Selective visualization of rat brain 5-HT2A receptors by autoradiography with [3H]MDL 100,907

The recently developed 5-HT2A receptor selective antagonist [3H]MDL100,907 ((+/-)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol]) has been characterized as a radioligand for the autoradiographic visualization of these receptors. [3H]MDL100,907 binding to rat brain tissue sections was saturable, had sub-nanomolar affinity (Kd = 0.2-0.3 nM), and presented a pharmacological profile consistent with its binding to 5-HT2A receptors (rank order of affinity for [3H]MDL100,907-labelled receptors: MDL100,907 > spiperone > ketanserin > mesulergine). The distribution of receptors labelled by [3H]MDL100,907 was compared to the autoradiographical patterns obtained with [3H]Ketanserin, [3H]Mesulergine, and [3H]RP62203 (N-[3-[4-(4-fluorophenyl)piperazin-1-y1]propyl]-1,8-naphtalenes ultam) and to the distribution of 5-HT2A receptor mRNA as determined by in situ hybridization. As opposed to the other radioligands, [3H]MDL100,907 labelled a single population of sites (5-HT2A receptors) and presented extremely low levels of non-specific binding. The close similarity of the distributions of [3H]MDL100,907-labelled receptors and 5-HT2A mRNA further supports the selectivity of this radioligand for 5-HT2A receptors and suggests a predominant somatodendritic localization of these receptors. The present results point to [3H]MDL100,907 as the ligand of choice for the autoradiographic visualization of 5-HT2A receptors.     

3H]MDL 100,907 labels 5-HT2A serotonin receptors selectively in primate brain

The selective antagonist for the 5-HT2A serotonin receptor MDL 100,907, recently characterized autoradiographically in rat brain, has been characterized as a radioligand for the visualization of this receptor in human and monkey brain. In both species [3H]MDL 100,907 binding to brain sections was saturable, had sub-nanomolar affinity (Kd = 0.14-0.19 nM in human brain; Kd= 0.16-0.19 nM in monkey brain) and presented a pharmacological profile consistent with its binding to 5-HT2A receptors (rank order of affinity for [3H]MDL 100,907-labeled receptors: MDL 100,907 > spiperone > ketanserin > mesulergine). The autoradiographical signal obtained with [3H]MDL 100,907 was compared to the signal obtained with [3H]ketanserin, [3H]RP62203 and [3H]mesulergine in both species, and to the distribution of 5-HT2A receptor mRNA as determined by in situ hybridization in monkey brain. At variance with the other radioligands, [3H]MDL 100,907 showed a single population of binding sites with extremely low levels of non-specific binding. As expected, mesulergine showed low affinity for [3H]MDL 100,907-labeled receptors and the autoradiographic pattern shown by [3H]mesulergine confirmed the lack of labeling of the 5-HT2A receptor by this radioligand in primate brain. The similarity of the distribution of [3H]MDL 100,907-labeled receptors and 5-HT2A mRNA in monkey brain, supports the selectivity of this radioligand for 5-HT2A receptors and suggests a somatodendritic localization of these receptors. The present results confirm [3H]MDL 100,907 as the radioligand of choice at present for the autoradiographic visualization of 5-HT2A receptors in mammalian brain including post-mortem human brain.     

Autoradiographic mapping of 5-HT1B- and 5-HT1D receptors in human brain using [3H]alniditan, a new radioligand

[3H]alniditan, a new potent non-indole serotonin 5-HT1B/1D agonist, was used as a radioligand to characterize 5-HT1B and 5-HT1D receptor (previously termed 5-HT1D beta and 5-HT1D alpha) in various regions of the human brain. Quantitative receptor autoradiography was applied for high anatomical resolution and sensitivity. Highest densities of 5-HT1B/1D receptors were found in the substantia nigra and in the globus pallidus. High to moderate densities were measured in the caudate nucleus, putamen, nucleus accumbens, central gray and hippocampal formation. Very low densities were detected in various cortical regions. In the cerebellum no [3H]alniditan binding was detected. Selective 5-HT1B receptor labeling was achieved using [3H]alniditan in the presence of 300 nM of ketanserin (sufficient to block 5-HT1D receptor labeling). The identity of the 5-HT1B binding sites under these conditions was corroborated by the pIC50 of sumatriptan, which corresponded to its affinity for cloned human 5-HT1B receptors expressed in cells. Surprisingly, the distribution of selective 5-HT1B receptor labeling was completely identical to the distribution of labeling of 5-HT1B + 5-HT1D receptors. The present data indicate that [3H]alniditan is a suitable radioligand for measuring 5-HT1B/1D receptor in the human brain and that the 5-HT1B binding sites are predominant in the presently investigated regions of the human brain.     

