Serotonin-lesion Myoclonic syndromes. I. Neurochemical profile and S-1 receptor binding

This paper and the following one describe the effects of L-5-hydroxytryptophan (5-HTP) (after 3 intracisternal injections of 5,7-dihydroxytryptamine (DHT], fenfluramine (FF), p-chloroamphetamine (PCA) and drug combinations on (i) brain regional amine concentration (HPLC with LEC) and serotonin S-1 receptor binding; and (ii) 'serotonergic' behaviors in the same adult rats. Serotonin (5-HT) neurotoxins produced significantly different regional profiles of 5-HT depletion. Multiple DHT injections caused a 90-100% depletion of 5-HT concurrently in neocortex, hippocampus, striatum, septum/accumbens, pons, cerebellum, and cervical cord. Only PCA significantly depleted midbrain. Drug combinations with DHT resembled DHT alone rather than additive depletions, except for PCA + DHT, which produced a hybrid pattern of depletion. The S-1 binding assay, using cold 5-HT to displace [3H]5-HT, was performed with and without ascorbate, EDTA, CaCl2, and pargyline. Without ascorbate, binding was specific, saturable, region-dependent, and non-linear with high (Kd 1-3 nM) and low affinity (10-20 nM) components but no cooperativity (0.8 less than nH less than 1.0). Bmax and Kd did not differ significantly between vehicle- and drug-treated animals in neocortex, hippocampus, striatum, thalamus, hypothalamus, midbrain, pons, medulla, cervical cord, cerebellum, or septum/accumbens two weeks after lesioning, while the assay did detect a 60% reduction in Bmax induced by ascorbic acid (1 mM). The effects of assay conditions exceeded the changes sometimes reported in S-1 receptor Bmax after 5-HT lesions.     

Spiroperidol binding sites on mouse lymphoid cells. Effects of ascorbic acid and psychotropic drugs

Since highly differentiated cells of mammalian immune systems reportedly have binding sites for a variety of neurohumoral agents, we investigated parameters related to possible existence of dopamine receptors on murine lymphoid cells. Using a dopamine antagonist, [3H]spiroperidol, we found evidence for displaceable binding on mouse spleen cells. Total and displaceable (10 microM haloperidol) binding of spiroperidol was markedly enhanced by the absence of ascorbic acid in the incubation medium. Displaceable binding of a dopamine receptor agonist [( 3H]ADTN) could not be found on lymphoid cells either in the presence or absence of ascorbic acid. In the absence of ascorbate, displaceable spiroperidol binding to mouse spleen cells revealed partially saturable, but complex, kinetics and we calculated positive cooperativity at low ligand concentrations.     

[3H]5-HT binding in post-mortem human cerebral cortex: methodological considerations

Summary The effects of different membrane preparations and assay conditions on [³H]5-HT binding to post-mortem human cortical tissue was studied. Optimal binding necessitated thorough removal of endogenous 5-HT and this was achieved either by hypotonic lysis or by preincubation of the membranes at 37C. Calcium chloride (4 mM) increased specific [³H]5-HT binding. The further addition of ascorbic acid (5.7 mM) or ascorbic acid and clorgyline (10 M) reduced specific [³H]5-HT binding.     

Spiroperidol binding sites on mouse lymphoid cells : Effects of ascorbic acid and phsychotropic drugs

Since highly differentiated cells of mammalian immune systems reportedly have binding sites for a variety of neurohumoral agents, we investigated parameters related to possible existence of dopamine receptors on murine lymphoid cells. Using a dopamine antagonist, [³H]spiroperidol, we found evidence for displaceable binding on mouse spleen cells. Total and displaceable (10 μM haloperidol) binding of spiroperidol was markedly enhanced by the absence of ascorbic acid in the incubation medium. Displaceable binding of a dopamine receptor agonist ([³H]ADTN) could not be found on lymphoid cells either in the presence or absence of ascorbic acid. In the absence of ascorbate, displaceable spiroperidol binding to mouse spleen cells revealed partially saturable, but complex, kinetics and we calculated positive cooperatively at low ligand concentrations.     

