[3H]Rauwolscine: an antagonist radioligand for the cloned human 5-hydroxytryptamine2B (5-HT2B) receptor

In previous reports, [³H]5-HT has been used to characterize the pharmacology of the rat and human 5-HT_2B receptors. 5-HT, the native agonist for the 5-HT_2B receptor, has a limitation in its usefulness as a radioligand since it is difficult to study the agonist low-affinity state of a G protein-coupled receptor using an agonist radioligand. When using [³H]5-HT as a radioligand, rauwolscine was determined to have relatively high affinity for the human receptor (K_i human = 14.3 ± 1.2 nM, compared to K_i rat = 35.8 ± 3.8 nM). Since no known high affinity antagonist was available as a radioligand, these studies were performed to characterize [³H]rauwolscine as a radioligand for the cloned human 5-HT_2B receptor expressed in AV12 cells. When [³H]rauwolscine was initially tested for its usefulness as a radioligand, complex competition curves were obtained. After testing several α₂-adrenergic ligands, it was determined that there was a component of [³H]rauwolscine binding in the AV12 cell that was due to the presence of an endogenous α₂-adrenergic receptor. The α₂-adrenergic ligand efaroxan was found to block [³H]rauwolscine binding to the α₂-adrenergic receptor without significantly affecting binding to the 5-HT_2B receptor and was therefore included in all subsequent studies. In saturation studies at 37° C, [³H]rauwolscine labeled a single population of binding sites, K_d = 3.75 ± 0.23 nM. In simultaneous experiments using identical tissue samples, [³H]rauwolscine labeled 783 ± 10 fmol of 5-HT_2B receptors/mg of protein, as compared to 733 ± 14 fmol of 5-HT_2B receptors/mg of protein for [³H]5-HT binding. At 0° C, where the conditions for [³H]5-HT binding should label mostly the agonist high affinity state of the human 5-HT_2B receptor, [³H]rauwolscine (B_max = 951 ± 136 fmol/ mg), again labeled significantly more receptors than [³H]5-HT (B_max = 615 ± 34 fmol/mg). The affinity of [³H]rauwolscine for the human 5-HT_2B receptor at 0° C did not change, K_d = 4.93 ± 1.27 nM, while that for [³H]5-HT increased greatly (K_d at 37° C = 7.76 ± 1.06 nM; K_d at 0° C = 0.0735 ± 0.0081 nM). When using [³H]rauwolscine as the radioligand, competition curves for antagonist structures modeled to a single binding site, while agonist competition typically resulted in curves that best fit a two site binding model. In addition, many of the compounds with antagonist structures displayed higher affinity for the 5-HT_2B receptor when [³H]rauwolscine was the radioligand. Typically, ∼ 85% of [³H]rauwolscine binding was specific binding. These studies display the usefulness of [³H]rauwolscine as an antagonist radioligand for the cloned human 5-HT_2B receptor. This should provide a good tool for the study of both the agonist high- and low-affinity states of the human cloned 5-HT_2B receptor. Received: 26 June 1997 / Accepted: 30 August 1997     

3H-DOB (4-bromo-2,5-dimethoxyphenylisopropylamine) labels a guanyl nucleotide-sensitive state of cortical 5-HT2 receptors

