Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [H]8-OH-DPAT and other serotonergic ligands

[³H]8-OH-DPAT is a selective ligand for labeling 5-HT_1A receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [³H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [¹²⁵I]RTI-55 and [³H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [³H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [³H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [¹²⁵I]cyanopindolol, [³H]ketanserin/[³H]mesulergine, [³H]GR-65630, [³H]GR-113808 and [³H]LSD) that specifically labeled 5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄ and 5-HT_5–7 receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.     

Characteristics of Binding of [3H]WAY100635 to Rat Hippocampal Membranes

Kinetic analysis of binding of [³H][N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide ([³H]WAY100635) to 5-HT_1A receptors in rat hippocampal membranes has revealed complex regulation mechanism for this radioligand. Saturation binding experiments revealed that [³H]WAY100635 binds to a single class of receptors with very high apparent affinity (K _D = 87 ± 4 pM, B _max = 15.1 ± 0.2 fmol/mg protein). The binding was almost irreversible, as the dissociation rate constant obtained k _off = (7.8 ± 1.1) × 10⁻³ min⁻¹, means that equilibrium with this radioligand cannot be achieved before 7.5 h incubation at 25°C. Systematic association kinetic studies of [³H]WAY100635 binding revealed sharp reaction acceleration at higher radioligand concentration, proposing mechanism of positive cooperativity. The affinities of antagonists determined from competition with [³H]WAY100635 did not coincide with their abilities to inhibit 5-HT-dependent activation of [³⁵S]GTPγS binding probably due to the ligand’s kinetic peculiarities. Thus, [³H]WAY100635 appears to be an excellent tool for determining receptor binding sites, but its applicability in equilibrium studies is strongly limited.     

Solubilization and characterization of [3H]Imipramine and [3H]Paroxetine binding sites from calf striatum

The serotonin (5-HT) transporter from calf striatum cerebral membranes was solubilized with digitonin and characterized by gel exclusion chromatography. [³H]Imipramine and [³H]paroxetine were utilized as markers for labeling it.³H-imipramine labels a high- and a low-affinity site on striaturn membranes, whereas it binds to a single high-affinity site on the solubilized fraction. [³H]Paroxetine binds with the same affinity to a single site on both membranes and solubilized preparations. After gel exclusion chromatography of the solubilizate both [³H]imipramine and [³H]paroxetine bind on an identical fraction of 205 kDa molecular weight, with a similar maximum number of binding sites (Bmax). Our results suggest that both³H-imipramine and [³H]paroxetine bind to a common site on the 5-HT transporter.     

Chemical integrity of [3H]GABA used in binding studies

A method which is claimed to be able to determine the proportion of true GABA within radiolabeled GABA used in binding studies was tested using [³H]GABA. The method was found to be unsuitable for³H-labeled GABA and, furthermore, both theoretical considerations and the present experimental data indicated that it could also produce misleading results with [¹⁴C]GABA.     

Neophobia and cortical and subcortical binding of the dopamine D2 receptor antagonist [3H]-raclopride

The potential role of dopamine system in response to novelty was analysed using the selective dopamine D2 receptor antagonist, raclopride, in behavioral and biochemical assays, in rats (the open field test, and specific binding of [3H]-raclopride, within different brain structures measured with autoradiography). It was found that raclopride at a low dose (50 microg/kg, IP) caused anxiolytic-like effect (increased the anti-thigmotactic index), whereas at a higher dose (500 microg/kg, IP) produced general inhibitory influence, and decreased the anti-thigmotactic index. Analysis of the behavioral and biochemical results of the experiment revealed a significant negative correlation between the ligand binding in the substantia nigra pars reticulata (SNR), and the number of entries into the central sector of the open field (r=-0.48, p<0.05), as well as the positive correlation between time spent in the central sector of the open field and [3H]-raclopride binding within nucleus accumbens septi (r=0.57, p<0.05). Factor analysis revealed a Factor 1 (eigenvalue=3.361) grouping parameters of central entries into the open field and [3H]-raclopride binding in the SNR (factor loadings are 0.814 and 0.703 respectively), indicating that both phenomena are under control of a similar central process. The above data are discussed in relation to the structure dependent dopamine D2 receptor mechanisms in a rat response to novelty.     