Acute acidosis elevates malonaldehyde in rat brain in vivo

Oxidative stress in brain tissue was measured experimentally in situ using microdialysis to sample the extracellular environment for a lipid peroxidation breakdown product and antioxidants. The extracellular concentrations of the lipid peroxidation product malonaldehyde (MDA) and the antioxidants ascorbic acid (AA) and uric acid (UA) were measured in rat cortex and striatum in vivo using microdialysis coupled to HPLC with UV detection. Tissue acidosis following ischaemia and epileptic seizures may contribute to neuronal damage, which may be mediated by reactive oxygen species. Perfusion of microdialysis probes with acidic artificial cerebrospinal fluid (pH 6) led to a significant increase in the sampled concentration of MDA and the antioxidant ascorbic acid. Simultaneous perfusion of ascorbate (5 mM) with acidic ACSF (pH 6) completely attenuated the rise in lipid peroxidation. This study provides in vivo evidence for acidosis induced oxidative stress in brain tissue and an antioxidant action of ascorbate. The methodology described here can provide direct in vivo information in respect of oxidative stress in experimental situations. The method could equally be applied to the assessment of oxidative stress in a number of pathological models not necessarily confined to the CNS     

Oxidative stress induced by ascorbate causes neuronal damage in an in vitro system

Of particular physiological interest, ascorbate, the ionized form of ascorbic acid, possesses strong reducing properties. However, it has been shown to induce oxidative stress and lead to apoptosis under certain experimental conditions. Ascorbate in the brain is released during hypoxia, including stroke, and is subsequently oxidized in plasma. The oxidized product (dehydroascorbate) is transported into neurons via a glucose transporter (GLUT) during a reperfusion period. The dehydroascorbate taken up by cells is reduced to ascorbate by both enzymatic and non-enzymatic processes, and the ascorbate is stored in cells. This reduction process causes an oxidative stress, due to coupling of redox reactions, which can induce cellular damage and trigger apoptosis. Ascorbate treatment decreased cellular glutathione (GSH) content, and increased the rates of lipid peroxide production in rat cortical slices. Wortmannin, a specific inhibitor of phosphatidylinositol (PI)-3-kinase (a key enzyme in GLUT translocation), prevented the ascorbate induced-decrease of GSH content, and suppressed ascorbate-induced lipid peroxide production. However, wortmannin was ineffective in reducing hydrogen peroxide (H(2)O(2))-induced oxidative stress. The oxidative stress caused ceramide accumulation, which was proportionally changed with lipid peroxides when the cortical slices were treated with ascorbate. These differential effects support the hypothesis that GLUT efficiently transports the dehydroascorbate into neurons, causing oxidative stress.     

Ascorbic acid protects SH-SY5Y neuroblastoma cells from apoptosis and death induced by beta-amyloid

beta-Amyloid causes apoptosis and death in cultured neurons that may be mediated by generation of reactive oxygen species. Since ascorbic acid concentrations are relatively high in brain, we tested whether and how this antioxidant might protect cultured SH-SY5Y neuroblastoma cells from apoptotic cell death. SH-SY5Y cells did not contain ascorbate in culture but readily took it up to achieve intracellular concentrations several-fold those of GSH. Treatment of cells with 2-10 microM beta-amyloid(25-35) decreased both intracellular ascorbate and GSH without affecting rates of ascorbate transport, which suggests that the peptide induces an oxidant stress in the cells. Overnight culture of cells with 10-20 microM beta-amyloid(25-35) induced apoptosis in SH-SY5Y cells when measured as externalization of phosphatidylserine by annexin V binding, as DNA fragmentation in the TUNEL assay, and as caspase-3 activity in cell lysates. Pre-loading cells with ascorbate substantially prevented apoptosis measured by these assays as well as cell death. In addition to preventing apoptosis, ascorbate loading of SH-SY5Y cells also decreased basal rates of generation of endogenous beta-amyloid. Together, these results support the notion that beta-amyloid induces apoptosis and death in neurons due to oxidant stress and suggest that intracellular ascorbate effectively prevents this toxicity.     

