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.     

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.     

Identification and Characterisation of 5-Hydroxytryptamine3 Recognition Sites in Human Brain Tissue

[3H]Zacopride displayed regional saturable specific binding to homogenates of human brain tissues, as defined by the inclusion of BRL43694 [endo-N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)-1-methylindazole-3- carboxamide] in the incubation media. Scatchard analysis of the saturation data obtained from amygdaloid and hippocampal tissues identified the binding as being of high affinity and to a homogeneous population of binding sites (KD = 2.64 +/- 0.75 and 2.93 +/- 0.41 nmol/L and Bmax = 55 +/- 7 and 44 +/- 9 fmol/mg of protein in the amygdala and hippocampus, respectively). 5-Hydroxytryptamine 3 (5-HT3) receptor agonists and antagonists competed for the [3H]zacopride binding site, competing with up to 40% of total binding with a similar rank order of affinity in both tissues; agents acting on various other neurotransmitter receptors failed to inhibit binding. Kinetic data revealed a fast association that was fully reversible (k+1 = 6.61 X 10(5) and 7.65 X 10(5)/mol/L/s and k-1 = 3.68 X 10(-3) and 3.45 X 10(-3)/s in the amygdala and hippocampus, respectively). It is concluded that [3H]zacopride selectively labels with high affinity 5-HT3 recognition sites in human amygdala and hippocampus and, if these binding domains represent 5-HT3 receptors, may provide the opportunity for 5-HT3 receptor antagonists to modify 5-HT function in the human brain.     

Adenosine binding sites of rat pheochromocytoma PC 12 cell membranes: partial characterization and solubilization

Rat pheochromocytoma PC 12 cell membranes were shown to possess A2-like adenosine binding sites as assessed by using 5'-N-ethylcarboxamide[3H]adenosine [( 3H]NECA). Specific [3H]NECA binding to PC 12 cell membrane at 0 degrees C was saturable and showed a monophasic saturation profile. In contrast, [3H]NECA binding to PC 12 cell membrane at 30 degrees C exhibited a biphasic profile suggesting the presence of two specific binding site. The rank order of potency for inhibition of [3H]NECA binding at 0 degrees C was NECA greater than 2-chloroadenosine greater than 2',5'-dideoxyadenosine greater than isobutylmethylxanthine much greater than phenylisopropyladenosine. These adenosine binding sites were solubilized with sodium cholate and the solubilized portion retained the same ligand binding characteristics as those of the membrane-bound form. Gel filtration experiments indicated an apparent Stokes radius of 6.7 nm for these adenosine binding sites/detergent complexes.     

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.     

Solubilisation of the 5-Hydroxytryptamine3 Receptor from Pooled Rat Cortical and Hippocampal Membranes

5-Hydroxytryptamine3 (5-HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205-930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested (1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X-100, and 1% octyl glucoside), deoxycholate (0.5%) yielded the best solubilisation (54.6 +/- 6% of receptor, 70.5 +/- 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205-930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G-25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205-930 to these soluble sites was saturable and specific (Bmax = 46.1 +/- 6 fmol/mg of protein; KD = 0.33 +/- 0.09 nM; n = 4) and was similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205-930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4 degrees C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists completed for the soluble receptors with a rank order of potency typical for binding at a 5-HT3 receptor site: zacopride (Ki = 0.26 nM) greater than quipazine (0.37 nM) = Q ICS 205-930 (0.33 nM) greater than ICS 205-930 (0.93 nM) greater than GR 38032F (2.2 nM) greater than BRL 24924 (4.1 nM) greater than MDL 72222 (23.4 nM) greater than ketanserin (6,000 nM). The agonists 5-HT and 2-methyl-5-HT also competed for [3H]Q ICS 205-930 binding with high affinity (39.6 and 55.6 nM, respectively). Therefore, we conclude that the 5-HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.     

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.     

(125I)iodo-zacopride: new ligand for the study by autoradiography of central 5-HT3 receptors

This paper describes the synthesis and the pharmacological characteristics of the first radioiodinated ligand of central 5-HT3 receptors: [125I]iodo-zacopride. Specific sites having a high affinity (Kd = 4.3 nM) for [125I]iodo-zacopride have been found in membranes from the rat entorhinal cortex. In addition, a highly significant correlation (r = 0.995) existed between the Ki of several 5-HT-related drugs for displacing both [125I]iodo-zacopride from its specific binding sites, and [3H]zacopride from 5-HT3 receptors. Finally, [125I]iodo-zacopride was successfully used for the autoradiographic mapping of 5-HT3 receptors in the rat central nervous system.     

