Dopamine receptor binding: differentiation of agonist and antagonist states with 3H-dopamine and 3H-haloperidol.

³H-Dopamine and ³H-haloperidol bind with high affinity and selectivity to synaptic dopamine receptors in membrane preparations of the calf caudate. Binding of both ligands shows marked regional variations with greatest density in caudate, putamen, globus pallidus, nucleus accumbens and olfactory tubercle, areas rich in dopamine nerve terminals. The rank-order of phenothiazines and related agents as well as catecholamines in displacing both dopamine and haloperidol binding closely parallels their pharmacological potencies and affinities for the dopamine-sensitive adenylate cyclase. Dopamine's affinity for specific ³H-dopamine binding sites is 100 times its apparent affinity for the dopamine sensitive adenylate cyclase. Agonists have about 50 times more affinity for dopamine than haloperidol sites, whereas antagonists display about 100 times greater affinity for haloperidol than dopamine sites.     

Stereospecific (3H)(minus)-alprenolol binding sites, beta-adrenergic receptors and adenylate cyclase

The binding of [³H](?)-alprenolol (a potent β-adrenergic antagonist) to sites in frog erythrocyte membranes has been studied by a centrifugal assay. The specificity of the binding sites is strikingly similar to what might be expected of the β-adrenergic receptor binding sites which mediate stimulation of adenylate cyclase by catecholamines in these membranes. The sites bind β-adrenergic antagonists and agonists with affinities which are directly related to their antagonist or agonist potency on the frog erythrocyte membrane adenylate cyclase. Binding shows strict stereospecificity with (?)-isomers exhibiting two orders of magnitude higher affinities than (+)-isomers. Dissociation constants for potent β-adrenergic antagonists are in the range of 10^?9 – 10^?8M whereas those for β-adrenergic agonists are about two orders of magnitude higher (≥ 10^?6M).     

[3H]2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen (ADTN)

The regional distribution and in vivo binding of the dopamine analog 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen (ADTN) was studied in the brain. The highest density of binding sites was in the striatum, with virtually no binding in the cerebellum. The binding of [³H]ADTN reflects an occupation of specific dopamine sites because the binding was diminished by the simultaneous administration of the dopamine antagonist haloperidol or the dopamine precursorl-3,4-dihydroxyphenylalanine (l-dopa). Chronic administration of haloperidol orl-dopa prior to assaying for in vivo binding resulted in an increase in the number of sites for [³H]ADTN which correlates to the increase observed in in vitro assays following long-term treatment with these agents. The subcellular distribution of in vivo labeled ADTN sites in the caudate nucleus indicate a high density of specific binding sites in the microsomal fraction, P₃. Overall, these data demonstrate that the aminotetralins, such as ADTN, which bind with high affinity to the dopamine receptor in the caudate nucleus in vitro and in vivo, can provide precise information on the topography of this receptor.     

Comparison of Neuroleptic Binding Characteristics in Rat Striatum and Renal Cortex

Abstract

The binding characteristics of the dopaminergic ligand, ³H- spiperone, were compared in renal cortical and striatal membrane homogenates of the rat. This ligand labelled a single class of high affinity binding sites in striatum with an apparent dissociation constant (Kd) of 0.13 nM and a maximal number of binding sites (Bmax) of 890 fmol/mg protein representing D-2 receptors. In the renal cortex, ³H-spiperone identified a population of binding sites with a Bmax and a Kd of 310 fmol/mg protein and 5.1 nM, respectively. The antagonist displacing profile suggests the dopaminergic nature of the renal binding site. The affinities of dopamine antagonists for the peripheral ³H-spiperone binding site were in general in the micromolar range while the affinities of D-2 or D-2/D-1 dopamine antagonists in striatum were in the nanomolar range. Moreover, these sites showed differential stereoselectivity for (+)- and (-)-isomers of sulpiride. In conclusion, the presence of a D-2/DA-2 dopamine receptor population in renal cortex could not be confirmed. The pharmacological properties of the peripheral ³H-spiperone binding site are also different from the DA-1 receptor but seem to resemble those previously reported for dopamine receptors in sympathetic ganglia and adrenal medulla.     