Serotonin binding in mouse brains. Some methodological aspects

The binding of (3H)5-hydroxytryptamine ([3H]5-HT, serotonin) to crude homogenates of brains from three different strains of mice has been studied. The strains, C57/BL, DBA and BALB did not show significant differences in the binding characteristics, with Kd values around 6-7 nM and Bmax 270-310 fmoles/mg protein. Various methodological aspects were investigated and found to be important for the binding assay. The presence of ascorbic acid (5.7 mM) thus caused a significant increase in Bmax by 30% without any change in the Kd values. This increase seemed to be due to a decrease in the non-specific binding rather than to an increase in the total binding of serotonin. The presence of a specific MAO-A inhibitor, clorgyline (10 microM), during the assay, resulted in a significant reduction of Bmax by 20% without any change in the affinity for serotonin, when tissue which had been frozen was used. Less than 2% of added serotonin was metabolized during the binding procedure in the absence of clorgyline. Thus, the decrease in binding capacity caused by clorgyline, may be due to a non-competitive blocking effect of the serotonin binding structure. These results indicate that the use of MAO inhibitors in (3H)5-HT binding assays on frozen tissue, rather than being necessary, might negatively affect the result. Neither storage of the brains at -70 degrees nor postmortem storage of the animals for 60 hours at 4 degrees resulted in obvious changes in the (3H)5-HT binding characteristics.     

Prooxidant effects of ascorbate in rat brain slices.

Ascorbate is a well-known reducing agent, but it can generate oxidative potential under appropriate condition. In rat cerebral cortex homogenate, 1 mM ascorbate decreased thiobarbituric acid-reactive substances (TBARS) content to 86% +/- 4% of control values, confirming that ascorbate is a reducing agent. However, ascorbate increased TBARS, in a dose-related manner, in slices prepared from cerebral cortex. Ferrous ion (Fe2+) had little effect on ascorbate-induced lipid oxidation in cortical slices, and EDTA did not have an influence on the ascorbate-induced oxidative action. Conversely, ascorbate plus Fe2+ elevated TBARS content to more than threefold over ascorbate alone in tissue homogenates. In summary, ascorbate is a reducing agent in the brain tissue homogenate but has an oxidizing effect in brain slices. A hypothesis is proposed to explain the oxidative effects of ascorbate in cortical slices, wherein extracellular ascorbate is oxidized to dehydroascorbate, which is rapidly carried into the cells via a glucose transporter (GLUT). The dehydroascorbate in cytosol is then reduced back to ascorbate, and, during the reduction process, cellular components are oxidized.     

Serotonergic aspects of the response of human platelets to immune-adjuvant muramyl dipeptide

The human platelet serotonergic responses of aggregation and uptake were shown to be modulated by muramyl peptides. Muramyl dipeptide inhibited serotonin uptake in a temperature dependent and stereospecific manner. It also blocked the binding to platelets of [3H]imipramine, a well-known inhibitor of serotonin uptake. Muramyl dipeptide decreased serotonin2 (5-HT2) mediated platelet aggregation, and blocked the binding of a 5-HT2-specific ligand: lysergic acid diethylamide. Since muramyl peptides are released upon degradation of bacteria, the findings offer a possible mechanism for neuro-immune modulation by emphasizing the interaction between 5-HT (a neurotransmitter) and muramyl peptides (immuno-adjuvants).     

Temperature-independent binding of [3H](+-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid in brain synaptic membranes treated by Triton X-100

Specific binding of [3H](+-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a highly selective antagonist for N-methyl-D-aspartic acid (NMDA) receptors, was examined in brain synaptic membranes treated with Triton X-100 by using a filtration assay method. Elevation of incubation temperature from 2 to 30 degrees C markedly diminished the binding. The binding reached a plateau within 5 min after the initiation of incubation at 2 degrees C, while the time required to attain an equilibrium was 1 min at 30 degrees C. The binding at 2 degrees C was rapidly dissociated by the addition of an excess of unlabeled CPP, NMDA and L-glutamic acid (L-Glu). The binding was also saturable with increasing concentrations of the ligand and displaced by various amino acids structurally related to L-Glu in a stereospecific manner. Competitive but not noncompetitive antagonists for the NMDA receptors invariably inhibited the binding. However, the binding was not prominently affected by agonists for the other subclasses of the brain excitatory amino acid receptors. Both reduced and oxidized forms of glutathione significantly displaced the binding. Scatchard analysis revealed that Triton treatment increased the affinity and density of binding sites which consisted of a single component. Among some endogenous tryptophan metabolites, kynurenic, anthranilic and quinolinic acids inhibited the binding. These results suggest that a filtration assay method is also useful to detect the binding of NMDA receptors in the brain.     

