No change in dopamine D1 receptor in vivo binding in rats after sub-chronic haloperidol treatment

A frequent side effect in the long-term treatment of schizophrenia with the dopamine D2 antagonist haloperidol (HAL) is the appearance of tardive dyskinesia or, in animals, of repetitive involuntary vacuous chewing movements (VCMs). In rats, chronic HAL-induced or D1 receptor-stimulated VCMs are suppressed by D1 antagonists, suggesting that this behavioral supersensitivity is mediated by D1 receptors. The goal of this study was to investigate in vivo the possible relationship between D1 receptor binding and D1-mediated behavioral supersensitivity, after subchronic HAL treatments. D1 agonist R-SKF 82957 and antagonist SCH 23390, both labeled with carbon-11, were used to assess in vivo D1 receptor binding. Rats were treated with HAL (1.5 mg/kg, i.p.) or vehicle for 21 days, followed by a 4 day washout period. No significant difference was found in the regional brain binding of either radioligand. D1 receptor-mediated behaviors including VCMs, grooming, and rearing were measured in control or HAL-treated rats. VCMs were significantly increased in HAL-treated rats, suggesting D1 receptor stimulation and possibly receptor supersensitivity. This study failed to link the purported D1 receptor-mediated behaviors with in vivo receptor binding measures of R-[11C]SKF 82957 or [11C]SCH 23390 in rat brain regions.     

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.     

Binding characteristics and sensitivity to endogenous dopamine of [11C]-(+)-PHNO, a new agonist radiotracer for imaging the high-affinity state of D2 receptors in vivo using positron emission tomography

[11C]-(+)-PHNO (4-propyl-9-hydroxynaphthoxazine) is a new agonist radioligand that provides a unique opportunity to measure the high-affinity states of the D2 receptors (D2-high) using positron emission tomography (PET). Here we report on the distribution, displaceablity, specificity and modeling of [11C]-(+)-PHNO and compare it with the well characterized antagonist D2 radioligand, [11C]raclopride, in cat. [11C]-(+)-PHNO displayed high uptake in striatum with a mean striatal binding potential (BP) of 3.95 +/- 0.85. Pre-treatment with specific D1 (SCH23390), D2 (raclopride, haloperidol) and D3 receptor (SB-277011) antagonists indicated that [11C]-(+)-PHNO binding in striatum is specific to D2 receptors. Within-subject comparisons showed that [11C]-(+)-PHNO BP in striatum was almost 2.5-fold higher than that measured with [11C]-(-)-NPA ([11C]-(-)-N-propyl-norapomorphine). Comparison of the dose-effect of amphetamine (0.1, 0.5 and 2 mg/kg; i.v.) showed that [11C]-(+)-PHNO was more sensitive to the dopamine releasing effect of amphetamine than [11C]raclopride. Amphetamine induced up to 83 +/- 4% inhibition of [11C]-(+)-PHNO BP and only up to 56 +/- 8% inhibition of [11C]raclopride BP. Scatchard analyses of [11C]-(+)-PHNO and [11C]raclopride bindings in two cats showed that the Bmax obtained with the agonist (29.6 and 32.9 pmol/mL) equalled that obtained with the antagonist (30.6 and 33.4 pmol/mL). The high penetration of [11C]-(+)-PHNO in brain, its high signal-to-noise ratio, its favorable in vivo kinetics and its high sensitivity to amphetamine shows that [11C]-(+)-PHNO has highly suitable characteristics for probing the D2-high with PET.     

Radiosynthesis and pharmacological evaluation of [11C]EMD-95885: a high affinity ligand for NR2B-containing NMDA receptors

EMD-95885, 6-[3-[4-(4-fluorobenzyl)piperidino]propionyl]-3H-benzoxazol-2-one (1) has been described as a selective antagonist for the NMDA receptors containing NR2B subunits, displaying an IC50 of 3.9 nM for this subtype. EMD-95885 (1) has been synthesized in good overall yield and labelled with carbon-11 ( T1/2 : 20.4 min) at its benzoxazolinone moiety using [11C]phosgene. The pharmacological profile of [11C]EMD-95885 ([11C]-1) was evaluated in vivo in rats with biodistribution studies and brain radioactivity monitored with intracerebral radiosensitive beta-microprobes. The brain uptake of [11C]-1 was homogeneous (0.4-0.6%ID/mL) across the different brain structures studied. This in vivo brain regional distribution of [11C]-1 was not consistent with the known distribution of NR2B subunits. Also as a measure of specificity the hippocampus/cerebellum ratio reached 0.8 throughout the time course of the experiment supporting the lack of specificity. Competition studies with the NR2B prototypic ligand ifenprodil and EMD-95885 (1), 30 min before the radioligand injection, displayed homogeneous reduction of [11C]-1 uptake of 40-60%. Pre-treatment of rats with DTG (sigma ligand), MDL105519 (glycine site antagonist) and MK801 (ion channel blocker) had no inhibitory effect on [11C]-1 uptake. Use of haloperidol as a blocking drug also resulted in a homogeneous inhibition of [11C]-1 uptake by 66-60%, which does not reflect binding to dopamine or sigma receptors. Due to the homogeneous radioligand uptake and inhibition and no measure of cerebral blood flow effects during these blocking studies it is uncertain whether any specific binding is observed. In view of these results, [11C]EMD-95885 ([11C]-1) does not have the required properties for imaging NR2B containing NMDA receptors using positron emission tomography.     

Radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide: a ligand for in vitro and in vivo studies of serotonin receptors

Radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide ([125I]-EIL) has been evaluated as a ligand for in vitro and in vivo studies of cerebral serotonin 5-HT2 receptors. [125I]-EIL exhibited high affinity (KD = 209 pM) for 5-HT2 receptors with a high degree of specific binding (80-95%) in membranes from rat prefrontal cortex. The regional distribution of [125I]-EIL binding in vivo to seven areas of mouse brain correlated significantly (Rs = 0.93) with known densities of 5-HT2 receptors. In vivo specificity, defined by tissue to cerebellum radioactivity ratios, reached a maximum for frontal cortex at 6 hr (21.2) and persisted through 16 hr (8.8). Ketanserin, a 5-HT2 receptor antagonist, fully inhibited binding in a dose dependent fashion in all brain regions except cerebellum. By contrast, blockers for dopamine D2, alpha- or beta-adrenergic receptors did not significantly inhibit radioligand binding in any region. [125I]-EIL selectively labels 5-HT2 receptors in vivo with the highest specificity of any serotonergic ligand reported to date, indicating that [123I]-EIL should prove applicable to single photon emission computed tomography studies in living brain.     

Adenosine and dopamine receptor antagonist binding in the rat ventral and dorsal striatum: lack of changes after a neonatal bilateral lesion of the ventral hippocampus.

There is experimental evidence from radioligand binding experiments for the existence of strong antagonistic interactions between different subtypes of adenosine and dopamine receptors in the striatum, mainly between adenosine A1 and dopamine D1 and between adenosine A2A and dopamine D2 receptors. These interactions seem to be more powerful in the ventral compared to the dorsal striatum, which might have some implications for the treatment of schizophrenia. The binding characteristics of different dopamine and adenosine receptor subtypes were analysed in the different striatal compartments (dorsolateral striatum and shell and core of the nucleus accumbens), by performing saturation experiments with the dopamine D1 receptor antagonist [125I]SCH-23982, the dopamine D2-3 receptor antagonist [3H]raclopride, the adenosine A1 receptor antagonist [3H]DPCPX and the adenosine A2A receptor antagonist [3H]SCH 58261. The experiments were also performed in rats with a neonatal bilateral lesion of the ventral hippocampus (VH), a possible animal model of schizophrenia. Both dopamine D2-3 and adenosine A2A receptors follow a similar pattern, with a lower density of receptors (40%) in the shell of the nucleus accumbens compared with the dorsolateral caudate-putamen. A lower density of adenosine A1 receptors (20%) was also found in the shell of the nucleus accumbens compared with the caudate-putamen. On the other hand, dopamine D1 receptors showed a similar density in the different striatal compartments. Therefore, differences in receptor densities cannot explain the stronger interactions between adenosine and dopamine receptors found in the ventral, compared to the dorsal striatum. No statistical differences in the binding characteristics of any of the different adenosine and dopamine receptor antagonists used were found between sham-operated and VH-lesioned rats.     

125]I-spectramide: a novel benzamide displaying potent and selective effects at the D2 dopamine receptor

The new substituted benzamide Spectramide, (N-[2-[4-iodobenzyl-N-methylamino]-2-methoxy-4-ethyl]-5-chloro- methylamine] benzamide) labelled with 125I was used as a potent and highly selective dopamine-D2 receptor antagonist in rat striatal homogenates for in vitro receptor binding. Kinetic experiments demonstrated the reversibility of the binding and the estimated Kd from saturation analysis was 25 pM, with a Bmax of 20 pmol/g of tissue. Competition studies showed that spectramide did not interact potently with the D1 or dopamine-uptake site. Drugs known to interact with other receptor systems were weak competitors of the binding, while binding was potently inhibited by other D2 antagonists, such as spiperone and eticlopride. These data indicate that Spectramide binds selectively and with high affinity to the dopamine D2 receptors, and may prove to be a useful tool for the study of these receptors in vivo using PET or SPECT.     

