Evidence for the Importance of a Carboxyl Group in the Binding of Ligands to the D2 Dopamine Receptor

A series of group specific modifying reagents were tested for their effects on [3H]spiperone binding to brain D2 dopamine receptors to identify amino acid residues at the binding site of the D2 dopamine receptor that are critical for ligand binding. The dependence of ligand binding to the receptor on the pH of the incubation medium was also examined. N-Acetylimidazole, 5,5'-dithiobis(2-nitrobenzoic acid), 1,2-cyclohexanedione, and acetic anhydride had no specific effect on [3H]spiperone binding, indicating the lack of participation of tyrosine, free sulphydryl, arginine, or primary amino groups in ligand binding to the receptor. N,N'-Dicyclohexylcarbodiimide (DCCD) potently reduced the number of [3H]spiperone binding sites, indicating that a carboxyl group is involved in ligand binding to the receptor. The effects of DCCD could be prevented by prior incubation of the receptor with D2 dopamine receptor selective compounds. The pH-binding profile for [3H]spiperone binding indicated the importance of an ionising group of pKa 5.2 for ligand binding which may be the same carboxyl group. Diethyl pyrocarbonate, the histidine modifying reagent, also inhibited [3H]spiperone binding, reducing the affinity of the receptor for this ligand but the effects were not at the ligand binding site. From the effects of pH changes on ligand binding some evidence was obtained for a second ionising group (pKa 7.0) that specifically affects the binding of substituted benzamide drugs to the receptor. It is concluded that the D2 dopamine receptor binding site contains separate but over-lapping binding regions for antagonists such as spiperone and substituted benzamide drugs. The former region contains an important carboxyl group; the latter region contains another group that may be a second carboxyl group or a histidine.     

Thymopoietin, a Thymic Polypeptide, Specifically Interacts at Neuronal Nicotinic α-Bungarotoxin Receptors

alpha-Bungarotoxin (alpha-BGT), a snake venom polypeptide, interacts potently and specifically with a nicotinic receptor population in neuronal tissue. However, the identity of this site is unclear, because, unlike at the neuromuscular junction and in electroplax, in nervous tissue the toxin does not block nicotinic cholinergic responses. Therefore, we sought endogenous compounds other than acetylcholine that could interact with the neuronal alpha-BGT site. In the present experiments, thymopoietin, a polypeptide isolated from the thymus, is shown to inhibit potently alpha-BGT binding to brain membranes in a dose-dependent manner (IC50 = 3.1 nM). This effect was not shared by a wide variety of other peptides, including thysplenin, a closely related polypeptide. Thymopoietin did not inhibit the binding of other radioligands known to interact with different populations of cholinergic receptors, such as [3H]nicotine and [3H]methylcarbachol, which bind to nicotinic receptors, or [3H]quinuclidinylbenzilate, which binds to muscarinic receptors. These results show that thymopoietin potently and specifically affects 125I-alpha-BGT binding to brain membranes and suggest that thymopoietin might be an endogenous ligand for alpha-BGT receptors in neuronal tissue.     

D1 and D2 Dopamine Receptors in Caudate-Putamen of Nonhuman Primates (Macaca fascicularis)

