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

ABSTRACT

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

Mechanisms of agonism and inverse agonism at serotonin 5-HT1A receptors

Mechanisms of agonist and inverse agonist action at the serotonin 5-HT1A receptor have been studied using the modulation of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding in membranes of Chinese hamster ovary (CHO) cells expressing the receptor (CHO-5-HTA1A cells). A range of agonists increased [35S]GTPgammaS binding with different potencies and to different maximal extents, whereas two compounds, methiothepin and spiperone, inhibited both agonist-stimulated and basal [5S]GTPgammaS binding, thus exhibiting inverse agonism. Potencies of agonists to stimulate [35S]GTPgammaS binding in membranes from CHO-5-HT1A cells were reduced by adding increasing concentrations of GDP to assays, whereas changes in sodium ion concentration did not affect agonist potency. The maximal effect of the agonists was increased by increasing sodium ion concentrations. The affinities of agonists in ligand binding assays were unaffected by changes in sodium ion concentration. Increasing GDP in the assays of the inverse agonists increased potency for spiperone to inhibit [35S]GTPgammaS binding and had no effect for methiothepin, in agreement with the sensitivity of these compounds to guanine nucleotides in ligand binding assays. Potencies for these inverse agonists were unaffected by changes in sodium ion concentration. These data were simulated using the extended ternary complex model. These simulations showed that the data obtained with agonists were consistent with these compounds achieving agonism by stabilising the ternary complex. For inverse agonists, the simulations showed that the mechanism for spiperone may be to stabilise forms of the receptor uncoupled from G proteins. Methiothepin, however, probably does not alter the equilibrium distribution of different receptor species; rather, this inverse agonist may stabilise an inactive form of the receptor that can still couple to G protein.     

A 5-HT7 Heteroreceptor-Mediated Inhibition of [3H]Serotonin Release in Raphe Nuclei Slices of the Rat: Evidence for a Serotonergic–Glutamatergic Interaction

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain, loaded with [3H]serotonin ([3H]5-HT), superfused, and the electrically induced efflux of radioactivity was determined. The nonselective 5-HT receptor agonist 5-carboxamido-tryptamine (5-CT; 0.001 to 1 microM) inhibited the electrically stimulated [3H]5-HT overflow from raphe nuclei slices (IC50 of 3.34 +/- 0.37 nM). This effect of 5-CT on [3H]5-HT overflow was antagonized by the 5-HT7 receptor antagonist SB-258719 (10 microM) and the 5-HT(1B/1D) antagonist SB-216641 (1 microM), the IC50 values for 5-CT in the presence of SB-258719 and SB-216641 were 94.23 +/- 4.84 and 47.81 +/- 4.66 nM. The apparent pA2 values for SB-258719 and SB-216641 against 5-CT were 6.43 and 7.12, respectively. The inhibitory effect of 5-CT on [3H]5-HT overflow was weakly antagonized by 10 microM of WAY-100635, a 5-HT1A receptor antagonist (IC50 6.65 +/- 0.56 nM, apparent pA2 4.99). The antagonist effect of SB-258719 (10 microM) on 5-CT-evoked [3H]5-HT overflow inhibition was also determined in the presence of 1 microM SB-216641 or 1 microM SB-216641 and 10 microM WAY-100635, and additive interactions were found between the antagonists of 5-HT7 and 5-HT1 receptor subtypes. Addition of the Na+ channel blocker tetrodotoxin (1 microM) in the presence of SB-216641 (1 microM) and WAY-100635 (10 microM) attenuated the inhibitory effect of 5-CT on KCl-induced [3H]5-HT overflow. These findings indicate that 5-CT inhibits [3H]5-HT overflow from raphe nuclei slices of the rat by stimulation of 5-HT7 and 5-HT(1B/1D receptors, whereas the role of 5-HT1A receptors in this inhibition is less pronounced. They also suggest that 5-HT7 receptors are probably not located on serotonergic neurons and thus may serve as heteroreceptors in regulation of 5-HT release in the raphe nuclei. 5-CT (0.1 microM) also inhibited [3H]glutamate release, and SB-258719 (10 microLM) suspended this effect. We therefore speculated that the axon terminals of the glutamatergic cortico-raphe neurons may possess 5-HT7 receptors that inhibit glutamate release, which consequently leads to decreased activity of serotonergic neurons. The postulated glutamatergic-serotonergic interaction in the raphe nuclei was further evidenced by the finding that N-methyl-D-aspartate and AMPA enhanced [3H]5-HT release.     

