The differential activities of R (+)- and S(-)-zacopride as 5-HT3 receptor antagonists

R(+)- and S(-)-zacopride were assessed as potential 5-HT3 receptor antagonists in behavioural and biochemical tests. The S(-)isomer was more potent than the R(+)isomer to antagonise the hyperactivity induced by the injection of amphetamine or the infusion of dopamine into the nucleus accumbens in the rat. In contrast, the R(+)isomer was more potent to reduce the aversive behaviour of mice to a brightly illuminated environment and in a marmoset human threat test, to facilitate social interaction in rats, to increase performance in a mouse habituation test and prevent a scopolamine-induced impairment, and to antagonise the inhibitory effect of 2-methyl-5-hydroxytryptamine to reduce [3H]acetylcholine release in slices of the rat entorhinal cortex. In binding assays, [3H]S(-)-zacopride and [3H]R(+)-zacopride labelled homogenous populations of high-affinity binding sites in the rat entorhinal cortex, R(+)-zacopride compete for a further 10 to 20% of the binding of [3H]R(+)/S(-)-zacopride or [3H]R(+)-zacopride in excess of that competed for by (S)(-)-zacopride. It is concluded that both isomers of zacopride have potent but different pharmacological activities, with the possibility of different recognition sites to mediate their effects.     

Agonist interactions with 5-HT3 receptor recognition sites in the rat entorhinal cortex labelled by structurally diverse radioligands.

1. The pharmacological properties of 5-HT3 receptor recognition sites labelled with [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 in membranes prepared from the rat entorhinal cortex were investigated to assess the presence of cooperativity within the 5-HT3 receptor complex. 2. In rat entorhinal cortex homogenates, [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 labelled homogeneous densities of recognition sites (defined by granisetron, 10 microM) with high affinity (Bmax = 75 +/- 5, 53 +/- 5, 92 +/- 6 and 79 +/- 6 fmol mg-1 protein, respectively; pKd = 9.41 +/- 0.04, 8.69 +/- 0.14, 8.81 +/- 0.06 and 10.14 +/- 0.04 for [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330, respectively, n = 3-8). 3. Quipazine and granisetron competed for the binding of each of the radioligands in the rat entorhinal cortex preparation at low nanomolar concentrations (pIC50; quipazine 9.38-8.51, granisetron 8.62-8.03), whilst the agonists, 5-hydroxytryptamine (5-HT), phenylbiguanide (PBG) and 2-methyl-5-HT competed at sub-micromolar concentrations (pIC50; 5-HT 7.16-6.42, PBG 7.52-6.40, 2-methyl-5-HT 7.38-6.09). 4. Competition curves generated with increasing concentrations of quipazine, PBG, 5-HT and 2-methyl-5-HT displayed Hill coefficients greater than unity when the 5-HT3 receptor recognition sites in the entorhinal cortex preparation were labelled with [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330. These competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]-(S)-zacopride. Competition for the binding of [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 by granisetron generated Hill coefficients around unity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation and autoradiographic localisation of 5-HT3 receptor recognition sites identified with [3H]-(S)-zacopride in the forebrain of the rat

The pharmacological characterisation and topographical distribution of [3H]-(S)-zacopride recognition sites in the forebrain of the rat was studied using homogenate and autoradiographic radioligand binding techniques. [3H]-(S)-Zacopride labelled a single, saturable, specific binding site (defined by 10.0 microM granisetron) in homogenates prepared from the entorhinal cortex of the rat (pKD = 9.51 +/- 0.08; Bmax = 104 +/- 7 fmol mg-1 protein; mean +/- SEM, n = 8). Pharmacological characterisation of the recognition site, within the entorhinal cortex, suggested that [3H]-(S)-zacopride selectively labelled the recognition site of the 5-HT3 receptor. Specific binding of [3H]-(S)-zacopride (defined by 1.0 microM granisetron) was differentially distributed throughout the forebrain of the rat; highest densities were located within sub-nuclei of the amygdala (cortical amygdaloid nucleus, amygdalohippocampal area, posterior medial cortical amygdaloid nucleus, posterior lateral amygdaloid nucleus), cortical areas (primary olfactory cortex, entorhinal cortex) and hippocampus. Non-specific binding was distributed homogeneously, although lower in myelinated structures. It is concluded that [3H]-(S)-zacopride selectively labels 5-HT3 receptor recognition sites within the forebrain of the rat; the topographical distribution of these sites, within the limbic nuclei, is consistent with the behavioural actions in animal models of the selective 5-HT3 receptor antagonists.     

