Influence of neonatal treatment with the pyrethroid insecticide cypermethrin on the development of dopamine receptors in the rat kidney

The influence of neonatal treatment with the pyrethroid insecticide cypermethrin ((R,S)alpha-cyano-3-phenoxybenzyl (1R,S)-cis-trans-3-(2,2-dichloro-vinyl)-2,2-dimethylcyclopropane carboxylate) on postnatal development of renal dopamine receptors was investigated by radioligand binding assay techniques. Treatment with cypermethrin was made on rats from the 10th to the 16th day after birth. Dopamine D1- and D2-like receptors were assayed in frozen sections of kidney of 21-, 30-, 60- and 90-day-old rats using as ligands of dopamine D1- and D2-like receptors [3H]([R](+)-(chloro-2,3,4,5,-tetrahydro-5-phenyl-1,4,-benzazepinal hemimaleate) (SCH 23390) and [3H]spiperone, respectively. Treatment with cypermethrin was without effect on the affinity (Kd value) or the density (Bmax value) of dopamine D1- and D2-like receptors of rats of 21 days of age. In older groups, treatment with the compound reduced the affinity and increased the density of dopamine D1-like receptors, whereas it was without effect on the affinity of dopamine D2-like receptors and decreased their density. These findings indicate that neonatal treatment with the pyrethroid insecticide cypermethrin induces long-lasting impairment of renal dopamine D1- and D2-like receptors and that kidney is a target of the toxic action of the compound. Renal dopamine receptor changes caused by cypermethrin are consistent with possible alterations of renal tubular function and of sympathetic neuroeffector modulation. The above data suggest also that, different from the adult, neonatal exposure to pyrethroid insecticides may induce toxic effects.     

In vitro and in vivo characterization of R(+)-FIDA2: a dopamine D2-like imaging agent

R(+)-FIDA2, (R)-(+)-2,3-dimethoxy-5-iodo-N-[(1-(4'-fluorobenzyl)-2-pyrrolid iny l)- methyl]benzamide, is a new dopamine D2-like receptor imaging agent that can be labeled with either 123I or 18F for SPECT or PET imaging. The purpose of this study was to characterize its in vitro and in vivo binding properties. METHODS: In vitro binding studies using [125I]R(+)-FIDA2 were performed in Sf9 cells expressing dopamine D2 or D3 receptors and in rat basal forebrain homogenates, which contain a high density of dopamine D2-like receptors. A series of in vivo SPECT imaging studies in nonhuman primates (cynomologous monkeys) were performed by intravenously injecting 7.1 +/- 1.0 mCi of [123I]R(+)-FIDA2. At least one control study and one displacement experiment, in which a cold compound was injected intravenously 90 min after tracer injection, was performed in each monkey. Data were acquired in 10-min frames for 180 min, and the activity in regions of interest (basal ganglia and cerebellum) were plotted versus time. RESULTS: Iodine-125-R(+)-FIDA2 displayed Kd values for D2 and D3 receptor subtypes expressed in Sf9 cells of 0.11 and 0.04 nM, respectively. As expected, SPECT images of monkey brain (transaxial sections, 2 mm) showed that the radioactivity was localized in the area of the basal ganglia and reached peak concentrations in 11.5 +/- 5.8 min postinjection. An injection of R(+)7-OH-PIPAT, a new ligand that is selective for dopamine D3 receptors and the high affinity state of dopamine D2 receptors, did not show significant displacement of [123I]R(+)-FIDA2 binding in the basal ganglia. CONCLUSION: These studies indicate that R(+)-FIDA2 may be a useful ligand for in vitro pharmacological characterization and in vivo imaging of CNS dopamine D2-like receptors.     