3H-(+/-)-4-Bromo-2,5-dimethoxyphenylisopropylamine (3H-DOB), a putative agonist radioligand, was synthesized and used to label 5-HT2 receptors in a particulate fraction prepared from rat frontal cortex tissue homogenates. The specific binding (defined by the difference in 3H-DOB binding in the presence and absence of 10(-6) M cinanserin, a potent and specific 5-HT2 antagonist) displayed high affinity (KD = 4.1 X 10(-10) M) and saturability with a Bmax of 17.9 fmol/mg of protein. The distribution of specific 3H-DOB binding in nine brain regions correlated closely with the distribution of 3H-ketanserin (an antagonist radioligand)-labeled 5-HT2 receptors. Competition studies in frontal cortex homogenates using a variety of compounds revealed a distinct 5-HT2 receptor pharmacology. A series of 5-HT2 antagonists exhibited high affinities in competition studies for specific 3H-DOB binding. The absolute potencies of these antagonists as well as their order of potencies closely correlated with their potencies in competing for 3H-ketanserin-labeled brain 5-HT2 receptors. A series of 5-HT2 agonists also exhibited high affinities in competition studies for specific 3H-DOB binding. Although the order of potencies of these agonists was similar to their order in competing for 3H-ketanserin-labeled brain 5-HT2 receptors, the agonists displayed 10-100-fold higher affinities for the 3H-DOB-labeled sites than for the 3H-ketanserin-labeled sites. The level of specific 3H-DOB binding in the frontal cortex homogenates was approximately 5% of the levels of 3H-ketanserin-labeled 5-HT2 receptors (358 fmol/mg of protein). Taken together, these results indicate that 3H-DOB labels a subset of brain 5-HT2 receptors that has high affinity for agonists as well as antagonists); 3H-ketanserin appears to label both subsets of brain 5-HT2 receptors. Antagonists apparently do not discriminate between these two subsets of 5-HT2 receptors. 3H-DOB specific binding to 5-HT2 receptors was potently inhibited by guanosine 5'-(beta, gamma-imido)triphosphate and guanosine 5'-O-(3-thio)triphosphate (nonhydrolyzable derivatives of GTP) with IC50 values of 42 and 21 nM, respectively, whereas adenosine 5'-(beta, gamma-imido)triphosphate and adenosine 5'-O-(3-thio)triphosphate (nonhydrolyzable derivatives of ATP) had no effect. In summary, 3H-DOB specific binding displays the pharmacological characteristics of a 5-HT2 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

4-[125I]iodo-(2,5-dimethoxy)phenylisopropylamine and [3H]ketanserin labeling of 5-hydroxytryptamine2 (5HT2) receptors in mammalian cells transfected with a rat 5HT2 cDNA: evidence for multiple states and not multiple 5HT2 receptor subtypes

Evidence has accumulated indicating that the radioactive hallucinogens 4-bromo-[3H](2,5-dimethoxy)phenylisopropylamine ([3H]DOB) and 4-[125I]iodo-(2,5-dimethoxy)phenylisopropylamine ([125I]DOI) label an agonist high affinity state of the 5-hydroxytryptamine2 (5HT2) receptor and [3H]ketanserin labels both agonist high and low affinity states. Recently, an alternative hypothesis has been put forward proposing that the radioactive hallucinogens are labeling a 5HT2 receptor subtype distinct from the receptor labeled by [3H]ketanserin. In order to provide definitive evidence as to which of these hypotheses is correct, the rat 5HT2 receptor gene was transfected into NIH-3T3 (mammalian fibroblast) cells and COS (green monkey kidney) cells. Neither nontransfected cell type expresses 5HT2 receptors; the transfected cells expressed high affinity binding sites for both [125I] DOI (KD = 0.8 nM and Bmax = 363 fmol/mg in NIH-3T3 cells; KD = 0.2 nM and Bmax = 26 fmol/mg in COS cells) and [3H]ketanserin (KD = 0.4 nM and Bmax = 5034 fmol/mg in NIH-3T3 cells; KD = 1.0 nM and Bmax = 432 fmol/mg in COS cells). The affinities of agonists and antagonists for the [125I]DOI-labeled receptor were significantly higher than for the [3H]ketanserin-labeled receptor. The affinities of agonists and antagonists for these binding sites were essentially identical to their affinities for the sites radiolabeled by these radioligands in mammalian brain homogenates. The [125I]DOI binding was guanyl nucleotide sensitive, indicating a coupling to a GTP-binding protein. These data indicate that the 5HT2 receptor gene product contains both the guanyl nucleotide-sensitive [125I]DOI binding site and the [3H]ketanserin binding site. Therefore, these data indicate that the 5HT2 receptor gene product can produce a high affinity binding site for the phenylisopropylamine hallucinogen agonists as well as for the 5HT2 receptor antagonists. These results strongly support the two-state hypothesis for the 5HT2 receptor and do not support the multiple 5HT2 receptor subtype hypothesis.     