Pharmacological and Biochemical Characterization of Rat Hippocampal 5-Hydroxytryptamine1A Receptors Solubilized by 3–[3–(Cholamidopropyl)dimethylammonio]-1-Propane Sulfonate (CHAPS)

Rat hippocampal 5-hydroxytryptamine1A (5-HT1A) binding sites were solubilized with a yield of 34% using 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS, 10 mM) as detergent. Kinetic analyses of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) binding indicated that the 5-HT1A sites exhibit the same properties in the soluble form as in the membrane-bound form. Furthermore, a positive correlation (r = 0.988) was found between the respective pIC50 values of a series of agonists and antagonists to inhibit [3H]8-OH-DPAT binding to either soluble or membrane-bound 5-HT1A sites. Gel filtration through Sephacryl S-400 as well as chromatography on wheat germ agglutinin (WGA)-agarose did not affect the modulation by guanine nucleotides (5'-guanylylimidodiphosphate) of [3H]8-OH-DPAT binding which suggests that the 5-HT1A binding subunit is a glycoprotein tightly attached to a G protein even in its soluble form. The [3H]8-OH-DPAT binding material eluted from Sephacryl S-400 had an apparent molecular mass of 155 kilodaltons, as expected from a heterodimer with one binding subunit (approximately 60 kilodaltons) and one G protein (approximately 80 kilodaltons). Marked enrichment in 5-HT1A binding sites relative to other soluble proteins was found in the peak fractions eluted from Sephacryl S-400 (by sixfold) and WGA-agarose (by 26-fold) columns, suggesting that these chromatographic steps might be of interest for the purification of central 5-HT1A receptors.     

Stimulation of Inositol Incorporation into Lipids of PC 12 Cells by Nerve Growth Factor and Bradykinin

The effects of bradykinin (BK) and lithium on the phosphatidylinositol cycle were examined in PC12 cells cultured for 20 h in the presence [PC12(+)] or in the absence [PC12(-)] of nerve growth factor (NGF). BK (1 microM) induced a small stimulation of the incorporation of myo-[2-3H]inositol into the lipids of PC12(-) cells and a three- to fourfold stimulation of such incorporation into the lipids of PC12 (+) cells. About 15 h of incubation with NGF and greater than 10 min of incubation with BK were needed for maximal stimulation of inositol incorporation by BK. In the presence of 25 mM LiCl, BK stimulated the inositol monophosphate levels nine-fold in PC12 (-) and 30-fold in PC12 (+) cells. After incubation for 20 h with NGF, an increased binding of [3H]BK to the PC12 (+) cells was observed at 4 degrees C. Exposure of the cells for 30 min to 25 mM LiCl enhanced the effect of BK on the inositol incorporation into total inositol lipids, especially in PC12(+) cells. In these cells, LiCl in the presence of BK also increased several-fold the intracellular levels of inositol bisphosphate and inositol trisphosphate.     

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.     

Changes of Ganglioside Pattern During Cerebellar Development of Normal and Staggerer Mice

Saturable and specific binding sites for 5-[3H]hydroxytryptamine (5-HT, serotonin) characterized by a KD of 3.5-4.5 nM were detected in the chick embryo brain and were shown to develop linearly as a function of age, weight, and protein content. Saturation and displacement studies using unlabeled 5-HT as the displacing ligand suggested a single population of binding sites. However, displacement studies using 5-methoxytryptamine, lysergic acid diethylamide (LSD), 2-bromo-lysergic acid diethylamide (BOL), methysergide, and spiperone as competing ligands suggested the existence of subclasses of [3H]5-HT binding sites because the Hill coefficients were less than unity. When compared with the reported [3H]5-HT binding sites (5-HT1) in the rat forebrain, the IC50 values of the competing ligands were similar. However, the Hill coefficients for LSD and methysergide were less than unity which suggested that the [3H]5-HT binding sites in the chick embryo brain may be more similar to those found in rat spinal cord than rat forebrain. To study [3H]5-HT binding site regulation and development, various serotonergic compounds were injected into the chorioallantoic fluid of the eggs at different times during embryonic development. Multiple pretreatments with d,l-5-hydroxytryptophan, 5-HT, or BOL were found to have no significant effects on either the affinity (KD) or number (Bmax) of specific [3H]5-HT binding sites. Multiple pretreatments with p-chlorophenylalanine were found to increase the Bmax of specific [3H]5-HT binding by 23% (p less than 0.01) whereas multiple pretreatments with LSD were found to decrease the Bmax of specific binding by 45% (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Adenosine Receptors in the PC 12 Pheochromocytoma Cell Line Using Radioligand Binding: Evidence for A-2 Selectivity

Examination of the binding characteristics of the adenosine agonist radioligands [3H]N6-cyclohexyladenosine [( 3H]CHA), [3H]cyclopentyladenosine [( 3H]CPA), and [3H]5'-N-ethylcarboxamido adenosine [( 3H]NECA) to membranes prepared from PC12 cells showed that the A-1-selective ligands (CHA and CPA) had minimal binding, which was not amenable to analysis using curve-fitting programs. However, [3H]NECA, a nonselective A-1/A-2 agonist, gave reproducible binding, which was enhanced by removal of endogenous adenosine, using the catabolic enzyme adenosine deaminase. This binding was of high affinity (KD = 4.7 nM) with limited capacity (263 fmol/mg of protein). Specific binding of [3H]NECA was unaffected by the presence of either CPA (50 nM) or MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM), a finding suggesting involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. Binding of [3H]NECA to PC12 cell membranes was stereo-selective, with the R isomer of N6-phenylisopropyladenosine (PIA) being approximately 12 times more active than S-PIA. The A-1-selective agonist CPA was a weak inhibitor of [3H]NECA binding (Ki = 251 nM). The rank order of activity of adenosine agonists in displacing specific [3H]NECA binding was NECA greater than or equal to 2-chloroadenosine greater than CHA greater than or equal to 5'-N-methylcarboxamido adenosine greater than or equal to R-PIA greater than CPA greater than S-PIA. Binding was also displaced by the marine adenosine agonist 1-methylisoguanosine and by a series of xanthine antagonists with the activity order being 1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine greater than 8-phenyltheophylline greater than 8-p-sulfophenyltheophylline.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Increase in serotonin 5-HT1A receptors in prefrontal and temporal cortices of brains from patients with chronic schizophrenia.