Profiles of interaction of R(+)/S(-)-zacopride and anxiolytic agents in a mouse model.

The mouse black and white test box was used to measure changes in behaviour in an aversive situation where the administration of R(+)-zacopride (but not S(-)-zacopride) alone decreased aversive responding to the white area. A similar anxiolytic profile of action was observed using parachlorophenylalanine (PCPA), whose effects were antagonised by a co-treatment with R(+)-zacopride and reversed by S(-)-zacopride to an exacerbation of the aversive response. An anxiolytic profile of action was also observed using ondansetron, granisetron, chlordiazepoxide, diazepam, ritanserin, 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), E4424 (2-[4-[4-(4-chloro-l-pyrazoyl)butyl]-l-piperazinyl]-pyrimidine), umepsirone, DuP753 (2-n-butyl-4-chloro-5-hydroxy-methyl-1-[2(1H-tetrazol-5-yl) biphenyl-4-yl)methyl)]-imidazole), SQ29,852 ((S)-1-[6-amino-2[hydroxy)(4-phenyl-butyl)phosphinyl]-oxy)-1- nexy]-2-proline), devazepide and guanfacine, and this was retained following co-treatment with PCPA. The anxiolytic profile of action of PCPA was also retained following co-treatment with renzapride which when administered alone failed to modify behaviour. However, the ability of chlordiazepoxide, diazepam, ondansetron and E4424 (but not devazepide, DuP753 or SQ29,852) to reduce aversive responding was inhibited by co-treatment with R(+) and/or S(-)-zacopride. It is concluded that the reduction in aversive responding caused by pharmacological manipulation at the benzodiazepine, 5-HT receptor subtypes 5-HT1A, 5-HT1C/5-HT2 and 5-HT3 (but not at the cholecystokin CCKA or angiotensin receptors or inhibition of angiotensin converting enzyme) can be inhibited by R(+) and S(-)-zacopride. The data is discussed in terms of zacopride having an agonist or partial agonist effect at the 5-HT3 receptor.     

Inhibitors of GlyT1 affect glycine transport via discrete binding sites

In the forebrain, synaptic glycine concentrations are regulated through the glycine transporter GlyT1. Because glycine is a coagonist of the N-methyl-D-aspartate (NMDA) receptor (NMDAR), which has been implicated in schizophrenia, inhibition of GlyT1 is thought to provide an option for the treatment of schizophrenia. In support of this hypothesis, GlyT1 inhibitors facilitate in vivo NMDAR function and demonstrate antipsychotic-like effects in animal models. Among the specific GlyT1 inhibitors, substituted N-methyl-glycine (sarcosine) derivatives (e.g., (R)-N[3-(4'fluorophenyl)-3-(4'phenyl-phenoxy)propyl]-sarcosine [NFPS], (R)-N[3-phenyl-3-(4'-(4-toluoyl)phenoxy)-propyl]sarcosine [(R)-NPTS], and (R,S)-(+/-)N-methyl-N-[(4-trifluoromethyl)phenoxy]-3-phenyl-propylglycine [Org24589]), and non-sarcosine-containing inhibitors, such as 2-chloro-N-[(S)-phenyl[(2S)-piperidin-2-yl] methyl]-3-trifluoromethyl benzamide, monohydrochloride (SSR504734), have been described. In the present study, we analyzed the mode of interaction of these compounds with GlyT1 by using electrophysiological measurements in Xenopus laevis oocytes, and with two binding assays, using [(3)H](R)-NPTS or 2-chloro-N-[(S)-phenyl[(2S)-N-methylpiperidin-2-yl]-methyl]-3-trifluoromethyl benzamide monohydrochloride ([(3)H]N-methyl-SSR504734) as radioligands. Inhibition of electrogenic glycine transport by sarcosine-based compounds was apparently irreversible and independent of glycine concentration. The latter indicates a noncompetitive mode of action. In contrast, both SSR504734 and N-methyl-SSR504734 exhibited reversible and competitive inhibition of glycine transport. In GlyT1-expressing membranes, the binding of the novel radioligand [(3)H]N-methyl-SSR504734 to a single site on GlyT1 was competitively displaced by glycine and SSR504734 but noncompetitively by sarcosine-based compounds. Inversely, [(3)H](R)-NPTS binding was competitively inhibited by sarcosine-based compounds, whereas glycine, SSR504734, and N-methyl-SSR504734 noncompetitively decreased maximal binding. Our data indicate that besides exerting an apparently irreversible or reversible inhibition, GlyT1 inhibitors differ by exhibiting either a noncompetitive or competitive mode of inhibition. The divergent modes of inhibition may significantly affect the efficacy and tolerability of these drugs.     

