[3H]Metergoline: A New Ligand of Serotonin Receptors in the Rat Brain

Abstract: A specific binding site for [³H]metergoline characterized by a K_D of 0.5–1.0 nM was detected in microsomal and synaptic plasma membranes from various areas of the adult rat brain. Experiments with 5,7-dihydroxy-tryptamine- and kainic acid-induced lesions indicated that this specific binding site was localized post-synaptically with respect to serotoninergic neurons. The pharmacological characteristics of [³H]metergoline binding to microsomal membranes from the whole forebrain strongly suggest that this ligand labels a class of serotonin receptors. This was particularly obvious in the hippocampus in which serotonin was about 400 times more potent than dopamine and norad-renaline for displacing bound [³H]metergoline. In the striatum, serotonin was only 10 times as potent as dopamine in inhibiting [³H]metergoline binding, suggesting that this ligand may also bind to dopamine receptors. Striking similarities between the binding sites for [³H]metergoline and [³H]serotonin were observed in the hippocampus. Thus, not only the total numbers of binding sites for these two ligands in control rats but also their respective increases following intracerebral 5,7-dihydroxytryptamine treatment were very similar. Therefore, at least in the hippocampus, [³H]metergoline might well be the appropriate ligand for studying the characteristics of the 'antagonist form' of serotonin receptors postulated by Bennett and Snyder.     

Histamine H1-Receptor Binding Sites in Guinea Pig Brain Membranes: Regulation of Agonist Interactions by Guanine Nucleotides and Cations

Abstract: Agonist, but not antagonist, interactions with histamine H₂-receptors labeled by [³H]mepyramine are regulated selectively by sodium, divalent cations, and guanine nucleotides. Sodium decreases the affinity of histamine and the agonist 2-amino-ethylpyridine for [³H]mepyramine sites in guinea pig brain membranes up to 10-fold. The effect of sodium is exerted to a lesser extent by lithium, while potassium and rubidium are much weaker. Guanine nucleotides also decrease the affinity of histamine for H₁ binding sites about twofold. GTP and its nonmetabolized analogue GMP-PNP as well as GDP exert similar effects, while GMP, ATP, ADP, and AMP are inactive. The effects of GTP and sodium on histamine interactions with H₁-receptors are additive. By contrast, certain divalent cations enhance the potency of histamine at H₁-receptors. Manganese is most potent, while magnesium is almost as active as manganese and calcium is essentially inactive. Sodium, divalent cations, and guanine nucleotides have negligible effects on the interactions of antihistamines with H₁-receptors.     

Differential Regulation by Guanine Nucleotides of Opiate Agonist and Antagonist Receptor Interactions

Abstract: Guanine nucleotides differentiate binding of tritium-labeled agonists and antagonists to rat brain membranes. In the absence of sodium, GTP (50 μM) decreased binding of [³H]-labeled agonists by 20–60% and [³H]-labeled antagonists by 0–20%. In the presence of 100 mM-NaCl, GTP had no effect on antagonist binding, but decreased agonist binding by 60–95%. GMP was less potent than either GTP or GDP in decreasing agonist binding. GTP (50 μM) reduced high-affinity [³H]dihydromorphine sites by 52% and low-affinity sites by 55%. Without sodium, GTP reduced high-affinity [³H]-naloxone sites by 36%; in the presence of 100 mM-NaCl, GTP had no effect on either high- or low-affinity [³H]naloxone sites. GTP increased the association rate of [³H]dihydromorphine twofold and the dissociation rate by fourfold, while having no effect on association or dissociation rates of the antagonist [³H]diprenorphine. The affinities of uniabeled antagonists in inhibiting [³H]-diprenorphine binding were not affected by GTP or sodium, but the affinities of agonists were reduced 40- 120-fold, with met- and leu-enkephalin affinities reduced by the greatest degree. GTP and sodium lowered [³H]dihydromorphine binding in an additive fashion, while divalent cations, especially manganese, reversed the effects of GTP on [³H]-labeled agonist binding by stimulating membrane-bound phosphatases that hydrolyze GTP to GMP and guanosine. These results suggest that by affecting binding of agonists, but not antagonists, GTP may regulate opiate receptor interactions with their physiological effectors.     