Effects of chronic antidepressant treatment on dopamine-related [3H]SCH 23390 and [3H]Spiperone binding in the rat striatum

1. The effects of chronic administration of antidepressants on dopamine-related [3H]SCH 23390 and [3H]spiperone binding to rat striatal membranes were assessed. 2. The monoamine oxidase inhibitors phenelzine (5 or 10 mg kg-1/day) and tranylcypromine (1 mg kg-1/day) and the tricyclic desipramine (10 mg kg-1/day) were administered for 28 days by constant subcutaneous infusion using Alzet (2ML4) osmotic minipumps. 3. These treatments did not alter Kd estimates for either [3H]SCH 23390 or [3H]spiperone binding sites. The monoamine oxidase inhibitors induced a decrease in the Bmax values for both [3H]SCH 23990 and [3H]spiperone binding sites. Desipramine induced a decrease in the Bmax value for [3H]SCH 23390 binding but had no effect on the Bmax value for [3H]spiperone binding.     

Effects of Valeriana Officinalis Extracts on [3H]Flunitrazepam Binding,Synaptosomal [3H]GABA Uptake,and Hippocampal [3H]GABA Release

Extracts of Valeriana officinalis have been used in folkloric medicine for its sedative, hypnotic, tranquilizer and anticonvulsant effects, and may interact with -aminobutyric acid (GABA) and/or benzodiazepine sites. At low concentrations, valerian extracts enhance [³H]flunitrazepam binding (EC₅₀ 4.13 × 10^–10 mg/ml). However, this increased [³H]flunitrazepam binding is replaced by an inhibition at higher concentrations (IC₅₀ of 4.82 × 10^–1 mg/ml). These results are consistent with the presence of at least two different biological activities interacting with [³H]flunitrazepam binding sites. Valerian extracts also potentiate K⁺ or veratridine-stimulated release of radioactivity from hippocampal slices preloaded with [³H]GABA. Finally, inhibition of synaptosomal [³H]GABA uptake by valerian extracts also displays a biphasic interaction with guvacine. The results confirm that valerian extracts have effects on GABA_A receptors, but can also interact at other presynaptic components of GABAergic neurons.     

Purification of endogenous inhibitors of [3H]flunitrazepam binding from bovine brain

Endogenous substances which inhibited the binding of [³H]flunitrazepam ([³H]FNZ) to bovine synaptosomal membranes have been purified from the hot acetic acid extracts of the bovine brain. Three peaks of inhibitory activity were obtained by Sephadex G-10 gel chromatography. Two of the peaks (Peak 2, and Peak 3) which had lower molecular weights that that of peak 1 were identified as inosine and hypoxanthine by TLC methods. Another peak (Peak 1) was further purified to homogeneity using both cation and anion ion-exchange chromatography and the following two-step reversed-phase HPLC. The purified substance inhibited the [³H]FNZ binding dose-dependently and competitively but did not have an effect on the binding of the peripheral-type BZ ligand [³H]Ro 5-4864. It was also shown that the substance was heat-stable and resistant to proteolytic degradation (trypsin, -chymotrypsin, pronase). However, a significant loss of inhibitory activity to [³H]FNZ binding was observed after acid hydrolysis. Molecular weight estimates based on gel filtration methods were less than 500 dalton, and the maximal ultraviolet absorption peak was at 314 nm. These results suggest that this substance is a new endogenous ligand for the central BZ receptor and may play an important role in regulating the GABAergic tone in the central nervous system.     

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.     

Allosteric modulation of the [3H]quinuclidinyl benzylate binding to M-cholinoreceptors in rat cerebral cortex by adrenotropic agents