Selectivity of serotonergic drugs for multiple brain serotonin receptors. Role of [3H]-4-bromo-2,5-dimethoxyphenylisopropylamine ([3H]DOB), a 5-HT2 agonist radioligand

The affinities of putative serotonin receptor agonists and antagonists for 5-HT1A, 5-HT1B, 5-HT1C, and 5-HT2 receptors were assayed using radioligand binding assays. The 5-HT1 sites were labeled with the agonist radioligands [3H]-8-hydroxy-2-(di-n-propylamino)-tetralin [3H]-8-OH-DPAT, [3H]-5-HT, and [3H]mesulergine. The 5-HT2 receptor was labeled with the antagonist radioligand [3H]ketanserin or the agonist radioligand [3H]-4-bromo-2,5-dimethoxyphenylisopropylamine ([3H]DOB). The apparent 5-HT1 receptor selectivity of agonist compounds was found to be 50- to 100-fold higher when the 5-HT2 receptor affinity was determined using the antagonist radioligand [3H]ketanserin than when the agonist radioligand [3H]DOB was used. Quipazine, a putative specific 5-HT2 agonist, appeared to be only 3-fold more potent at 5-HT2 than at 5-HT1A receptors when [3H]ketanserin was used as the 5-HT2 radioligand. When [3H]DOB was used as the 5-HT2 radioligand, quipazine was determined to be 100-fold more potent at 5-HT2 receptors than at 5-HT1A receptors. 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a putative specific 5-HT1B receptor agonist was apparently 10-fold more potent at 5-HT1B receptors than at 5-HT2 receptors when [3H]ketanserin was used as the 5-HT2 radioligand. When [3H]DOB was used as the 5-HT2 radioligand, TFMPP was found to be equipotent at 5-HT1B and 5-HT2 receptors. Using the 5-HT2 antagonist radioligand [3H]ketanserin, a similar pattern of underestimating 5-HT2 receptor selectivity and/or overestimating 5-HT1A or 5-HT1B receptor selectivity was observed for a series of serotonin receptor agonists. Antagonist receptor selectivity was not affected significantly by the nature of the 5-HT2 receptor assay used. These data indicate that, by using an antagonist radioligand to label 5-HT2 receptors and agonist radioligands to label 5-HT1 receptors, the 5-HT1 receptor selectivity may be overestimated. This may be an especially severe problem in serotonin drug development as drugs that interact potently with 5-HT2 receptors have been reported to be psychoactive and/or hallucinogenic.     

(R)-(-)-[77Br]4-bromo-2,5-dimethoxyamphetamine labels a novel 5-hydroxytryptamine binding site in brain membranes

(R)-(-)-[77Br]4-Bromo-2,5-dimethoxyamphetamine [(R)-(-)-[77Br] DOB] was synthesized to a high specific activity (1875 +/- 50 Ci/mmol) and used to label membrane-associated recognition sites in rat brain. (R)-(-)-[77Br]DOB displayed high affinity (KD = 0.60 +/- 0.08 nM) for a relatively low density of binding sites (Bmax = 1.2 +/- 0.08 pmol/g of tissue) in rat cortical membranes as compared with [3H]ketanserin (KD = 0.65 +/- 0.1 nM; Bmax = 6.2 +/- 0.6 pmol/g of tissue). Guanine, but not adenine, nucleotides were found to inhibit specific (R)-(-)-[77Br]DOB binding. GTP (10(-4) M) did not eliminate specific (R)-(-)-[77Br]DOB binding but caused a competitive inhibition of the radioligand. Drug competition studies of 5-hydroxytryptamine (5-HT) and related agents indicate that both putative agonists and antagonists display nanomolar potency for these sites. A significant correlation (p less than 0.01) exists between drug potencies for (R)-(-)-[77Br]DOB-labeled sites and both 5-HT2 (r = 0.64) and 5-HT1C (r = 0.68) binding sites. However, the sites do not appear to be identical. Moreover, a significant correlation exists between drug potencies for (R)-(-)-[77Br]DOB-labeled sites and human hallucinogenic drug potencies (r = 0.89; p less than 0.01). We conclude that (R)-(-)-[77Br]DOB labels a unique 5-HT recognition site in rat brain that does not coincide with previously described 5-HT binding site subtypes. The (R)-(-)-[77Br]DOB site does not appear to be a high affinity "state" of the 5-HT2 receptor but may label a subset of heterogeneous 5-HT2 recognition sites.     

