Down-Regulation of [3H]5-Hydroxytryptamine Binding Sites in Chick Embryo Brain by Monoamine Oxidase Inhibitors or Fenfluramine and Potentiation by d,l-5-Hydroxytryptophan

Three monoamine oxidase (MAO) inhibitors--pargyline, clorgyline and deprenyl--as well as the serotonin (5-HT, 5-hydroxytryptamine) releasing agent fenfluramine were administered to developing chick embryos and the effects on [3H]5-HT binding parameters and endogenous 5-HT levels were assessed. Multiple, but not acute, pretreatments with any of the three MAO inhibitors significantly increased 5-HT levels (p less than 0.01) and decreased receptor number (Bmax) to a maximum of 20% (p less than 0.01) without affecting the affinity (KD). When d,l-5-hydroxytryptophan (d,l-5-HTP) was similarly administered there were large increases in 5-HT levels (p less than 0.01), but no significant effects on either Bmax or KD. However, if d,l-5-HTP was co-administered with any of the MAO inhibitors there was a significant (p less than 0.01) enhancement of the MAO inhibitor-induced down-regulation to a maximum of about 40%. Multiple pretreatments with fenfluramine resulted in dose-related decreases in 5-HT levels (p less than 0.01) and Bmax (p less than 0.01) without affecting KD. The largest decrease in [3H]5-HT binding sites inducible by fenfluramine treatment alone was also about 40%. When given in combination with d,l-5-HTP, there was a potentiation of the down-regulation capabilities of fenfluramine at several different dosage levels; however, maximal down-regulation was also limited to 40%. Evidence was presented suggesting that these effects were not due to endogenous 5-HT or drugs remaining in the tissue preparation. The overall evidence implies that merely increasing endogenous 5-HT levels, as by precursor administration, does not necessarily induce down-regulation unless the 5-HT is also made available as functional 5-HT.     

Comparison of [125I]Iodolysergic Acid Diethylamide Binding in Human Frontal Cortex and Platelet Tissue

The human platelet contains a functional 5-hydroxytryptamine (5-HT) receptor that appears to resemble the 5-HT2 subtype. In this study, we have used the iodinated derivative [125I]iodolysergic acid diethylamide ([125I]iodoLSD) in an attempt to label 5-HT receptors in human platelet and frontal cortex membranes under identical assay conditions to compare the sites labelled in these two tissues. In human frontal cortex, [125I]iodoLSD labelled a single high-affinity site (KD = 0.35 +/- 0.02 nM). Displacement of specific [125I]iodoLSD binding indicated a typical 5-HT2 receptor inhibition profile, which demonstrated a significant linear correlation (r = 0.97, p less than 0.001, n = 17) with that observed using [3H]ketanserin. However, [125I]iodoLSD (Bmax = 136 +/- 7 fmol/mg of protein) labelled significantly fewer sites than [3H]ketanserin (Bmax = 258 +/- 19 fmol/mg of protein) (p less than 0.001, n = 6). In human platelet membranes, [125I]iodoLSD labelled a single site with affinity (KD = 0.37 +/- 0.03 nM) similar to that in frontal cortex. The inhibition profile in the platelet showed significant correlation with that in frontal cortex (r = 0.96, p less than 0.001, n = 16). We conclude that the site labelled by [125I]iodoLSD in human platelet membranes is biochemically similar to that in frontal cortex and most closely resembles the 5-HT2 receptor subtype, although the discrepancy in binding capacities of [125I]iodoLSD and [3H]ketanserin raises a question about the absolute nature of this receptor.     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

[3H]1-[2-(4-Aminophenyl)Ethyl]-4-(3-Trifluoromethylphenyl)Piperazine: A Selective Radioligand for 5-HT1A Receptors in Rat Brain

1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5-hydroxytryptamine (5-HT, serotonin) binding to 5-HT1A and 5-HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5-HT1A receptor radioligand was evaluated. With either 10 microM 5-HT or 1 microM 8-hydroxy-2-(di-n-propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d-Lysergic acid diethylamide and 5-HT, two nonselective inhibitors of [3H]5-HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5-HT1 receptors. Spiperone and 8-hydroxy-2-(di-n-propylamino)tetralin, two compounds that discriminate [3H]5-HT binding to 5-HT1A and 5-HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5-HT1A receptor subtype. Furthermore, the ability of N-(m-trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5-HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin-sensitive [3H]PAPP sites correlated with the densities of 5-HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5-HT1A receptor sites in rat brain.     