The allosteric effects of adrenotropic drugs and the membranotropic agent cocaine on the kinetics of [³H]quinuclidinyl benzylate ([³H]QNB) binding to muscarine cholinoceptors of synaptosomal membranes of rat cerebral cortex were studied. In control, the best results were obtained for the model that assumes the existence of two receptor pools (with high and low affinity) with calculated parameters of the activity (K _d), amount (B _max), and mono- to dimer receptors ratio (n). For the high-affinity receptors these parameters were K _d1 = 0.18 ± 0.08 nM, B _m1 = 221.2 ± 56.7 fmol/mg protein, n ₁ = 2, and for low-affinity receptors, K _d2 = 1.33 ± 0.11 nM, B _m2 = 748.7 ± 53.3 fmol/mg protein, n ₂ = 2. Allosteric modulation of the activity of specific neurotransmitter receptors can be accomplished by changing the receptor affinity and amount as well as the proportion of mono- and dimer receptors. Under control conditions, muscarine receptors exist as dimers. In the presence of α-adrenoreceptor agonist noradrenaline and β-adrenoreceptor antagonist propranolol, only one pool of the dimer muscarine receptors remains. The number of binding sites for noradrenaline and propranolol decreases approximately by 40% and 20%, respectively. The agonist of α₂-adrenoreceptors clonidine, the antagonist of α₂-adrenoreceptors yohimbine, and a membranotropic agent cocaine change the ligand binding so that only one receptor pool remains but some of the dimer receptors become monomeric (1 < n < 2). The amount of binding sites reduces by 20%, 25%, and 45% for clonidine, yohimbine, and cocaine, respectively.     

NAD-299 ANTAGONISES 5-HT-STIMULATED AND SPIPERONE-INHIBITED [35S]GTPγS BINDING IN CLONED 5-HT1A RECEPTORS

ABSTRACT

In Chinese Hamster Ovary (CHO) cells expressing cloned human 5-hydroxy-tryptamine_1A (5-HT_1A) receptors, (R)-3-N,N-dicyclobutylamino-8-fluoro-[6-³H]-3,4-dihydro-2H-1-benzopyran-5-carboxamide ([³H]NAD-299) exhibited high affinity (K_d = 0.16?nM) and labeled 34% more receptors than 8-hydroxy-2-([2,3-³H]di-n-propylamino)tetralin ([³H]8-OH-DPAT). NAD-299 behaved as a silent antagonist in [³⁵S]GTPγS binding similar to N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide (WAY-100635) and (S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((S)UH-301). 5-HT and 5-carboxamidotryptamine (5-CT) stimulated [³⁵S]GTPγS binding 2.5-fold while spiperone and methiothepin inhibited [³⁵S]GTPγS binding 1.4-fold. Furthermore, NAD-299 antagonised both the 5-HT stimulated and the spiperone inhibited [³⁵S]GTPγS binding to basal levels. The K_iL/K_iH ratios for spiperone (0.66), methiothepin (0.39), WAY-100635 (0.32), (S)UH-301 (0.94), NAD-299 (1.29), NAN-190 (1.23), (S)pindolol (5.85), ipsapirone (13.1), buspirone (24.6), (±)8-OH-DPAT (47.3), flesinoxan (55.8), 5-HT (200) and 5-CT (389) correlated highly significantly with the intrinsic activity obtained with [³⁵S] GTPγS (r = 0.97).     

Development of a scintillation proximity assay binding method for the human 5-hydroxytryptamine 6 receptor using intact cells

We describe the first validated scintillation proximity assay (SPA) binding method for quantitation of ³H-labeled d-lysergic acid diethylamide (LSD) binding to recombinant human 5-hydroxytryptamine 6 (5-HT₆) receptors expressed in Chinese hamster ovary (CHO)-Dukx and HeLa cells. The assay was developed using intact cells as a receptor source because membrane fractions derived from these cells failed to discern specific binding from a high level of nonspecific binding. The pharmacological binding profile of seven 5-HT₆ agonists and antagonists using intact CHO-Dukx/5-HT₆ cells in the SPA format was similar to data obtained from a filtration binding assay using HeLa/5-HT₆ membranes. K_i values and rank order of potencies obtained in the SPA format were consistent with published filtration data as follows: SB-271046 (K_i = 1.9 nM) > methiothepin (K_i = 6.2 nM) > mianserin (K_i = 74.3 nM) > 5-methoxytryptamine (5-MeOT, K_i = 111 nM) > 5-HT (K_i = 150 nM) > ritanserin (K_i = 207 nM) > 5-carboxamidotryptamine (5-CT, K_i = 704 nM). Additional evaluation with four antipsychotics demonstrated strong agreement with previous literature reports. A high specific binding signal and low assay variability, as determined by Z′ = 0.81 ± 0.017, make the SPA format amenable to automation and higher throughput; hence, this assay can be a viable alternative to the more labor-intensive filtration and centrifugation methods.     