D1 and D2 dopamine receptors were characterized in the caudate-putamen region of nonhuman primate brains (Macaca fascicularis). D1 dopamine receptors were identified with [3H]SCH 23390 and D2 receptors with [3H]-spiperone. Scatchard analysis of [3H]SCH 23390 saturation data using washed membranes revealed a single high-affinity binding site (KD, 0.352 +/- 0.027 nM) with a density (Bmax) of 35.7 +/- 2.68 pmol/g original wet tissue weight (n = 10). The affinity of [3H]spiperone for the D2 site was 0.039 +/- 0.007 nM and the density was 25.7 +/- 1.97 pmol/g original wet tissue weight (n = 10). D1 and D2 receptors in nonhuman primates may be differentiated on the basis of drug affinities and stereoselectivity. In competition experiments, RS-SKF 38393 was the most selective D1 agonist, whereas (+)-4-propyl-9-hydroxynaphthoxazine [(+)-PHNO] was the most selective D2 agonist. Apomorphine was essentially nonselective for D1 or D2 binding sites. Of the antagonists, R-SKF 83566 and SCH 23390 were the most selective for the D1 site, whereas YM-09151-2 was the most selective for the D2 site. cis-Flupentixol and (S)-butaclamol were the least selective dopamine antagonists. D1 receptors bound benzazepine antagonists (SCH 23390/SCH 23388, R-SKF 83692/RS-SKF 83692) stereoselectively whereas D2 receptors did not. Conversely D2 receptors bound (S)-sulpiride and (+)-PHNO more potently than their enantiomers whereas D1 receptors showed little stereoselectively for each of these isomeric pairs. These binding characteristics may be utilized for evaluation of individual receptor function in vivo.     

Structural analogues of 5-OMe-BPAT: synthesis and interactions with dopamine D2, D3, and serotonin 5-HT1A receptors.

Several structural analogues of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1), a representative of a series of 2-aminotetralin-derived benzamides with potential atypical antipsychotic properties, were synthesized and evaluated for their ability to bind to dopamine D2A, D3, and serotonin 5-HT1A receptors in vitro. The structure affinity relationships revealed that the aromatic ring of the benzamide moiety of 1 contributes to the high affinities for all three receptor subtypes. Furthermore, 1 may interact with the dopamine D2 and D3 receptors through hydrogen bond formation with its carbonyl group. Investigation of the role of the amide hydrogen atom by amide N-alkylation was not conclusive, since conformational aspects may be responsible for the decreased dopaminergic affinities of the N'-alkylated analogues of 1. The effects of the amide modifications on the serotonin 5-HT1A receptor affinity were less pronounced, suggesting that the benzamidoethyl side-chain of 1 as a whole enhances the affinity for this receptor subtype probably through hydrophobic interactions with an accessory binding site. The structural requirements for the substituents at the basic nitrogen atom supported the hypothesis that the 2-aminotetralin moieties of the 2-aminotetralin-derived substituted benzamides may share the same binding sites as the 2-(N,N-di-n-propylamino)tetralins.     

Modulation of [3H]quinpirole binding at striatal D2 dopamine receptors by a monoamine oxidaseA-like site: evidence from radioligand binding studies and D2 receptor- and MAO(A)-deficient mice.

[3H]Quinpirole is a dopamine agonist with high affinity for the D2 and D3 dopamine receptors. A variety of monoamine oxidase inhibitors (MAOIs) inhibit equilibrium binding of [3H]quinpirole binding in rat striatal membranes suggesting that MAOIs interact with a novel binding site that is labeled by [3H]quinpirole or that allosterically modulates [3H]quinpirole binding. To determine whether the D2 receptor is essential for [3H]quinpirole binding and/or modulation of [3H]quinpirole binding by MAOIs, D2 receptor-deficient mice were studied. [3H]Quinpirole binding was decreased in D2 receptor-deficient mice to 3% of that observed in wild-type controls indicating that [3H]quinpirole binding is associated with the D2 dopamine receptors. Then, in an attempt to label the site mediating the modulation of [3H]quinpirole binding, binding of the MAOI [3H]Ro 41-1049 was characterized in rat striatal membranes. [3H]Ro-41-1049 labeled a single binding site with a pharmacological profile with respect to MAOIs that was similar to both [3H]quinpirole binding (Spearman r=0.976) and MAO(A) activity. To determine whether MAO(A) plays a role in the modulation of [3H]quinpirole binding by MAOIs, MAO(A)-deficient mice were examined. In these mice, [3H]Ro-41-1049 binding was decreased to 7% of wild-type control. [3H]Spiperone binding was unaltered. Spiperone-displaceable [3H]quinpirole binding was decreased to 43% of wild-type control; however, the remaining [3H]quinpirole binding in MAO(A)-deficient animals was inhibited by Ro 41-1049 similar to wild-type. [3H]Ro-41-1049 binding was not decreased in D2 receptor-deficient mice. These data suggest that [3H]Ro-41-1049 labels multiple sites and that MAOIs modulate [3H]quinpirole binding to the D2 receptor via interactions at a novel, non-MAO binding site with MAO(A)-like pharmacology.     