Preliminary validation of the Child Abuse Potential Inventory in Argentina

Experiments were performed to characterize the pharmacology of Sch 50971 ((+)-trans-4-(4(R)-methyl-3(R)-pyrolidinyl)-1H-imidazole dihydrochloride, CAS 167610-28-8), a novel histamine H3 receptor agonist. The activity of Sch 50971 was compared with that of (R)-alpha-methylhistamine (CAS 75614-87-8), a potent and moderately selective agonist of histamine H3 receptors, in a series of in vitro and in vivo assays. Sch 50971 is a high affinity, selective H3 receptor agonist in vitro and in vivo. Sch 50971 inhibits [3H]-N-alpha-methylhistamine (CAS 673-50-7) binding to the histamine H3 receptor in human brain (Ki = 5.0 nmol/l) and guinea pig brain (Ki = 2.5 nmol/l). Sch 50971 also inhibits electric field stimulated guinea pig ileum contractions (pD2 = 7.47) and decreases [3H]-norepinephrine (CAS 51-41-2) release (pD2 = 7.48) from guinea pig pulmonary artery by activation of presynaptic inhibitory H3 receptors. The in vitro effects of Sch 50971 are antagonized by low concentrations of a selective H3 antagonist, thioperamide (CAS 106243-16-7). Sch 50971 has low affinity (IC50's > 10 mumol/l) for histamine H1, dopamine D1 and D2, serotonin 5-HT2 and muscarinic cholinergic receptors. It also does not exhibit histamine H2-antagonist activity. In guinea pigs and cats, Sch 50971 exhibits in vivo H3 agonist activity. Sch 50971 inhibits sympathetic hypertension evoked by stimulation of the medulla oblongata in anesthetized guinea pigs (ED30 = 0.3 mg/kg i.v., ED30 = 1.0 mg/kg i.d.). Sch 50971 also inhibits the effects of sympathetic nerve stimulation on nasal resistance in cats. In these assays, Sch 50971 exhibits an efficacy and potency comparable to H3-agonist (R)-alpha-methylhistamine. However, under in vivo conditions, Sch 50971 does not exhibit histamine H1-mediated responses that are seen with (R)-alpha-methylhistamine at doses close to those that produce H3 effects. Therefore, Sch 50971 is a novel, potent and selective agonist of histamine H3 receptors with an improved in vitro and in vivo receptor profile selectivity compared with (R)-alpha-methylhistamine.     

Biological profile of L-745,870, a selective antagonist with high affinity for the dopamine D4 receptor

L-745,870,(3-([4-(4-chlorophenyl)piperazin-1-yl]methyl)-1H- pyrollo[2,3-b] pyridine, was identified as a selective dopamine D4 receptor antagonist with excellent oral bioavailability and brain penetration. L-745,870 displaced specific binding of 0.2 nM [3H] spiperone to cloned human dopamine D4 receptors with a binding affinity (Ki) of 0. 43 nM which was 5- and 20-fold higher than that of the standard antipsychotics haloperidol and clozapine, respectively. L-745,870 exhibited high selectivity for the dopamine D4 receptor (>2000 fold) compared to other dopamine receptor subtypes and had moderate affinity for 5HT2, sigma and alpha adrenergic receptors(IC50 < 300 nM). In vitro, L-745,870 (0.1-1 microM) exhibited D4 receptor antagonist activity, reversing dopamine (1 microM) mediated 1) inhibition of adenylate cyclase in hD4HEK and hD4CHO cells; 2) stimulation of [35S] GTPgammaS binding and 3) stimulation of extracellular acidification rate, but did not exhibit any significant intrinsic activity in these assays. Although standard antipsychotics increase dopamine metabolism or plasma prolactin levels in rodents, L-745,870 (相似文献   

Solubilized rabbit striatal A2a-adenosine receptors: stability and antagonist binding

The A2a-adenosine binding subunit from rabbit striatal membranes was solubilized using 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and was characterized using the antagonist radioligand [3H]8-[4-[[[[2-aminoethyl)amino]carbonyl]methyl]oxy] phenyl]-1,3-dipropylxanthine (XAC). The solubilized receptor was very stable, with 55% of the specific [3H]XAC binding remaining after storage for 15 days at 4 degrees C. The dissociation constant (Kd) for binding of [3H]XAC to solubilized A2 receptors was determined in saturation studies to be 4.0 nM, with a Bmax of 600 fmol/mg protein. Xanthine inhibitors displaced the specific binding of the adenosine antagonist [3H]XAC (in the presence of 50 nM 8-cyclopentyl-1,3-dipropylxanthine) at 25 degrees C, with Ki values consonant with the expected affinities at A2a receptors. Binding of [3H]XAC (1 nM) or the adenosine agonist [3H]2-(carboxyethylphenylethylamino)adenosine-5'-N-ethyl carboxamide (5 nM) to A2a receptors was diminished in the presence of 0.1 M Na+ in both membranes and solubilized preparations. Agonist binding was increased (by 280% for membranes and 180% for solubilized receptors), and antagonist binding was decreased in the presence of 10 mM Mg2+. Displacement of [3H]XAC by the agonist (R)-N6-phenylisopropyladenosine was biphasic, corresponding to high (IC50 = 188 nM, RH = 30%) and low (IC50 = 9730 nM, RL = 70%) affinity sites. Preincubation with 100 microM GTP (10 mM Mg2+) converted the high affinity binding to low affinity, suggesting that receptor and G-protein are dissociated by the guanine nucleotide. The solubilized receptor was more easily inactivated by exposure to the reducing agent dithiothreitol (IC50 = 3 mM) than in membranes (IC50 = 220 mM), suggesting increased accessibility of structurally essential disulfide bridges.     