The enantiomers of zacopride: an intra-species comparison of their potencies in functional and anxiolytic models.

1. The 5-HT3 receptor antagonist, zacopride, and its enantiomers, R(+)-zacopride and S(-)-zacopride, were examined in three pharmacological models: (i) 5-HT-induced depolarization of the mouse isolated vagus nerve preparation, (ii) the 5-HT-evoked von Bezold-Jarisch reflex in the mouse, and (iii) the mouse light:dark box model of anxiety. Other standard 5-HT3 receptor antagonists were also included for comparison in these studies. 2. Racemic zacopride, and both of the enantiomers, displayed potent 5-HT3 receptor antagonist activity in the isolated vagus nerve and in the von Bezold-Jarisch model. No 5-HT3 receptor agonist or partial agonist effects of these compounds were detected. 3. In the isolated vagus nerve, R(+)-zacopride and ondansetron were surmountable 5-HT3 receptor antagonists (pA2 values of 9.3 and 8.3, respectively), whereas racemic zacopride, S(-)-zacopride and tropisetron were insurmountable antagonists, markedly suppressing the maximum response to 5-HT. 4. In vivo, racemic zacopride, R(+)-zacopride, S(-)-zacopride and WAY100289 were potent antagonists of the 5-HT-evoked von Bezold-Jarisch reflex, with minimum effective doses (lowest dose required to reduce the reflex by > or = 85%; MED85) of 1.0, 3.0, 0.3 and 3.0 micrograms kg-1, s.c., respectively. 5. Racemic zacopride, R(+)-zacopride and S(-)-zacopride were active in the mouse light:dark box model of anxiety, with similar potencies (minimum effective dose 1 microgram kg-1, s.c.) and similar active dose-ranges (1-1000 micrograms kg-1, s.c.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential modulation of extracellular levels of 5-hydroxytryptamine in the rat frontal cortex by (R)- and (S)-zacopride.

1. The ability of various anxiolytic and potential anxiolytic agents to modify 5-hydroxytryptamine (5-HT) release in the frontal cortex of the rat was assessed by the microdialysis technique. 2. The benzodiazepine receptor agonist, diazepam (2.5 mg kg-1, i.p.), the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.32 mg kg-1, s.c.) and the 5-HT1A receptor partial agonist buspirone (4.0 mg kg-1, i.p.) maximally reduced extracellular levels of 5-HT in the rat frontal cortex by approximately 50-60%, 70-80% and 30-40%, respectively. 3. (R)-zacopride (1.0-100 micrograms kg-1, i.p.) dose-dependently reduced extracellular levels of 5-HT in the rat frontal cortex (approximately 80% maximal reduction) whereas the other 5-HT3 receptor antagonists ondansetron (10 micrograms kg-1, i.p.) and (S)-zacopride (10-100 micrograms kg-1, i.p.) were ineffective. 4. In contrast to (S)-zacopride (100 nM; administered via the microdialysis probe), (R)-zacopride (1.0-100 nM; administered via the microdialysis probe) induced a concentration-dependent reduction in extracellular levels of 5-HT in the rat frontal cortex (approximately 70% maximal reduction). 5. In contrast to ondansetron (100 micrograms kg-1, i.p.), (S)-zacopride (10-100 micrograms kg-1, i.p.) dose-dependently reversed the (R)-zacopride (10 micrograms kg-1, i.p.) induced reduction in extracellular levels of 5-HT in the rat frontal cortex. The highest dose of (S)-zacopride (100 micrograms kg-1, i.p.) completely prevented the (R)-zacopride response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Up-regulation of GABA(B) receptors by chronic administration of the GABA(B) receptor antagonist SCH 50,911

