, the KD was 1.1 +/- 0.2 nM and the Bmax was 1130 +/- 70 fmoles/mg protein. Results of competition binding analyses yielded a pharmacological profile that was characteristic of dopamine D-1 receptor interaction. Competition studies of dopamine agonists against the potent antagonist radioligand indicated multiple affinities of agonist binding to the D-1 receptor. Displacement was best fit to a two-site model of ligand binding and high and low affinities were subject to regulation by guanine, but not adenine, nucleotides. Antagonist binding was not complex and was unaffected by guanine nucleotides. The role of monovalent cations in regulating D-1 receptor binding was evaluated by comparing effects of Na+, Li+, and K+ on binding of the antagonist [3H]SK&F R-83566 and the agonist [3H]fenoldopam (SK&F 82526). Whereas agonist binding was reduced in a concentration dependent fashion by monovalent cations with a ranking of potency Li+ greater than Na+ greater than K+, antagonist binding was enhanced by the cation Na+ but little affected by Li+ or K+. This effect of relatively low concentrations of Na+ to decrease agonist binding and increase antagonist binding suggests similarities between the D-1 receptor which is positively-coupled to adenylate cyclase and other receptors, e.g. alpha 2 adrenergic receptors, which are negatively-coupled to adenylate cyclase.     

Neophobia and cortical and subcortical binding of the dopamine D2 receptor antagonist [3H]-raclopride

The potential role of dopamine system in response to novelty was analysed using the selective dopamine D2 receptor antagonist, raclopride, in behavioral and biochemical assays, in rats (the open field test, and specific binding of [3H]-raclopride, within different brain structures measured with autoradiography). It was found that raclopride at a low dose (50 microg/kg, IP) caused anxiolytic-like effect (increased the anti-thigmotactic index), whereas at a higher dose (500 microg/kg, IP) produced general inhibitory influence, and decreased the anti-thigmotactic index. Analysis of the behavioral and biochemical results of the experiment revealed a significant negative correlation between the ligand binding in the substantia nigra pars reticulata (SNR), and the number of entries into the central sector of the open field (r=-0.48, p<0.05), as well as the positive correlation between time spent in the central sector of the open field and [3H]-raclopride binding within nucleus accumbens septi (r=0.57, p<0.05). Factor analysis revealed a Factor 1 (eigenvalue=3.361) grouping parameters of central entries into the open field and [3H]-raclopride binding in the SNR (factor loadings are 0.814 and 0.703 respectively), indicating that both phenomena are under control of a similar central process. The above data are discussed in relation to the structure dependent dopamine D2 receptor mechanisms in a rat response to novelty.     

Synthesis and activity of 2-[4-(4-[3H]-2-cyanophenyl)piperazinyl]-N-(2,4,6-[3H]3-3-methylphenyl)acetamide: a selective dopamine D4 receptor agonist and radioligand

The first selective dopamine D4 agonist radioligand is described. The synthesis of these piperazine radioligands relied on the transformation of brominated precursors 4a and 4b with tritium gas in the presence of a sensitive cyano functional group. The specific activity of these two radioligands was measured and [3H]6b found to be suitable for use in D4 saturation and competition binding studies. The synthesis, biological, and radioactivity of this new agonist radioligand as well as preliminary SAR will be discussed.     

Ascorbic acid and striatal transport of [3H] 1-methyl-4-phenylpyridine (MPP+) and [3H] dopamine

The inhibition of uptake of [3H] dopamine and [3H] 1-methyl-4-phenylpyridine (MPP+) was examined in mouse striatal synaptosomal preparations. Kinetic analysis indicated that ascorbic acid is a noncompetitive inhibitor of [3H] MPP+ uptake. No inhibition of [3H] dopamine uptake is observed. The dopamine uptake blockers, GBR-12909, cocaine, and mazindol strongly inhibit (IC50 less than 1 uM) both [3H] dopamine and [3H] MPP+ transport. Nicotine, its metabolites, and other tobacco alkaloids are weak inhibitors (IC50 greater than 1 mM) except 4-phenylpyridine and lobeline, which are moderate inhibitors (IC50 = 3 to 40 uM) of both [3H] dopamine and [3H] MPP+ uptake. These similarities in potencies are in agreement with the suggestion that [3H] MPP+ and [3H] dopamine are transported by the same carrier. The differences observed in the alteration of dopaminergic transport and mazindol binding by ascorbic acid suggest that ascorbic acid's effects on [3H] MPP+ transport are related to translocation and/or dissociation processes occurring subsequent to the initial binding event.     

