Comparison of the Binding of [3H]Spiperone and [3H]Domperidone in Homogenates of Mammalian Retina and Caudate Nucleus

Abstract— The specific binding of [³H]spiperone and [³H]domperidone, as defined by 1 μ m -(+)butaclamol, was compared in homogenates of bovine retina and caudate nucleus. Scatchard analyses of saturation data for [³H]spiperone binding yielded dissociation constants ( K _d) of 0.35 n m in the retina and 0.64 n m in the caudate nucleus. Comparison of the maximum number of binding sites (B_max) present in each tissue indicated that the density of sites in bovine caudate nucleus (270 fmol/mg protein) was approximately three times higher than in bovine retina (92 fmol/mg protein). This difference was even more marked in guinea pig tissues, with a ratio of 7:1 between corpus striatum and retina. The pharmacological analysis of [³H]spiperone binding in both the bovine retina and caudate nucleus indicated an interaction with dopaminergic rather than serotonergic sites. However, inhibition curves obtained to dopaminergic agonists in the bovine retina were significantly steeper than those observed in the bovine caudate nucleus, as reflected in the greater Hill coefficients obtained for these agents in the retina. Furthermore, only a small amount of specific [³H]domperidone binding was observed in either the bovine caudate nucleus or the guinea pig striatum, whilst no specific [³H]domperidone binding was detectable in homogenates of either bovine or guinea pig retina. These data suggest that the retina possesses only a small population of dopaminergic D2 sites and that these binding sites may differ from those present in the caudate nucleus.     

Characterization of [3H]Met-Enkephalin-Arg6-Phe7 Binding to Opioid Receptors in Frog Brain Membrane Preparations

Abstract: A tritiated heptapeptide, [³H]Tyr-Gly-Gly-Phe-Met-Arg-Phe ([³H]Met-enkephalin-Arg⁶-Phe⁷), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent K _D value of the radioligand calculated from homologous displacement experiments was 3.4 n M , and the maximal number of specific binding sites was 630 fmol/mg of protein. The K _D determined from equilibrium saturation binding studies was found to be 3.6 n M . However, the Hill coefficient was far below unity ( n _H = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [³H]Met-enkephalin-Arg⁶-Phe⁷ binding reflects a κ₂- and/or δ-subtype specificity of the heptapeptide. Binding to a κ₁ and/or μ site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met-enkephalin-Arg⁶-Phe⁷ in frogs cannot be ruled out. The [³H]Met-enkephalin-Arg⁶-Phe⁷ binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide.     

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 .     

The [3H]Tryptamine Receptor in Human Brain: Kinetics, Distribution, and Pharmacologic Profile

Abstract: The kinetics and distribution of [³H]tryptamine binding sites in human brain were investigated. Specific [³H]tryptamine binding in frontal cortex was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. A heterogeneous distribution for this binding site was demonstrated, with the highest density observed in hippocampus, thalamus ≫ caudate nucleus, frontal cortex, pons, temporal cortex > globus pallidus/putamen, cerebellum. The similarities in kinetics and distribution of the [³H]tryptamine binding site in human and rat brain indicate that these two binding sites represent homologous structures. However, the present displacement studies using various ligands (indoleamines and other tryptophan metabolites, phenylethylamines, and miscellaneous drugs) and salts (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cu²⁺) indicate stereospecific displacement as well as a rank-order potency profile that is different from that reported for the rat [³H]tryptamine binding site. This suggests the presence of distinct species-dependent [³H]tryptamine binding site subtypes. Taken together with the documented electrophysiological and behavioral evidence of tryptamine-mediated effects in the rat and the recent report of a significant loss of these binding sites in human portal systemic encephalopathy, as well as the present demonstration of an effect of guanine nucleotides on [³H]-tryptamine binding affinity, these findings suggest that these binding sites might be functional receptors. The implied role of tryptamine in neuropsychiatric disorders is supported by this demonstration of a receptor for [³H]-tryptamine in human brain.     

Characterization of 5,7-Dichlorokynurenate-Insensitive d-[3H]Serine Binding to Synaptosomal Fraction Isolated from Rat Brain Tissues

Abstract: To explore target sites for endogenous d -serine that are different from the glycine site of the N -methyl- d -aspartate (NMDA) type glutamate receptor, we have studied the binding of d -[³H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 µ M ) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 m M unlabeled d -serine. Association, dissociation, and saturation experiments indicated that d -[³H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K _D of 614 n M and a B _max of 2.07 pmol/mg of protein. d -Serine, l -serine, and glycine produced a total inhibition of the specific DCK-insensitive d -[³H]serine binding to the cerebellum with similar K _i values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 µ M . The profiles of displacement of the DCK-insensitive d -[³H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive d -[³H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive d -[³H]serine binding site could be a novel candidate for a target for endogenous d -serine in mammalian brains.     