3H]N-propylapomorphine and [3H]spiperone binding in brain indicate two states of the D2-dopamine receptor

[3H]N-propylapomorphine ([3H]NPA) a dopaminergic catecholamine derivative, labels a sub-set of D2-dopamine receptors in bovine caudate particulate preparation. [3H]Spiperone, a dopamine receptor antagonist, labels twice as many sites as [3H]NPA. Dopaminergic ergots and potent neuroleptics compete for both radioactive ligands with similar high affinities. Catecholamines and catecholamine derivatives compete more potently for [3H]NPA binding than for [3H]spiperone binding. Guanyl nucleotides reduce both [3H]NPA binding and the high affinity phase of catecholamine and catecholamine derivative competition for [3H]spiperone binding. These results are similar to binding results reported in studies of two-state receptors linked to adenylate cyclase such as the beta-adrenergic receptors. These observations indicate that the D2-dopamine receptor in the brain may exist in two states and may be inversely coupled to brain adenylate cyclase activity.     

Multiple states of the 5-hydroxytryptamine1 receptor as indicated by the effects of GTP on [3H]5-hydroxytryptamine binding in rat frontal cortex

In several monoamine receptor systems, agonist but not antagonist binding is found to be associated with both a high- and a low-affinity state of the receptor. Guanine nucleotides, like GTP, can eliminate the high-affinity receptor state such that only the low-affinity state of the receptor is present. Since [3H]5-hydroxytryptamine ([3H]5-HT) is an agonist, its binding may also be associated with multiple states of a receptor. To evaluate this, the binding of [3H]5-HT to rat frontal cortical membranes was measured in the absence and presence of GTP. Three different types of binding experiments were performed: (a) saturation experiments, (b) dissociation experiments, and (c) competition experiments with 5-hydroxytryptamine (5-HT) agonists. When all three types of binding experiments were carried out in the absence of GTP, results from either graphical representations or computer analysis of the data indicated that a two-component model of binding described the data better than a single-component model. By contrast, in the presence of GTP, a one-component model adequately described the data obtained from either saturation or dissociation experiments. Competition of [3H]5-HT (15 nM) binding by three 5-HT agonists (5-methoxytryptamine, 5-HT, and d-lysergic acid diethylamide) was adequately described by a single-component system in the presence of GTP as well, even though all three agonists produced biphasic inhibition curves in the absence of the guanine nucleotide. These experimental results are consistent with the idea that the binding of [3H]5-HT in the absence of GTP is associated with multiple receptor states. Since the presence of multiple states can confound the interpretation of inhibition curves of [3H]5-HT binding caused by agonists, it is important to eliminate the high-affinity state of the receptor by including GTP in the binding assay.     

Pharmacological characterization of [(3)H]-prostaglandin E(2) binding to the cloned human EP(4) prostanoid receptor

Prostaglandin (PG) E(2) (PGE(2)) is a potent prostanoid derived from arachidonic which can interact with EP(1), EP(2), EP(3) and EP(4) prostanoid receptor subtypes. Recombinant human EP(4) receptors expressed in human embryonic kidney (HEK-293) cells were evaluated for their binding characteristics using [(3)H]-PGE(2) and a broad panel of natural and synthetic prostanoids in order to define their pharmacological properties. [(3)H]-PGE(2) binding was optimal in 2-[N-Morpholino]ethanesulphonic acid (MES) buffer (pH 6.0) yielding 98+/-0.7% specific binding. The receptor displayed high affinity (K(d)=0.72+/-0.12 nM; n=3) for [(3)H]-PGE(2) and interacted with a saturable number of binding sites (B(max)=6.21+/-0.84 pmol mg(-1) protein). In competition studies, PGE(2) (K(i)=0.75+/-0.03 nM; n=12) and PGE(1) (K(i)=1.45+/-0.24 nM; n=3) displayed high affinities, as did two derivatives of PGE(1), namely 11-deoxy-PGE(1) (K(i)=1.36+/-0.34 nM) and 13,14-dihydro-PGE(1) (K(i)=3.07+/-0.29 nM). Interestingly, synthetic DP receptor-specific agonists such as BW245C (K(i)=64.7+/-1.0 nM; n=3) and ZK118182 (K(i)=425+/-42 nM; n=4), and the purported EP(3) receptor-specific ligand enprostil (K(i)=43.1+/-4.4 nM), also displayed high affinity for the EP(4) receptor. Two known EP(4) receptor antagonists were weak inhibitors of [(3)H]-PGE(2) binding akin to their known functional potencies, thus: AH23848 (K(i)=2690+/-232 nM); AH22921 (K(i)=31,800+/-4090 nM). These studies have provided a detailed pharmacological characterization of the recombinant human EP(4) receptor expressed in HEK-293 cells.     