Effect of neuropeptide-EI on the binding of [3H]SCH 23390 to the dopamine D1 receptor in rat striatal membranes

We have previously demonstrated that neuropeptide-EI, at high doses, stimulates the production of cAMP, in caudate putamen, through the activation of adenylate cyclase coupled to specific D₁ receptors. The aim of the present work was to find evidences for a probable interaction between this neuropeptide and the dopamine D₁ receptor in the mammalian central nervous system. The present data show that neuropeptide-EI, at high concentrations, affected both the maximum binding and the apparent affinity of [n-methyl-³H] (R)-(+)-8 chloro-2,3,4,5- tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hemimaleate to the dopamine D₁ receptor in a concentration-dependent manner.     

Pharmacological evaluation of (+)-2- [123I]A-69024: a radioligand for in vivo studies of dopamine D1 receptors

(+)-2-[123I] A-69024, [(+)-1-(2-[123I] iodo-4,5-dimethoxy-benzyl)-7-hydroxy-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline], is a specific and enantioselective dopamine D1 receptor radioligand. The in vivo biodistribution of this radioligand in rats showed high brain uptake and a distribution consistent with the density of dopamine D1 receptors. Highest uptake was observed in the striatum (0.65 %ID/g) at 5 min followed by clearance. As a measure of specificity the striatum/cerebellar ratio reached a maximum of 3.9 at 30 min post-injection. Radioactivity in the striatum was reduced by 68% by pre-administration of the D1 antagonist SCH 23390. Pre-administration of other dopamine binding drugs, spiperone (D2), 7-OH-DPAT (D3), and clozapine (D4) displayed no inhibitory effect on (+)-2-[123I]A-69024 accumulation in any brain region. Ketanserin (5-HT2/5-HT2C) and haloperidol (D2 receptor antagonist/sigma receptor ligand) also displayed no inhibitory effect in any brain region studied. With the pharmacologically inactive enantiomer, (-)-2-[123I]A-69024, the brain uptake was determined to be non-specific since a striatum/cerebellar ratio of near 1 was observed throughout the time course of the experiment. (+)-2-[123I]A-69024 displays enantioselectivity for dopamine D1 receptors and may deserve further investigation as a possible SPECT radioligand.     

Assessment of striatal D1 and D2 dopamine receptor-G protein coupling by agonist-induced [35S]GTP gamma S binding

The dopamine receptor-mediated modulation of guanosine 5'-O-(gamma-[35S]thio)triphosphate ([35S]GTP gamma S) binding has been characterized in rat striatal membranes. In optimized experimental conditions, the potency of dopamine was 4.47 microM [3.02-6.61 microM] and a maximal response representing 54.8 +/- 4.5% increase above basal level was observed. Data obtained with different agonists and antagonists clearly revealed that the most important fraction of this response was reflecting D2 receptor activation. Further analysis with specific antagonists also supported evidence for the involvement of D1 dopamine receptors. The potencies of compounds interacting with D1 and D2 receptors were deduced from [35S]GTP gamma S binding experiments and compared with their binding affinities for these receptors measured in similar experimental conditions. A good correlation between these parameters was observed, supporting the applicability of this technique for the study of dopamine receptors in the central nervous system.     