Survey on the pharmacodynamics of the new antipsychotic risperidone

This review reports on the pharmacodynamics of the new antipsychotic risperidone. The primary action of risperidone is serotonin 5-HT2 receptor blockade as shown by displacement of radioligand binding (Ki: 0.16 nM), activity on isolated tissues (EC50: 0.5 nM), and antagonism of peripherally (ED50: 0.0011 mg/kg) and centrally (ED50: 0.014 mg/kg) acting 5-HT2 receptor agonists in rats. Risperidone is at least as potent as the specific 5-HT2 receptor antagonist ritanserin in these tests. Risperidone is also a potent dopamine D2 receptor antagonist as indicated by displacement of radioligand binding (Ki: 1.4 nM), activity in isolated striatal slices (IC50: 0.89 nM), and antagonism of peripherally (ED50: 0.0057 mg/kg in dogs) and centrally acting D2 receptor agonists (ED50: 0.056-0.15 mg/kg in rats). Risperidone shows all effects common to D2 antagonists, including enhancement of prolactin release. However, some central effects such as catalepsy and blockade of motor activity occur at high doses only. Risperidone is 4-10 times less potent than haloperidol as a central D2 antagonist in rats and it differs from haloperidol by the following characteristics: predominant 5-HT2 antagonism; LSD antagonism; effects on sleep; smooth dose-response curves for D2 antagonism; synergism of combined 5-HT2/D2 antagonism; pronounced effects on amphetamine-induced oxygen consumption; increased social interaction; and pronounced effects on dopamine (DA) turnover. Risperidone displays similar activity at pre- and postsynaptic D2 receptors and at D2 receptors from various rat brain regions. The binding affinity for D4 and D3 receptors is 5 and 9 times weaker, respectively, than for D2 receptors; interaction with D1 receptors occurs only at very high concentrations. The pharmacological profile of risperidone includes interaction with histamine H1 and alpha-adrenergic receptors but the compound is devoid of significant interaction with cholinergic and a variety of other types of receptors. Risperidone has excellent oral activity, a rapid onset, and a 24-h duration of action. Its major metabolite, 9-hydroxyrisperidone, closely mimics risperidone in pharmacodynamics. Risperidone can be characterized as a potent D2 antagonist with predominant 5HT2 antagonistic activity and optimal pharmacokinetic properties.     

Opisthorchis viverrini metacercaria in Thai freshwater fish

N-chloroethyl derivatives of 7-hydroxy-1,2,3,4-tetrahydronaphthalene (7-OH-DPAT), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), or fluphenazine were microinjected into rat nucleus accumbens (Acc), and receptor binding quantified autoradiographically after 24 h. EEDQ reduced [3H]nemonapride (D2-like receptors) binding in Acc (by 84%) and islands of Calleja (IC; 44%), without affecting [3H](+)-7-OH-DPAT (D3); N-chloroethyl-7-OH-DPATs blocked both radioligands in Acc and IC (30%-70%); fluphenazine had no effect.     