Determination of the Equilibrium Dissociation Constants and Number of Glycine Binding Sites in Several Areas of the Rat Central Nervous System, Using a Sodium-Independent System

Parameters affecting the binding of [3H]glycine to membrane fractions isolated from the cerebral cortex, midbrain, cerebellum, medulla oblongata, and spinal cord of the rat were investigated in a Na+-free medium. A [3H]glycine binding assay was established in which the binding was specific, saturable, pH-sensitive, and reversible. Conditions were chosen in an effort to minimize binding to glycine uptake sites. From data on specific [3H]glycine binding Scatchard plots were prepared and the KD and Bmax values were calculated. Two glycine binding sites (high and low affinity) were identified only in the medulla (KD: 44, 211 nM; Bmax: 361, 1076 fmol/mg protein) and spinal cord (KD: 19, 104 nM; Bmax: 105, 486 fmol/mg protein). The ranges of the KD and Bmax values for the other three areas studied were 59 to 144 nM and 882 to 3401 fmol/mg protein, respectively. When the glycine content of each area, expressed as fmol/neuron, was plotted against the respective KD (high affinity), a negative correlation was found (r = --0.90; p less than 0.05). A similar negative correlation was found between the glycine content and Bmax (r = --0.88; p less than 0.05). Hill plots indicated a slope of essentially 1.0 for all areas. GABA, taurine, strychnine, diazepam, bicuculline, and imipramine had little or no effect on [3H]glycine binding.     

beta-Adrenergic receptors of brain cells. Membrane integrity implies apparent positive cooperativity and higher affinity

Beta-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [3H]dihydro-L-alprenolol ([3H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [3H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, beta-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of Bmax = 12.01 fmol/10(6) cells (7200 sites/cell), Kd = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with L-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k+ = 8.31 X 10(7) M-1 X min-1 and k- = 0.28 min-1 from the association results, and k- = 0.24 min-1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; Kd, as (k-/k+)1/n was found to be 101 pM. Analysis of the equilibrium binding of [3H]DHA to rat and mouse living brain cells resulted in values of Bmax = 13.04 fmol/10(6) cells (7800 sites/cell), Kd = 43.85 pM and n = 2.52, and Bmax = 8.08 fmol/10(6) cells (4800 sites/cell), Kd = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the beta-receptor in mammalian objects, too. The [3H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, Kd in the range 1-2 nM, and a Bmax of 10(3) - 10(4) sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the beta-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.     

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)     

Characterization of [3H]Quipazine Binding to 5-Hydroxytryptamine3 Receptors in Rat Brain Membranes

[3H]Quipazine was used to label binding sites in rat brain membranes that display characteristics of a 5-hydroxytryptamine3 (5-HT3) receptor. The radioligand binds with high affinity (KD, 1.2 +/- 0.1 nM) to a saturable population of sites (Bmax, 3.0 +/- 0.4 pmol/g of tissue) that are differentially located in the brain. Specific [3H]quipazine binding is not affected by guanine or adenine nucleotides. ICS 205-930, BRL 43964, Lilly 278584, and zacopride display less than nanomolar affinity for these sites whereas MDL 72222 is approximately one order of magnitude less potent. The pharmacological profile of the binding site is in excellent agreement with that of 5-HT3 receptors characterized in peripheral physiological models. We conclude that [3H]quipazine labels a 5-HT3 receptor in the rat CNS.     

"Specific" Binding of [3H]Imipramine to Protease-Sensitive and Protease-Resistant Sites

A number of 5-hydroxytryptamine (5-HT) uptake inhibitors have been shown to displace the binding of [3H]imipramine to rat cortical membranes in a complex manner with Hill slopes less than unity. Norzimeldine displaced the binding of [3H]imipramine in a biphasic manner with IC50 values for the two components of about 30 nM and 30 microM. This latter site alone was found in tissues that had been treated with a protease. Binding to both of these sites was displaced by 10 microM desipramine. The protease-sensitive [3H]imipramine binding sites were found to be saturable, high-affinity binding sites with a KD of 8 nM. The number of these sites varied between brain regions and was positively correlated with the regional distribution of [14C]5-HT but not [3H]noradrenaline uptake. This was not the case however for the protease-resistant but desipramine-displaceable binding sites. Since most previous [3H]imipramine binding studies have been performed with high concentrations of desipramine (10 microM) to define "specific binding," these data would suggest that either protease-sensitivity or displacability by 1 microM norzimeldine would give more reliable estimates of the specific binding.     