Identification of a Novel N-Methyl-D-Aspartate Receptor Population in the Rat Medial Thalamus

To evaluate the possibility of pharmacologically distinct N-methyl-D-aspartate (NMDA) receptor subtypes, quantitative autoradiography was used to determine the potency of several compounds as inhibitors of L-[3H]glutamate or [3H]MK-801 binding to rat brain NMDA receptors in 10 brain regions. Competitive NMDA receptor antagonists displayed differing pharmacological profiles in the forebrain, cerebellum, and medial regions of the thalamus (midline nuclei). For example, compared with other competitive antagonists, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonate (CPP) and LY-233536 were especially weak displacers of L-[3H]glutamate binding in the cerebellum. In the the medial thalamus, CPP and D-2-amino-5-phosphonopentanoate displayed relatively low affinities, whereas LY-233536 was relatively potent. The noncompetitive NMDA receptor antagonists also displayed regional variations in their pharmacological profiles. Relative to other regions, [3H]MK-801 binding in the cerebellum was weakly displaced by MK-801 and potently displaced by dextromethorphan and SKF-10047. In the medial thalamus, 1-[1-(2-thienyl)-cyclohexyl]piperidine was relatively potent and SKF-10047 was relatively weak. These results confirm previous suggestions that the cerebellum contains a distinct NMDA receptor subtype and indicate that nuclei of the medial thalamus contain a novel NMDA receptor subtype that is distinct from both those found in the cerebellum and in the forebrain.     

High-and Low-Affinity States of Striatal D2 Receptors Are Not Affected by 6-Hydroxydopamine or Chronic Haloperidol Treatment

Specific D2 binding in rat striatum was characterized and then the effects of chronic disruption of dopaminergic activity on antagonist and agonist binding to these sites were studied. D2 receptors were defined as those sites capable of binding [3H]spiperone in the presence of cinanserin, a 5-HT2 antagonist, but not in the presence of (+)-butaclamol, a D2 and 5-HT2 blocker. Saturation, competition, and kinetic analyses suggested that D2 receptors are a homogeneous population exhibiting more complex interactions with agonists than antagonists. Antagonist binding was monophasic and guanine nucleotide-insensitive whereas agonist binding was biphasic and guanine nucleotide-sensitive. D2 receptor density was elevated by more than 40% following dopamine depletion by 6-hydroxydopamine or chronic receptor blockade by haloperidol. However neither treatment altered the affinities or magnitudes of the high- and low-affinity components associated with agonist binding to the D2 receptor.     

In vitro and in vivo pharmacological profile of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562), a novel and putative atypical antipsychotic

In vitro and in vivo pharmacological properties of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562), a novel atypical antipsychotic, were investigated. NRA0562 showed high affinities for human cloned dopamine D(1), D(2), D(3) and D(4) receptors with Ki values of 7.09, 2.49, 3.48 and 1.79 nM. In addition, NRA0562 had high affinities for the 5-HT(2A) receptor and the alpha(1) adrenoceptor with Ki values of 1.5 and 0.56 nM, and moderate affinity for the histamine H(1) receptor. Using in vivo and ex vivo receptor binding studies in rats, we showed NRA0562 occupied frontal cortical 5-HT(2A) receptors and alpha(1) adrenoceptor potently, while occupancy of striatal dopamine D(2) receptor was moderate as were other atypical antipsychotics. NRA0562 dose-dependently inhibited methamphetamine (MAP)-induced locomotor hyperactivity in rats. At higher dosage, NRA0562 dose-dependently antagonized MAP-induced stereotyped behavior and induced catalepsy dose-dependently and significantly in rats. But, the ED(50) value in inhibiting MAP-induced locomotion hyperactivity was 10 times lower than that in inhibiting MAP-induced stereotyped behavior, and 30 times lower than that in inducing catalepsy. In addition, the potency of NRA0562 in antagonizing MAP-induced hyperactivity in rats was higher than that of other antipsychotics, clozapine, risperidone and olanzapine. NRA0562 had favorable properties in view of prediction of extrapyramidal side effects. As this antipsychotic has a unique profile with affinity and occupancy for receptors, we propose that NRA0652 may have unique atypical antipsychotic activities, and a moderate liability of extrapyramidal motor side effects seen in the treatment with classical antipsychotics.     