Studies of oxidative stress mechanisms using a morphine / ascorbate animal model and novel N-stearoyl cerebroside and laurate sensors

Summary. The field of oxidative stress, free radicals, cellular defense and antioxidants is a burgeoning field of research. An important biomarker of oxidative stress is ascorbate and alterations in ascorbate have been shown to be a reliable measure of oxidative stress mechanisms. The purpose of this pharmacological study was to assess changes in ascorbate in a morphine/ascorbate animal model using novel sensors which selectively detect electrochemical signals for ascorbate, dopamine (DA) and serotonin (5-HT). Studies were also performed to show reversal of morphine-induced effects by the opioid antagonist, naloxone. In vivo studies were modeled after (Enrico et al. 1997, 1998) in which the oxidative biomarker, ascorbate, was reported to compensate for free radicals produced by morphine-induced increases in DA and 5-HT. In vivo studies consisted of inserting the Laurate sensor in ventrolateral nucleus accumbens (vlNAcc), in anesthetized male, Sprague-Dawley rats. In separate studies, laboratory rats were injected with (1) ascorbate, (5–35 mg/kg, ip) or (2) dehydroascorbate (DHA) (20–100 mg/kg, ip). In another study, (3) morphine sulfate (10–20 mg/kg, sc) was injected followed by a single injection of naloxone (5 mg/kg, ip) in the same animal. Results showed that in vlNAcc, (1) neither ascorbate nor DHA injections produced ascorbate release, (2) morphine significantly increased DA and 5-HT release, but did not alter ascorbate release, and (3) naloxone significantly reversed the increased DA and 5-HT release produced by morphine. Moreover, the sensors, N-stearoyl cerebroside and laurate were studied in vitro, in separate studies, in order to assess selective and separate electrochemical detection of ascorbate, DA and 5-HT, neuromolecules involved in oxidative stress mechanisms. In vitro studies consisted of pretreatment of each sensor with a solution of phosphotidylethanolamine (PEA) and bovine serum albumin (BSA) which simulates the lipid/protein composition of brain. Each new sensor was tested for stability, sensitivity and selectivity by pipetting graduated increases in concentration of ascorbate, DA and 5-HT into an electrochemical cell containing saline/phosphate buffer. Multiple and repetitive images of electrochemical signals from ascorbate, DA and 5-HT were recorded. Results showed that both sensors produced three well-defined cathodic, selective and separate electrochemical signals for ascorbate, DA and 5-HT at characteristic oxidation potentials. Dopamine and 5-HT were detected at nM concentrations while ascorbate was detected at μM concentrations. In summary, the data show that very low concentrations of ascorbate occurred in vlNAcc since novel sensors detected ascorbate at high concentrations in vitro. The data indicate that little or no change in oxidative stress mechanisms occurred in vlNAcc after morphine or naloxone administration since the oxidative biomarker, ascorbate, was not signifi cantly altered. Thus, oxidative stress mechanisms and novel N-stearoyl cerebroside and laurate sensors, which selectively detect and separate neuromolecules involved in these mechanisms, may be potentially clinically relevant. Correspondence: Patricia A. Broderick, Department of Physiology and Pharmacology, The City University of New York Medical School, Convent Ave. & West 138th St., (Office: Suite 310F; MailRoom: Harris 01), NY 10031, USA     

Decrease of Na(+)-Ca2+ exchange activity by ascorbate in rat brain membrane vesicles

Na(+)-dependent Ca2+ uptake in rat brain microsomal membrane vesicles was inhibited by preincubating the vesicles with ascorbic acid at 0.1-10 mM. The inhibitory effect of ascorbate was blocked by simultaneous addition of ascorbate oxidase. The decrease in activity was not reversed upon removing the ascorbate. The kinetic study showed that the treatment with ascorbate decreased Bmax without a change in Km for Ca2+. The inhibitory effect by ascorbate was also observed in membrane vesicles derived from osmotically shocked synaptosomes and in reconstituted membrane vesicles. The effect by ascorbate was specific: it did not affect either ATP-dependent Ca2+ uptake in the presence of o-phenanthroline, an inhibitor of lipid peroxidation, or Na(+)-dependent glutamate uptake in the membrane vesicles. The activity of Na(+)-Ca2+ exchange was also decreased by isoascorbic acid, but not by ascorbate 2-sulfate at 1 mM. The treatment with glutathione or 2-mercaptoethanol did not affect the Na(+)-Ca2+ exchange activity, while 1 mM dithiothreitol caused the inhibition which was completely blocked by o-phenanthroline. The effect of ascorbate on Na(+)-dependent Ca2+ uptake was observed even under the conditions which suppress peroxidation of membrane phospholipids.     