Multiple dopamine binding sites: subcellular localization and biochemical characterization.

Abstract— The biochemical and pharmacological characteristics of dopamine agonist and antagonist binding to rat striatal subcellular fractions were studied and compared to the localization of dopamine–sensitive adenylate cyclase activity. The highest specific activity of adenylate cyclase sensitive to dopamine was associated almost exclusively with the crude synaptic membrane fraction (P₂). Using [³H]-haloperidol, [³H]apomorphine and [³H]spiroperidol as markers for the dopamine receptor, high affinity and stereoselective specific binding was observed for the crude synaptic fraction and the microsomal fraction (P₃). Analysis of the binding of [³H]haloperidol to the striatal microsomal preparation revealed a homogeneous receptor site with a K_d value of 3.0 nm . The data for [³H]haloperidol binding to the crude synaptosomal fraction showed two saturable binding sites with K_d values of 2.5 nm and 12.5 nm . A similar heterogeneous binding profile was observed in the P₂ fraction using [³H]apomorphine. The K_d values for [³H]apomorphine in this fraction were determined to be 1.2 nm and 7.2 nm . The effects of various biochemical parameters including ionic strength, salt concentration and pH on the binding of [³H]haloperidol to the P₂ fraction were also studied. Overall, these data show that the subcellular localization of multiple binding sites in the crude synaptosomal fraction and the identification of specific binding to purified synaptosomes correlate with the subcellular distribution of striatal dopamine-sensitive adenylate cyclase activity.     

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.     

Comparison of the effects of substituted benzamides and standard neuroleptics on the binding of 3H-spiroperidol in the rat pituitary and striatum with in vivo effects on rat prolactin secretion.

The abilities of sulpiride, metoclopramide, clozapine, loxapine, chlorpromazine, thioridazine, fluphenazine, haloperidol, (+)-butaclamol and RMI 81,582 to displace ³H-spiroperidol from rat pituitary and striatal membranes in vitro were compared to their abilities to stimulate rat prolactin secretion in vivo. There was a significant correlation between the abilities of clozapine, chlorpromazine, thioridazine, fluphenazine, RMI 81,582, haloperidol and (+)-butaclamol to bind to pituitary and striatal spiroperidol binding sites and to stimulate rat prolactin secretion. Loxapine was somewhat more potent and sulpiride and metoclopramide were markedly more potent in their abilities to stimulate prolactin secretion than would be predicted on the basis of their abilities to bind to pituitary dopamine receptors as measured by antagonism of ³H-spiroperidol binding. The abilities of metoclopramide and sulpiride to increase prolactin secretion and to produce anti-psychotic and extrapyramidal effects may be mediated by action at dopamine receptors which differ from those at which classical neuroleptics act, and they may also be mediated by non-dopaminergic mechanisms. Potency as inhibitors of ³H-neuroleptic binding in the rat pituitary or striatum appears to have heretofore unappreciated limitations to predict physiological functions such as prolactin stimulation and anti-psychotic activity.     

Dopamine receptors in bovine retina : characterization of the 3H-spiroperidol binding and its use for screening dopamine receptor affinity of drugs.

³H-spiroperidol bound in a saturable, stereospecifically displaceable manner to homogenates of bovine retina. Scatchard analysis of saturation experiments showed a K_D of 1.35 nM and a density of binding sites of 107 fmoles · mg protein^?1. Stereospecifically displaceable binding was pH and temperature dependent and linear with tissue concentration. Spiroperidol, pimozide, haloperidol and d-butaclamol were the most potent compounds in drug displacement curves (8.74 > pIC₅₀ > 7.61 M). Other neuroleptics such as cisflupenthixol, fluphenazine, clozapine, chlorpromazine and pipamperone, were one order of magnitude less potent. Among dopamine agonists, apomorphine (pIC₅₀ = 7.08 ± 0.19 M) was about 50 times more potent than dopamine itself, epinine and ADTN. Serotonin, α- and ß-adrenergic receptors agonists and antagonists were inactive. These results indicate that the sites labelled by ³H-spiroperidol in retina are dopaminergic; moreover the rank order of various antagonists and agonists observed in displacement curves suggests that this preparation could also provide a useful tool to reveal the selective affinity of drugs for the CNS dopamine receptor linked to the enzyme adenylylcyclase (D₁-receptors).     