Characterization of a Novel Serotonin Receptor Subtype (5-HTls) in Rat CNS: Interaction with a GTP Binding Protein

Three pharmacologically distinct high-affinity [3H]serotonin ([3H]5-HT) binding sites were identified in spinal cord synaptosomes. [3H]5-HT competition studies using selective 5-HT1A receptor ligands indicated that approximately 25% of high-affinity synaptosomal [3H]5-HT binding was inhibited by 5-HT1A-selective compounds, an estimate consistent with [3H](+-)-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) saturation experiments in which 5-HT1A receptors were directly labeled. [3H]5-HT competition studies using high-affinity 5-HT1B compounds performed in the presence of 100 nM 8-OH-DPAT (to block 5-HT1A receptors) indicated that approximately 26% of all specific, high-affinity [3H]5-HT binding to spinal cord synaptosomes was to 5-HT1B receptors. [3H]5-HT competition studies performed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (to block 5-HT1A and 5-HT1B receptors, respectively) indicated that the remaining 49% of [3H]5-HT binding did not possess the pharmacologic profile previous reported for 5-HT1C, 5-HT1D, 5-HT1E, 5-HT2, or 5-HT3 receptors. This residual 49% of [3H]5-HT binding to spinal cord synaptosomes observed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (subsequently referred to as "5-HT1S") displayed high affinity and saturability (KD = 4.7 nM) in association/dissociation and saturation experiments. Addition of 300 microM GTP or the nonhydrolyzable form of GTP, 5'-guanylylimidodiphosphate, inhibited [3H]5-HT binding to 5-HT1S receptors in saturation experiments by 35 and 57%, respectively, whereas ATP was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of Serotonin1a and Serotonin1b Binding Sites from Bovine Brain

Serotonin1 (5-hydroxytryptamine1, 5-HT1) binding sites have been solubilized from bovine brain cortex using a mixture of 0.1% Nonidet P-40 and 0.3% digitonin in a low-salt buffer containing 0.1% ascorbic acid. The affinity of [3H]5-HT for the soluble cortical binding sites (2.1 nM) is identical to its affinity at membrane-bound binding sites (2.1 nM). [3H]8-Hydroxy-2-(di-n-propylamino)tetralin ([3H]DPAT), a selective 5-HT1a radioligand, also binds to soluble cortical binding sites with high affinity (1.8 nM) comparable with its affinity in the crude membranes (1.7 nM). A significant correlation exists in the rank order potency of serotonergic agents for [3H]5-HT binding and for [3H]DPAT binding to crude and soluble membranes. The density of [3H]DPAT binding sites relative to the [3H]5-HT sites in the solubilized cortical membranes (35%) corresponds well with the proportion of 5-HT1a sites in the crude membranes determined by spiperone displacement (33%), suggesting that both the 5-HT1a and 5-HT1b binding sites have been cosolubilized. [3H]5-HT binding in the soluble preparations was inhibited by GTP, suggesting that a receptor complex may have been solubilized. [3H]Spiperone-specific binding was not detectable in this preparation, suggesting that 5-HT2 sites were not cosolubilized.     

WAY-100635 antagonist-induced plasticity of 5-HT receptors: regulatory differences between a stable cell line and an in vivo native system

We present evidence that the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor antagonist, N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY-100635), can induce receptor internalization in a human (h)5-HT(1A) receptor Chinese hamster ovary (CHO-K1) cell system. Exposure of h5-HT(1A) CHO cells to WAY-100635 decreased the cell-surface h5-HT(1A) receptor density in a way that was both time (24-72 h) and concentration (1-100 nm) dependent.[(3)H]WAY-100635 and [(3)H]8-hydroxy-dipropylaminotetralin ([(3)H]8-OH-DPAT) saturation analyses demonstrated a significant reduction (50-60%) in total h5-HT(1A) receptor number in the WAY-100635-treated (100 nm; 72 h) compared with control cells. In WAY-100635-treated cells, the 8-OH-DPAT-mediated inhibition of forskolin (FSK)-stimulated cAMP accumulation was right-shifted and the maximal inhibitory response of 8-OH-DPAT was impaired compared with control cells. Similar results were obtained for 8-OH-DPAT-mediated Ca(2+) mobilization after WAY-100635 treatment. h5-HT(1A) receptors labeled with [(3)H]WAY-100635, as well as [(3)H]4-(2'-Methoxy)-phenyl-1-[2'-(N-2'-pyridinyl)-p-fluorobenzamido]ethyl-piperazine (MPPF), exhibited a time-dependent rate of cellular internalization that was blocked by endocytotic suppressors and was pertussis-toxin insensitive. In contrast, quantitative autoradiographic studies demonstrated that chronic treatment of rats with WAY-100635 for two weeks produced a region-specific increase in the 5-HT(1A) receptor density. In conclusion, prolonged exposure of an h5-HT(1A) cell-based system to the 5-HT(1A) antagonist, WAY-100635, induced a paradoxical internalization of cell surface receptor resulting in depressed functional activity. This suggests that an antagonist can influence 5-HT(1A) receptor recycling in vitro differently to in vivo regulatory conditions.     