Regulation of tyrosine hydroxylase and aromatic L-amino acid decarboxylase by dopaminergic drugs

We provide evidence that dopamine receptors differentially modulate tyrosine hydroxylase and aromatic L-amino acid decarboxylase in the mouse striatum. The dopamine D1 receptor family (D1-like) antagonist, R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benazepine (SCH 23390), elevated aromatic L-amino acid decarboxylase activity and protein content in striatum, as well as the mRNA for the enzyme in midbrain. The dopamine D1-like receptor agonist, (+/-)-1-phenyl-2,3,4,5-tetrahydro-(1 H)-3-benzazepine-7,8-diol (SKF 38393), had no effect on aromatic L-amino acid decarboxylase. The dopamine D1-like drugs had no effect on tyrosine hydroxylase. In contrast, the dopamine D2 receptor family (D2-like) antagonists haloperidol and spiperone elevated both tyrosine hydroxylase and aromatic L-amino acid decarboxylase activities. The increase in aromatic L-amino acid decarboxylase activity was accompanied by elevated enzyme protein content but not mRNA. The dopamine D2-like receptor agonists, bromocriptine, quinpirole and (+/-)-7-hydroxydipropylaminotetralin (7-OH-DPAT), all decreased striatal tyrosine hydroxylase. Under the conditions used, bromocriptine and 7-OH-DPAT, but not quinpirole, decreased aromatic L-amino acid decarboxylase activity of striatum. Both the dopamine D1- and D2-like receptor antagonists enhanced the turnover of striatal dopamine to differing degrees, as judged by the ratio of acid metabolites of dopamine to dopamine. Taken together our results indicate that aromatic L-amino acid decarboxylase can be modulated independently of tyrosine hydroxylase.     

CI-1007, a dopamine partial agonist and potential antipsychotic agent. I. Neurochemical effects

The receptor binding and biochemical effects of the putative dopamine (DA) partial agonist CI-1007 ([R(+)-1,2,3,6-tetrahydro-4-phenyl- 1-[(3-phenyl-3-cyclohexen-1-yl)methyl]pyridine] maleate) and potential antipsychotic were evaluated with a variety of biochemical methods. In receptor binding studies, CI-1007 bound to rat striatal DA receptors exhibiting a Ki of 3 nM as assessed by inhibition of [3H]N-propylnorapomorphine binding. CI-1007 also exhibited high affinity for cloned human D2L (Ki = 25.5 nM) and D3 (Ki = 16.6 nM) receptors with less affinity for D4.2 receptors (Ki = 90.9 nM). The affinity for serotonin-1A (5-HT-1A), alpha-2 adrenergic and 5-HT-2 receptors was moderate (submicromolar range) and slight or negligible for alpha-1, DA D1 and various other receptors. Unlike dopamine, the inhibition of [3H]spiperone binding was monophasic for CI-1007 and only slightly affected by the addition of Gpp-(NH)p. In vitro CI-1007 antagonized the forskolin-induced increases in cyclic AMP levels in GH4C1 cells expressing the human D2L receptor, having an intrinsic activity of 53% of that seen with the full agonist quinpirole. In vivo CI-1007 antagonized the gamma-butyrolactone (GBL)-induced accumulation of L-3,4-dihydroxyphenylalanine in striatum and mesolimbic regions of rat brain, causing a maximal 64% reversal in striatum, consistent with a partial agonist profile. In microdialysis studies it decreased DA overflow in both striatum and nucleus accumbens, indicating decreased release of DA. CI-1007 also reduced brain DA synthesis (DOPA accumulation), metabolism (DOPAC and HVA) and utilization (after tyrosine hydroxylase inhibition with alpha-methyl-p-tyrosine). CI-1007 did not affect striatal acetylcholine levels indicating lack of potent postsynaptic DA actions. CI-1007 seemed to be selective for DA neurons as it did not alter rat brain norepinephrine (NE) synthesis in the NE-enriched brainstem or NE utilization in the mesolimbic region. In addition, it did not affect in general 5-HT synthesis and metabolism in striatum and mesolimbic regions. These neurochemical results demonstrate that CI-1007 is a selective potent brain dopamine partial agonist with limited agonist activity at postsynaptic DA receptors.     

N-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide: a potent and selective dopamine D4 ligand

A series of new 1-aryl-4-alkylpiperazines containing a terminal benzamide fragment or a tetralin-1-yl nucleus on the alkyl chain were synthesized and tested for binding at cloned human dopamine D4 and D2 receptor subtypes. A SAFIR (structure-affinity relationship) study on this series is herein discussed. The most relevant D4 receptor affinities were displayed by N-[omega-[4-arylpiperazin-1-yl]alkyl]-methoxybenzamides (compounds 5, 16-20), their IC50 values ranging between 0.057 and 7.8 nM. Among these, N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (17) emerged since it exhibited very high affinity for dopamine D4 receptor (IC50 = 0.057 nM) with selectivity of >10 000 for the D4 versus the D2 receptor; compound 17 was also selective versus serotonin 5-HT1A and adrenergic alpha1 receptors.     