Differential distribution of [3H]sumatriptan binding sites (5-HT1B, 5-HT1D and 5-HT1F receptors) in human brain: focus on brainstem and spinal cord

We report on the autoradiographic distribution of 5-HT1B, 5-HT1D and 5-HT1F receptor subtypes in human brain, focusing on the brainstem and cervical spinal cord. We have used [3H]sumatriptan as a radioligand in the presence of suitable concentrations of 5-CT (5-carboxamidotryptamine) to define 5-HT1F receptors, and ketanserin, to discriminate between 5-HT1B and 5-HT1D receptors. In the brainstem the highest concentrations of [3H]sumatriptan binding sites were seen in substantia nigra. The spinal trigeminal nucleus, substantia gelatinosa of the spinal cord, nucleus of the tractus solitarius and periaqueductal grey, also showed significant levels of [3H]sumatriptan binding sites. In the brainstem and spinal cord the total population of 5-CT-insensitive receptors, corresponding to 5-HT1F receptors, ranged from 9.8% in the periaqueductal grey to 53.4% in the substantia gelatinosa. This population represented 67.0% of binding in layer V of the frontal cortex. The decrease in [3H]sumatriptan binding in the presence of 200 nM ketanserin, indicative of the presence of 5-HT1D receptors, was very limited throughout the human brain, only reaching 20% of total specific binding over the periaqueductal grey. The proportion of [3H]sumatriptan binding sites displaced by 5-CT and insensitive to ketanserin, corresponding to 5-HT1B receptors, was, in general, the most abundant, ranging from 43.8% in substantia gelatinosa to 69.9% in the periaqueductal grey. Significant levels of 5-HT1B and 5-HT1D receptors found in migraine control pain areas suggest their involvement in antinociceptive mechanisms.     

Multiple subtypes of serotonin receptors are expressed in rat sensory neurons in culture

[3H]5-HT revealed the presence of serotonin receptors in cultured rat sensory neurons. [3H]5-CT binding was inhibited by cyanopindolol with an IC50 of 0.87 +/- 0.30 nM, suggesting the expression of the 5-HT1B receptor in these neurons. The presence of 5-HT1B receptors was confirmed by the displacement of [125I]Iodocyanopindolol binding by cyanopindolol with an IC50 of 2.43 +/- 0.81 nM. 5-HT1B receptors are the predominant type of serotonin receptors labeled by [3H]5-HT in cultured DRG neurons, representing approximately 60% of the specific [3H]5-HT binding sites. In addition, 5-HT1D and 5-HT2A receptor binding was also found in these neurons. RT-PCR analysis of RNA isolated from embryonic sensory neurons in culture confirmed the expression of 5-HT1B, 5-HT1D and 5-HT2A receptor mRNA. It also demonstrated the presence of 5-HT1F, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A and 5-HT5B receptor mRNA and the absence of 5-HT1A, 5-HT1E, 5-HT2B, 5-HT6 and 5-HT7 mRNA. The identification of multiple subtypes of serotonin receptors expressed in cultured embryonic sensory neurons suggests that DRG neuronal cultures may be an excellent model to examine the direct effects of serotonin on the activity of these sensory neurons.     

Detailed mapping of serotonin 5-HT1B and 5-HT1D receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function

The similar pharmacology of the 5-HT1B and 5-HT1D receptors, and the lack of selective compounds sufficiently distinguishing between the two receptor subtypes, have hampered functional studies on these receptors. In order to provide clues for differential functional roles of the two subtypes, we performed a parallel localization study throughout the guinea-pig brain and the trigeminal ganglia by means of quantitative in situ hybridization histochemistry (using [35S]-labelled riboprobes probes for receptor messenger RNA) and receptor autoradiography (using a new radioligand [3H]alniditan). The anatomical patterns of 5-HT1B and 5-HT1D receptor messenger RNA were quite different. While 5-HT1B receptor messenger RNA was abundant throughout the brain (with highest levels in the striatum, nucleus accumbens, olfactory tubercle, cortex, hypothalamus, hippocampal formation, amygdala, thalamus, dorsal raphe and cerebellum), 5-HT1D receptor messenger RNA exhibited a more restricted pattern; it was found mainly in the olfactory tubercle, entorhinal cortex, dorsal raphe, cerebellum, mesencephalic trigeminal nucleus and in the trigeminal ganglion. The density of 5-HT(1B/1D) binding sites (combined) obtained with [3H]alniditan autoradiography was high in the substantia nigra, superior colliculus and globus pallidus, whereas lower levels were detected in the caudate-putamen, hypothalamus, hippocampal formation, amygdala, thalamus and central gray. This distribution pattern was indistinguishable from specific 5-HT1B receptor labelling in the presence of ketanserin under conditions to occlude 5-HT1D receptor labelling; hence the latter were below detection level. Relationships between the regional distributions of the receptor messenger RNAs and binding sites and particular neuroanatomical pathways are discussed with respect to possible functional roles of the 5-HT1B and 5-HT1D receptors.     