Endogenous ligands for sigma opioid receptors in the brain ("sigmaphin"): evidence from binding assays

Two endogenous ligands which interact preferentially with the sigma opioid receptors were identified from the guinea-pig brain extract in a Sephadex G-50 fractionation. These two ligands inhibited more potently the binding of [3H]SKF-10047 to sigma opioid receptors than [3H]naloxone to mu opioid receptors, [3H]ethylketocyclazocine to kappa opioid receptors and [3H]DADLE to delta opioid receptors. In the phencyclidine receptor assay, these two ligands were almost inactive. Incubation of these ligands with trypsin destroyed at least 50% of the activities in the sigma opioid receptor assay. Both ligands inhibited the sigma binding in a dose-dependent manner. The inhibition could be eliminated when the two ligands were removed from incubation media by extensive washings. It is therefore concluded that sigma opioid receptors are not phencyclidine receptors and that endogenous ligands for sigma opioid receptors may exist in the brain.     

Characterization of High-Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate-Solubilized Bovine Striatal Preparations

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.     

Dopamine D4-like binding sites labeled by [3H]nemonapride include substantial serotonin 5-HT2A receptors in primate cerebral cortex

Dopamine D4-like binding sites are abundant in human cerebral cortex as detected by [3H]nemonapride. The extremely low density of D4 mRNA in human cerebral cortex is inconsistent with the high amount of D4-like binding sites. To investigate the nature of the D4-like receptors, [3H]nemonapride binding sites in the nonhuman primate cerebral cortex were characterized. Although [3H]nemonapride binding sites were D4-like, displaceable by clozapine but not raclopride, [3H]nemonapride binding was not displaced by selective D4 antagonists but was displaced by the selective 5-HT2A antagonist MDL100907. Using [3H]ketanserin as a 5-HT2A ligand, nemonapride showed high affinity for monkey (Ki = 10.4 nM) and cloned human (Ki = 9.4 nM) 5-HT2A receptors, while its affinity for rat receptors was lower (Ki = 140 nM). The present study demonstrates that cerebral cortical D4-like binding sites labeled by [3H]nemonapride in nonhuman primates consist of a very small portion of D4, but a substantial portion of 5-HT2A receptors. The unexpectedly high affinity of nemonapride for primate 5-HT2A receptor suggests reconsidering previous data from other studies using [3H]nemonapride, particularly those on D4-like receptors.     

In vitro binding properties and autoradiographic imaging of 3-iodobenzamide ([125I]-IBZM): a potential imaging ligand for D-2 dopamine receptors in SPECT

The in vitro binding properties of the [125I] labeled benzamide (S(-)-N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-hydroxy-3-iodo-6-methoxy- benzamide, IBZM) were determined in bovine and mouse caudate membrane homogenates and by autoradiography of mouse brain slices. [125I]-IBZM binding is saturable and reversible with a Bmax of 373 +/- 51 fmol/mg protein and a Kd of 3.1 +/- 0.62 nM (mean +/- SD, Scatchard analyses) and 0.56 nM as calculated by association and dissociation time constants. In competition experiments, Ki values for the D-2 antagonists YM-09151-2 and spiperone are 4 orders of magnitude lower than the Ki value for the D-1 antagonist SCH-23390 and S(-)-IBZM is ten-fold more potent than R(+)-IBZM. [125I]-IBZM has a low affinity for serotonin S-2 and for alpha receptors. Therefore, it is a highly selective ligand for dopamine D-2 receptors. Autoradiographic images of brain sections incubated with [125I]-IBZM show the dopamine D-2 receptors of the striatum, nucleus accumbens and olfactory tubercle with a high ratio of specific to nonspecific binding. Thus, S(-)-IBZM, when labeled with [123I], may be useful for in vivo imaging of dopamine D-2 receptors by single photon emission computerized tomography (SPECT).     