Decreased agonist, but not antagonist, binding to the naturally occurring Thr92Lys variant of the h5-HT7(a) receptor

In the present study on transfected human embryonic kidney (HEK)293 cells, we aimed at establishing whether expression of the naturally occurring Thr92Lys variation of the Gs-coupled h5-HT7(a) receptor leads to changes of ligand binding properties, of agonist-evoked cAMP formation and/or of antagonist-mediated blockade of the latter. Binding of [3H]5-carboxamidotryptamine ([3H]5-CT) to membranes and stimulated [3H]cAMP accumulation in whole cells were determined. Saturation binding experiments in membranes of transiently transfected cells expressing either the wild-type or the variant receptor revealed a single binding site in both cases and no difference in Bmax between both receptor isoforms. In competition binding experiments in membranes of stably transfected cells, the Thr92Lys variant exhibited a 2.8-11 times lower binding affinity of the ligands 5-hydroxytryptamine (5-HT), 5-CT, 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4yl)-1H-indole (RU24969), (+/-)-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT) and sumatriptan compared to the wild-type receptor. However, the variant did not differ from the wild-type with respect to the binding properties of the antagonists (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)-pyrrolodine-1-sulfonyl)phenol hydrochloride (SB-269970), risperidone, mesulergine and clozapine. In agreement with the decreased binding affinity of 5-HT, 5-CT, RU24969 and 8-OH-DPAT for the variant receptor, these agonists were less potent in stimulating [3H]cAMP accumulation in cells stably expressing the Thr92Lys h5-HT7(a) receptor. Sumatriptan did not stimulate cAMP accumulation in spite of its affinity for both receptor isoforms pointing to a putative weak antagonistic property of this drug at the h5-HT7 receptor. SB-269970 and clozapine were equipotent at both the variant and the wild-type receptor in producing a rightward shift of the 5-HT concentration-response curve for its stimulant effect on [3H]cAMP accumulation. In view of, e.g., the purported involvement of the 5-HT7 receptor in the regulation of circadian rhythm, it may be concluded that the decrease in affinity of 5-HT and other 5-HT receptor agonists at the (Thr92Lys) h5-HT7 receptor may be associated with changes of sleep physiology and of actions of new 5-HT7 receptor agonists designed to treat circadian dysregulation.     

Effects of 5-HT1A and 5-HT2A receptor stimulation on temporal differentiation performance in the fixed-interval peak procedure

We examined the effects of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and 5-HT(2A/2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on performance on the fixed-interval peak procedure, and the sensitivity of these effects to 5-HT1A and 5-HT2A receptor antagonists (N-[2-(4-[2-methoxyphenyl]-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide [WAY-100635] and ketanserin). Rats were trained to press a lever for food reinforcement in 50 min sessions consisting of 32 trials in which the lever was continuously available, separated by 10 s inter-trial intervals. In 16 trials, reinforcement was delivered following the first response after 30 s had elapsed since trial onset (fixed-interval 30 s). In 16 randomly interposed (peak/probe) trials, reinforcement was omitted, and the lever remained in the operant chamber for 120 s. Response rate in probe trials was plotted against time from trial onset. Time to peak response rate (t(peak)) and the Weber fraction were derived from modified Gaussian curves fitted to each rat's data. 8-OH-DPAT (0.05 mg kg(-1)) reduced t(peak) and increased the Weber fraction; the effect on t(peak) was antagonized by WAY-100635 (0.1 mg kg(-1)). DOI (0.25 mg kg(-1)) also reduced t(peak) and increased the Weber fraction; the reduction of t(peak) was antagonized by ketanserin (2 mg kg(-1)). Stimulation of 5-HT1A and 5-HT2A receptors alters temporal differentiation in qualitatively similar ways.     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

