High-Affinity Binding of [3H]Desipramine to Rat Brain: A Presynaptic Marker for Noradrenergic Uptake Sites

Abstract: High-affinity binding sites (apparent K _D= 1.5 nM) for [³H]desipramine have been demonstrated and characterized in membranes prepared from rat brain. The binding of [3H]desipramine was found to be saturable, reversible, heat-sensitive, sodium-dependent, and regionally distributed among various regions of the brain. High concentrations of [³H]desipramine binding sites were found in the septum, cerebral cortex, and hypothalamus, whereas lower concentrations were found in the medulla, cerebellum, and corpus striatum. A very good correlation ( r = 0.81, P < 0.001) was observed between the potencies of a series of drugs in inhibiting high-affinity [³H]desipramine binding and their capacity to block norepinephrine uptake into synaptosomes. In 6-hydroxydopamine-lesioned rats there was a marked decrease in [³H]norepinephrine uptake and [3H]desipramine binding with no significant alterations in either [³H]serotonin uptake or [³H]imipramine binding. These results suggest that the high-affinity binding of [³HJdesipramine to rat brain membranes is pharmacologically and biochemically distinct from the high-affinity binding of [3H]imipramine, and that there is a close relationship between the high-affinity binding site for [³H]desipramine and the uptake site for norepinephrine.     

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.     

Localisation of Tricyclic Antidepressant Binding Sites on Serotonin Nerve Terminals

Abstract: High-affinity specific [³H]imipramine binding has been demonstrated in the brain and platelets of various species including man. Electrolytic lesion of the rat dorsal raphe, which resulted in a significant decrease in the endogenous levels of serotonin produced a reduction in the density of [³H]imipramine binding sites in the hypothalamus and cortex. The affinity constants were unchanged. These results suggest that [³H]imipramine binding sites are located on serotonin nerve terminals.     

[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.     

[3H]Imipramine Is Accumulated but Not Released from Slices of the Rabbit Caudate and Hypothalamus

Abstract: Slices of rabbit caudate and hypothalamus take up and accumulate [³H]imipramine. In superfused slices of both structures electrical stimulation or exposure to tyramine failed to release recently taken up [³H]imipramine. De-polarization by exposure to 30–60 mm-potassium caused only a small release of [³H]imipramine that was not concentration-dependent. The release of [³H]imipramine by high potassium was independent of the presence of calcium ions in the superfusion medium. These results contrasted with those obtained for the release of [³H]dopamine from the caudate and [³H]noradrenaline from the hypothalamus, where tyramine, electrical stimulation, and high potassium caused a significant release of the labeled neurotransmitters. The release of [³H]dopamine from the caudate and [³H]noradrenaline from the hypothalamus elicited by electrical stimulation or high potassium was entirely calcium-dependent. It is concluded that [³H]imipramine is taken up into the two brain regions and is accumulated in a nonvesicular site from which it is not released by calcium-dependent depolarizing stimuli.     

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] 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.     

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.     

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.     

Distribution and Ontogeny of 1S,3R-1-Aminocyclopentane-1,3-Dicarboxylic Acid-Sensitive and Quisqualate-Insensitive [3H]Glutamate Binding Sites in the Rat Brain

Abstract: Displacement of [³H]glutamate by 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid and quisqualate (in the presence of saturating concentrations of ionotropic glutamate receptor agonists) was used to characterize optimal ionic conditions, distribution, and the ontogeny of glutamate receptor binding sites in rat brain. Using rat forebrain membranes or receptor autoradiography, optimal 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive [³H]glutamate binding was found in the presence of 100 m M bromide ions and in the absence of calcium ions. Under these conditions, [³H]glutamate binding was relatively quisqualate insensitive. In regions of the neonatal (11-day-old) and adult rat brain, this [³H]glutamate binding was highest in forebrain (striatum, cerebral cortex, and hippocampus) and hypothalamus/midbrain but was lower in the cerebellum, olfactory bulb, and pons/medulla regions. 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive and quisqualate-insensitive [³H]glutamate binding was present in the rat forebrain at 1 day of age and gradually increased more than twofold by day 50 (adult). Thus, in the presence of bromide ions and in the absence of calcium ions, [³H]glutamate labels a subpopulation of metabotropic glutamate receptors that are sensitive to 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid but insensitive to quisqualate. Expression of [³H]glutamate binding under these conditions was both regionally and developmentally regulated in rat brain, suggesting that [³H]glutamate is labeling a distinct population of metabotropic glutamate receptors.     