Effects of neuroleptics on 3H-haloperidol and 3H-cis(Z)-flupenthixol binding and on adenylate cyclase activity in vitro.

The subcellular localization of dopamine-sensitive adenylate cyclase was studied in rat brain striatum and compared to the distribution of dopamine binding sites. The highest specific activity of adenylate cyclase activities sensitive to dopamine was associated almost exclusively with synaptic membranes (mithchondrial fraction; P₂). Using [³H] haloperidol and [³H] apomorphine as markers for the dopamine receptor, specific binding was observed in both the mitochondrial (P₂) and microsomal (P₃) fractions. Data for the mitochondrial fraction revealed a heterogeneity of binding sites. Two saturable sites for [³H] haloperidol were observed with K_d values of 2.5nM and 12.5nM respectively. Overall, the localization of multiple binding sites in the crude synaptosomal fraction correlates well with the localization of dopamine-sensitive adenylate cyclase in this fraction.     

Characterization of the binding of 3H-SCH 23390, a selective D-1 receptor antagonist ligand,in rat striatum

A novel benzazepine, SCH 23390, has recently been described as a very potent and selective dopamine D-1 receptor antagonist based on its potent inhibition of dopamine sensitive adenylate cyclase and its selective displacement of ³H-piflutixol from rat striatal receptor sites. In the present study, the in vitro binding of ³H-SCH 23390 to specific striatal receptor sites has been characterized. Binding was saturable and stereospecific, and the results of both saturation and competition studies are consistent with the binding of ³H-SCH 23390 to a single striatal site. A K_D of 0.53 nM was obtained through Scatchard analysis. Relative potencies of a variety of neuroleptics in competing with ³H-SCH 23390 nd also ³H-spiperone support an interpretation that the single site to which ³H-SCH 23390 binds is the D-1 dopamine receptor. Also, the binding capacity of ³H-SCH 23390 (69 pmoles/gm wet weight) is in agreement with published values for the binding capacities of ³H-piflutixol and ³H-flupentixol. These data, coupled with the low level of non-specific binding encountered with this radioligand (4–8% of total binding at normally employed ligand concentration of 0.3 nM), its high specific activity and its negligible binding to plastic and glass surfaces make it ideally suited for studying interactions with this receptor.     

Dopamine receptor sites in the anterior pituitary.

An immunocytochemical method was developed to visualize dopamine receptor sites on dispersed anterior pituitary cells of the rat. Dopamine receptors were labeled with the antagonist haloperidol. Some cells were incubated with haloperidol and a 100-fold excess of the potent antagonist D-butaclamol to determine nonspecific binding. The labeled sites were stained with an antibody against haloperidol and the peroxidase anti-peroxidase (PAP) technique. PAP complexes which served as markers for dopamine binding sites appeared on the outer plasmalemmal surface of the vast majority of mammotrophs. PAP complexes attached to the inner surface of endocytotic vesicle membrane suggested internalization of receptor-rich portions of the plasmalemma. Some gonadotrophs and somatotrophs were specifically stained to a lesser extent. However, high receptor site density and internalization of PAP complexes were never observed on cell types other than mammotrophs. The presence of dopamine receptors on the plasmalemma of mammotrophs provides strong additional evidence that dopamine acts upon these cells as a prolactin inhibitory hormone.     