Dopamine receptor subtype density as a function of age in Aplysia californica

The age-associated changes in dopamine subtype receptors were examined in Aplysia californica. The density of the subtype receptors D1, D2, D3 and D4 was examined in the ganglia from 4.5-, 6-, 8-, 9- and 12-month animals. Receptor analysis was performed by examining the binding of radiolabeled ligands to the individual subtypes. [³H]SCH23390 and [³H]Clozapine were used to analyze D1 and D4 specific binding. [³H]Quinpirole was used for determining D2 and D3 specific binding. Specific binding was found to be present for all four receptor subtypes. All receptor subtypes showed an increase in density from 4.5 to 6 months. From 6 to 8 months D2 and D3 decreased, while D1 and D4 increased. D4 showed the strongest increase. All four subtypes examined showed decreases from 8 to 12 months. ANOVA results indicated age was a significant factor in the subtype receptor density for all receptor types.     

Monoclonal anti-idiotypic antibodies to platelet activating factor (PAF) and their interaction with PAF receptors.

Monoclonal anti-idiotypic antibodies (3C3F3E4 and 10D3F8H7) that interact with platelet activating factor (PAF) receptors were generated using an auto-anti-idiotypic approach by immunizing mice with an aldehydic analog of PAF coupled to bovine thyroglobulin. The resulting hybridomas were screened for anti-idiotypic antibody (anti-anti-PAF) with F(ab')2 fragments of affinity-purified polyclonal rabbit anti-PAF antibody. These antibodies displayed internal image properties of PAF and were considered as Ab2 beta according to the following criteria: (a) they bound to F(ab')2 fragments of the affinity-purified rabbit polyclonal anti-PAF antibody that had high affinity for PAF; (b) they inhibited [3H]PAF binding to rabbit polyclonal anti-PAF antibody and its F(ab')2 fragment in a concentration-dependent manner; (c) they displaced [3H]PAF from the anti-PAF antibody/[3H]PAF complex specifically; (d) they inhibited [3H]PAF binding to PAF receptors on rabbit platelet membranes dose dependently; (e) they displaced [3H]PAF from the [3H]PAF/PAF receptor complex specifically; and (f) they stimulated rabbit platelets to aggregate, and this aggregation could be inhibited or totally blocked by specific PAF receptor antagonists WEB 2086 and SRI 63-441. All of the above are consistent with the first successful production of monoclonal antibodies that mimic PAF and interact specifically with the PAF binding domain of PAF receptors on rabbit platelet membranes.     

Improvement in Conditions for Solubilisation and Characterisation of Brain D2 Dopamine Receptors Using Various Detergents

A series of detergents of varying chemical properties has been tested for solubilisation of bovine caudate nucleus D2 dopamine receptors using [3H]spiperone binding to assay the solubilised sites. The properties of the lysophosphatidylcholine (LPC)- and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulphonate (CHAPS)-solubilised preparations are described in detail. The preparations are truly solubilised, and sucrose density gradient and gel filtration data are reported. Specific [3H]spiperone binding in the LPC-solubilised preparation assayed at 4 degrees C is solely to D2 dopamine receptors. If the assay temperature is raised to 25 degrees C, the amount of specific [3H]spiperone binding is largely unchanged, but it forms a greater proportion of the total [3H]spiperone binding owing to a reduction in nonstereospecific (spirodecanone) [3H]spiperone binding at the higher temperature. The effect of raising the assay temperature is important as it enables more precise determinations of specific [3H]spiperone binding to be made. Part of the specific [3H]spiperone binding at 25 degrees C is to solubilised S2 serotonin receptors in addition to D2 dopamine receptors. Good correlations are observed between the affinities for binding of ligands to the solubilised D2 receptors and corresponding data obtained on membrane-bound receptors. Agonist binding in LPC-solubilised preparations is insensitive to guanine nucleotides. It is speculated that the spirodecanone sites represent, in part, proteolysed or damaged D2 dopamine, or S2 serotonin, receptors. In the CHAPS-solubilised preparation the pharmacological profile of [3H]spiperone binding is unclear when assayed at 4 degrees C, but in assays at 25 degrees C a clear serotonin S2 receptor component of specific [3H]spiperone binding can be discerned.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of protease inhibitors on 1,25-dihydroxyvitamin D3 receptors