NMDA Receptors in Rat Cerebellum and Forebrain: Subtle Differences in Pharmacology and Modulation

Abstract: Binding of [³H]glutamate, [³H]glycine, and the glutamate antagonist [³H]CGS-19755 to NMDA-type glutamate receptors was examined in homogenates of rat forebrain and cerebellum. Most glutamate agonists had a higher affinity at the [³H]glutamate binding site of cerebellar NMDA receptors as compared with forebrain, whereas all the glutamate antagonists examined showed the reverse relationship. The [³H]glycine binding site of forebrain and cerebellar NMDA receptors showed a similar pharmacology in both brain regions. In the cerebellum, however, [³H]glycine bound to a second site with a 10-fold lower affinity and with a pharmacology that resembled that of the glycine/strychnine chloride channel. [³H]Glutamate binding was not affected by glycine agonists or antagonists, nor was [³H]glycine binding affected by glutamate agonists in either forebrain or cerebellum. Both CGS-19755 and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, glutamate antagonists, reduced [³H]glycine binding in cerebellum, whereas only CGS-19755 was effective in forebrain. Glycine agonists and antagonists modulated [³H]CGS-19755 binding in forebrain and cerebellum to different extents in the two brain regions. From these studies we conclude that the cerebellar NMDA receptor has a different pattern of modulation at glutamate and glycine sites and that glycine may play a more important role in the control of NMDA function in the cerebellum as compared with forebrain.     

[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.     

Differential Profiles of Binding of a Radiolabeled Agonist and Antagonist at a Glycine Recognition Domain on the N- Methyl-D-Aspartate Receptor lonophore Complex in Rat Brain

Abstract: Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [³H] 5, 7-dichlorokynurenic acid ([³H]- DCKA) but not of the agonist ligand [³H] glycine ([³H] Gly) to a Gly recognition domain on the N -methyl-D-aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [³H] DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (±)-α-(4-chlorophenyl)-4- [(4-fluorophenyl)methyl]-1-piperidine ethanol, with [³H] Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil. In addition, spermidine significantly attenuated the abilities of four different antagonists at the Gly domain to displace [³H] DCKA binding virtually without affecting those of four different agonists. Phospholipases A₂ and C and p -chloromercuribenzosulfonic acid were invariably effective in significantly inhibiting [³H] DCKA binding with [³H] Gly binding being unaltered. Moreover, the densities of [³H] DCKA binding were not significantly different from those of [³H]- Gly binding in the hippocampus and cerebral cortex, whereas the cerebellum had more than a fourfold higher density of [³H] Gly binding than of [³H] DCKA binding. These results suggest that the Gly domain may have at least two different forms based on the preference to agonists and antagonists in the rodent brain.     