2,2-二甲基-4-(2,5-二氟苯基)-5-[(4-氨基磺酰基)苯基]-3(2H)-呋喃酮的合成

用2,5-二氟苯乙酸与茴香硫醚经傅-克反应、与3-溴-3-甲基-2-氧代丁腈成环及硝酸氧化制得2,2-二甲基-4-(2,5-二氟苯基)-5-[(4-甲磺酰基)苯基]-3(2H)-呋喃酮(7),7与乙酐反应后再经过硫酸氢钾复合盐氧化、氢氧化钠水解得4-[2,2-二甲基-3-氧代-4-(2,5-二氟苯基)-3(2H)-呋喃-5-基]苯磺酸钠(9),最后依次与磺酰氯和氨水反应制得2,2-二甲基-4-(2,5-二氟苯基)-5-[(4-氨基磺酰基)苯基]-3(2H)-呋喃酮,总收率约46％.     

High affinity binding of [3H]6-nitroquipazine to cortical membranes in the rat: inhibition by 5-hydroxytryptamine and 5-hydroxytryptamine uptake inhibitors

[3H]6-Nitroquipazine is a new, suitable radioligand for studying the uptake system for 5-hydroxytryptamine (5-HT; serotonin). In the present study, inhibition by drugs of the binding of [3H]6-nitroquipazine to uptake sites for 5-HT in the cerebral cortex of the rat was investigated. The inhibition of 5-HT and several inhibitors of the uptake of 5-HT (paroxetine, clomipramine, citalopram, Z-norzimelidine, fluoxetine, imipramine, desipramine and 5-methoxytryptoline) against the binding of [3H]6-nitroquipazine to membranes from the cortex of the rat were the same and competition curves indicated a single population of binding sites. The addition of 5-HT and the tricyclic inhibitors of the uptake of 5-HT, imipramine, clomipramine and desipramine, all produced changes in the apparent dissociation constant (Kd), without changes in the number of binding sites (Bmax). Also, the non-tricyclic inhibitors of the uptake of 5-HT, paroxetine, citalopram, fluoxetine and Z-norzimelidine, and 5-methoxytryptoline, all produced changes in Kd values without changes in the Bmax. These results suggest that all the drugs used in this experiment exhibited competitive interactions with the binding of [3H]6-nitroquipazine to uptake sites for 5-HT in the brain of the rat. These drugs may bind to common binding sites, which are likely to represent the substrate recognition sites for the uptake of 5-HT.     

Antihypertensive effects of chronic 5-hydroxytryptamine (5-HT2) receptor blockade with ketanserin in the spontaneously hypertensive rat

Summary The effects of chronic oral treatment with the 5-hydroxytryptamine (serotonin) receptor blocking agent ketanserin (17 mg/100 g dry food) on blood pressure, heart weight, peripheral vascular reactivity, baroreceptor sensitivity, central cardiovascular reactivity and central catecholamine turnover were investigated in the spontaneously hypertensive rat. Blood pressure measurements were performed in conscious rats 24 h after insertion of catheters. After 6 weeks treatment basal blood pressure was reduced (16%) compared to control rats (given identical food, except for ketanserin). Both heart weight and body weight were reduced (both to 93% of control values) leaving heart weight/body weight ratio unchanged. Pressor responses to phenylephrine and depressor responses to isoprenaline (after pretreatment with reserpine and atropin) were not different while the blood pressure increase to 5-hydroxytryptamine was inhibited, indicating that after 6 weeks treatment the blood pressure reduction is not directly related to -adrenoceptor blockade. Cardiovascular response to stress (jet air), baroreceptor sensitivity (bradycardia to phenylephrine) and central catecholamine synthesis rates (accumulation of 5-hydroxytryptophan and dihydroxyphenylalanine after synthesis inhibition) were unchanged supporting earlier evidence that central mechanisms probably do not contribute to the hypotensive effects of ketanserin.     