M1 Muscarinic Acetylcholine Receptor in Cultured Rat Neostriatum Regulates Phosphoinositide Hydrolysis

Abstract: : Muscarinic acetylcholine receptor expression and function in cultured rat neostriatal neurons were examined. All experiments were performed on intact neurons grown in vitro for 12-14 days. The muscarinic antagonist N-[³H]methylscopolamine ([³H]NMS) binds to a single site in cultures with a K_D of 89 pM and a B_max of 187 fmol/mg of protein, or 32,000 sites/neuron. Competition studies using [³H]NMS were performed to determine what receptor sur > types were present. Nonlinear analysis of competition curves was best described with a single binding site for atropine, pirenzepine, and AF-DX 116 {11-[[2-[(diethylamino)-methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one}, with K_i values of 0.6, 62, and 758 nM, respectively. These results indicate that the muscarinic receptors present in neostriatal cultures are of the M₁subtype, having high affinity for pirenzepine and low affinity for AF-DX 116. In contrast with antagonists, carbachol displaced [³H]NMS from two sites with K_i values of 6.5 and 147 μM, with the higher-affinity form predominant (83% of sites). The M₁ receptor subtype was linked to phosphoinositide turnover. Carbachol stimulated the formation of phosphoinositides with an EC₅₀ of 37 μM and was antagonized by atropine. At equimolar doses, pirenzepine was more potent than AF-DX 116 at antagonizing the response.     

Characterization of [3H]CP-96,501 as a Selective Radioligand for the Serotonin 5-HT1B Receptor: Binding Studies in Rat Brain Membranes

Abstract: 3-(1,2,5,6-Tetrahydro-4-pyridyl)-5- n -propoxyindole (CP-96,501) was found to be a more selective ligand at the serotonin 5-HT_1B receptor than the commonly used 5-HT_1B agonist, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [³H]CP-96,501, was found to bind with a high affinity ( K _D, 0.21 n M ) to a single binding site ( n _H, 1.0). The receptor density of this site ( B _max, 72 fmol/mg of protein) matched that of the 5-HT_1B receptor determined with [³H]5-HT. Competition curves of 16 serotonergic compounds in [³H]CP-96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5-HT_1B receptor determined with [³H]5-HT or [¹²⁵I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [³H]CP-96,501 binding. In the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p], [³H]CP-96,501 binding was reduced, while the potency of CP-96,501 to displace [¹²⁵I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP-96,501. The results of this study suggest that [³H]CP-96,501 is a useful agonist radioligand for the 5-HT_1B receptor.     

[3H]Nemonapride and [3H]Spiperone Label Equivalent Numbers of D2 and D3 Dopamine Receptors in a Range of Tissues and Under Different Conditions

Abstract: [³H]Nemonapride and [³H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [³H]nemonapride and [³H]spiperone give markedly different B _max values for preparations of D₂ dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D_2(short), D_2(long), and D₃] in different buffers. B _max values derived using either radioligand differ by an average of <20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [³H]spiperone and [³H]nemonapride do not differentiate between different forms or populations of D₂-like receptors.     

[3H]Spiperone Labels Non-Cyclase-Linked Dopamine Receptors in the Ventral Tegmental Area of Rat Brain

Abstract: The binding of [³H]spiperone, a neuroleptic/dopamine receptor ligand, to membranes of the ventral tegmental area of the rat was studied in vitro and found to be rapid, saturable, reversible, and of high affinity. Specific binding was displaced by the dopaminergic agonists dopamine, apomorphine, and 2-amino-6,7-dihydroxytetralin, and stereospecifically by the neuroleptic drugs butaclamol and flupenthixol. Bromocryptine and other ergots displaced the binding, as did the D-2 antagonists domperidone, molindone, metoclopramide, and sulpiride. Noradrenergic, histaminergic, and serotonergic components of the binding were not detected in displacement studies with various agonists and antagonists. These data are consistent with the hypothesis that [³H]spiperone labels dopamine receptors in the ventral tegmental area that are not linked to adenylate cyclase and are therefore likely to be of the D-2 type.     