A Trifluoromethylphenyl Piperazine Derivative with High Affinity for 5-Hydroxytryptamine-1A Sites in Rat Brain

The inhibition of [3H]5-hydroxytryptamine [( 3H]5-HT) binding in rat brain by 1-[2-(3-bromoacetamidophenyl)ethyl]-4-(3-trifluoromethylphenyl) piperazine (BrAcTFMPP) and that by spiperone were compared. Spiperone inhibition of [3H]5-HT binding in cortex was consistent with displacement from two sites with dissociation constants (KD) of 24 nM (5-HT-1A site) and 19 microM (5-HT-1B site) for spiperone. BrAcTFMPP also discriminated two subpopulations of [3H]5-HT binding sites with dissociation constants of 0.5 nM and 146 nM for the compound. The proportion of high-affinity sites for each compound represented about 35% of the specific [3H]5-HT binding. In the presence of 1 microM spiperone, a concentration that saturates the 5-HT-1A sites while having a minimal effect on 5-HT-1B sites, BrAcTFMPP displaced [3H]5-HT from a single site with a KD for BrAcTFMPP of 145 nM. The inhibition of [3H]5-HT binding by spiperone in the presence of 30 nM BrAcTFMPP was best fit by a single-site model with a KD of 21 microM for spiperone. In corpus striatum, 5-HT-1A sites, as defined with spiperone, represented 15% of the specific [3H]5-HT binding and 30 nM BrAcTFMPP also blocked about 15% of the binding. A significant difference between spiperone and BrAcTFMPP was their affinity for 5-HT-2 receptors. BrAcTFMPP (KD = 41 nM) had an 80-fold lower affinity for these sites than spiperone (KD = 0.5 nM). Thus, BrAcTFMPP and spiperone discriminate the same two subpopulations of [3H]5-HT binding sites and BrAcTFMPP displays a high affinity and a selectivity for 5-HT-1A sites versus both 5-HT-1B and 5-HT-2 sites.     

Pharmacological Differentiation and Characterization of 5-HT1A, 5-HT1B, and 5-HT1C Binding Sites in Rat Frontal Cortex

Drug interactions with 5-HT1 (5-hydroxytryptamine type 1) binding site subtypes were analyzed in rat frontal cortex. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) displays high affinity (Ki 3.3 +/- 1 nM) for 29 +/- 3% of total [3H]5-HT binding in rat frontal cortex and low affinity (Ki 9,300 +/- 1,000) for 71 +/- 4% of the remaining 5-HT1 sites. Therefore, non-5-HT1A binding in rat frontal cortex was defined as specific [3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. 5-Methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl) 1 H indole (RU 24969), 1-(m-trifluoromethylphenyl)piperazine (TFMPP), mianserin, and methysergide produce shallow competition curves of [3H]5-HT binding from non-5-HT1A sites. Addition of 10(-3) M GTP does not increase the apparent Hill slopes of these competition curves. Computer-assisted iterative curve fitting suggests that these drugs can discriminate two distinct subpopulations of non-5-HT1A binding sites, each representing approximately 35% of the total [3H]5-HT binding in the rat frontal cortex. All three 5-HT1 binding site subtypes display nanomolar affinity for 5-HT and 5-methoxytryptamine. A homogeneous population of 5-HT1A sites can be directly labeled using [3H]8-OH-DPAT. These sites display nanomolar affinity for 8-OH-DPAT, WB 4101, RU 24969, 2-(4-[4-(2-pyrimidinyl)-1-piperazinyl] butyl)-1,2-benzisothiazol-3-(2H)one-1, 1-dioxidehydrochloride (TVX Q 7821), 5-methoxydimethyltryptamine, and d-lysergic acid diethylamide. The potencies of RU 24969, TFMPP, and quipazine for [3H]5-HT binding are increased by addition of 100 nM 8-OH-DPAT and 3,000 nM mianserin to the [3H]5-HT binding assay. Moreover, the drugs have apparent Hill slopes near 1 under these conditions. This subpopulation of total [3H]5-HT binding is designated 5-HT1B. By contrast, methysergide and mianserin become more potent inhibitors of residual [3H]5-HT binding to non-5-HT1A sites in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969. The drug competition curves under these conditions have apparent Hill slopes of near unity and these sites are designated 5-HT1C. Drug competition studies using a series of 24 agents reveals that each 5-HT1 subtype site has a unique pharmacological profile. These results suggest that radioligand studies can be used to differentiate three distinct subpopulations of 5-HT1 binding sites labeled by [3H]5-HT in rat frontal cortex.     