Chronic treatment of mice with the specific gamma-aminobutyric acid(B) (GABA(B)) receptor antagonist (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH 50,911) increased both the number of GABA(B) receptors in the whole brain (measured as [3H]CGP 54626 [S-(R,R)]-3-[[1-(3,4-dichlorophenyl)amino]-2-hydroxypropyl](cyclohexylmethyl)phosphinic acid hydrochloride binding) and the ability of baclofen to activate GABA(B) receptor coupled G-protein (measured as % reduction of the EC50 of baclofen to activate [35S]GTP(gamma)S binding). The results indicate that persistent blockade of GABA(B) receptors leads to their compensatory up-regulation and suggest that GABA(B) receptors are tonically activated by endogenous GABA.     

Quantitative autoradiography of [3H]piquindone binding sites (dopamine D2 receptors) in rat brain

(-)Piquindone is a new antipsychotic pyrroloisoquinoline derivative that binds to dopamine D2 receptors. We used in vitro quantitative autoradiography to determine the distribution of [3H](-)piquindone binding sites in rat forebrain. [3H](-)Piquindone binding to brain slices was sodium dependent, saturable and of high affinity (Kd = 5 nM at 0 degree C). In autoradiographic experiments, there was a good signal to noise ratio for [3H](-)piquindone binding with nonspecific binding representing only about 20% of total binding in caudate putamen. The D2 antagonists (-)sulpiride and raclopride were much more potent inhibitors of [3H](-)piquindone binding than the D1 antagonist SCH 23390. Dopamine inhibited binding with a potency similar to that previously found with standard membrane binding procedures. Autoradiography indicated that binding sites [3H](-)piquindone are localized to olfactory tubercle, accumbens nucleus, caudate putamen, cell bridges between caudate putamen and olfactory tubercle, and substantia nigra. Binding in these areas is stereoselective since we found no specific binding with [3H](+)piquindone, the biologically inactive enantiomer. Within caudate putamen, there was a lateral to medial gradient in the optical density of [3H](-)piquindone autoradiograms which might, in part, be attributable to white matter density rather than to D2 receptors.     

Mutation studies of Ser7.39 and Ser2.60 in the human CB1 cannabinoid receptor: evidence for a serine-induced bend in CB1 transmembrane helix 7

Ligands of structurally diverse natures are able to bind at the CB(1) cannabinoid receptor, suggesting the existence of multiple binding sites on the receptor. Modeling studies have implicated Ser2.60(173) and Ser7.39(383) as possible interaction site(s) for CB(1) agonists. To test the importance of these residues for receptor recognition, recombinant human CB(1) receptors, stably expressed in human embryonic kidney 293 cells, were used to investigate the consequences of mutating Ser2.60 (to S2.60A) or Ser7.39 (to S7.39A) in radioligand binding and guanosine 5'-3-O-(thio)triphosphate functional assays. The S7.39A mutant resulted in a total ablation of [(3)H](-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP55,940) high-affinity binding. However, [(3)H](R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55,212-2) binding properties at S7.39A were comparable with those of the wild-type (WT) receptor. The binding affinity of (-)-11beta-hydroxy-3-(1',1'-dimethylheptyl)hexahydrocannabinol (AM4056) and (-)-11-hydroxydimethylheptyl-Delta(8)-tetrahydrocannabinol (HU210) were drastically reduced (50- to 100-fold) at the S7.39A mutant. Likewise, the EC(50) for HU210 and AM4056-mediated activation of the S7.39A receptor was increased by >200-fold. In contrast, the binding affinity and potency of WIN55,212-2, CP55,940, HU210, and AM4056 were unaltered at the S2.60A mutant compared with WT human CB(1) receptors. These results clearly suggest that Ser7.39, but not Ser2.60, plays a crucial role in mediating ligand specific interactions for CP55,940, HU210, and AM4056 at the human CB(1) receptor. Our modeling studies predict that Ser7.39 in a g-chi1 conformation may induce a helix bend in TMH7 that provides docking space for CP55,940 binding; the S7.39A mutation may alter this binding space, precluding CP55,940 binding.     