Zinc Inhibition of t-[3H]Butylbicycloorthobenzoate Binding to the GABAA Receptor Complex

Abstract: The effect of Zn²⁺ on t -[³H]butylbicycloorthobenzoate ([³H]TBOB) binding to the GABA_A receptor complex was studied autoradiographically in rat brain. Zn²⁺ inhibited [³H]TBOB binding in a dose-dependent manner at physiological concentrations. Saturation analysis revealed noncompetitive inhibition in various brain regions. The inhibitory effect of Zn²⁺ had regional heterogeneity; regions showing the greatest inhibition of [³H]TBOB binding were cortical laminae I–III, most areas of hippocampus, striatum, septum, and cerebellar cortex. Regions with relatively less inhibition of [³H]TBOB binding included cortical laminae V–VI, thalamus, superior colliculus, inferior colliculus, and central gray matter. The effect of Zn²⁺ and those of other GABA_A ligands, such as benzodiazepines, bicuculline, isoguvacine, and picrotoxin, on [³H]TBOB binding seemed to be additive. Ni²⁺, Cd²⁺, and Cu²⁺ also inhibited [³H]TBOB binding with a regional heterogeneity similar to that produced by Zn²⁺. These results are consistent with Zn²⁺ acting at the previously detected recognition site on the GABA_A receptor complex, distinct from the picrotoxin, GABA, and benzodiazepine sites. The regional heterogeneity of the Zn²⁺ effect may reflect differential regional distribution of GABA_A receptor subtypes among brain regions. Other divalent cations probably act at the Zn²⁺ binding site.     

Inhibitory Effects of Ascorbic Acid on the Binding of [3H]Dopamine Antagonists to Neostriatal Membrane Preparations : Relationship to Lipid Peroxidation

Abstract: Ascorbic acid, sodium ascorbate, and isoascorbic acid (the stereo-isomer of ascorbic acid) inhibited the stereospecific binding of [³H]spiroperidol to neostriatal membrane preparations. Greater inhibitory effects were obtained at intermediate concentrations of the three ascorbic acid analogs (i.e., 0.06 and 0.6 mM) than at higher (6 m M ) or lower (0.006 m M ) concentrations. In parallel experiments, the three ascorbic acid analogs induced lipid peroxidation, which was also greater at the two intermediate than at higher or lower concentrations. Several known inhibitors of lipid peroxidation, including propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, α-naphthol, and cobalt chloride, as well as the iron chelating agents EDTA and DETAPAC (diethylenetriaminepentaacetic acid) were able to counteract the effects of the ascorbic acid analogs on both lipid peroxidation and on [³H]spiroperidol binding. These data strongly suggest that an iron-catalyzed lipid peroxidation is responsible for the observed inhibitory effects on binding. In other experiments, neostriatal membrane preparations that were preincubated with ascorbic acid (0.6 m M ) and subsequently washed still had greatly diminished capacity to bind [³H]spiroperidol, indicating that ascorbic acid need not be physically present during the binding assay in order to affect binding. This experimental procedure also appears to be a way in which [³H]spiroperidol binding sites can be inactivated and washed free of the inactivating agent.     

Comparison of [3H]WIN 35,428 Binding, a Marker for Dopamine Transporter, in Embryonic Mesencephalic Neuronal Cultures with Striatal Membranes of Adult Rats

Abstract: In contrast to striatal membranes of adult rats, where high- ( K _D1= 34 n M ) and low- ( K _D2= 48,400 n M ) affinity binding sites for [³H]WIN 35,428 are present, in primary cultures of ventral mesencephalon neurons (CVMNs) only low-affinity binding sites were found ( K _D= 336,000 n M ). The binding of [³H]WIN 35,428 in CVMNs prepared from rat embryos was reversible, saturable, and located in cytosol. Although dopamine (DA) uptake blockers inhibited [³H]DA uptake at nanomolar concentrations in CVMNs, the displacement of [³H]WIN 35,428 binding in CVMNs by DA uptake inhibitors required 100-8,000 times higher concentrations than were needed to displace [³H]WIN 35,428 binding in striatal membranes. Piperazine derivatives, e.g., GBR-12909, GBR-12935, and rimcazole, inhibited [³H]WIN 35,428 binding in CVMNs more effectively than did cocaine, WIN 35,428, mazindol, nomifensine, or benztropin. A positive correlation ( r = 0.779; p < 0.001) was found between drug affinities for the striatal membrane sites labeled by [³H]WIN 35,428 and their abilities to inhibit DA uptake in CVMNs, whereas no correlation existed between the IC₅₀ values of drugs that inhibited [³H]WIN 35,428 binding and [³H]DA uptake in CVMNs. The cytosolic [³H]WIN 35,428 binding sites may be a piperazine acceptor and may not be involved in the regulation of the DA transporter.     