We describe herein two different effects of protease inhibitors and substrates on receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) obtained from the intestinal mucosa of vitamin D-deficient chicks: inhibition of binding of 1,25(OH)2D3 to its receptor and stabilization of the receptor. Both L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, block [3H]1,25(OH)2D3 binding to the receptor. Fifty per cent inhibition of binding occurs at 20 microM TPCK, and 100% inhibition at 100-200 microM; TLCK is about 25-fold less effective. At higher concentrations (10-100 mM), the chymotrypsin substrates N alpha-p-tosyl-L-arginine methyl ester and tryptophan methyl ester and the cathepsin B inhibitor leupeptin also inhibit [3H] 1,25(OH)2D3 binding to its receptor. Different inhibitors and substrates interact with the receptor differently: TPCK (20 microM) and N alpha-p-tosyl-L-arginine methyl ester (10 mM) are reversible, noncompetitive inhibitors, L-tryptophan methyl ester (20 mM) is a reversible competitive inhibitor, and phenylmethylsulfonyl fluoride (300 microM) shows no effect on [3H]1,25(OH)2D3 binding to its receptor. The most stable form of unoccupied 1,25(OH)2D3 receptors from chick intestinal mucosa was that obtained from a low salt chromatin preparation (t 1/2 = 6.0 h). The presence of KCl drastically decreased receptor stability (t 1/2 = 1.8 h); and the addition of 2.5 mM CaCl2 further reduced their stability. Phenylmethylsulfonyl fluoride and Trasylol inhibited the KCl-induced receptor instability, but did not prevent the additional instability in the presence of CaCl2. In summary, TPCK and TLCK exert direct effects on the 1,25(OH)2D3 receptor molecule, independent of their protease inhibitor function. These compounds may prove useful as covalent affinity labels for the receptor. On the other hand, phenylmethylsulfonyl fluoride and Trasylol stabilize 1,25(OH)2D3 receptors, probably via inhibition of KCl-activated nuclear protease(s). This receptor stabilization will be advantageous in receptor assays and/or purification procedures.     

GBR-12909 and fluspirilene potently inhibited binding of [3H] (+)3-PPP to sigma receptors in rat brain

Fluspirilene and GBR-12909, two compounds structurally similar to BMY-14802 and haloperidol, were assessed for their ability to interact with sigma receptors. Fluspirilene, an antipsychotic agent that interacts potently with dopamine receptors, inhibited the binding of [3H]-(+) 3-PPP (IC50 = 380 nM) more potently than rimcazole, a putative sigma antagonist that was tested clinically for antipsychotic activity. GBR-12909, a potent dopamine uptake blocker, also inhibited the binding of [3H]-(+) 3-PPP with an IC50 of 48 nM. However, other compounds that block the re-uptake of catecholamines, such as nomifensine, desipramine, imipramine, xylamine, benztropine and cocaine, were much weaker than GBR-12909 as sigma ligands. Thus, GBR-12909 and fluspirilene, compounds structurally similar to BMY-14802, are potent sigma ligands.     