Physicochemical Properties of Serotonin 5-HT3 Binding Sites Solubilized from Membranes of NG 108-15 Neuroblastoma-Glioma Cells

Specific binding sites with pharmacological properties typical of serotonin 5-HT3 receptors were identified in membranes of the murine hybridoma cell line NG 108-15, using [3H]zacopride as a ligand. Optimal solubilization of these sites (yield, 50%) could be achieved using the detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 24 mM plus 0.5 M NaCl in 25 mM Tris-HCl, pH 7.4. Specific [3H]zacopride binding to soluble sites in the 100,000-g CHAPS extract was saturable and showed characteristics (Bmax = 425 +/- 81 fmol/mg of protein; KD = 0.19 +/- 0.02 nM) closely related to those of membrane-bound sites (Bmax = 932 +/- 183 fmol/mg of protein; KD = 0.60 +/- 0.03 nM). Determination of association (k+1 = 0.17 nM min-1) and dissociation (k-1 = 0.02 min-1) rate constants for the soluble sites gave a KD value of 0.12 nM, a result consistent with that calculated from saturation studies. As assessed from the displacement potencies (IC50) of 10 different drugs, the pharmacological profile of [3H]zacopride specific binding sites was essentially the same (r = 0.99) in the CHAPS-soluble extract and in cell membranes, although some increase in the affinity for 5-HT3 antagonists (zacopride, ICS 205-930, and MDL 72222) and decrease in the affinity for 5-HT3 agonists (2-methyl-5-hydroxytryptamine and phenylbiguanide) were noted for the soluble sites. Sucrose density gradient sedimentation of the CHAPS-soluble extract gave a Svedberg coefficient of 12S for the material with [3H]zacopride specific binding capacity. Chromatographic analyses using Sephacryl S-400 and wheat germ agglutinin-agarose columns indicated marked enrichment (by 2.5- and 10-fold, respectively) in [3H]zacopride specific binding activity in the corresponding eluates compared with the starting soluble extract, a finding suggesting that both steps are of potential interest for the partial purification of solubilized 5-HT3 receptors. Two soluble materials with apparent molecular masses of approximately 600 and approximately 36 kDa were found to bind [3H]zacopride specifically in the Sephacryl S-400 eluate. Interestingly, molecular mass determination by radiation inactivation of [3H]zacopride binding sites in frozen NG 108-15 cells gave a value of approximately 35 kDa.     

Ontogeny of Calcium Antagonist Binding Sites in Chick Brain and Heart

The ontogeny of chick brain and heart ventricle calcium antagonist binding sites was determined, using [3H]nitrendipine ([3H]NDP), as the ligand. The binding of [3H]NDP to adult heart and brain was kinetically very similar, with the former displaying a KD of 0.28 +/- 0.02 nM and a Bmax of 138 +/- 17 fmol/mg protein, and the latter a KD of 0.30 +/- 0.02 nM and a Bmax of 160 +/- 12 fmol/mg protein. The binding site in both brain and heart was highly specific for dihydropyridine calcium antagonists, such as nifedipine, nimodipine, and nisoldipine, since these drugs were several orders of magnitude more potent as inhibitors of [3H]NDP binding than verapamil, methoxyverapamil, or diltiazem. The developmental appearance of [3H]NDP binding sites in brain was rather gradual, with adult levels being attained just prior to birth. This was in contrast to the profile in heart ventricle which showed essentially adult levels at seven days gestation. The acquisition of [3H]NDP binding sites in chick brain roughly paralleled the onset of neuronal maturation and functional activity. In both chick brain and heart, verapamil and methoxyverapamil were weak inhibitors of [3H]NDP binding. However, the inhibition of binding in both tissues was markedly biphasic, with only 50% of the binding sites being susceptible to inhibition by each agent, suggesting that multiple calcium antagonist binding sites may exist in both tissues.     