Pharmacological characterization of mammalian D1 and D2 dopamine receptors expressed in Drosophila Schneider-2 cells

Mammalian D1 and D2 dopamine receptors were stably expressed in Drosophila Schneider-2 (S2) cells and screened for their pharmacological properties. Saturable, dose-dependent, high affinity binding of the D1-selective antagonist [3H]SCH-23390 was detected only in membranes from S2 cells induced to express rat dopamine D1 receptors, while saturable, dose-dependent, high affinity binding of the D2-selective antagonist [3H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D2 receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D1 and D2 receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D1 receptor bound the (+)-enantiomer of the D1-selective agonist SKF38393 with higher affinity than the (-)-enantiomer, while the dopamine D2 receptor bound the (-)-enantiomer of the D2-selective agonist norpropylapomorphine with higher affinity than the (+)-enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPgammaS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D1 and D2 receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D1 and D2 dopamine receptors free of promiscuous G protein contaminants.     

Stereospecific binding of a new benzazepine, [3H]SCH23390, in cortex and neostriatum

The binding of the D1 antagonist SCH23390 to membrane preparations from rat cerebral cortex was examined using enantiomers of dopamine agonists and antagonists to compete with the bound [3H]SCH23390 at its Kd value. The competition curves were compared with those obtained with preparations from the neostriatum. The results demonstrate that specific [3H]SCH23390 binding in the cerebral cortex has the same pharmacological profile as in the neostriatum, so that this radioligand can be used to label dopamine D1 receptors in brain regions with a sparse dopaminergic innervation.     

Synthesis and evaluation of [¹¹C]Cimbi-806 as a potential PET ligand for 5-HT₇ receptor imaging

2-(2',6'-Dimethoxy-[1,1'-biphenyl]-3-yl)-N,N-dimethylethanamine has been identified as a potent ligand for the serotonin 7 (5-HT(7)) receptor. In this study, we describe the synthesis, radiolabeling and in vivo evaluation of [(11)C]2-(2',6'-dimethoxy-[1,1'-biphenyl]-3-yl)-N,N-dimethylethanamine ([(11)C]Cimbi-806) as a radioligand for imaging brain 5-HT(7) receptors with positron emission tomography (PET). Precursor and reference compound was synthesized and subsequent (11)C-labelling with [(11)C]methyltriflate produced [(11)C]Cimbi-806 in specific activities ranging from 50 to 300 GBq/μmol. Following intravenous injection, brain uptake and distribution of [(11)C]Cimbi-806 was assessed with PET in Danish Landrace pigs. The time-activity curves revealed high brain uptake in thalamic and striatal regions (SUV ～2.5) and kinetic modeling resulted in distribution volumes (V(T)) ranging from 6 mL/cm(3) in the cerebellum to 12 mL/cm(3) in the thalamus. Pretreatment with the 5-HT(7) receptor antagonist SB-269970 did not result in any significant changes in [(11)C]Cimbi-806 binding in any of the analyzed regions. Despite the high brain uptake and relevant distribution pattern, the absence of appropriate in vivo blocking with a 5-HT(7) receptor selective compounds renders the conclusion that [(11)C]Cimbi-806 is not an appropriate PET radioligand for imaging the 5-HT(7) receptor in vivo.     

Stimulated D(1) dopamine receptors couple to multiple Galpha proteins in different brain regions

Previous studies have revealed that activation of rat striatal D(1) dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G(s) and G(q), respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D(1) dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D(1) dopamine receptors couple differentially to multiple Galpha protein subunits. Antisera against Galpha(q) blocks dopamine-stimulated PIP(2) hydrolysis in hippocampal and in striatal membranes. The binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(i) was enhanced in all brain regions. Dopamine also increased the binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(q) in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine-stimulated binding of [(35)S]GTPgammaS to Galphas only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Galpha proteins. Dopamine induced increases in GTPgammaS binding to Galpha(s) and Galpha(q) was blocked by the D(1) antagonist SCH23390 but not by D(2) receptor antagonist l-sulpiride, suggesting that D(1) dopamine receptors couple to both Galpha(s) and Galpha(q) proteins. Co-immunoprecipitation of Galpha proteins with receptor-binding sites indicate that in the frontal cortex, D(1) dopamine-binding sites are associated with both Galpha(s) and Galpha(q) and, in hippocampus or amygdala, D(1) dopamine receptors couple solely to Galpha(q). The results indicate that in addition to the D(1)/G(s)/adenylyl cyclase system, brain D(1)-like dopamine receptor sites activate phospholipase C through Galpha(q) protein.     