Binding studies with [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT), a specific serotonin1A (5-HT1A) receptor agonist, were done on the autopsied brains from control subjects and from patients with chronic schizophrenia. All the patients and controls were of the Japanese race. In the controls, representative Scatchard plots for the specific [3H]8-OH-DPAT bindings in the prefrontal cortex and hippocampus revealed a single component of high affinity binding site (Kd value = 5.7 and 5.9 nM, Bmax value = 80.1 and 101.0 fmol/mg protein, respectively). The [3H]8-OH-DPAT bindings to the prefrontal cortex and hippocampus were potently inhibited by serotonin (IC50 = 6.3 x 10(-9) M) and 5-HT1A agonists (IC50 = 5.0 x 10(-9) - 2.3 x 10(-7) M), while other neurotransmitters, 5-HT2 and 5-HT3 related compounds did not inhibit the binding (IC50 greater than 10(-5) M). The bindings were decreased in the presence of 0.1mM GTP and 0.1mM GppNHp but not in the presence of 0.1mM GMP. In the prefrontal and temporal cortices of schizophrenics, there was a significant increase in the specific [3H]8-OH-DPAT binding, by 40% and 60%, respectively, with no change in the hippocampus, amygdala, cingulum, motor cortex, parietal or occipital cortex, as compared to findings in the controls. Scatchard analysis showed that this increased binding reflects changes in the number of sites but not in the affinity. The effect of 0.1mM GppNHp on the binding to prefrontal cortex was observed in both controls and schizophrenic patients. The bindings were significantly greater in the schizophrenic patients than in controls, in the presence of 0.1mM GppNHp. Our findings suggest that there are GTP-sensitive 5-HT1A sites in the human brain and that selective increases in GTP-sensitive 5-HT1A sites in the prefrontal and temporal cortices of schizophrenics relate to the pathophysiology of schizophrenia.     

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.     

Discrimination of Multiple [3H]5-Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain

Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [³H]5-hydroxytryptamine ([³H]5-HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000-fold difference between its dissociation constants (2-12 nM versus 35,000 nM) for the high- and low-affinity sites, respectively. Inhibition of [³H]5-HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two-site model in each brain region. In the frontal cortex the high-affinity neuroleptic sites comprised about 60% of the total [^3/H]5-HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [³H]5-HT binding assayed in the absence or presence of 1 μM-spiperone (a concentration that completely blocks the high-affinity site while having minimal activity at the low-affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [³H]5-HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [³H]5-HT binding sites having a differential regional distribution in rat brain.     

[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.     

Identification of Multiple Binding Sites for [3H]5-Hydroxytryptamine in the Rat CNS

Recent studies indicate that there may be multiple subtypes of [3H]5-hydroxytryptamine ([3H]5-HT) binding sites. Mianserin and spiperone inhibited the specific binding of [3H]5-HT (2-3 nM) to rat brain cortical membranes with shallow displacement curves. The displacement data for spiperone were best described by the presence of three independent binding sites, for which spiperone had high, medium, and low affinities. The displacement data for mianserin were best fitted by two independent, high- and low-affinity sites. The inclusion of mianserin (250 nM) to inhibit [3H]5-HT binding to the mianserin-sensitive site selectively blocked one of the sites discriminated by spiperone. These results suggest the presence of three binding sites for [3H]5-HT, one blocked by low concentrations of spiperone (5-HT1A), one blocked by low concentrations of mianserin (5-HT1C), and one blocked only by high concentrations of both mianserin and spiperone (5-HT1B). Regional differences in the relative densities of the three sites were observed. The hippocampus was rich in 5-HT1A sites, whereas the striatum contained mainly 5-HT1B and 5-HT1C sites. Selective degeneration of 5-HT-containing nerve terminals induced by the neurotoxin 5,7-dihydroxytryptamine increased binding to all three sites in the cerebral cortex. Binding of [3H]5-HT to the three sites was differentially modulated by CaCl2 and guanylimidodiphosphate. The present data suggest the presence of three independent 5-HT1 binding sites having different affinities for mianserin and spiperone and having different regional distributions.     

The GTP-Insensitive Component of High-Affinity [3H]8-Hydroxy-2-(Di-n-Propylamino)tetralin Binding in the Rat Hippocampus Corresponds to an Oxidized State of the 5-Hydroxytryptamine1A Receptor

Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]-G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]-G protein complex.