Modulation by D1 and D2 dopamine receptors of ATP-induced release of intracellular Ca2+ in cultured rat striatal neurons

The aim of the present study was to investigate, whether dopamine D1 and/or D2 receptors are able to interfere with the ATP-induced increase of the intracellular Ca2+ concentration ([Ca2+]i) in cultured striatal neurons identified by their morphological characteristics and their [Ca2+]i transients in response to a high-K+ superfusion medium. ATP appeared to release Ca2+ mostly from an intracellular pool, since its effect was markedly depressed in the presence of cyclopiazonic acid, which is known to deplete such storage sites [Rubini, P., Pinkwart, C., Franke, H., Gerevich, Z., N?renberg, W., Illes, P., 2006. Regulation of intracellular Ca2+ by P2Y1 receptors may depend on the developmental stage of cultured rat striatal neurons. J. Cell. Physiol. 209, 81-93]. The mixed D1/D2 receptor agonist dopamine increased the ATP-induced [Ca2+]i transients in a subpopulation of neurons. At the same time, dopamine did not alter the responses to K+ in these cells. The selective D1 (SKF 83566) and D2 (sulpiride) receptor antagonists failed to modify the effect of ATP, but unmasked in the previously unresponsive neurons an inhibitory and facilitatory effect of dopamine, respectively. A combination of the two antagonists resulted in a failure of dopamine to modulate the [Ca2+]i responses in any cell investigated. In conclusion, D1 and D2 receptors may modulate in an opposite manner the signalling pathways of P2Y1 receptors in striatal neurons and thereby alter their development/growth or their cellular excitability and/or the release of GABA from their terminals.     

Dopamine-stimulated cyclic AMP and binding of [3H] dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [3H] dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1.10(-8) M and was half-maximal at 7.9 +/- 3.4 10(-7) M. The increase at 1.10(-5) M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1.10(-9) M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC50 value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1.10(-5) M dopamine was 2.3 +/- 0.9.10(-6) M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [3H] Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37 degrees C, it was rapid, reversible, saturable and stereospecific. The Kd value for high affinity binding sites was 0.43 +/- 0.1.10(-7) M and 4.7 +/- 1.6.10(-7) M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2 +/- 0.4.10(-7) M epinine, 4.1 +/- 1.8.10(-6) M fluphenazine, 8.0 +/- 1.6.10(-6) M haloperidol, 4.2 +/- 1.2.10(-6) M cis-flupenthixol, 2.7 +/- 4.0.10(-5) M trans-flupenthixol, less than 1.10(-5) M apomorphine, sulpiride, naloxone and isoproterenol.     

Inhibition by theanine of binding of [3H]AMPA, [3H]kainate,and [3H]MDL 105,519 to glutamate receptors

In an investigation of the mechanisms of the neuroprotective effects of theanine (gamma-glutamylethylamide) in brain ischemia, inhibition by theanine of the binding of [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, and [3H](E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519) to glutamate receptors was studied in terms of its possible inhibiting effects on the three receptor subtypes (AMPA, kainate, and NMDA glycine), with rat cortical neurons. Theanine bound the three receptors, but its IC50 of theanine was 80- to 30,000-fold less than that of L-glutamic acid.     

Fluoroethoxy-1,4-diphenethylpiperidine and piperazine derivatives: Potent and selective inhibitors of [3H]dopamine uptake at the vesicular monoamine transporter-2

A small library of fluoroethoxy-1,4-diphenethyl piperidine and fluoroethoxy-1,4-diphenethyl piperazine derivatives were designed, synthesized and evaluated for their ability to inhibit [³H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and dopamine transporter (DAT), [³H]serotonin (5-HT) uptake at the serotonin transporter (SERT), and [³H]dofetilide binding at the human-ether-a-go-go-related gene (hERG) channel. The majority of the compounds exhibited potent inhibition of [³H]DA uptake at VMAT2, Ki changes in the nanomolar range (K_i?=?0.014–0.073?µM). Compound 15d exhibited the highest affinity (K_i?=?0.014?µM) at VMAT2, and had 160-, 5-, and 60-fold greater selectivity for VMAT2 vs. DAT, SERT and hERG, respectively. Compound 15b exhibited the greatest selectivity (>60-fold) for VMAT2 relative to all the other targets evaluated, and 15b had high affinity for VMAT2 (K_i?=?0.073?µM). Compound 15b was considered the lead compound from this analog series due to its high affinity and selectivity for VMAT2.     