Inhibition of [3H]quinpirole binding by a monoamine oxidase inhibitor in subcellular fractions of rat striatum

[3H]Quinpirole is a dopamine agonist with high affinity for D2-like dopamine receptors. A number of non-dopaminergic compounds, most notably monoamine oxidase inhibitors (MAOIs), inhibit the binding of [3H]quinpirole, but not other D2-like agonists and antagonists, in rat striatal membranes by a mechanism that does not involve the enzymatic activity of MAO. To further characterize this novel interaction, the subcellular distribution of spiperone-displaceable, "D2-like" [3H]quinpirole-labeled sites in rat striatum was assessed and compared with the distribution of MAOI-displaceable [3H]quinpirole binding (MQB). "D2-like" [3H]quinpirole binding exhibited similar nanomolar affinity in the crude synaptosomal (P2), crude microsomal (P3), and ribosomal, post-microsomal (P4) fractions. Total binding activity (fmol bound/fraction) of "D2-like" [3H]quinpirole binding was concentrated in the synaptosomal fraction (P2B). The subcellular distribution of MQB paralleled that of "D2-like" [3H]quinpirole binding. This suggests that "D2-like" [3H]quinpirole binding and MQB occur at a common membrane-bound binding site.     

An assessment of the genetic toxicology of antimony trioxide

Dopamine receptor expression in human fetal forebrain (between 6 and 20 weeks of gestation) was measured using tissue-slice receptor autoradiography with the D1-like and D2-like antagonists [3H]-SCH23390 and [3H]-YM09151-2, respectively. Tissue sections were assayed in saturation studies and examined for age- and sex-related changes in Bmax. We made the following observations: (1) the ages at which D1- and D2-like receptors were first expressed in whole forebrain sections could be reliably identified but were not significantly different from one another (gestational age 65 days for D1- vs. 72 days for D2-like receptors); (2) age-related increases in both D1- and D2-like receptors were demonstrated in forebrain and, from the middle of the first to the middle of the second trimester, the Bmax for each ligand increased by an order of magnitude after the onset of the specific binding site's expression; (3) age-related increases in D1-like receptors, but not D2-like receptors, could be demonstrated in cortex; and, (4) in one case of trisomy 18, the Bmax for [3H]-SCH23390 was significantly elevated above the 95% confidence interval when compared to an age-regressed normal sample. Although D2-like receptor density significantly increased with age in forebrain, age-regressed changes in D2-like receptor expression in cortex and striatum did not reach statistical significance. Likewise, a comparison of the mean Bmax's by sex for both ligands in midgestational striatum failed to reach significance. These data corroborate the findings of other investigators who have delineated the ontogeny of dopaminergic systems in other animal species. The regional differences in the expression of dopamine receptor families may be relevant to the role which dopamine may play during normal gestational brain development. Moreover, significant deviations in dopamine receptor expression during gestation (as seen in this one case of trisomy 18) may signify underlying pathological processes that ultimately are manifested by abnormal psychological development and/or cognitive functioning.     

Regional distribution of dopamine D4 receptors in rat forebrain

Binding of the D2-like (D2/D3/D4) radioligand [3H]nemonapride under selective conditions (with 300 nM S[-]-raclopride and other masking agents to occlude D2/D3 receptors and non-specific binding sites) revealed a subset of raclopride-insensitive binding sites considered D4-like receptors. These sites were stereoselective to R(-)-N-n-propylnorapomorphine (NPA) over its S(+)-NPA in a similar fashion to cloned D4 receptors expressed in cell lines. In addition, the highly D4-selective agent L-745,870 displaced 74-83% of these sites in rat brain regions, suggesting that most were D4 receptors. These apparent D4 receptors represented a relatively high proportion of D2-like receptors in hippocampus, dorsolateral frontal, medial prefrontal and entorhinal cortex, but fewer in caudate-putamen and nucleus accumbens.     