Photoaffinity Labeling of the 5-HydroxytryptamineIA Receptor in Rat Hippocampus

1-[2-(4-Azidophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (p-azido-PAPP) inhibits [3H]5-hydroxytryptamine [( 3H]5-HT) binding to 5-HT1A and 5-HT1B sites in rat brain with equilibrium dissociation constants (KD) of 0.9 nM and 230 nM, respectively. [3H]p-Azido-PAPP was synthesized and its reversible and irreversible binding properties to the hippocampal 5-HT1A site characterized. [3H]p-Azido-PAPP labeled a single class of sites in rat hippocampal membranes with a KD of 1 nM and a maximal binding density of 370 fmol/mg protein. The pharmacological profile of [3H]p-azido-PAPP binding was consistent with the radioligand's selective interaction with the 5-HT1A receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes preincubated with [3H]p-azido-PAPP and irradiated showed a major band of incorporation of radioactivity at approximately 55,000 daltons. This incorporation could be blocked when membranes were incubated with 1 microM of several agents that have high affinity for 5-HT1A sites [5-HT, 8-hydroxy-2-(di-n-propylamino)tetraline, TVX Q 7821, spiperone, buspirone, d-lysergic acid diethylamide, metergoline]. The results indicate that on photolysis [3H]p-azido-PAPP irreversibly labels a polypeptide that is, or is a subunit of, the 5-HT1A receptor in rat hippocampus.     

Characterization of Multiple [3H]5–Hydroxytryptamine Binding Sites in Rat Spinal Cord Tissue

Abstract: High-affinity [³H]5-hydroxytryptamine ([³H]5-HT) binding in the rat spinal cord is similar to that demonstrated in the frontal cortex. [³H]5-HT binds with nearly the same affinity to sites in both tissues. Furthermore, similar patterns of displacement of [³H]5–HT were seen in both tissues, with either spiperone or LSD as the unlabeled ligand. This high-affinity binding appears to be to multiple sites, since displacement studies using 2 nM [³H]5–HT result in Hill coefficients less than unity for spiperone, LSD, and quipazine [Hill coefficients (n_H): 0.44, 0.39, 0.40, respectively]. These sites apparently have an equal affinity for [³H]5-HT, since unlabeled 5-HT did not discriminate between them. Thus, the high-affinity [³H]5-HT binding in the spinal cord may be analogous to that observed in the frontal cortex, where two populations of sites have previously been described (5-HT_IA, 5-HT_IB). In addition to the multiple high-affinity spinal cord binding sites, a low-affinity [³H]5-HT binding component was also identified. A curvilinear Scatchard plot results from saturation studies using [³H]5-HT (0.5–100 nM) in the spinal cord. The plot can be resolved into sites having apparent dissociation constants of 1.4 nM and 57.8 nM for the high-and low-affinity components, respectively. Additional support for a change in affinity characteristics at higher radioligand concentrations comes from the displacement of 30 nM [³H]5-HT by the unlabeled ligand. A nonparallel shift in the dissociation curve was seen, resulting in a Hill coefficient less than unity (0.32). None of the specifically bound [³H]5-HT in the spinal cord is associated with the 5-HT uptake carrier, since fluoxetine, an inhibitor of 5-HT uptake, does not alter binding characteristics. In addition, a 5-HT binding site analogous to the site designated 5-HT, was not apparent in the spinal cord. Ketanse-rin and cyproheptadine, drugs that are highly selective for 5-HT, sites, did not displace [³H]5-HT from spinal tissue, and [³H]spiperone, a radioligand that binds with high affinity to 5-HT₂ sites, did not exhibit saturable binding in the tissue. Thus, the 5-HT₂ binding site reported in other regions of the central nervous system, and the serotonin uptake carrier do not appear to contribute to the multiple binding sites demonstrated in the spinal cord.     

Effects of Prenatal Phenobarbital on Benzodiazepine Receptor Development

Phenobarbital (PB) was administered to pregnant mice during days 9-21 of gestation. Forebrain and cerebellar [3H]flunitrazepam ([3H]FLU) binding was assayed in the offspring at birth and at 21 days of age. Prenatal treatment produced a decrease in the number (Bmax) of [3H]FLU receptors in both the forebrain and cerebellum at birth. A small decrease in the [3H]FLU dissociation constant (KD) values in the forebrain was also detected at birth, but no changes were seen in the [3H]FLU KD values in the cerebellum. No changes were observed in forebrain and cerebellar [3H]FLU Bmax or KD values at 21 days of age, indicating that the effects of prenatal exposure to PB on [3H]FLU binding are eliminated during the postnatal development of the forebrain and cerebellum. The receptor affinity for the triazolopyridazine CL 218,872, which distinguishes the type I and type II benzodiazepine (BDZ) receptors, was not altered by prenatal PB treatment. The coupling of the BDZ receptor to the gamma-aminobutyric acid and pentobarbital binding sites was unaffected by exposure to PB in utero.     