Dopamine D1 receptors characterized with [3H]SCH 23390. Solubilization of a guanine nucleotide-sensitive form of the receptor

Dopamine D1 receptors were solubilized from canine and bovine striatal membranes with the detergent digitonin. The receptors retained the pharmacological characteristics of membrane-bound D1 receptors, as assessed by the binding of the selective antagonist [3H]SCH 23390. The binding of [3H]SCH 23390 to solubilized receptor preparations was specific, saturable, and reversible, with a dissociation constant of 5 nM. Dopaminergic antagonists and agonists inhibited [3H]SCH 23390 binding in a stereoselective and concentration-dependent manner with an appropriate rank order of potency for D1 receptors. Moreover, agonist high affinity binding to D1 receptors and its sensitivity to guanine nucleotides was preserved following solubilization, with agonist dissociation constants virtually identical to those observed with membrane-bound receptors. To ascertain the molecular basis for the existence of an agonist-high affinity receptor complex, D1 receptors labeled with [3H] dopamine (agonist) or [3H]SCH 23390 (antagonist) prior to, or following, solubilization were subjected to high pressure liquid steric-exclusion chromatography. All agonist- and antagonist-labeled receptor species elute as the same apparent molecular size. Treatment of brain membranes with the guanine nucleotide guanyl-5'-yl imidodiphosphate prior to solubilization prevented the retention of [3H]dopamine but not [3H]SCH 23390-labeled soluble receptors. This suggests that the same guanine nucleotide-dopamine D1 receptor complex formed in membranes is stable to solubilization and confers agonist high affinity binding in soluble preparations. These results contrast with those reported on the digitonin-solubilized dopamine D2 receptor, and the molecular mechanism responsible for this difference remains to be elucidated.     

Pituitary and brain D2 receptor density measured in vitro and in vivo in EEDQ treated male rats.

The effect of the alkylating compound N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (20 mg/kg, 24 h) on dopamine D2 receptor density in rat pituitary and brain was measured using in vitro and in vivo radioligand binding techniques. In the in vitro radioligand binding experiments EEDQ was found to reduce the density (Bmax) of [3H]-spiperone binding sites in the striatum by 86% while in the pituitary the corresponding decrease was only 37%. The affinity (KD) of the remaining striatal and pituitary D2 receptors was not different in EEDQ treated animals as compared to controls. When D2 receptor density was measured in vivo the effect of EEDQ was less pronounced. Thus, in rats given EEDQ the specific binding of either of the two D2 ligands [3H]-raclopride or [3H]-spiperone (administered in a single dose) in striatum and in the limbic forebrain was reduced by 45-62%; moreover, no significant decrease in pituitary D2 receptor density was observed. The data are discussed in relation to the finding (presented in a separate paper) that the same dose of EEDQ that failed to influence pituitary D2 receptor density as measured in vivo effectively antagonizes the prolactin decreasing effect of the partial D2 agonist (-)-3-(3-hydroxyphenyl)-N-n-propyl-piperidine [(-)-3-PPP].     

11C]cyclopropyl-FLB 457: a PET radioligand for low densities of dopamine D2 receptors

(S)-5-bromo-N-[(1-cyclopropylmethyl-2-pyrrolidinyl)methyl]-2,3-dimethoxybenzamide (4) has pico-molar in vitro binding affinity to D(2) receptor (K(i) (D(2))=0.003 nM) with lower affinity to D(3) receptor (K(i) (D(3))=0.22 nM). In this study, we describe radiosynthesis of [(11)C]4 and evaluation of its binding characteristics in post-mortem human brain autoradiography and with PET in cynomolgus monkeys. The (11)C labelled 4 was synthesized by using [(11)C]methyltriflate in a methylation reaction with its phenolic precursor with good incorporation yield (64+/-11%, DCY) and high specific radioactivity >370 GBq/micromol (>10,000 Ci/mmol). In post-mortem human brain autoradiography [(11)C]4 exhibited high specific binding in brain regions enriched with dopamine D(2)/D(3) receptors and low level of non-specific binding. In cynomolgus monkeys [(11)C]4 exhibited high brain uptake reaching 4.4% ID at 7.5 min. The binding in the extrastriatal low density D(2)-receptor regions; thalamus and frontal, parietal, temporal, and occipital cortex, was clearly visible. Pre-treatment with raclopride (1 mg/kg as tartrate) caused high reduction of binding in extrastriatal regions, including cerebellum. [(11)C]4 is a promising radioligand for imaging D(2) receptors in low density regions in brain.     