Alpha-melanotropin hormone inhibits the binding of [3H]SCH 23390 to the dopamine D1 receptor in vitro

We have previously demonstrated that the simultaneous presence of alpha-melanocyte stimulating hormone (alpha-MSH) and dopamine resulted in a reduction in cyclic AMP (cAMP) levels in slices containing caudate putamen and accumbens nuclei as compared to those treated only with dopamine or alpha-MSH. This study was carried out to explore if the interaction between alpha-MSH and dopamine could be explained on the basis of a direct interaction between alpha-MSH and the dopamine D1 receptor. Saturation curves for [n-methyl-3 H](R)-(+)-8 chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1 H-3-benzazepin-7-o] hemimaleate ([3H]SCH 23390) binding in the presence of increasing concentrations of alpha-MSH were performed. Nonlinear regression in the presence of alpha-MSH revealed an increased dissociation constant (Kd). The binding capacity (Bmax) was not affected by the peptide. These data suggest an apparent competitive interaction between alpha-MSH and [3H]SCH 23390 in striatal membranes on the dopamine D1 receptor; (Ki = 1.2 X 10(-7) M). The present data show that alpha-MSH could interact with the dopamine D1 receptor modulating allosterically the affinity of [3H]SCH 23390 for the receptor or by causing a change in the lipid environment of the dopamine receptor, resulting in an inhibition of the ligand binding to it.     

Liposomal amikacin: improved treatment of Mycobacterium avium complex infection in the beige mouse model

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.     

Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists

1. The pharmacological profile of adenosine A1 receptors in human, guinea-pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). 2. The affinity of [3H]-DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea-pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea-pig or human cortical membranes. 3. The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5'-N-ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5-26.2 fold higher in rat cortical membranes than in human and guinea-pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 microM) reduced 2-chloro-N6-cyclopentyladenosine (CCPA) affinity 7-13.9 fold, whereas the affinity of DPCPX was unaffected. 4. The affinity of six xanthine-based adenosine receptor antagonists was 2.2-15.9 fold higher in rat cortical membranes compared with human or guinea-pig membranes. The rank order of potency was species-independent. In contrast, three pyrazolopyridine derivatives, (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea-pig, rat and mouse brain membranes. pKi values (M) for [3H]-DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively. 5. Drug affinity for adenosine A2A receptors was determined in a [3H]-2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido ade nosine ([3H]-CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]-DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]-DPCPX binding sites in human, rat, mouse and guinea-pig brain membranes.     

Adenosine A2A receptors modulate the binding characteristics of dopamine D2 receptors in stably cotransfected fibroblast cells

In membrane preparations from rat striatum, where adenosine A2A and dopamine D2 receptors are coexpressed, stimulation of adenosine A2A receptors was found to decrease the affinity of dopamine D2 receptors for dopamine agonists. We now demonstrate the existence of this antagonistic interaction in a fibroblast cell line (Ltk-) stably transfected with the human dopamine D2 (long-form) receptor and the dog adenosine A2A receptor cDNAs (A2A-D2 cells). In A2A-D2 cells, but not in control cells only containing dopamine D2 receptors (D2 cells), the selective adenosine A2A agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamido adenosine (CGS 21680) induced a 2-3-fold decrease in the affinity of dopamine D2 receptors for dopamine, as shown in competition experiments with dopamine versus the selective dopamine D2 antagonist [3H]raclopride. By contrast, activation of the constitutively expressed adenosine A2B receptors with 5'-N-ethyl-carboxamidoadenosine (NECA) did not modify dopamine D2 receptor binding. In A2A-D2 cells CGS 21680 failed to induce or induced only a small increase in adenosine-3',5'-cyclic-monophosphate (cAMP) accumulation. In D2 cells NECA- or forskolin-induced adenylyl cyclase activation was not associated with any change in dopamine D2 receptor binding. These results indicate that adenylyl cyclase activation is not involved in the adenosine A2A receptor-mediated modulation of the binding characteristics of the dopamine D2 (long-form) receptor.     