High-Affinity Choline Transport Sites: Use of [3H]Hemicholinium-3 as a Quantitative Marker

Abstract: High-affinity choline transport (HAChT), the rate-limiting and regulatory step in acetylcholine (ACh) synthesis, is selectively localized to cholinergic neurons. Hemicholinium-3 (HC3), a potent and selective inhibitor of HAChT, has been used as a specific radioligand to quantify HAChT sites in membrane binding and autoradiographic studies. Because both HAChT velocity and [³H]HC3 binding change as in vivo activity of cholinergic neurons is altered, these markers are also useful measures of cholinergic neuronal activity. Evidence that [³H]HC3 is a specific ligand for HAChT sites on cholinergic terminals is reviewed. The ion requirements of HAChT and [³H]HC3 binding indicate that sodium and chloride are required for recognition of both choline and [³H]HC3. A common recognition site is also indicated by the close correspondence of the potency of HC3 and choline analogues for inhibiting both HAChT and [³H]HC3 binding. The parallel regional distributions of both markers in adult brain, during development and after specific lesions, all indicate specific cholinergic localization. The close association of HAChT and [³H]HC3 binding sites is also supported by parallel regulatory changes occurring after in vivo drug treatments and in vitro depolarization. Overall, the data indicate a close association between HAChT and [³H]HC3 binding and are consistent with the sites being identical. Methodologic considerations in using [³H]HC³ as a ligand and considerations in interpretation of results are also discussed.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Distribution of Specific High-Affinity Binding Sites for [3H]Imipramine in Human Brain

Abstract: [³H]Imipramine binds with high affinity to membranes from different regions of the human brain. The highest density of binding sites was observed in the hypothalamus and substantia nigra and the lowest density in the white matter and cerebellum. As found in rat brain, tricyclic antidepressant drugs are potent inhibitors of [³H]imipramine binding. Atypical antidepressants are, however, much weaker at inhibiting the specific binding. The [³H]imipramine binding site in human cortex is apparently identical to the site already described in the rat brain and in human platelets.     

Desensitization of Nicotine-Stimulated [3H]Dopamine Release from Mouse Striatal Synaptosomes

Potential desensitization of brain nicotinic receptors was studied using a [³H]dopamine release assay. Nicotine-stimulated [³H]dopamine release from mouse striatal synaptosomes was concentration-dependent with an EC₅₀ of 0.33 ± 0.13 μ M and a Hill coefficient of 1.44 ± 0.18. Desensitization by activating concentrations of nicotine had a similar EC₅₀ and a half-time of 35 s. Concentrations of nicotine that evoked little release also induced a concentration-dependent desensitization (EC₅₀=6.9 plusmn; 3.6 n M , t_1/2= 1.6-2.0 min, n _H=1.02 ± 0.01). Both types of desensitization produced a maximum 75% decrease in [³H]dopamine release. Recovery from desensitization after exposure to low or activating concentrations of nicotine was time-dependent with half-times of 6.1 min and 12.4 min, respectively. Constants determined for binding of [³H]nicotine to striatal membrane at 22°C included a K _Dof 3.7 ± 0.5 n M , B_max of 67.5 ± 2.2 fmol/mg, and Hill coefficient of 1.07 ± 0.06. Association of nicotine with membrane binding sites was biphasic with half-times of 9 s and 1.8 min. The fast rate process contributed 37% of the total reaction. Dissociation was a uniphasic process with a half-time of 1.6 min. Comparison of constants determined by the release and binding assays indicated that the [³H]-nicotine binding site could be the presynaptic receptor involved in [³H]dopamine release in mouse striatal synaptosomes.     

NEUROCHEMICAL PARAMETERS IN THE HYPERRESPONSIVE PHASE AFTER A SINGLE DOSE OF NEUROLEPTICS TO MICE

Abstract— Four days after a single dose of teflutixol (5 mg/kg i.p.), at which time mice are superresponsive to dopamine agonists, e.g. apomorphine, the specific binding of [³H]haloperidol, [³H]cis (Z)-flupenthixol, [³H]apomorphine, [³H]dopamine, [³H]propylbenzilylcholine mustard and [³H]GABA to striatal membranes in vitro is equal to that of saline-treated mice. Specific binding of [³H]haloperidol is also unchanged 3 days following a single dose of fluphenazine (5mg/kg i.p.) and 2 days following haloperidol (5 mg/kg i.p.), but slightly decreased 3 days following cis(Z)-flupenthixol (5 mg/kg i.p.).
The possibility that remaining neuroleptic or active metabolites could obscure a slight increase in dopamine receptor binding was rejected, since remaining amounts of [³H]teflutixol in the final binding assay 4 days after intraperitoneal injection of [³H]teflutixol (5 mg/kg) were too small to influence the binding of [³H]haloperidol in vitro .
It is concluded that the pharmacological superresponsiveness and the decrease in dopamine synthesis and release seen after the initial receptor blockade following a single dose of neuroleptic drugs in mice are nor accompanied by changes in dopamine, muscarine or GABAergic receptor characteristics in corpus striatum. The possibility that changes occur in a small number of functional operative dopamine receptors cannot be excluded, however.     