Presence of D2-dofamine Receptors in Human Term Placenta

Abstract

We studied the binding of [³H]-spiperone on human term placental membranes. This binding reached plateau level after 30 min incubation at 37°C and was reversed (t_1/2 ～ 5 min) by addition of an excess of unlabeled spiperone. Scatchard analysis of saturation experiments with increasing doses of [³H]-spiperone (0–25 nM) showed one class of high affinity binding sites with a dissociation constant (Kd) of 14 ± 2 nM and a maximal binding capacity (Bmax) of 222 ± 9 fmoles/mg protein. The affinity of 5 competitors was determined in competitive binding assays. The D₂-dopamine antagonists were the most potent inhibitors: Ki for spiperone and haloperidol were 8 ± 2 and 56 ± 22 nM respectively. Dopamine inhibited [³H]-spiperone binding with a Ki of 570 ± 50 μM whereas Schering 23390 (D₁ antagonist) and propranolol (β-adrenergic antagonist) were without effect. The binding was also inhibited by 100 μM GTPγS (38 ± 8% inhibition), indicating that the dopamine receptor is coupled with a GTP binding protein. These results demonstrate for the first time the presence of D₂-dopamine receptors in human placenta.     

An Evaluation of the Interactions of Antiestrogens with Pituitary and Striatal Dopamine Receptors

Abstract

We have examined the ability of various antiestrogens (AE's) to compete with ³H-spiroperidol for binding to membrane preparations from striatal tissue and anterior pituitary glands of immature female rats in order to determine the affinity of binding of AE's to D-2 dopamine receptors. Scatchard analyses revealed the presence of a single class of high affinity receptor sites in both the striatum and pituitary with a dissociation constant (K_d) of 0.33 nM and 0.40 nM, respectively, for the dopamine antagonist spiroperidol. The AE's tamoxifen, 4-hydroxy-tamoxifen (TAM-OH), CI-628, LY 117018, and a structurally related compound t-butyl-phenoxyethyl diethylamine (BPEA) were all able to compete with spiroperidol for binding to D-2 receptors and demonstrated relative binding affinities of 0.4-0.06%, with spiroperidol set at 100%. Dopamine displayed a lower affinity, 0.01%. Estradiol failed to compete with spiroperidol for D-2 receptor binding while the non-steroidal estrogen diethylstilbestrol (DES) showed very week competition. For the lipophilic AE's, alteration of the level of their non-specific binding greatly affected their relative affinities in these competitive binding assays. The amine side chain on an aromatic ring appears to be a critical structural requirement in allowing the AE's to bind to the dopamine receptor. The relatively low affinity of AE's for the dopamine receptor and the high degree of interaction of AE's with other proteins suggest that only limited occupancy of D-2 receptors by AE's is likely in vivo.     

Dopamine receptors in the goldfish retina: 3H-spiroperidol and 3H-domperidone binding; and dopamine-stimulated adenylate cyclase activity

Dopamine receptors in the goldfish retina have been examined by binding studies using ³H-spiroperidol and ³H-domperidone as specific ligands, and by measuring retinal adenylate cyclase activities in the presence and absence of dopamine. Our results indicate that washed membranes from goldfish retinal homogenate bind a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain, with the exception that in this retina there is virtually no binding of the specific D₂ receptor antagonist, ³H-domperidone. In addition, there is a very low basal activity of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree of coupling between this enzyme and the dopamine receptor. Taken together, our findings indicate that the goldfish retina contains a high density of D₁ type dopamine receptors and few, if any, D₂ type receptors.     

Evidence for essential thiol groups and disulfide bonds in agonist and antagonist binding to the dopamine receptor

Dithiothreitol (DTT), a disulfide reducing agent, diminished the specific binding of [³H] dopamine to partially purified calf striatal membranes (P₂) but did not have an effect on [³H] spiroperidol binding. The thiol reagents, p-chloromercuribenzoate (PCMB), N-ethylmaleimide (NEM) and iodoacetamide (IA), were also tested for inhibitory effects on agonist and antagonist binding to the dopamine receptor. PCMB inhibited both [³H] dopamine and [³H] spiroperidol binding by changing the affinity (K_d) and the number of binding sites (B_max) for both of these ligands. This effect of PCMB was reversed by the addition of DTT. NEM inhibited binding to the dopamine agonist site but not to the antagonist site, while IA was ineffective on either site. These results indicate that a DTT-reducible disulfide bond may be an essential component for agonist binding to the dopamine receptor. Furthermore, the experiments with PCMB, NEM and IA suggest that the exposure of thiol groups in the dopamine receptor may play an important role in agonist and antagonist binding.     