Comparison of (R)-[3H] Tomoxetine and (R/S)-[3H] Nisoxetine Binding in Rat Brain

Abstract: ( R )-[³H]Tomoxetine is a radioligand that binds to the norepinephrine (NE) uptake site with high affinity but also binds to a second, lower-affinity site. The goal of the present study was to identify the nature of this low-affinity site by comparing the binding properties of ( R )-[³H]tomoxetine with those of ( R/S )-[³H]nisoxetine, a highly selective ligand for the NE uptake site. In homogenate binding studies, both radioligands bound to the NE uptake site with high affinity, whereas ( R )-[³H]tomoxetine also bound to a second, lower-affinity site. The autoradiographic distribution of binding sites for both radioligands is consistent with the known distribution of NE-containing neurons. However, low levels of ( R )-[³H]-tomoxetine binding were seen in the caudate-putamen, globus pallidus, olfactory tubercle, and zona reticulata of the substantia nigra, where ( R/S )-[³H]nisoxetine binding was almost absent. In homogenates of the caudate-putamen, the NE uptake inhibitors desipramine and ( R )-nisoxetine and the serotonin (5-HT) uptake inhibitor citalopram produced biphasic displacement curves. Autoradiographic studies using 10 n M ( R )-nisoxetine to mask the binding of ( R )-[³H]tomoxetine to the NE uptake site produced autoradiograms that were similar to those produced by [³H]citalopram. Therefore, ( R )-[³H]tomoxetine binds to the NE uptake site with high affinity and the 5-HT uptake site with somewhat lower affinity.     

Pharmacology and Regional Distribution of the Binding of 6-[3H]Nitro-7-Sulphamoylbenzo[f]-Quinoxaline-2,3-Dione to Rat Brain

Abstract: 6-Nitro-7-sulphamoylbenzo[ f ]quinoxaline-2,3-dione (NBQX) is a competitive antagonist selective for α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. Here we report the pharmacological characteristics and anatomical distribution of [³H]NBQX binding to rat brain. The association rate of [³H]NBQX to rat cerebrocortical membranes was rapid, with peak binding occurring within 10 min at 0°C. The off-rate was also rapid, with near-complete dissociation of the radioligand within 5 min of addition of 1 m M unlabelled l -glutamate. [³H]NBQX bound to a single class of sites with K _D and B _max values of 47 n M and 2.6 pmol mg⁻¹ of protein, respectively. The rank order of inhibition of [³H]NBQX binding by AMPA receptor ligands was NBQX ≫ 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) ≥ ( S )-5-fluorowillardiine ≥ AMPA ≫ l -glutamate. The chaotrope KSCN had no effect on the IC₅₀ value of unlabelled NBQX displacement of [³H]NBQX binding. The kainate receptor-selective ligands NS102 and kainate were only very weak displacers. It is interesting that NBQX and CNQX displaced significantly more [³H]NBQX than any of the agonists tested. Autoradiographic analysis of the binding of [³H]NBQX to coronal sections showed a distribution compatible with that of [³H]AMPA binding. These data indicate that [³H]NBQX provides a useful novel tool to characterise the antagonist binding properties of AMPA receptors.     

[3H]Imipramine Binding of Protein Nature in Human Platelets: Inhibition by 5-Hydroxytryptamine and 5-Hydroxytryptamine Uptake Inhibitors

Abstract: The nature of [³H]imipramine binding to human platelets was investigated. Desipramine and 5-hydroxytryptamine (5-HT) displaced the same amount of binding and the binding was sensitive to protease treatment. The nature of pharmacological inhibition of [³H]imipramine binding was investigated in saturation experiments. Increases in K d without changes in B _max were noted with the addition of 5-HT, desipramine, norzimeldine, or 5-methoxytryptoline. Reductions in B _max without alterations in K _D were obtained when citalopram or clomipramine was added. It is concluded that the [³H]imipramine binding site in human platelets is of protein nature and that this binding site contains the substrate recognition site for 5-HT uptake. In addition, [³H]imipramine and other 5-HT uptake inhibitors have bonds to other parts of the 5-HT uptake carrier or to the surrounding lipid membrane. This additional binding outside the substrate recognition site is not one single site but most likely represents sites that are specific for the chemical structure of each uptake inhibitor, respectively.     