[3H]SCH 23390 Binding to Human Putamen D-1 Dopamine Receptors: Stereochemical and Structure-Affinity Relationships Among l-Phenyl-1H-3-Benzazepine Derivatives as a Guide to D-l Receptor Topography

Abstract: A series of l-phenyl-1 H -3-benzazepine analogues were assessed for enantiomeric and structure-affinity relationships at human putamen D-1 dopamine receptors labelled with [³H]SCH 23390. Substitution at the 7-position of both 3-H and 3-methyl benzazepine molecules critically affected affinity for these receptors over a 500-fold range. The general rank order of potency of 7-substituents was Cl = Br ≫ CH₃ > OH ≥ H. 3-Methyl substituents increased the affinity of 7-H and 7-OH compounds two- to fivefold compared to desmethyl counterparts. The displacement of [³H]SCH 23390 binding showed substantial enantioselec-tivity; the R-enantiomer of SKF 83566 was 500-fold more potent that its S-antipode. However, the displacement of [³H]spiperone binding from D-2 sites in the same tissue showed negligible enantioselectivity. Through such structure-affinity relationships, these studies may help to define the topography of the human brain D-1 dopamine receptor and guide the design of more selecive agents for functional studies.     

Aminergic Receptors in Drosophila melanogaster: Properties of [3H]Dihydroergocryptine Binding Sites

Abstract: [³H]Dihydroergocryptine ([³H]DHE) binds to a particulate preparation from Drosophila melanogaster heads at a level of 2.4 ± 0.4 pmol/mg protein, with an apparent dissociation constant of 2.0 ± 0.5 n M . The binding sites are inactivated by heat, pronase treatment, detergents, and sulfhydryl and disulfide reagents. [³H]DHE binding is inhibited by low concentrations of serotonergic and α-adrenergic ligands. The specificity of the binding sites, as revealed by displacement studies, differs from that of [³H]DHE binding sites in various vertebrate tissues. The [³H]DHE binding sites may correspond to serotonergic receptors, and possibly, to additional classes of receptors for putative neurotransmitters in Drosophila .     

Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D₁ and D₂ receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D₁ receptor antagonist, [³H]SCH 23390, and a dopamine D₂ receptor antagonist, [³H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant ( K _D) of 0.65 and 0.5 n M , respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [³H]SCH 23390 and [³H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D₁ or D₂ receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [³²P]NAD demonstrated predominant labeling of bands of M_r 47,000–52,000, 42,000–45,000, and 40,000-39,000, which corresponded to the known molecular weights of the α subunits of G_s and G_i proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D₂ receptor agonists, was present in the CVs. These findings suggest that the dopamine D₁ and D₂ receptors in the CVs couple with adenylate cyclase via G_s/G_i protein.     

Uptake of [3H]Dopamine into Dopaminergic and Noradrenergic Neurones of the Isolated Neurointermediate Lobe of the Rat Hypophysis. Effects of Desipramine and Nomifensine

Abstract: The isolated neurointermediate lobe (NIL) of the rat hypophysis accumulates [³H]dopamine from the incubation medium. Column chromatographic analysis showed that 92% of the tissue radioactivity was contained in the catecholamine fraction. [³H]Dopamine represented 70% and [³H]noradrenaline 30% of the [³H]catecholamines. Desipramine (1 μM) prevented the formation of [³H]noradrenaline without affecting the storage of [³H]dopamine. Nomifensine (10 μM) blocked the storage of [³H]dopamine and [³H]noradrenaline. Thus, in the NIL, [³H]dopamine is taken up into dopaminergic and noradrenergic neurones. In the latter, [³H]dopamine is converted to [³H]noradrenaline, indicating a significant dopamine β-hydroxylase activity in the NIL tissue. A selective labeling of the dopamine stores with [³H]dopamine can be achieved in the presence of desipramine.     