8-[3H]Hydroxy-2-(di-n-propylamino) tetralin binding sites in goldfish retina

The binding sites of 8-[³H]hydroxy-2-(di-n-propylamino)tetralin ([³H]DPAT) were characterized in the retina of goldfish in order to evaluate the selectivity of the ligand for serotonin_1A (5HT_1A) receptors. Specificity of the binding was performed in the presence of serotonergic and dopaminergic agonists and antagonists. Buspirone, spriroxatrine and 5-methoxy-N,N-dimethyltryptamine were potent inhibitors, followed by propranolol, citalopram, imipramine and desipramine. Serotonin was not a potent inhibitor, and its interaction with the binding sites of [³H]DPAT was complex. Nomifensine displayed an important inhibition, however, other dopamine uptake blockers, such as bupropion and GBR-12909, were less potent. Haloperidol was also a good inhibitor, but the D₁ receptor agonist, SKF-38393, the D₂ receptor antagonist, sulpiride, and dopamine did not inhibit the binding. GppNHp inhibited the binding in the micromolar range. The analysis of saturation experiments by isotopic dilution, using buspirone to determine nonspecific binding, revealed two sites. The number of binding sites defined by buspirone were higher than the ones defined by nomifesine. The specific binding, using buspirone for definition, was reduced by the intraocular injection of 6-hydroxydopamine. This investigation demonstrates that [³H]DPAT labels 5HT_1A receptors in goldfish retina, but also interacts with a non-5HT receptor site. These receptors seem to be localized in dopaminergic neurons.     

CHARACTERISTICS AND REGIONAL DISTRIBUTIONS OF TWO DISTINCT [3H]NALOXONE BINDING SITES IN THE RAT BRAIN

Using [³H]naloxone at a concentration of 4.5 nm , the potent opiate agonist etorphine as well as the potent antagonist diprenorphine displace only about 75% of specific naloxone binding P₂ fractions from rat whole forebrain, without additive effect. Several other opiates and antagonists completely displace specific naloxone binding. This indicates that etorphine and diprenorphine specifically bind to one and the same naloxone binding site (type I) while leaving another naloxone binding site (type II) unaffected. Type I binding sites are much more thermo-labile than type II. [³H]Naloxone binding to type I sites is unaffected by incubation temperature in the range 10 to 25°C. while binding type II sites decreases rapidly with increasing incubation temperature, no specific type II binding being detectable at or above 20°C. The two naloxone receptor types also differ with respect to pH dependence, and affinity for naloxone with types I and II having affinity constants (K_d) of 2 and 16 nm , respectively, at 0°C. The two binding sites have different regional distributions with high relative levels of type II receptors in cerebellum and low relative levels in pons-medulla and striatum. In whole rat brain there are about 4 times as many type II receptors as type I. These results suggest that naloxone and several other opiate agonists and antagonists bind to two distinct receptor types which are probably not agonist/antagonist aspects of the same receptor.     

The effect of chronic L-DOPA administration on supersensitive pre- and postsynaptic dopaminergic receptors in rat brain

Chronic administration of haloperidol induced supersensitivity of the pre- and postsynaptic dopaminergic receptors in rat brain. The response of the presynaptic receptors was determined by an enhanced inhibitory effect of apomorphine on dopamine synthesis after gamma-butyrolactone injection. This change in the receptor function was detected both in the nigrostriatal and mesolimbic pathways. Haloperidol also increased the ³H-spiperone binding sites in striatal membranes, indicating supersensitivity of the postsynaptic receptors. Subsequent prolonged treatment with high doses of L-DOPA/carbidopa resulted in a decrease in ³H-spiperone binding sites, but had no effect on the supersensitive presynaptic receptors. It is suggested that tardive dyskinesia may be a state of both pre- and postsynaptic dopamine receptor supersensitivity and that chronic L-DOPA treatment may have a differential effect on these sites.     