Differential effect of cholesterol on two types of 5-hydroxytryptamine binding sites

The specific binding of [3H]5-hydroxytryptamine ([3H]5-HT, [3H]serotonin) to rat cerebral cortex is increased approximately 1.5 to 2.0 fold by cholesterol hydrogen succinate (CHS) and is solubilized into the supernatant fraction by 12 mM CHS. [3H]5-HT binding sites can be constituted by incubating the supernatant fraction obtained from CHS-treated cerebral cortex with cerebellum in which no significant [3H]5-HT binding is detectable. The constituted [3H]5-HT binding could be displaced by unlabeled 5-HT, d-lysergic acid diethylamide (d-LSD) and spiperone as could the binding to cortex membranes. Unlabeled 5-HT, d-LSD and spiperone each inhibited specific [3H]5-HT binding to constituted binding sites by 50% at about 1 X 10(-9) M. Specific [3H]spiperone binding was not detectable in the constituted membranes. Stearic acid which is reported to have similar effects on membrane fluidity as cholesterol also increased specific [3H]5-HT binding in cortical membranes. Stearic acid does not affect specific [3H]spiperone binding. These results suggest that [3H]5-HT and [3H]spiperone binding sites are affected differently by membrane fluidity.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

5-Hydroxytryptamine Uptake and Imipramine Binding Sites in Neurotumor NCB-20 Cells

NCB-20 cells (neuroblastoma X fetal Chinese hamster brain hybrids) are equipped with a [3H]5-hydroxytryptamine [( 3H]5-HT) uptake system and [3H]imipramine recognition sites. Approximately 80% of the radioactivity taken up by cells incubated with [3H]5-HT was identified with 5-HT. [3H]5-HT uptake was temperature-dependent, partially sodium-dependent, saturable (Km = 7.3 +/- 0.6 microM; Vmax = 2.0 +/- 0.6 pmol/min/mg), and inhibited by clomipramine, imipramine, fluoxetine, and desipramine, but not by iprindole, mianserin, or opipramol. Lineweaver-Burk plots showed a competitive type of inhibition by imipramine and fluoxetine. [3H]5-HT uptake was not inhibited by nisoxetine or benztropine. [3H]Imipramine binding sites had a KD of 12 +/- 2 nM and a Bmax of 22 +/- 7 pmol/mg protein. The binding was sodium-sensitive although to a lesser extent than that found with brain membranes. Imipramine binding was displaced by tricyclic antidepressants with the following order of potency: clomipramine greater than imipramine greater than fluoxetine greater than desipramine much greater than iprindole = mianserin greater than opipramol. These results suggest that imipramine binding sites are present together with the 5-HT uptake sites in NCB-20 cells and that these sites interact functionally but are different biochemically.     

Serotonin 5-HT1D Receptors in Human Prefrontal Cortex and Caudate: Interaction with a GTP Binding Protein

Radioligand binding studies were performed to characterize serotonin 5-HT1D receptors in postmortem human prefrontal cortex and caudate homogenates. [3H]5-HT binding, in the presence of pindolol (to block 5-HT1A and 5-HT1B receptors) and mesulergine (to block 5-HT1C receptors), was specific, saturable, reversible, and of high affinity. Scatchard analyses of [3H]5-HT-labeled 5-HT1D sites in human prefrontal cortex produced a KD value of 4.2 nM and Bmax of 126 fmol/mg protein. In competition experiments, 8-hydroxydipropylaminotetralin, trifluoromethylphenylpiperazine, mesulergine, 4-bromo-2,5-dimethoxyphenylisopropylamine, and ICS 205-930 had low affinity for [3H]5-HT-labeled 5-HT1D sites, indicating that the pharmacology of the 5-HT1D site is distinct from that of previously identified 5-HT1A, 5-HT1B, 5-HT1C, 5-HT2, and 5-HT3 sites. 5-HT1D sites in human brain have a similar pharmacology to the 5-HT1D sites previously identified in rat, porcine and bovine brains. Guanyl nucleotides, guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) and guanosine 5'-(beta, gamma-imido)-triphosphate (Gpp(NH)p), modulated the binding of [3H]5-HT to 5-HT1D sites, whereas adenyl nucleotides had no effect. These findings are supportive of the presence of serotonin 5-HT1D receptors in human prefrontal cortex and caudate which appear to be coupled to a GTP binding protein.