Characterization of a Human NK1 Tachykinin Receptor in the Astrocytoma Cell Line U 373 MG

Abstract: The human NK₁ tachykinin receptor in the astrocytoma cell line U 373 MG was characterized using selective agonists and antagonists described for this receptor in the rat. Specific [³H]substance P binding sites were present on cell homogenates, whereas [³H]neurokinin A or [³H]-senktide binding sites were absent. The binding was saturable and reversible. The binding of [³H]substance P was inhibited by very low concentrations of [L-Pro⁹]substance P and [Sar⁹,Met(O₂)¹¹]substance P; septide was ∼ 1,000-fold less potent. The most potent peptide antagonist was trans -4-hydroxy-1-(1 H -indol-3-ylcarbonyl)-L-prolyl- N -methyl- N -(phenylmethyl)-L-tyrosineamide. The rank order of potency for the nonpeptide antagonists was ( S , S )-CP 96,345 > (±)-CP 96,345 > (±)-2-chlorobenzylquinuclidinone > ( R , R )-CP 96,345 > RP 67580 > RP 68651. In [³H]-inositol-labeled cells, substance P stimulated phosphatidylinositol turnover. A good correlation was found when the abilities of NK₁ receptor agonists for stimulating inositol phosphate production and for inhibiting [³H]substance P binding were compared. Similarly, the binding and functional assays were well correlated for the antagonists. As a result of its high sensitivity and selectivity, the U 373 MG cell line thus appears an excellent tool for investigating the pharmacology of the human NK₁ receptor.     

[3H] GBR-12935 Binding to Cytochrome P450 in the Human Brain

Abstract: The presence of multiple [³H] GBR-12935 binding sites in the human brain has been revealed in several recent studies. One site represents the dopamine uptake site. In rat brain it was demonstrated that [³H] GBR-12935 also binds to nondopaminergic "piperazine acceptor sites." One of these sites has been identified as cytochrome P450IID1 in canine brain. [³H] GBR-12935 binding to the piperazine acceptor sites in the human brain was investigated in the present study. A pharmacological definition of the piperazine acceptor sites is presented: the [³H]- GBR-12935 binding fraction that could be discriminated by 10 μ M GBR-12909 in the presence of 0.3 μ M mazindol. This binding fraction was saturable, with binding affinity in the range of 3–8 n M. It was also demonstrated that the piperazine acceptor or cytochrome P450-sensitive drugs cis -flupentixol and proadifen (SKF 525 A) compete for the same binding sites, suggesting the cytochrome P450 nature of the binding. The findings presented support the proposal that at least part of this fraction represents cytochrome P450IID6, the human form of P450IID1. The distribution of [³H] GBR-12935 binding to the suggested P450IID6-site in 12 brain regions was examined, without significant differences in binding densities between the regions. The significance of the present findings on the cytochrome P450 system in brain is discussed.     

The Peripheral-Type Benzodiazepine Receptor Ligands [3H]Ro 5-4864 and [3H]PK 11195 Bind to the Retina of Rabbit, but Not of Turtle

Abstract: The binding of [³H]flunitrazepam, [³H]RO 5-4864, and [³H]PK 11195 to membrane preparations of the retina was studied in the turtle and rabbit. Only a single population of [³H]flunitrazepam binding sites was detected in the turtle, whereas two populations appeared to be present in the rabbit. No specific binding for [³H]RO 5-4864 and [³H]PK 11195 could be detected in the turtle. In rabbit, both ligands bound with high affinity, revealing a significant population of binding sites (K_D values of 24 ± 2.3 and 2.2 ± 0.8 nM, and B_max values of 440 ± 35 and 1,482 ± 110 fmol/mg of protein, respectively). The binding was temperature - and protein-dependent. Displacement studies showed a similar rank order of potency of various unlabeled ligands against both [³H]RO 5-4864 and [³H]PK 11195 (PK 11195 > Ro 5-4864 > flunitrazepam > flumazenil). These results suggest that peripheral-type benzodiazepine receptors are present in the retina of the rabbit, but not of the turtle.     

Allosteric Interactions Among Agonists and Antagonists at 5-Hydroxytryptamine3 Receptors

Abstract: Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine₃ (5-HT₃) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT₃ receptors. The affinities of competitive 5-HT₃ receptor antagonists were similar regardless of whether the receptors were labeled with [³H]RS-42358, [³H]granisetron, or 1-( m -[³H]chlorophenyl)biguanide ([³H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [³H]mCPG. The dissociation of [³H]mCPG, [³H]RS-42358, and [³H]RS-25259, but not [³H]granisetron, from both cloned and native 5-HT₃ receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT₃ receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.     

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.