Beta-oxygenated analogues of the 5-HT2A serotonin receptor agonist 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane

Activation of 5-HT(2A) serotonin receptors represents a novel approach to lowering intraocular pressure. Because 5-HT(2A) serotonin receptor agonists might also produce undesirable central effects should sufficient quantities enter the brain, attempts were made to identify 5-HT(2) serotonin receptor agonists with reduced propensity to penetrate the blood-brain barrier. 1-(4-Bromo-2,5-dimethoxyphenyl)-2-aminopropan-1-ol (6), an analogue of the 5-HT(2) serotonin receptor agonist 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB; 1a) bearing a benzylic hydroxyl group, was identified as a candidate structure. Of the four optical isomers of 6, the 1R,2R-isomer (6d; K(i) = 0.5 nM) was found to bind at 5-HT(2A) receptors with an affinity similar to that of R(-)DOB (K(i) = 0.2 nM). Like R(-)DOB, 6d behaved as a partial agonist (efficacy ca. 50%) in a 5-HT(2)-mediated calcium mobilization assay. However, in an in vivo test of central action (i.e., stimulus generalization with rats as subjects), 6d was >15 times less potent than R(-)DOB. O-Methylation of 6d (i.e., 7d; 5-HT(2A) K(i) = 0.3 nM) resulted in an agent that behaved as a full (93% efficacy) agonist. Intraocular administration of 300 microg of 6d and 7d to ocular hypertensive monkeys was shown to reduce intraocular pressure by 20-27%. Given the route of administration (i.e., topical), and concentrations necessary to reduce intraocular pressure, compounds such as 6d should demonstrate minimal central effects at potentially useful therapeutic doses and offer useful leads for further development.     

Synthesis and benzodiazepine receptor affinity of derivatives of the new tricyclic heteroaromatic system pyrido[3',2':5,6]thiopyrano[4,3-c]pyridazin-3(2H,5H)-one

Derivatives 7-13 of a new tricyclic heteroaromatic system, pyrido[3',2':5,6]thiopyrano[4,3-c]pyridazin-3(2H,5H)-one, were prepared as potential ligands at the benzodiazepine receptor, in view of their structural analogy with potent ligands such as the pyrazoloquinolines of the CGS series II, and especially with the benzothiopyrano[4,3-c]pyridazinones VI. They were obtained starting from the versatile ketones 2,3-dihydrothiopyrano[2,3-b]pyridin-4(4H)-one 1 and the corresponding 7-methyl derivative 2, via condensation with glyoxylic acid, and reaction of the intermediate acid mixtures with hydrazine or substituted phenylhydrazines. When evaluated for their binding affinity at the benzo diazepine receptor in bovine cortical membranes, the target compounds 8-13 displayed an affinity in the micromolar/submicromolar order. A hypothesis is presented to rationalize these results.     

Disposition of 4-bromo-2,5-dimethoxyphenethylamine (2C-B) and its metabolite 4-bromo-2-hydroxy-5-methoxyphenethylamine in rats after subcutaneous administration

The psychedelic compound 4-bromo-2,5-dimethoxyphenethylamine (2C-B) has appeared as an agent in drug abuse or overdose cases in humans. The human pharmacokinetics of this drug is unknown and only partial information is available on its metabolites. Our experimental study was focused on the disposition and kinetic profile of 2C-B in rats after subcutaneous administration using a GC-MS validated method. One of the major metabolites 4-bromo-2-hydroxy-5-methoxyphenethylamine (2H5M-BPEA) was confirmed in rat tissues of lung, brain, liver and was quantitatively evaluated as well. The disposition of 2C-B was characterized by its estimated half-life 1.1h and estimated volume of distribution 16L/kg. The lung susceptibility for drug retention and gradual temporal release parallel to the brain were ascertained. The drug penetrating the blood/brain barrier was without significant delay. 2C-B brain to serum ratio attained a maximum value of 13.9 and remained over the value of 6.5 to the end of our observation (6h after the dose). The distribution of the hydroxylated metabolite 2H5M-BPEA into the lipophilic brain tissue was less efficient in relation to the parent compound. The kinetics of the drug partitioning between blood to brain may be important for the subsequent assessment of its psychotropic or toxic effects.     