Positron-labeled dopamine agonists for probing the high affinity states of dopamine subtype 2 receptors

It is well documented that guanidine nucleotide-coupled dopamine subtype 2 receptors (D2) are configured in high and low affinity states for the dopamine agonist in vitro. However, it is still unclear whether these functional states exist in vivo. We hypothesized that positron-labeled D2 agonist and Positron Emission Tomography can be used to probe these functional states noninvasively. Recently, we demonstrated in nonhuman primates that N-[11C]propyl-norapomorphine (NPA), a full D2 agonist, is a suitable tracer for imaging the high affinity states of D2 receptors in vivo. We also developed kinetic modeling method to derive receptor parameters, such as binding potential (BP) and specific uptake ratios (V3'). When coupled with a dopamine releasing drug, amphetamine, NPA was found to be more sensitive than antagonist tracers, such as [11C]raclopride (RAC), to endogenous dopamine concentration changes (by about 42%). This finding suggests that NPA is a superior tracer for reporting endogenous DA concentration. In addition, the difference of the BP or V3' of NPA and RAC under control and amphetamine challenge conditions could be used to estimate the functional states of D2 receptors in vivo. On the basis of our findings and the assumptions that NPA binds only to the high affinity states and RAC binds equally to both affinity states, we proposed that about 70% of the D2 receptors are configured in the high affinity states in vivo.     

In vitro and in vivo binding of (E)- and (Z)-N-(iodoallyl)spiperone to dopamine D2 and serotonin 5-HT2 neuroreceptors

Apparent affinities (Ki) of (E)- and (Z)-N-(iodoallyl)spiperone [E)- and (Z)-NIASP) for dopamine D2 and serotonin 5-HT2 receptors were determined in competition binding assays. (Z)-NIASP (Ki 0.35 nM, D2; Ki 1.75 nM, 5-HT2) proved slightly more potent and selective for D2 sites in vitro than (E)-NIASP (Ki 0.72 nM, D2; Ki 1.14 nM, 5-HT2). In vivo, radioiodinated (E)- and (Z)-[125I]-NIASP showed regional distributions in mouse brain which are consonant with prolonged binding to dopamine D2 receptors accompanied by a minor serotonergic component of shorter duration. Stereoselective, dose-dependent blockade of (E)-[125I]-NIASP uptake was found for drugs binding to dopamine D2 sites, while drugs selective for serotonin 5-HT2, alpha 1-adrenergic and dopamine D1 receptors did not inhibit radioligand binding 2 hr postinjection. Specific binding in striatal tissue was essentially irreversible over the time course of the study, and (E)-[125I]-NIASP gave a striatal to cerebellar tissue radioactivity concentration of 16.9 to 1 at 6 hr postinjection. Thus, (E)-[125I]-NIASP binds with high selectivity and specificity to dopamine D2 sites in vivo.     

(+)-[C-11]-cis-N-benzyl-normetazocine: A selective ligand for sigma receptors in vivo

The in vivo biodistribution profile of the novel sigma (σ) receptor ligand (+)-[C-11]-cis-N-benzyl-normetazocine ([C-11]-(+)-NBnNM) in mouse brain was examined. This radioligand displayed high brain uptake and a distribution consistent with the density of σ receptors. Brain radioactivity levels peaked at 15 min postinjection and were largely maintained (ca. 80% of maximal values) up to 90 min postinjection. Pretreatment with several different σ ligands (haloperidol, (+)-pentazocine, DuP 734, ifenprodil) effectively inhibited [C-11]-(+)-NBnNM binding in a dose-dependent manner in all brain regions. [C-11]-(+)-NBnNM binding sites were shown to be saturable with unlabeled (+)-NBnNM (ED50 = 0.02 mg/kg) and enantioselectively inhibited by the optical isomers of pentazocine. A blocking dose of the dopamine D2 antagonist spiperone (1 mg/kg) did not significantly inhibit [C-11]-(+)-NBnNM binding. Pretreatment with the phencyclidine (PCP) blocker 1-[1-(2-thienyl)cyclohexyl] piperidine (TCP) did not significantly alter total brain tissue radioactivity. Thus, [C-11]-(+)-NBnNM binds with high specificity and selectivity to σ receptors in vivo and offers excellent potential to study σ receptors in living human brain via positron emission tomography.     

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.     

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.     

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.