Differential radioligand binding properties of [3H]5-hydroxytryptamine and [3H]mesulergine in a clonal 5-hydroxytryptamine1C cell line.

[3H]5-Hydroxytryptamine ([3H]5-HT) and [3H]mesulergine were used to label 5-HT1C receptors expressed in NIH 3T3 mouse fibroblast cells. Using a rapid filtration assay, saturation analysis of the [3H]5-HT radioligand data indicate that the binding is biphasic. Based on computerized analysis of the data, a 2-site model of radioligand binding is significantly more consistent with the data than a one-site model (P less than 0.01). The KD values of [3H]5-HT for the 2 populations are 0.5 +/- 0.1 nM and 31 +/- 15 nM, while the Bmax values are 400 +/- 90 pmol/g protein and 3,000 +/- 600 pmol/g protein, respectively. A biphasic binding pattern is also observed with [3H]5-HT using a centrifugation assay (KD1 = 0.6 +/- 0.06 nM, KD2 = 60 +/- 10 nM; Bmax1 = 740 +/- 90 pmol/g, Bmax2 = 4,000 +/- 700 pmol/g). By contrast, saturation analysis of [3H]mesulergine binding is monophasic (KD = 4.7 +/- 0.7 nM) with a Bmax value (6,800 +/- 1,000 pmol/g protein) that is significantly greater than that obtained using [3H]5-HT (P less than 0.01). Drug competition studies confirm that both [3H]5-HT and [3H]mesulergine label at least 2 subpopulations of expressed 5-HT1C receptors in NIH 3T3 cells. 10(-4) M GTP eliminates the high affinity [3H]5-HT-labeled binding sites with minimal effect on the low affinity [3H]5-HT-labeled sites and no effect on [3H]mesulergine-labeled sites. These data demonstrate that at least 2 distinct subpopulations of 5-HT1C receptors in NIH 3T3 cells can be differentiated using radioligand binding techniques.     

Regional and subcellular localization in human brain of [3H]paroxetine binding, a marker of serotonin uptake sites

The characteristics of the binding of [3H]paroxetine, a selective serotonin (5-HT) uptake blocker, were investigated in human brain. The Kd value was 0.23 +/- 0.07 nM, and the Bmax value was 190 +/- 39 fmol/mg protein in the putamen. The capacity of various antidepressive drugs to inhibit [3H]paroxetine-specific binding in human brain was well correlated with their capacity to inhibit [3H]5-HT uptake in rat brain. The highest concentrations of [3H]paroxetine-specific binding sites were found in the substantia nigra, hypothalamus, and hippocampus. Lower values were obtained in the basal ganglia and the thalamus. The specific binding was very low in cerebral and cerebellar cortices. The regional distribution of [3H]paroxetine binding sites differs from that of [3H]ketanserin binding to S2 serotonin receptors. The subcellular distribution of the [3H]paroxetine-specific binding sites obtained by differential centrifugation revealed a synaptosomal enrichment in the frontal cortex and striatum, whereas an enrichment in the microsomal fraction was found in striatum. The results show that [3H]paroxetine is a ligand of choice to label the 5-HT uptake molecular complex in human brain.     

Binding characteristics of [3H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCl: the recovery of binding activity by dilution

This paper describes the binding properties of [3H]peptidergic opioid ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [3H]-[D-Ala2-, Met5]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl2 and 100 mM NaCl at 0 degree C (on ice) for 3 h. With this assay condition, the significant binding of following [3H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin (mu-type), [3H]-[D-Ala2, D-Leu5]enkephalin (delta-type) and [3H]dynorphin1-9 (kappa-type). The number of binding sites in solubilized preparations for each [3H]ligand corresponded to 40-50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [3H]ligand. Apparent dissociation constants between solubilized receptors and [3H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycodeoxycholate and NaCl and the dilution process.     

Effect of ascorbic acid on the synaptosomal uptake of [3H]MPP+, [3H]dopamine, and [14C]GABA

The effects of ascorbic acid on the synaptosomal uptake of [3H]MPP+, [3H]dopamine, and [14C]GABA were examined in attempts to understand the mechanism of ascorbic acid attenuation of MPTP neurotoxicity. [3H]Dopamine uptake was increased at lower levels (0.01 and 0.1 mM) and decreased at higher levels (10 mM) of ascorbic acid. Ascorbic acid inhibited [3H]MPP+ uptake (IC50 = 0.1 mM) and [14C]GABA uptake (IC50 = 10 mM). Washout of ascorbic acid restored uptake of [3H]dopamine and [3H]MPP+, suggesting that ascorbic-acid-induced lipid peroxidation was not involved in the effect on uptake. In addition to the possible involvement of antioxidant mechanisms in the in vivo attenuation of the neurotoxicity of MPTP by ascorbic acid, the present results indicate a direct effect of ascorbic acid in inhibiting the uptake of MPP+ into dopaminergic nerve terminals.     