The interaction of R(+)- and S(-)-zacopride with PCPA to modify rodent aversive behaviour.

The interaction of R(+)- and S(-)zacopride (0.00001-10 mg/kg i.p.) with parachlorophenylalanine (PCPA, 3 day treatment 100 mg/kg i.p.) to modify behaviour in an aversive situation was investigated in the mouse black and white test box and rat social interaction test. R(+)-Zacopride (but not S(-)zacopride) and PCPA had an anxiolytic profile of action to reduce aversive responding in both species. Their established anxiolytic profiles were abolished by a subsequent treatment with S(-)zacopride. In contrast, S(-)-zacopride was less or ineffective if administered simultaneously with R(+)-zacopride. A co-treatment of PCPA with R(+)-zacopride also inhibited the anxiolytic profiles observed to the individual treatments. It is concluded that there is a complex interaction between the isomers of zacopride to modify responding to an aversive situation that is dependent on the temporal sequence of drug administration, and which may be modified by changes in basal 5-hydroxytryptamine (5-HT) tone and agonist, partial agonist and antagonist effects at the 5-HT3 receptor.     

Labelling of human platelet alpha 2-adrenoceptors with the full agonist [3H](-)adrenaline

[3H](-)Adrenaline has been used to characterize alpha 2-adrenoceptors on human platelets. Although (-)adrenaline is a good substrate for the platelet enzyme MAO-B, enzymatic inhibition was not a prerequisite to quantify the specific binding of the radioligand to platelet membranes. At 25 degrees C the binding was rapid (t1/2 of association: 10.3 min), reversible (t1/2 of dissociation: 4.0 min) and linearly dependent on the amount of protein present in the assay. The binding sites for [3H](-)adrenaline showed the specificity required for an alpha 2-adrenoceptor. The rank order of potency of inhibitors of [3H](-)adrenaline binding was oxymetazoline greater than idazoxan congruent to phentolamine congruent to clonidine congruent to (-)adrenaline greater than (-)noradrenaline greater than yohimbine much much greater than phenylephrine much greater than prazosin greater than (+)propranolol. Moreover, the nucleotide guanosine triphosphate (GTP) inhibited in a concentration-dependent manner (10(-9)-10(-4) M) the specific binding of [3H](-)adrenaline, suggesting that the radioligand preferentially labelled the high affinity state of the alpha 2-adrenoceptor. Linear (Scatchard) and non-linear analyses of [3H](-)adrenaline binding indicated the existence of a single population of non-interacting sites (KD = 2.5-2.7 nM; Bmax = 49-53 fmol/mg protein). The binding characteristics for [3H](-)adrenaline were not affected by age and sex of the donors or by freezing of platelet-rich plasma. In the same subjects alpha 2-adrenoceptor density (Bmax) for the full agonist [3H](-)adrenaline was 2.9-fold lower than that quantitated by the selective antagonist [3H]yohimbine. The inhibition constants (Ki) of adrenergic drugs and of various antidepressant drugs in competing with [3H](-)adrenaline were correlated with the inhibition constants of these drugs in competing with [3H]clonidine (r = 0.96; P less than 0.001) which suggests that both radioligands labelled the same alpha 2-adrenoceptor on the human platelet. The binding of the full agonist [3H](-)adrenaline to human platelet membranes might be a useful tool for the study of dysfunctions related to the high affinity state of the alpha 2-adrenoceptor.     

Pseudoproline-containing analogues of morphiceptin and endomorphin-2: evidence for a cis Tyr-Pro amide bond in the bioactive conformation.