Involvement of a Disulfide Bond in the Binding of Flunitrazepam to the Benzodiazepine Receptor from Bovine Cerebral Cortex

Abstract: The effects of chemical modification of a disulfide bond(s) (-SS-) or sulfhydryl group(s) (-SH) on the [³H]-flunitrazepam ([³H]FNZ) binding to membrane-bound or immunoprecipitated benzodiazepine (BZD) receptors (BZD-R) from bovine cerebral cortex were examined. Reduction of -SS- with dithiothreitol (DTT) brought about a reversible, time- and dose-dependent inhibition of [³H]FNZ binding to the membrane-bound BZD-R. Alkylation of the membranes with the -SH-modifying reagent iodoacetamide (IAA) or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) produced a slight inhibition of [³H]FNZ binding in a dose-dependent manner. Scatchard analysis of saturation curves of [³H]FNZ binding in the presence and absence of 5 m M DTT revealed changes in affinity without modification in the maximal binding capacity, thus indicating a competitive mode of interaction. DTT pretreatment of both the membrane-bound and the immunoprecipitated BZD-R led to [³H]FNZ binding inhibition. Consistent with the modification of a binding site is the observation that reduction of -SS- does not bear on the binding affinity, but rather reduces the number of sites. Complete protection from DTT inhibition of [³H]FNZ binding by FNZ (an agonist) or by Ro 15–1788 (an antagonist) suggests the presence of -SS- at, or very close to, the BZD recognition binding site. No protection against IAA or DTNB inhibition was provided by FNZ. Photoaffinity labeling experiments with [³H]FNZ revealed a clear-cut band of 50 kDa in native and alkylated membranes but an extremely weak label in 5 m M DTT/IAA-treated membranes. The present results provide evidence for the participation of a disulfide bond in the recognition binding site of the bovine cerebral cortex BZD-R.     

[3H]Dizocilpine Association Kinetics Distinguish Stimulatory and Inhibitory Polyamine Sites of N-Methyl-d-Aspartate Receptors

Abstract: Spermine and other polyamines both stimulate and inhibit N -methyl- d -aspartate receptor function, probably by interacting with two separate sites. To characterize these two actions, the effect of spermine on the binding kinetics of the channel blocker [³H]dizocilpine was studied in the presence of glutamate and glycine. Low concentrations (10 µ M ) of spermine increased the association and dissociation rates without modifying equilibrium binding, indicating that spermine increases the accessibility of [³H]dizocilpine to the channel by interacting with a high-affinity, stimulatory site. At higher concentrations (1 m M ), spermine markedly decreased equilibrium [³H]-dizocilpine binding by decreasing both affinity and B _max, indicating that spermine allosterically inhibits binding by interacting with a second, low-affinity site. The presumed polyamine antagonists arcaine, diethylenetriamine, and 1,10-diaminodecane completely inhibited equilibrium [³H]dizocilpine binding, probably by interacting with the inhibitory polyamine site or other sites, but not with the stimulatory polyamine site. Low concentrations (10 µ M ) of ifenprodil completely reversed the increase in association rate produced by spermine, whereas higher concentrations (IC₅₀ = 123 µ M ) inhibited equilibrium binding, indicating that ifenprodil is both a potent antagonist of the stimulatory site and a low-affinity ligand of the inhibitory site. The polyamine agonists spermine, spermidine, and neomycin interacted with the inhibitory site, but produced only partial inhibition of equilibrium [³H]dizocilpine binding.     

Regulation by Cations of [3H]Spiroperidol Binding Associated with Dopamine Receptors of Rat Brain

Abstract: The effects of monovalent and divalent cations on binding of [³H]spiroperidol to dopamine receptors in rat corpus striatum were studied. Both monovalent and divalent cations as well as several chelating agents increase the number of [³H] spiroperidol binding sites. Manganese is most potent, enhancing binding at 1 μ m concentration, while magnesium and calcium are at least two orders of magnitude less potent and the monovalent cations sodium, potassium and lithium are still weaker. Divalent cations enhance the potency of dopaminergic agonists in competing for [³H]spiroperidol binding, an effect which appears to be independent of the ionic augmentation of [³H]spiroperidol binding. Divalent cations decrease both the association and dissociation rates of [³H]spiroperidol binding to dopamine receptor sites.     

Properties of [3H]Prazosin-Labeled α1Adrenergic Receptors in Rat Brain and Porcine Neurointermediate Lobe Tissue

Abstract: [³H]Prazosin binding to α₁ receptors in homogenates of rat prefrontal cortical tissue and porcine pituitary neurointermediate lobe tissue was investigated. Competition curves produced by coincubating adrenergic agonists and antagonists with 0.5 n M [³H]prazosin and tissue revealed some anomalous binding properties. In the brain and pituitary tissue, agonist competition curves produced "shallow" slopes, with Hill coefficients significantly lower than unity. The IC₅₀ of the agonists epinephrine, norepinephrine, and clonidine for inhibition of 0.5 n M [³H]prazosin binding were significantly lower in the porcine pituitary than in the rat brain. Most antagonists, such as prazosin, chlorpromazine, and piperoxan, produced "steep" competition curves with Hill coefficients close to unity, with two notable exceptions. WB-4101 and phentolamine produced competition curves with Hill coefficients significantly less than unity in the rat brain preparation. Ketanserin, an antagonist, displayed a sevenfold higher affinity for the a, sites in the pituitary tissue than in the brain tissue. These anomalies in the binding results may indicate the presence of an endogenous modulatory factor affecting agonist and antagonist affinities for the a, receptor.     