Effect of temperature and ionic environment on the specific binding of 3H(—)sulpiride to membranes from different rat brain regions

Sulpiride is an antipsychotic drug endowed with the properties of a dopamine antagonist. The failure of sulpiride to inhibit neostriatal dopamine stimulated adenylate cyclase activity indicated that this drug is a selective D₂ receptor antagonist. In this study we used a novel synthesized ²H(—)sulpiride with very high specific activity (72 Ci/mol) and characterized the temperature sensitivity of the binding sites labeled by this compound. Kinetic analysis of ³H(—)sulpiride binding in rat striatum showed unstable behavior when incubation was performed at 37 or 30°C. However when experiments were carried out at 15 or 10°C, binding reached a stable steady-state within 10 min. Scatchard analysis of binding isotherms obtained at 10°C showed a 5-fold increase in the maximum number of binding sites and a decrease in K_d values to one-third those obtained at 37°C. Pharmacological characterization of the binding sites labeled by ³H(—)sulpiride at 10°C showed a greater affinity for antagonists but not for agonists than 37°C. Under both experimental condition, ³H(—)sulpiride binding sites were Na⁺ and GTP-sensitive. The temperature sensitive binding phenomenon appeared to be area specific. ³H(—)sulpiride binding sites in tissues other than from striatum were influenced less or not at all by changes in incubation temperature.     

Solubilization and Characterization of Striatal Dopamine Receptors

Abstract: Dopamine receptor binding proteins were sol-ubilized with the detergent 3–(3–cholamidopropyl) dimethylammonio - 2 - hydroxy - 1– propanesulfonate (CHAPSO) from bovine and rat striatal membranes. The binding of the dopamine antagonist [³H]spiroperidol ([³H]Spi) to the solubilized dopamine receptors was determined by the polyethyleneglycol method. The CHAPSO-solubilized dopamine receptor binding proteins remain in the supernatant fraction following centrifuga-tion at 100,000 ×g for 2 h. The CHAPSO-solubilized dopamine receptor proteins, as well as the prelabeled [³H]Spi-receptor protein complex, bind specifically to wheat germ agglutinin (WGA)-agarose columns, which is consistent with an identification as glycoproteins. HPLC analysis of the CHAPSO-solubilized, prelabeled [³H]Spi-receptor protein complex (CHAPSO preparation) reveals association with a high molecular weight form, indicating the formation of aggregates and/or micelles. Treatment of the WGA-agarose-bound [³H]Spi-receptor protein complex with digitonin (CHAPSO-digitonin preparation) results in dissociation of the high molecular weight form into lower molecular weight forms. The HPLC profile of the prelabeled [³H]Spi-receptor complex in the CHAPSO-digitonin preparation reveals two radioactive peaks. The major peak had a retention time of 16 min, corresponding to an apparent MW of 175,000, whereas the minor peak had a retention time of 21 min, corresponding to an apparent MW of 49,000. The CHAPSO-solubilized dopamine receptor binding proteins are sensitive to modulation by GTP, indicating that the association with the GTP binding component is preserved in the “soluble” state. The potencies of dopamine antagonists and agonists for inhibiting the binding of [³H]Spi to CHAPSO-solubilized dopamine receptor proteins are similar to those for membrane-bound proteins. Chronic treatment with haloperidol increases the B_max, and does not change the K_D for [³H]Spi in the CHAPSO-solubilized and in the membrane-bound preparations. Thus, the CHAPSO-solubilized dopamine receptor proteins retain the binding characteristics of the supersensitive membrane-bound dopamine receptors.