Characterization of [3H]LS‐3‐134, a novel arylamide phenylpiperazine D3 dopamine receptor selective radioligand

LS‐3‐134 is a substituted N‐phenylpiperazine derivative that has been reported to exhibit: (i) high‐affinity binding (K_i value 0.2 nM) at human D3 dopamine receptors, (ii) > 100‐fold D3 versus D2 dopamine receptor subtype binding selectivity, and (iii) low‐affinity binding (K_i > 5000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin‐dependent activation of the adenylyl cyclase inhibition assay, LS‐3‐134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [³H]‐labeled LS‐3‐134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10–15 min at 37°C) and binds with high affinity to both human (K_d = 0.06 ± 0.01 nM) and rat (K_d = 0.2 ± 0.02 nM) D3 receptors expressed in HEK293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [³H]LS‐3‐134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies, we propose that [³H]LS‐3‐134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype.     

Effects of mercury compounds on the spontaneous and potassium-evoked release of [3H]dopamine from mouse striatal slices

The effects of mercury compounds on the spontaneous and potassium-evoked release of [3H]dopamine from mouse striatal slices have been examined. All mercury compounds examined produced concentration-dependent increases in the spontaneous release of [3H]dopamine, with an order of potency of methylmercury greater than mercuric (Hg2+) mercury greater than p-choloromercuribenzene sulfonic acid. Methylmercury had no effect on the 25 mM potassium evoked release of [3H]dopamine in the presence of 1.3 mM calcium. However, in calcium-free conditions, methylmercury significantly increased the potassium-evoked release of [3H]dopamine. Mercuric mercury significantly reduced the 25 mM potassium evoked release of [3H]dopamine in the presence of 1.3 mM calcium, and this response was not reversible with brief washing of the tissue. In calcium-free conditions, mercuric mercury significantly elevated the evoked release of [3H]dopamine, similar to the result obtained with methylmercury. It is suggested that mercury compounds alter dopaminergic synaptic function, possibly by disrupting calcium homeostasis or calcium-dependent processes, and that methylmercury and mercuric mercury can have differential effects to alter dopaminergic neurotransmission.     

Analogs of SR-141716A (Rimonabant) alter d-amphetamine-evoked [3H] dopamine overflow from preloaded striatal slices and amphetamine-induced hyperactivity

The CB(1) cannabinoid receptor antagonist SR-141716A (Rimonabant) markedly diminishes the behavioral effects of opiates and nicotine and has been an important tool to ascertain the role of cannabinoid receptors in drug addiction. The present goal was to determine the less-explored interaction of SR-141716A and d-amphetamine in neurochemical and behavioral assays. Additionally, the effect of the substituents and substitution patterns on the phenyl ring located at the 5 position of SR-141716A (4-chlorophenyl), and of the CB(1)/CB(2) cannabinoid receptor agonist WIN-55,212-2, was determined. SR-141716A, AM-251 (4-iodophenyl) and NIDA-41020 (4-methoxyphenyl) did not alter amphetamine-evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine. MRI-8273-30-1 (4-fluorophenyl; 0.1-10 microM) attenuated amphetamine (3 microM)-evoked [(3)H]overflow, and MRI-8273-59 (3,4-dichlorphenyl; 0.01-10 microM) augmented amphetamine (0.3-3 microM)-evoked [(3)H]overflow. WIN-55,212-2 was without effect. In a locomotor activity experiment, SR-141716A and MRI-8273-30-1 did not alter amphetamine-induced hyperactivity. However, MRI-8273-59 (1-3 mg/kg) dose-dependently attenuated amphetamine (1 mg/kg)-induced hyperactivity. The present results suggest that SR-141716A is less efficacious to alter amphetamine effects than its reported efficacy to diminish the effects of opiates and nicotine. Modification of the 5-phenyl position of SR-141716A affords compounds that do interact with amphetamine in vitro and in vivo.