Allosteric Modulation of [3H]CGP 39653 Binding by Glycine in Rat Brain

Abstract: D,L-(E)-2-Amino-4-propyl-5-phosphono-3-pen-tenoic acid (CGP 39653). a new, high-affinity, selective NMDA receptor antagonist, interacts with rat cortical membranes in a saturable way and apparently to a single binding site, with a K_D of 10.7 nM and a receptor density of 2.6 pmol/mg of protein. Displacement analysis of [³H]CGP 39653 binding shows a pharmacological profile similar to that reported for another NMDA antagonist, 3-[(±)-2-carboxypiperazin-4-yI]propyl-1-phosphonic acid (CPP). Glycine, however, is able to discriminate between the two ligands; in fact, it does not affect [³H]CPP binding but inhibits [³H]CGP 39653 binding in a biphasic way. D-Serine, another agonist at the strychnine-insensitive glycine binding site of the NMDA receptor complex, inhibits [³H]CGP 39653 binding in the same way as glycine, with a potency that correlates with its binding affinity at the glycine site. In addition, 7-chlorokynurenic acid, an antagonist at the glycine site, is able to reverse the displacement of [³H]CGP 39653 by glycine in a dose-dependent manner. Furthermore, the dissociation rate constant of [³H]CGP 39653 is enhanced in the presence of glycine, whereas the presence of NMDA receptor ligands does not modify the rate of dissociation of [³H]CGP 39653 from the receptor. These results indicate that part of the binding of the NMDA antagonist CGP 39653 can be potently modified by glycine through an allosteric mechanism, and suggest the existence of two antagonist preferring NMDA receptor subtypes that are differentially modulated through the glycine binding site.     

Binding Characteristics and Interactions of Depressant Drugs with [35S]t-Butylbicyclophosphorothionate, a Ligand that Binds to the Picrotoxinin Site

Abstract: [³⁵S]r-Butylbicyclophosphorothionate (TBPT), a cage convulsant with picrotoxinin-like activity, binds to rat brain membranes to a single site with an apparent K_D of 25.1 ± 5.6 n M and a B_max of 1.40 ± 0.22 pmol/mg protein. TBPT binding to rat brain membranes was inhibited by a variety of convulsant, depressant, anxiolytic, and anticonvulsant drugs that had previously been shown to inhibit [³H]a-dihydropicrotoxinin binding. Depressant drugs such as pentobarbital and the nonbarbiturate (+)etomidate inhibited TBPT binding in an uncompetitive manner. Thus, pentobarbital and (+)etomidate decreased both the affinity and the number of binding sites of TBPT to whole brain membranes. The IC₅₀ values of (+)etomidate (9 μ M ) and pentobarbital (90 μ M ) are similar to the EC₅₀ values at which they enhance both [³H]-γ-aminobutyric acid and [³H]diazepam binding in cerebral cortex membranes. RO5–4864, which has recently been shown to be a convulsant, also inhibited TBPT binding (IC₅₀= 10 μ M ). These results suggest that TBPT binds to the picrotoxinin site and further supports the notion that the picrotoxinin site is an important modulatory site at the benzodiazepine-GABA receptor-ionophore complex.     

Characterization of the Binding of [3H]NS 257, a Novel Competitive AMPA Receptor Antagonist, to Rat Brain Membranes and Brain Sections

Abstract: The binding of [³H]NS 257 {1,2,3,6,7,8-hexahydro-3-(hydroxyimino)- N,N -[³H]dimethyl-7-methyl-2-oxobenzo[2,1- b :3,4- c '] dipyrrole-5-sulfonamide} to rat cortical membranes was characterized in the absence and presence of thiocyanate. Specific [³H]NS 257 binding was saturable and reversible, and the stimulating effect of thiocyanate on binding was optimal at 100 m M . In the presence of thiocyanate [³H]NS 257 bound to a single population of binding sites with an affinity of 225 ± 8 n M and a binding site density of 0.61 ± 0.04 pmol/mg of original tissue. Thiocyanate increased the affinity of the binding site labeled by [³H]NS 257 for both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and l -glutamate by a factor of 20 and 5, respectively. However, the affinity of the agonist domoate and the antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo( f )-quinoxaline (NBQX) was decreased in the presence of thiocyanate. Apparently, the affinities of antagonists as well as agonists for the AMPA receptor can be either increased or decreased by thiocyanate. The rank order of potency of the putative agonists quisqualate > AMPA > l -glutamate > domoate > kainate and of the antagonists NBQX > CNQX is consistent with the labeling of AMPA receptors. Autoradiographic studies showed that the distribution of [³H]NS 257 binding sites in rat brain was similar to that of [³H]AMPA binding sites. NS 257 is the first AMPA antagonist to be described showing an increased affinity for the AMPA receptor in the presence of thiocyanate.     