High affinity binding of [3H]paroxetine and [3H]imipramine to human platelet membranes

Paroxetine, one of the most potent and specific serotonin uptake inhibitors, was tritiated and used for binding studies with human platelet membranes. Specific, high affinity binding was demonstrated. The binding was compared with [3H]imipramine binding; it was found that the maximal binding (Bmax) was the same for [3H]paroxetine and [3H]imipramine, whereas the affinity was much higher for [3H]paroxetine (KD 0.08 nM and 0.56 nM for paroxetine and imipramine binding, respectively). IC50 was calculated for the inhibition of [3H]paroxetine and [3H]imipramine binding by a number of antidepressants; the corresponding Hill coefficients were also calculated.     

Synthesis, in vitro binding profile, and central nervous system penetrability of the highly potent 5-HT3 receptor antagonist [3H]-4-(2-methoxyphenyl)-2-[4(5)-methyl-5(4)-imidazolylmethyl]thiazole

4-(2-Methoxyphenyl)-2-[4(5)-methyl-5(4)-imidazolylmethyl]thiazole (5) is a highly potent member of a structurally novel series of selective serotonin-3 receptor antagonists. The synthesis of tritiated 5 and its binding profile in neuroblastoma-glioma 108-15 cells are described. Furthermore, in vivo studies in rat with this radioligand indicate that it effectively penetrates the blood-brain barrier upon peripheral administration. Thus, 5 should be a useful pharmacological tool for both in vitro and in vivo studies of this class of compounds.     

High-affinity binding of [3H]6-nitroquipazine to 5-hydroxytryptamine transporter in human platelets

The characteristics of the binding [3H]6-nitroquipazine, a very potent and selective inhibitor of 5-hydroxytryptamine (5-HT; serotonin) uptake, to human platelet membranes were studied at a physiological temperature of 37 degrees C. The presence of a single saturable high-affinity binding component for [3H]6-nitroquipazine was demonstrated Non-specific binding was estimated in the presence of 1 microM paroxetine. Scatchard analysis revealed an apparent equilibrium dissociation constant (Kd) of 0.450 +/- 0.04 nM and a maximal number of binding sites (Bmax) of 2508 +/- 360 fmol/mg protein (mean +/- S.D., n = 4). The kinetically derived dissociation constant (Kd) was 0.431 nM. [3H]6-Nitroquipazine binding was inhibited selectively by 5-HT uptake inhibitors, and the potency of various drugs to inhibit [3H]6-nitroquipazine binding closely correlated with their inhibitory effects on [3H]5-HT uptake into synaptosome. Moreover, Ki values for drug inhibition of [3H]6-nitroquipazine binding to human platelet membranes were significantly correlated with the corresponding Ki values for inhibition of [3H]paroxetine binding at 37 degrees C. The present results suggest that the binding sites for [3H]6-nitroquipazine are associated with the 5-HT transporter in human platelets.     

Nicotine-evoked [3H]5-hydroxytryptamine release from rat striatal synaptosomes

The aim of this study was to characterize the pharmacology of presynaptic nicotinic cholinoceptors (nAChRs) that modulate release of 5-hydroxytryptamine (5-HT) from superfused rat brain synaptosomes preloaded with [3H]5-HT. Nicotine increased 5-HT release from striatal synaptosomes (maximally by 15-30%) but not from cerebral cortex or hippocampal synaptosomes. Release of striatal 5-HT was increased in a concentration-dependent manner by nicotine, epibatidine, cytisine, and ACh (with added esterase inhibitor and muscarinic antagonist). Respective EC50 values were: 0.5, 0.003, 0.1 and 0.7 microM. The maximal effect of each agonist was virtually completely blocked by a high concentration of the insurmountable nicotinic antagonist mecamylamine; at a higher concentration of epibatidine (3 microM), a mecamylamine-insensitive effect was revealed. Nicotine, ACh and epibatidine appeared equally efficacious, whereas cytisine was of lower efficacy (60-70% of ACh). Release evoked by a half-maximal concentration of nicotine was inhibited by the nicotinic antagonists dihydro-beta-erythroidine (IC50 0.04 microM) and methyllycaconitine (IC50 0.06 microM). Nicotine-evoked 5-HT release was not reduced by tetrodotoxin given in a concentration that blocked veratridine-evoked release. These findings provide functional evidence for a direct action of nicotine on 5-HT neurons in the brain. The presynaptic nAChRs that modulate striatal 5-HT release appear to possess a novel pharmacological profile.