Analogues of the opioid peptides [D-Phe(3)]morphiceptin (H-Tyr-Pro-D-Phe-Pro-NH(2)) and endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH(2)) containing the pseudoproline (Psi Pro) (4R)-thiazolidine-4-carboxylic acid (Cys[Psi(R1,R2)pro]) or (4S)-oxazolidine-4-carboxylic acid (Ser[Psi(R1,R2)pro]) in place of Pro(2) were synthesized. The pseudoproline ring in these compounds was either unsubstituted (R(1), R(2) = H) or dimethylated (R(1), R(2) = CH(3)) at the 2-C position. 2-C-dimethylated pseudoprolines are known to be quantitative or nearly quantitative inducers of the cis conformation around the Xaa(i-1)-Xaa(i)[Psi(CH(3),CH)(3)pro)] imide bond. All dihydropseudoproline-containing analogues (R(1), R(2) = H) showed good mu opioid agonist potency in the guinea pig ileum (GPI) assay, high mu receptor binding affinity in the rat brain membrane binding assay, and, like their parent peptides, excellent mu receptor binding selectivity. (1)H NMR spectroscopic analysis of the Cys[Psi(H,H)pro](2)- and Ser[Psi(H,H)pro](2)-containing analogues in DMSO-d(6) revealed that they existed in a conformational equilibrium around the Tyr-Xaa[Psi(H,H)pro] peptide bond with cis/trans ratios of 40:60 and 45:55, respectively. The dimethylated thiazolidine- and oxazolidine-containing [D-Phe(3)]morphiceptin- and endomorphin-2 analogues (R(1), R(2) = CH(3)) all retained full mu agonist potency in the GPI assay and displayed mu receptor binding affinities in the nanomolar range and high mu receptor selectivity. As expected, no conformers of the latter analogues with a trans conformation around the Tyr-Xaa[Psi(CH(3),CH(3)pro)] imide bond were detected by (1)H NMR spectral analysis, indicating that in these compounds the cis conformation is highly predominant (>98%). These results represent the most direct evidence obtained so far to indicate that morphiceptin and endomorphin-2 have the cis conformation around the Tyr-Pro peptide bond in their bioactive conformations.     

Synthesis, biodistribution, and primate imaging of fluorine-18 labeled 2beta-carbo-1'-fluoro-2-propoxy-3beta-(4-chlorophenyl)tr opanes. Ligands for the imaging of dopamine transporters by positron emission tomography

2beta-(R)-Carbo-1-fluoro-2-propoxy-3beta-(4-chlorophenyl) tro pane ((R)-FIPCT, R-6) and 2beta-(S)-carbo-1-fluoro-2-propoxy-3beta-(4-chlorophenyl) tro pane ((S)-FIPCT, S-6) were prepared and evaluated in vitro and in vivo for dopamine transporter (DAT) selectivity and specificity. High specific activity [(18)F](R)-FIPCT and [(18)F](S)-FIPCT were synthesized in 5% radiochemical yield (decay-corrected to end of bombardment (EOB)) by preparation of the precursors 2beta-carbo-R-1-mesyloxy-2-propoxy-3beta-(4-chlorop hen yl)tropane (R-12) and 2beta-carbo-S-1-mesyloxy-2-propoxy-3beta-(4-chlorop hen yl)tropane (S-12) followed by treatment with no carrier-added potassium[(18)F]fluoride and kyrptofix K222 in acetonitrile. Competition binding in cells stably expressing the transfected human DAT and serotonin transporter (SERT) labeled by [(3)H]WIN 35428 and [(3)H]citalopram, respectively, demonstrated the following order of DAT affinity (K(i) in nM): GBR 12909 (0.36) > CIT (0.48) > (S)-FIPCT (0.67) > (R)-FIPCT (3.2). The affinity of (S)-FIPCT and (R)-FIPCT for SERT was 127- and 20-fold lower, respectively, than for DAT. In vivo biodistribution studies were performed in male rats and demonstrated that the brain uptake of [(18)F](R)-FIPCT and [(18)F](S)-FIPCT were selective and specific for DAT rich regions (caudate and putamen). PET brain imaging studies in monkeys demonstrated high [(18)F](R)-FIPCT and [(18)F](S)-FIPCT uptake in the caudate and putamen which resulted in caudate-to-cerebellum and putamen-to-cerebellum ratios of 2.5-3.5 at 115 min. [(18)F](R)-FIPCT uptake in the caudate/putamen achieved transient equilibrium at 75 min. In an imaging experiment with [(18)F](S)-FIPCT in a rhesus monkey with its left hemisphere lesioned with MPTP, radioactivity was reduced to background in the caudate and putamen of the lesioned hemisphere. The high specific activity one-step radiolabeling preparation and high specificity and selectivity of [(18)F](R)-FIPCT and [(18)F](S)-FIPCT for DAT indicate [(18)F](R)-FIPCT and [(18)F](S)-FIPCT are potential radioligands for mapping brain DAT in humans using PET.     