Ethanol and the γ-Aminobutyric Acid-Benzodiazepine Receptor Complex

Abstract: Ethanol appears to enhance γ-aminobutyric acid (GABA)-mediated synaptic transmission. Using radioligand binding techniques, we investigated the possibility that the GABA-benzodiazepine receptor complex is the site responsible for this effect. Ethanol at concentrations up to 100 m M failed to alter binding of [³H]flunitrazepam (FNZ), [³H]Ro 15-1788, or [³H]methyl-γ-carboline-3-carboxylate (MBCC) to benzodiazepine receptors, or of [³H]muscimol to GABA receptors in rat brain membranes. Scatchard analyses of the binding of these radioligands at 4°C and 37°C revealed no significant effects of 100 m M ethanol on receptor affinity or number. A variety of drugs as well as chloride ion increased binding of [³H]FNZ and/or [³H]muscimol, but these influences were not modified by ethanol. These findings indicate that ethanol probably potentiates GABAergic neurotransmission at a signal transduction site beyond the GABA-benzodiazepine receptor complex.     

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.     

Lindane Administration to the Rat Induces Modifications in the Regional Cerebral Binding of [3H]Muscimol, [3H]-Flunitrazepam, and t-[35S]Butylbicyclophosphorothionate: An Autoradiographic Study

Abstract: The effect of lindane administration on the specific binding of ligands to different sites on the GABA_A receptor-ionophore complex was studied in the rat brain by receptor mapping autoradiography. [³H]Muscimol (Mus), [³H]flunitrazepam (Flu), and t -[³⁵S]butylbicyclophosphorothionate (TBPS) were used as specific ligands of GABA, benzodiazepine, and picrotoxinin binding sites, respectively. Rats received a single oral dose of 30 mg/kg lindane and they were classified into two groups according to the absence or presence of convulsions. Vehicle-treated groups acted as controls. The effect of the xenobiotic on ligand binding was measured in different brain areas and nuclei 12 min or 5 h after its administration. Lindane induced a generalized decrease in [³⁵S]TBPS binding, which was present shortly after dosing. In addition, [³H]Flu binding was increased in lindane-treated animals, this modification also appearing shortly after administration but diminishing during the studied time. Finally, lindane induced a decrease in [³H]Mus binding, which became more evident over time. These modifications were observed both in the presence and in the absence of convulsions. However, an increase in [³H]-Mus binding was detected shortly after lindane-induced convulsions. The observed decrease in [³⁵S]TBPS binding is in agreement with the postulated action of lindane at the picrotoxinin binding site of the GABA_A receptor chloride channel. The effects observed on the binding of [³H]Flu and [³H]Mus may be secondary to the action of lindane as an allosteric antagonist of the GABA_A receptor.     

Characterization of Serotonin Transporter in Goldfish Retina by the Binding of [3H]Paroxetine and the Uptake of [3H]Serotonin: Modulation by Light

Abstract: The serotonin (5-HT) uptake system of goldfish retina was evaluated by the binding of [³H]paroxetine to membrane preparations and the uptake of [³H]5-HT into isolated cells from goldfish retina. The order of potency of inhibitors of [³H]paroxetine binding was imipramine > 5-methoxy- N,N -dimethyltryptamine > desipramine > fluoxetine > citalopram > 5-HT. The saturation experiments indicated a high-affinity binding site, and positive cooperativity with Hill coefficient higher than unity. The association reached equilibrium at about one hour of incubation and was efficiently displaced by imipramine. The equilibrium dissociation constants calculated by the antilog of the log concentration of ligand giving 50% of occupation, and by the ratio of dissociation/association constants, were similar: 5.84 and 2.34 n M , respectively. The binding was not significantly reduced by decreasing the temperature of incubation and was sodium dependent. The lesion with 5,7-dihydroxytryptamine reduced the binding to 60%. The uptake of [³H]5-HT into isolated cells also showed positive cooperativity. The order of potency of inhibitors was similar to the one obtained for the binding of [³H]paroxetine. Darkness increased the uptake of 5-HT. The allosteric regulation of the 5-HT transporter and the modulation by light could be related to the physiological role of the monoamine, as a neurotransmitter and as a precursor of melatonin synthesis in the retina.