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [³H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [³H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent K_D for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 M) with no change in B_max. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the B_max was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [³H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [³H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [³H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The B_max values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [³H]SR 95531 binding whereas the treatment increases apparent affinity of [³H]SR 95531 and [³H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein. These results also suggest that properties of cerebellar GABA_A receptors are different from those in other regions.Abbreviations used GABA -aminobutyric acid - FC frontal cortex - CB cerebellum - ST striatum - SN substantia nigra     

[3H]Spiroxatrine: A 5-HT1A Radioligand with Agonist Binding Properties

Spiroxatrine has been reported to be a 5-HT1A serotonin receptor antagonist. Therefore [3H]spiroxatrine was synthesized and its 5-HT1A receptor binding properties in homogenates of rat hippocampal membranes were characterized with the expectation that it would be the first 5-HT1A antagonist radioligand. [3H]8-Hydroxydipropylaminotetralin [( 3H]8-OH-DPAT), a well-characterized 5-HT1A agonist radioligand, was studied in parallel for comparative purposes. Scatchard analyses of saturation studies of [3H]spiroxatrine and [3H]8-OH-DPAT binding produced KD values of 0.9 nM and 1.8 nM, with Bmax values of 424 and 360 fmol/mg protein, respectively. A highly significant correlation (r = 0.98; p less than 0.001) exists between Ki values obtained for a series of drugs in competing for [3H]-spiroxatrine and [3H]8-OH-DPAT binding. Of special interest was the observation that 5-HT1A agonists such as serotonin, 8-OH-DPAT, and ipsapirone competed with equal high affinities for [3H]spiroxatrine or [3H]8-OH-DPAT-labelled 5-HT1A receptors. [3H]Spiroxatrine and [3H]8-OH-DPAT binding to 5-HT1A receptors was inhibited by guanosine 5'-(beta,gamma-imido)triphosphate (a nonhydrolyzable analog of GTP) in a concentration-dependent manner whereas adenosine 5'-(beta,gamma-imido)triphosphate (a nonhydrolyzable analog of ATP) had no effect. The similarities in the 5-HT1A receptor radiolabelling properties of [3H]spiroxatrine and [3H]8-OH-DPAT, i.e., the high affinities of agonists and the guanyl nucleotide sensitivity, indicate that [3H]spiroxatrine has "agonist-like" binding properties in its interaction with the 5-HT1A receptor.     

An [3H]oxotremorine binding method reveals regulatory changes by guanine nucleotides in cholinergic muscarinic receptors of cerebral cortex

A rapid, reliable filtration method for [³H]oxotremorine binding to membranes of the cerebral cortex that allows the direct study of regulation by guanine nucleotides of muscarinic receptors was developed. [³H]Oxotremorine binds to cerebral cortex membranes with high affinity (K _D, 1.9 nM) and low capacity (B _max, 187 pmol/g protein). These sites, which represent only about 18% of those labeled with [³H]quinuclidinyl benzilate, constitute a population of GTP-sensitive binding sites. Association and dissociation binding experiments revealed a similar value ofK _D (2.3 nM). Displacement studies with 1–4000 nM oxotremorine showed the existence of a second binding site of low affinity (K _D, 1.2 M) and large capacity (B _max, 1904 pmol/g protein). Gpp(NH)p, added in vitro, produced a striking inhibition of [³H]oxotremorine binding with an IC 50 of 0.3 M. Saturation assays, in the presence of 0.5 M Gpp(NH)p, revealed a non-competitive inhibition of the binding with little change in affinity. These results are discussed from the viewpoint of conflicting reports in the literature about guanine nucleotide regulation of muscarinic receptors in reconstituted systems and membranes from different tissues.     