Potent cannabinergic indole analogues as radioiodinatable brain imaging agents for the CB1 cannabinoid receptor

A series of novel aminoalkylindoles was synthesized in an effort to develop compounds that are potent agonists at the CB1 cannabinoid receptor and that are also easily labeled with radioisotopes of iodine for biochemical and imaging studies. 2-Iodophenyl-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl]methanone (8, AM2233) had a very high affinity for the rat CB1 receptor, with most of the affinity residing with the (R)-enantiomer. Radioiodinated 8, (R)-8, and (S)-8 were prepared by radioiododestannylation of the tributyltin analogues in high yields, radiochemical purities, and specific radioactivities. In a mouse hippocampal membrane preparation with [131I](R)-8 as radioligand, racemic 8 exhibited a K(i) value of 0.2 nM compared with 1.6 nM for WIN55212-2. In autoradiographic experiments with mouse brain sections, the distribution of radioiodinated 8 was consistent with that of brain CB1 receptors. Again, very little specific binding was seen with the (S)-enantiomer [131I](S)-8 and none occurred with the (R)-enantiomer [131I](R)-8 in sections from CB1 receptor knockout mice. Radioiodinated 8 thus appears to be a suitable radioligand for studies of CB1 cannabinoid receptors.     

Zacopride and its optical isomers produce stereoselective antagonism of a 2-methylserotonin discriminative stimulus.

The serotonin 5-HT3 receptor antagonist effects of zacopride and its optical isomers were evaluated in rats trained to discriminate 5.0 mg/kg of 2-methylserotonin (2-Me 5-HT) from saline in a 2-lever operant task. Zacopride and its enantiomers potently antagonized the 2-Me 5-HT stimulus; on the basis of 50% inhibition (ID50) of drug lever responding the order of potency is S(-)-zacopride (ID50 = 0.05 micrograms/kg) greater than (+/-)-zacopride (ID50 = 0.60 micrograms/kg) greater than R(+)-zacopride (ID50 = 1.6 micrograms/kg). The stereoselectivity and potency ratio of zacopride's isomers for antagonizing the 2-Me 5-HT stimulus parallels their previously reported results in binding studies. It is concluded that zacopride's isomers produce antagonism of a 2-Me 5-HT stimulus by stereoselective interaction at 5-HT3 receptors and that stereochemical factors are important in evaluating the stimulus properties of 5-HT3 agents.     

Effects of in vivo and in vitro treatments with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline on putative muscarinic receptor subtypes in rat brain

N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was found to irreversibly decrease the Bmax of [3H](-)-quinuclidinyl-benzilate [(-)QNB] binding in rat brain following in vivo administration or by incubation of tissue homogenates with EEDQ in vitro. A greater reduction in the Bmax of [3H](-)QNB binding was observed in the hippocampus and cortex than in the brainstem following in vivo or in vitro treatment with EEDQ. Competition of pirenzepine for [3H](-)QNB binding was best described by computer-derived models assuming two binding sites in all brain regions. However, following EEDQ treatment there was a rightward shift in the pirenzepine competition curves for the remaining [3H](-)QNB-binding sites in all brain regions. Computer analysis of the pirenzepine competition curves indicated that this was due to a selective decrease in the number of [3H](-)QNB-binding sites having high affinity for pirenzepine. Although the binding of [3H](-)QNB to the site having lower affinity for pirenzepine was apparently unaltered, the affinity of pirenzepine for this binding site was significantly lowered following both in vivo and in vitro treatment with EEDQ. Thus, EEDQ differentially modifies muscarinic receptor-binding sites having high and low affinity for pirenzepine. The reduction in the Bmax of [3H](-)QNB binding and the rightward shift in the pirenzepine competition curve elicited by EEDQ both in vivo and in vitro could be prevented by coadministration of reversible muscarinic antagonists, thereby demonstrating that EEDQ interacts at the ligand recognition site of muscarinic receptors. These data suggest that the putative muscarinic receptor subtypes discriminated by pirenzepine may represent differences in the accessibility of pirenzepine and EEDQ to a homogeneous population of [3H](-)QNB-binding sites or, alternatively, that these muscarinic receptor-binding sites discriminated by pirenzepine and EEDQ represent structurally distinct molecular entities.     