Effect of Guanine Nucleotides on Dopaminergic Agonist and Antagonist Affinity for [3H]Sulpiride Binding Sites in Rat Striatal Membrane Preparations

Abstract: [³H]Sulpiride bound to rat striatal membrane preparations with a saturable, high affinity component. This binding was displaced potently by dopamine antagonists (both classic neuroleptics and the benzamide, sulpiride) and less potently by dopamine agonists. GTP and its stable analogue Gpp(NH)p did not affect [³H]sulpiride binding to the membranes but altered the affinity for dopaminergic agonists. This effect was specific in that antagonist binding was not affected and only GTP, GDP, and Gpp(NH)p produced the effect. Similar alterations in ligand binding affinity caused by guanine nucleotides have been observed for binding sites linked to an adenylate cyclase. Such an interpretation for the case of [³H]sulpiride is contrary to suggestions that sulpiride labels only those dopamine receptors that are not cyclase linked.     

Pharmacology of [3H]R(+)-7-OH-DPAT binding in the rat caudate-putamen

Dopamine D3 receptors may be involved in drug addiction and in disorders such as schizophrenia and Parkinson's disease. To determine the pharmacological properties of dopamine D3 receptors in the rat caudate-putamen, we have investigated R(+)-[3H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin ([3H]R(+)-7-OH-DPAT) binding to membrane preparations from the rat caudate-putamen. Kinetic analyses showed that [3H]R(+)-7-OH-DPAT binding reached equilibrium in approximately 1 h and that both association and dissociation curves were composed of at least two components. Likewise, saturation curves showed at least two binding components with a combined Bmax value of about 600 fmol/mg protein, which is three times higher than what is present in the subcortical limbic area. Competition curves were performed with agonists such as R(-)-propylnorapomorphine, dopamine, PD 128907, quinpirole, and bromocriptine, and antagonists such as haloperidol, raclopride, clozapine, GR 218231x, remoxipride, and U99194A. These experiments revealed that [3H]R(+)-7-OH-DPAT binding could be resolved into three specific binding sites (R1-R3) and one nonspecific binding site, with R1-R2 probably representing D3 receptor binding and the minor R3 representing D2 receptor binding. The low affinities of (+/-)-8-OH-DPAT and 1,3-di(2-tolyl)guanidine to inhibit [3H]R(+)-7-OH-DPAT binding indicate negligible involvement of 5-HT1A or sigma binding sites, respectively. The pharmacological profile of [3H]R(+)-7-OH-DPAT (2 nM) binding in the caudate-putamen was similar to that of dopamine on [125I]iodosulpride binding in the cerebellar lobule X, which contain D3 but not D2 receptors. Mg2+ increased and GTP and Na+ decreased the binding of [3H]R(+)-7-OH-DPAT, suggesting a coupling of endogenous D3 receptors to G proteins. Taken together, these results suggest that dopamine D3 receptors display multiple agonist binding states, and that D3 receptors are present in high concentrations in the rat caudate-putamen. These results may have implications for the physiological and pathological roles of dopamine D3 receptors in the brain.     

''Molten-globule'' state accumulates in carbonic anhydrase folding

Binding characteristics of [3H]BAY K 8644, a new class of pharmacologically potent compounds, the calcium channel activating dihydropyridines (DHP), were demonstrated in cultured myocardial cells. [3H]BAY K 8644 exhibited reversible and saturable binding to myocytes, and specific binding was Ca2+-dependent. The equilibrium dissociation constant, Kd, was 35.2 nM, and maximal binding capacity, Bmax, was 1.07 pmol/mg protein. Binding of the 3H-ligand was highly specific for various potently displacing DHP derivatives (either the calcium channel activating BAY K 8644, or the Ca2+ entry blockers of the nifedipine type) with inhibition constants (Ki values) in the nanomolar range. BAY K 8644, on the other hand, showed very low affinity to other receptors tested in brain and heart membranes. Displacement potency of BAY K 8644 correlated well with data of the functional pharmacology; e.g., the enhanced myocardial contractility. Results from competition studies using [3H]BAY K 8644 and [3H]nimodipine support the conclusion that both the channel activating and inhibiting DHP structures interact with the same specific receptor site that might be associated with the putative Ca2+-channel.