Hippocampal 8-[3H]Hydroxy-2-(Di-n-Propylamino)Tetralin Binding Site Densities, Serotonin Receptor (5-HT1A) Messenger Ribonucleic Acid Abundance, and Serotonin Levels Parallel the Activity of the Hypothalamopituitary-Adrenal Axis in Rat

We have previously demonstrated that susceptibility of the Lewis rat to inflammatory disease, compared with the relatively resistant Fischer F344/N rat, is related to a hyporesponsive hypothalamopituitary-adrenal axis to inflammatory and other stress mediators. Because serotonin (5-HT) and the 5-HT1A receptor are important stimulators of this axis, we have investigated the levels of 8-[3H]-hydroxy-2,3-(di-n-propylamino)tetralin binding sites, 5-HT1A mRNA, 5-HT, and 5-hydroxyindoleacetic acid in various brain regions of Lewis, outbred Harlan Sprague Dawley, and Fischer F344/N rats. Lewis rats expressed significantly fewer hippocampal and frontal cortical 8-[3H]-hydroxy-2,3-(di-n-propylamino)tetralin binding sites and less 5-HT1A mRNA than Harlan Sprague Dawley and Fischer F344/N rats. Adrenalectomy increased the number of 8-[3H]hydroxy-2,3-(di-n-propylamino)tetralin binding sites and 5-HT1A mRNA expression in the hippocampus of all three strains. Levels of hippocampal 5-HT in Fischer F344/N rats were significantly greater than levels detected in the same regions from Lewis and Harlan Sprague Dawley rats. Hypothalamic 5-HT and 5-hydroxyindoleacetic acid levels in Harlan Sprague Dawley rats were higher than the same area from the other two strains. Adrenalectomy increased the levels of 5-hydroxyindoleacetic acid in the hypothalamus of all three strains. We conclude that hippocampal 5-HT1A receptor densities and 5-HT levels in the rat parallel the activity and responsiveness of the hypothalamopituitary-adrenal axis.     

[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.     

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.     

Further Characterization of 5-HT1A Receptors in the Goldfish Retina: Role of Cyclic AMP in the Regulation of the In Vitro Outgrowth of Retinal Explants

The presence of serotonin 5-HT_1A receptors and their physiological role were further characterized in the goldfish retina. The effects of the 5-HT_6/7 receptor antagonists pimozide, fluphenazine and amoxapine, the 5-HT_1A receptor antagonist WAY-100,135, and the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, on the 5-HT_1A receptor agonist [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes, were evaluated. In addition, the effects of serotonin, 8-hydroxy-2-(di-n-propylamino)tetralin, WAY-100,135, the adenylate cyclase inhibitors SQ22536 and MDL12330A, and the cyclic AMP analog 8-bromoadenosine-3:5 cyclic monophosphate were also studied on neuritic outgrowth from retinal explants. WAY-100,135 but not 5-HT_6/7receptor antagonists inhibited [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline decreased [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding sites up to 70%, while receptor turnover was similar to that reported in other tissues. Serotonin and 8-hydroxy-2-(di-n-propylamino)tetralin stimulated cyclic AMP production, both ex vivo and in vitro, and these increases were related to inhibition of neuritic outgrowth. The inhibitory effect was reduced by SQ22536 and by WAY-100,135, and was mimicked by 8-bromoadenosine-3:5cyclic monophosphate. This study supports previous findings about the role of serotonin as a regulator of axonal outgrowth during in vitro regeneration of the goldfish retina and demonstrates that this effect is mediated, at least in part, by 5-HT_1A receptors through a mechanism which involves an increase of cyclic AMP levels.     

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.     

Identification and role of serotonin 5-HT1A and 5-HT1B receptors in primary cultures of rat embryonic rostral raphe nucleus neurons

Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10(-8) M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), and the 5-HT(1D/1B) agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionam ide dihydrochloride quaternary-hydrate (WAY 100135) (10(7) M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-p iperazinyl]-1Hindole-2-carboxylate (SDZ 216-525) (10(-7)M), and that of CP-93,129 was blocked by methiothepin (10(-7) M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10(-6) M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.     

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.     