Muscarinic cholinoceptors in the rabbit's myometrium: a study of the relationship between binding and response

The relationship between [3H](-)-quinuclinidyl benzilate [( 3H](-)QNB) binding and muscarinic cholinoceptors in the myometrium of the oestrogen treated rabbit was studied. [3H](-)QNB binding was specific, saturable and reversible. The ability of muscarinic agonists and antagonists and some of their stereoisomers to inhibit [3H](-)QNB binding and to stimulate or inhibit contraction of the myometrium were almost identical suggesting that [3H](-)QNB binds specifically to the muscarinic cholinoceptor in this tissue. There appeared to be no receptor reserve in this tissue and whilst structural and stereospecific requirements for binding were almost identical to those for the muscarinic cholinoceptor in the guinea pig ileum requirements for activation were not. Competition binding experiments with pirenzepine suggested that antagonist binding sites were heterogeneous whilst those with 4-diphenylacetoxy-N-methyl-piperidine MeBr did not. Results were discussed with regard to simple and complex models of muscarinic cholinoceptor interactions.     

Alpha 2-adrenoceptor in HT29 human colon adenocarcinoma cell-line: study of [3H](-)-adrenaline binding

The HT29 human adenocarcinoma cell-line was used to investigate the binding of [3H](-)-adrenaline at alpha 2-adrenoceptors. All aspects of the study indicated that alpha 2-adrenoceptors were specifically labeled. [3H](-)-adrenaline bound with high affinity (KD = 2.28 +/- 0.41 nM) to a single population of non-interacting sites. The rank order of adrenoceptor antagonists (yohimbine greater than phentolamine much greater than prazosin) and agonists (UK-14,304 greater than clonidine greater than (-)-adrenaline greater than (-)-noradrenaline greater than (+)-adrenaline greater than (+)-noradrenaline greater than amidephrine) to compete with [3H](-)-adrenaline binding showed that the labeled sites were alpha 2-selective and stereospecific. Comparison of the binding parameters of [3H](-)-adrenaline with those of [3H]clonidine (partial-agonist) and [3H]yohimbine (antagonist) indicated that [3H](-)-adrenaline and [3H]clonidine labeled a similar number of sites (156 +/- 13 versus 175 +/- 21 fmol/mg protein) and that [3H]yohimbine (Bmax = 246 +/- 22 fmol/mg protein) labeled more sites than the 3H-agonists. Data on the inhibition of [3H]yohimbine binding by (-)-adrenaline was better fitted to a two-site model and revealed (1) that the KiL/KiH ratio was higher for (-)-adrenaline than for clonidine (2) that both agonists recognized the same percentage of high-affinity receptors. The results from a kinetic study of [3H](-)-adrenaline binding were apparently inconsistent with the equilibrium data. Both the association and dissociation were bi-exponential, suggesting a relative heterogeneity of the labeled sites. The tritiated physiological full-agonist was moreover able to induce tight-binding. The practical consequences of this property are discussed.     

Kappa-opioid receptors are differentially labeled by arylacetamides and benzomorphans

Using Chinese Hamster Ovary cell membranes that stably expressed the human kappa-opioid receptor, we investigated the hypothesis that kappa(1)- and kappa(2)-opioid receptors, historically defined by their pharmacological selectivity for either arylacetamides or benzomorphans are, in fact, different affinity states or binding sites on the same kappa-opioid receptors. Receptor binding studies showed that GTP gamma S potently inhibited [3H](5 alpha,7 alpha,8 beta)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro [4.5]dec-8-yl)-benzeneacetamide (U69,593) binding, compared to virtually no inhibition of [3H]bremazocine binding. Saturation binding experiments showed a three-fold decrease in [3H]U69,593 affinity in the presence of GTP gamma S, but GTP gamma S had no effect on [3H]bremazocine affinity. The kappa-opioid receptor antagonist nor-binaltorphimine had a four-fold higher affinity for [3H]U69,593-labeled receptors than for [3H]bremazocine-labeled receptors. Functional selectivity studies, measuring the stimulation of [35S]GTP gamma S agonist-induced binding, showed a significantly higher U69,593-induced G protein-receptor activation in comparison to the stimulation observed with bremazocine. These results suggest that pharmacologically defined 1 kappa-opioid receptor subtypes may be different affinity states of the same receptor.