Artifactual High-Affinity and Saturable Binding of [3H]5-Hydroxytryptamine Induced by Radioligand Oxidation

The binding of [3H]5-hydroxytryptamine (5-HT, serotonin) to cerebellar membranes was examined after preincubation of [3H]5-HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homogenates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT1 binding sites. In the presence of ascorbate during a 4-h preincubation period, minimal specific binding of 2 nM [3H]5-HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (butylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14-fold following a 4-h preincubation of [3H]5-HT in the absence of ascorbate. The increase in apparent specific [3H]5-HT binding is time-dependent and plateaus after 4-6 h of preincubation. When ascorbate is present during the 4-h preincubation, Scatchard analysis of [3H]5-HT binding reveals a KD value of 3.0 +/- 0.3 nM and a Bmax value of 1.9 +/- 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially unchanged at 3.6 +/- 0.1 nM but the Bmax is significantly increased to 36.5 +/- 7 pmol/g tissue. Drug competition studies reveal that the apparent specific "[3H]5-HT binding" in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5-HT, bufotenine) but not by nonhydroxylated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that [3H]5-HT is essentially destroyed by a 4-h incubation at 22 degrees C in the absence of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potent,extra-channel influence of several calcium-channel modulators on striatal binding of [3H]tyramine

A number of Ca²⁺-, K⁺-, and Na⁺-channel modulators has been tested with respect to their effects on [³H]tyramine (TY) binding, as a putative marker for the vesicular dopamine (DA) transporter in striatal membrane preparations containing vesicle ghosts. Among organic Ca²⁺-channel modulators, the diphenylalkylamines tested consistently inhibited TY binding: the order of potency was prenylamine>lidoflazine>flunarizine>cinnarizine, with K_i values of 0.1, 0.2, 0.5 and 1.2 M, respectively. Low (up to 100 nM) concentrations of prenylamine did competitively inhibit TY binding, and higher concentrations provoked a mixed-type inhibition. Furthermore, LIGAND-analysis of competition curves revealed a high- and a low-affinity binding site for prenylamine and flunarizine. The TY binding process was also sensitive to selected K⁺- and Na⁺-channel modulators. Since several Ca²⁺-antagonists are known to affect H⁺-ATPase and the bioenergetics of catecholamine storage vesicles in chromaffin granules, thus affecting monoamine storage, the energy requirements for the formation of the TY/carrier complex were here assessed, assuming similarity between chromaffin granules and synaptic vesicles. TY binding, though not reflecting endovesicle-sequestered TY, was indeed strongly sensitive (with K_i coefficients in the fM or low nM range) to the dissipation of the vesicular transmembrane proton concentration ( pH), electrical ( ), and proton electrochemical ( H⁺) gradients, provoked by a number of specifically targeted agents. It is concluded that Ca²⁺-channel agents of the diphenylalkylamine group may directly affect striatal TY binding due to an extrachannel-regulated competition with TY for the vesicular carrier of DA, as well indirectly, by disruption of the transmembrane energization of the reserpine-sensitive carrier.     

Synthesis and preliminary evaluation of [H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors

The selective antagonist radioligand [³H]2-propylthioadenosine-5′-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([³H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74 Ci/mmol. In preliminary saturation binding studies, [³H]PSB-0413 showed high affinity for platelet P2Y₁₂ receptors with a K_D value of 4.57 nM. Human platelets had a high density of P2Y₁₂ receptors exhibiting a B_max value of 7.66 pmol/mg of protein.     

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.     

Characterization of [(3)H]Quisqualate binding to recombinant rat metabotropic glutamate 1a and 5a receptors and to rat and human brain sections

We have investigated the binding properties of [(3)H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K:(D) values of 27 +/- 4 and 81 +/- 22 nM: for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently. (S:)-4-Carboxyphenylglycine, (S:)-4-carboxy-3-hydroxyphenylglycine, and (R,S)-1-aminoindan-1,5-dicarboxylic acid, which completely inhibited [(3)H]quisqualate binding to the mGlu5a receptor, were inactive in a functional assay using this receptor. The distribution and abundance of binding sites in rat and human brain sections were studied by quantitative receptor radioautography and image analysis. Using 10 nM: [(3)H]quisqualate, a high density of binding was detected in various brain regions with the following rank order of increasing levels: medulla, thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactory tubercle, dentate gyrus molecular layer, CA1-3 oriens layer of hippocampus, striatum, and cerebellar molecular layer. The ionotropic component of this binding could be inhibited by 30 microM: kainate, revealing the distribution of mGlu1+5 receptors. The latter were almost completely inhibited by the group I agonist (S:)-3,5-dihydroxyphenylglycine. The binding profile correlated well with the cellular sites of synthesis and regional expression of the respective group I receptor proteins revealed by in situ hybridization histochemistry and immunohistochemistry, respectively.