Characterization of [125I-Tyr10]human growth hormone-releasing factor (1-44) amide binding to rat pituitary: evidence for high and low affinity classes of sites

A sensitive binding assay was developed to determine binding characteristics of commercially available [125I-Tyr10]human growth hormone-releasing factor (hGRF) (1-44)NH2 in rat pituitary using 0.1 gland homogenate (70-75 micrograms protein) per incubation tube. Under standard assay conditions, addition of 5 mM EDTA efficiently prevented the degradation of both human and rat GRF for at least 3 h. Association of the ligand was time-dependent: equilibrium was reached within 30 min of incubation at 23 degrees C and remained stable for an additional 150 min (K1 = 5.01 +/- 0.86 nM-1.min-1). Specific binding increased linearly with the amount of protein present in the assay, from 15 to 170 micrograms per incubation tube. This binding was reversible, dissociation occurring almost completely after a 120-min period (K-1 = 8.13 +/- 0.29 x 10(-3) min-1). A concentration of 5-10 mM Mg2+ was required for optimal specific binding whereas 50 mM Mg2+ or monovalent cations such as Na+, K+, Li+ decreased it. Scatchard analysis of cold saturation studies by the Ligand program statistically revealed the presence of two distinct classes of binding sites; the first was of high affinity (0.68 +/- 0.11 nM) and low capacity (140 +/- 22 fmol/pituitary), the second was of lower affinity (590 +/- 347 nM) and higher capacity (38.7 +/- 18.7 pmol/pituitary). Similar values were obtained with various bovine serum albumin (BSA) concentrations and when using crude or washed pituitary homogenates, suggesting that the second low affinity site was not BSA or a soluble protein from the homogenate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective labeling of 5-HT1A and 5-HT1B binding sites in bovine brain

Drug interactions with serotonin(1A) 5-HT1A and serotonin(1B) (5-HT1B) binding sites were analyzed in bovine brain membranes. 5-HT1A binding sites were directly labeled with [3H]8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) in bovine hippocampal membranes. 5-HT1B binding sites were labeled by [3H]5-HT in bovine striatal membranes where less than 15% of specific binding sites are sensitive to nanomolar concentrations of 8-OH-DPAT. Each of the 12 agents tested was more potent at the 5-HT1A than 5-HT1B binding site. 5-HT, bufotenine, N,N-dimethyltryptamine (DMT) and quipazine were only slightly more potent at the 5-HT1A binding site. By contrast, 8-OH-DPAT, TVX Q 7821 and buspirone were significantly more potent at [3H]8-OH-DPAT binding sites in bovine hippocampus than at [3H]5-HT binding sites in bovine striatum. These findings suggest that 5-HT1A, and 5-HT1B binding sites have distinct pharmacological profiles and can be directly labeled with appropriate [3H]ligands in specific brain regions.     

Cortical and striatal variations in drug competition studies with putative 5-hydroxytryptamine1D binding sites.

The ability of 5-hydroxytryptamine (5-HT), 5-carboxytryptamine (5-CT) and sumatriptan to compete for [3H]5-HT binding sites in various brain tissues was analyzed in bovine, guinea pig, pig and human cortex and caudate. Radioligand binding conditions were designed to allow for the selective labeling of putative 5-HT1D binding sites. 5-HT competed monophasically with putative 5-HT1D binding sites in each of the 8 tissues studied. By contrast, both 5-CT and sumatriptan competed with markedly shallow displacement curves in each of the 8 tissues. In the case of sumatriptan, complete displacement of [3H]5-HT could not be achieved even at concentrations as high as 2000 times its IC50 value. These data indicate that 10(-5) M 5-HT should not be used to define specific binding to 5-HT1D receptors in radioligand binding assays. Instead, 5-HT1D receptor binding sites should be redefined as [3H]5-HT-labeled binding sites displaced by 10(-5) M sumatriptan.