Dopamine D3 and D4 receptor antagonists: synthesis and structure--activity relationships of (S)-(+)-N-(1-Benzyl-3-pyrrolidinyl)-5-chloro-4- [(cyclopropylcarbonyl) amino]-2-methoxybenzamide (YM-43611) and related compounds

In this study, we synthesized a series of (S)-N-(3-pyrrolidinyl)benzamide derivatives, 1, 2a-d, 5a-1, and 7, and their enantiomers, (R)-1 and (R)-5c-e, and evaluated their binding affinity for cloned dopamine D2, D3, and D4 receptors and their inhibitory activity against apomorphine-induced climbing behavior in mice. The results indicate that D2, D3, and D4 receptors have different bulk tolerance (D4 > D3 > D2) for the substituent of the 4-amino group (R1) on the benzamide nuclei and that cyclopropyl-, cyclobutyl-, and cyclopentylcarbonyl groups likely possess adequate bulkiness with respect to D3 and D4 affinity and selectivity over D2 receptors in this series. The results also suggested that the N-substituent (R2) on the pyrrolidin-3-yl group performs an important role in expressing affinity for D2, D3, and D4 receptors and selectivity among the respective subtypes. One of the compounds, (S)-(+)-N-(1-benzyl-3-pyrrolidinyl)-5-chloro-4-[(cyclopropylcarbonyl+ ++) amino]-2-methoxybenzamide (5c) (YM-43611), showed high affinity for D3 and D4 receptors (Ki values of 21 and 2.1 nM, respectively) with 110-fold D4 selectivity and 10-fold D3 preference over D2 receptors and weak or negligible affinity for representative neurotransmitter receptors. Compound 5c displayed potent antipsychotic activity in inhibiting apomorphine-induced climbing behavior in mice (ED50 value, 0.32 mg/kg sc).     

RLH-033, a novel, potent and selective ligand for the sigma 1 recognition site

RLH-033 [2-(4-phenylpiperidinyl)ethyl 1-(4-nitrophenyl)cyclopentanecarboxylate HCl] is a rationally designed ligand that was synthesized and evaluated for its binding affinities at sigma 1 and sigma 2 sites in guinea pig brain. RLH-033 has high affinity (Ki = 50 pM) for sigma 1 sites labeled by [3H](+)-pentazocine, while it was over 2000-fold less affinity at sigma 2 sites labeled by [3H]1,3-di(2-tolyl)guanidine (DTG) in the presence of 500 nM (+)-pentazocine (Ki = 105 nM). Unlike its potent sigma activity, the compound has little affinity for dopamine D1 (Ki = 2.9 microM), D2 (Ki = 0.35 microM), muscarinic M1 (Ki = 0.88 microM) or M2 (Ki = 1.7 microM) receptors, and none at all for N-methyl-D-aspartate, phencyclidine and opioid receptors. Thus, RLH-033 is the most potent sigma 1 ligand reported to date, and its very high affinity suggests it may be a useful radioligand to characterize the pharmacology of sigma 1 recognition sites.     

Dynamic changes in striatal dopamine D2 and D3 receptor protein and mRNA in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) denervation in baboons

Loss of nigrostriatal neurons leads to striatal dopamine deficiency and subsequent development of parkinsonism. The effects of this denervation on D2-like receptors in striatum remain unclear. Most studies have demonstrated increases in striatal dopamine D2-like receptors in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated denervation, but others have found either decreases or no change in binding. To clarify the response to denervation, we have investigated the time-dependent changes in dopamine D2, D3, and D4 receptor protein and mRNA levels in unilaterally MPTP-lesioned baboons. MPTP (0.4 mg/kg) was infused into one internal carotid artery, producing a contralateral hemi-parkinsonian syndrome. After MPTP treatment, the animals were maintained for 17-480 d and then euthanized. MPTP decreased ipsilateral dopamine content by >90%, which did not change with time. Ipsilateral D2-like receptor binding in caudate and putamen initially decreased then increased two- to sevenfold over the first 100 d and returned to near baseline levels by 480 d. Relative levels of D2 mRNA were essentially unchanged over this period. D4 mRNA was not detected. In contrast, D3 mRNA increased sixfold by 2 weeks and then decreased. At the peak period of increase in binding sites, all D2-like receptors were in a micromolar affinity agonist-binding state, implying an increase in uncoupled D2 but not D3 receptor protein. Taken together, these data suggest that MPTP-induced changes in D2-like dopamine receptors are complex and include translational or post-translational mechanisms.