[1251]2α-BUNGAROTOXIN AND [3H]QUINUCLIDINYLBENZILATE BINDING IN CENTRAL NERVOUS SYSTEMS OF DIFFERENT SPECIES

Abstract— [¹²⁵I]Diiodo α-bungarotoxin ([¹²⁵I]₂BuTx) and [³H]quinuclidinylbenzilate ([³H]QNB) binding sites were measured in post-nuclear membrane fractions prepared from whole brains or brain regions of several species. Species studied included Drosophila melanogaster (fruit fly), Torpedo californiea (electric ray), Carassius auratus (goldfish), Ram pipiens (grass frog), Kana cutesheiana (bullfrog), Rattus norvegicus (rat, Sprague-Dawley), Mus muscalus (mouse, Swiss random, C58/J, LG/J), Oryctolagus cuniculus (rabbit, New Zealand Whitc), and Bos (cow). Acetyl-CoA: choline O -acetyltransferase (EC 2.3.1.6) levels were also determined in the post nuclear supernatants and correlated with the number of binding sites.
All species and regions except Drosophila had 16–150 fold more [³H]QNB binding sites than [¹²⁵I]₂BuTx binding sites. Brain regions with the highest levels of [¹²⁵I]₂BuTx binding were Drosophila heads (300 fmol/mg), goldfish optic tectum (80fmol/mg), and rat and mouse hippocampus (3040 fmol/mg). The highest levels of [³H]QNB binding were seen in rat and mouse caudate (1.3–1.6 pmol/mg). Lowest levels of [³H]QNB and [¹²⁵I]₂BuTx binding were seen in cerebellum. The utility of [¹²⁵I]₂BuTx and [³H]QNB binding as quantitative measures of nicotinic and muscarinic acetylcholine receptors in CNS is discussed.     

Interaction of Guanine Nucleotides with [3H]Kainate and 6-[3H]Cyano-7-Nitroquinoxaline-2,3-dione Binding in Goldfish Brain

Abstract— Recent reports have suggested that a major proportion of [³H]kainate binding in goldfish brain is to a novel form of G-protein-linked glutamate receptor. Here we confirm that guanine nucleotides decrease [³H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G-protein modulation should be minimised. Inclusion of GTPγS resulted in an approximately twofold decrease in the affinity of [³H]kainate binding and a 50% reduction in the apparent B _max values in both Mg²⁺/Na⁺ and Mg²⁺/Na⁺-free buffer when assayed at 0°c. The pharmacology of [³H]kainate binding is similar to that of well-characterised ionotropic kainate receptors but unlike that of known me-tabotropic glutamate receptors, with neither 1 S ,3 R -amino-1,3-cyclopentanedicarboxylic acid (1 S ,3 R -ACPD) nor ibo-tenic acid being effective competitors. The molecular mass of the [³H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand-gated ion channels. Furthermore, GTP-γS also inhibited the binding of the non-NMDA receptor-selective antagonist 6-[³H]cyano-7-ni-troquinoxaline-2,3-dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [³H]kainate and 6-[³H]cyano-7-nitroquinoxaline-2,3-dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G-protein-mediated modulation.     

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.     

Ontogenetic Development of Histamine H1- Receptor Binding in Rat Brain

Abstract: Histamine H_1- receptors labeled with [³H]mepyramine in rat brain show an age-dependent development. [³H]Mepyramine receptor density and histidine decarboxylase activity in whole rat brain reach adult levels at 25–30 days after birth and they attain 50% of adult level at day 10 and 17, respectively. The apparently later development of histidine decarboxylase activity in whole rat brain is partly accounted for by a biphasic developmental increase of this enzymatic activity in cerebral cortex. For all other brain regions examined, the development of histamine H_1- receptors parallels that of histidine decarboxylase. The increase in [³H]mepyramine binding can be accounted for by an absolute increase in the numbers of the receptor sites, with no change in affinity. Subcellular fractionation studies indicate that histamine H_1- receptors are predominantly associated with synaptosomal fractions derived from both newborn and adult rat.