Modulation of 5-Hydroxytryptamine1A Receptor Density by Nonhydrolyzable GTP Analogues

Co-incubation of rat cortical membranes with 10(-4) M GTP results in a competitive inhibition of 5-hydroxytryptamine1A (5-HT1A) receptor binding sites labeled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT). Preincubation of cortical membranes with 10(-4) M GTP does not significantly change either KD or Bmax values, indicating that the effect of GTP is reversible. By contrast, GTP gamma S and 5'-guanylylimidodiphosphate (GppNHp) are nonhydrolyzable analogues of GTP which lengthen the time course of guanine nucleotide activation of guanine nucleotide binding proteins (G proteins) and thereby alter G protein-receptor interactions. These nonhydrolyzable GTP analogues were used to characterize the effects of persistent alterations in G proteins on [3H]8-OH-DPAT binding to 5-HT1A receptors. Co-incubation of rat cortical membranes with either 10(-4) M GTP gamma S or GppNHp results in a decrease in both the affinity and apparent density of 5-HT1A binding sites. Co-incubation with the nonhydrolyzable nucleotides reduces the affinity of [3H]8-OH-DPAT binding by 65-70% and lowers the density of the binding site by 53-61%. Similarly, preincubation of membranes with a 10(-4) M concentration of either GTP gamma S or GppNHp significantly increases the KD value and reduces the Bmax value of [3H]8-OH-DPAT binding. These results indicate that GTP gamma S and GppNHp induce persistent changes in 5-HT1A receptor-G protein interactions that are reflected as a decrease in the density of binding sites labeled by [3H]8-OH-DPAT.     

Characterization of the 5-Hydroxytryptamine Receptor Modulating the Release of 5-[3H]Hydroxytryptamine in Slices of the Human Neocortex

In the rat brain, the presynaptic 5-hydroxytryptamine (5-HT) autoreceptors located on 5-HT terminals correspond to the 5-HT1B subtype. The presence of a 5-HT receptor probably located on 5-HT nerve endings and modulating transmitter release in the human neocortex has been reported, but its detailed pharmacological characterization is not yet available. On the other hand, receptor binding and autoradiographic results indicate that the 5-HT1B receptor subtype is not present in the human brain. We, therefore, studied the modulation of the electrically evoked release of [3H]5-HT by various 5-HT receptor agonists and antagonists in preloaded slices of human neocortex obtained from 18 patients undergoing neurosurgery. The nonselective 5-HT1A/1B/1D receptor agonist 5-carboxamidotryptamine produced a potent inhibition (70% at 0.03 microM) of the electrically evoked release of [3H]5-HT which was blocked by 5-HT receptor antagonists with the following relative order of potency: methiothepin greater than metergoline = methysergide greater than propranolol. The selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin at 0.1 microM did not modify the electrically evoked release of [3H]5-HT. The 5-HT1A/1B receptor agonist RU 24969 was 10 times more potent at inhibiting [3H]5-HT overflow in the rat frontal cortex than in the human neocortex. The potent 5-HT1B receptor antagonist cyanopinodolol did not modify the 5-carboxamidotryptamine-induced inhibition of the electrically evoked release of [3H]5-HT in slices of the human neocortex, but produced by itself a small inhibition of [3H]5-HT overflow.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-Hydroxytryptamine3 Receptors Sited on Cholinergic Axon Terminals of Human Cerebral Cortex Mediate Inhibition of Acetylcholine Release

Synaptosomes prepared from freshly obtained human cerebral cortex and labeled with [3H]choline have been used to investigate the modulation of [3H]acetylcholine ([3H]ACh) release by 5-hydroxytryptamine (5-HT). The Ca(2+)-dependent release of [3H]-ACh occurring when synaptosomes were exposed in superfusion to 15 mM KCl was inhibited by 5-HT (0.01-1 microM) in a concentration-dependent manner. The effect of 5-HT was mimicked by 1-phenylbiguanide, a 5-HT3 receptor agonist, but not by 8-hydroxy-2-(di-n-propylamino)tetralin, a 5-HT1A receptor agonist. The 5-HT3 receptor antagonists tropisetron and ondansetron blocked the effect of 5-HT, whereas spiperone and ketanserin were ineffective. It is suggested that cholinergic axon terminals in the human cerebral cortex possess 5-HT receptors that mediate inhibition of ACh release and appear to belong to the 5-HT3 type.     

Identification of the 5-HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8-Methoxy-2-[N-n-Propyl, N-3-(2-Nitro-4-Azidophenyl)Aminopropyl]Aminotetralin

The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3'-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3'-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI. As expected of a possible correspondence between PI and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline- and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of PI were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).