Chronic alnespirone-induced desensitization of somatodendritic 5-HT1A autoreceptors in the rat dorsal raphe nucleus

The effects of long-term (7, 14 or 21 days) administration of the 5-HT1A receptor agonist alnespirone [5 mg/(kg day), i.p.] on the binding characteristics of 5-HT1A, 5-HT2A and 5-HT3 receptors, and the functional status of 5-HT1A autoreceptors were assessed using biochemical and electrophysiological approaches in rats. Whatever the treatment duration, the specific binding of [3H]8 hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT), [3H]trans,4-[(3Z)3-(2-dimethylaminoethyl) oxyimino-3(2-fluorophenyl) propen-1-yl] phenol hemifumarate ([3H]SR 46349B), and [3H]S-zacopride to 5-HT1A, 5-HT2A and 5-HT3 receptors, respectively, were unaltered in all the brain areas examined. In contrast, in vitro electrophysiological recordings performed 24 h after the last injection of alnespirone showed that the potency of the 5-HT1A receptor agonist, 8-OH-DPAT, to depress the firing of serotoninergic neurons in the dorsal raphe nucleus, was significantly reduced after a 21-day treatment with alnespirone. However, no changes were noted after a 7-day or 14-day treatment. These data indicate that desensitization of somatodendritic 5-HT1A autoreceptors is a selective but slowly developing adaptive phenomenon in response to their chronic stimulation in rats.     

3H]Alnespirone: a novel specific radioligand of 5-HT1A receptors in the rat brain

Determination of the optimal assay conditions for the specific binding of a tritiated derivative of the novel potential anxiolytic drug alnespirone (S-20499, (+)-4-[N-(5-methoxy-chroman-3-yl)-N-propylamino]butyl-8-azaspiro-( 4,5)-decane-7,9-dione) allowed the demonstration that this radioligand bound with a high affinity (Kd = 0.36 nM) to a homogeneous class of sites in rat hippocampal membranes. The pharmacological properties of [3H]alnespirone specific binding sites matched exactly (r = 0.95) those of 5-HT1A receptors identified with [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) as radioligand. Furthermore, membrane binding experiments and autoradiographic labeling of tissue sections showed that the regional distribution of [3H]alnespirone specific binding sites in the rat brain and spinal cord superimposed over that of 5-HT1A receptors specifically labeled by [3H]8-OH-DPAT. However, the differential sensitivity of [3H]alnespirone and [3H]8-OH-DPAT specific binding to various physicochemical effectors (temperature, pH, Mn2+, N-ethyl-maleimide) supports the idea that these two agonist radioligands did not recognize 5-HT1A receptors exactly in the same way. These differences probably account for the reported inability of alnespirone, in contrast to 8-OH-DPAT, to induce some 5-HT1A receptor-mediated behavioural effects in rats.     

Atypical in vitro and in vivo binding of [3H]S-14506 to brain 5-HT1A receptors

The tritiated derivative of the potent 5-HT1A receptor agonist S-14506 ?1[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl)pipera zine? was tested for its capacity to selectively label the serotonin 5-HT1A receptors both in vitro in the rat and the mouse brain, and in vivo in the mouse. In vitro studies showed that the pharmacological profile and the distribution of [3H]S-14506 specific binding sites (Kd = 0.15 nM) in different brain regions matched perfectly those of the prototypical 5-HT1A receptor ligand [3H]8-OH-DPAT. However, in the three regions examined (hippocampus, septum, cerebral cortex), the density of [3H]S-14506 specific binding sites was significantly higher (+66-90%) than that found with [3H]8-OH-DPAT. Whereas the specific binding of [3H]8-OH-DPAT was markedly reduced by GTP and Gpp(NH)p and increased by Mn2+, that of [3H]S-14506 was essentially unaffected by these compounds. In addition, the alkylating agent N-ethylmaleimide was much less potent to inhibit the specific binding of [3H]S-14506 than that of [3H]8-OH-DPAT. Measurement of in vivo accumulation of tritium one hour after i.v. injection of [3H]S-14506 to mice revealed marked regional differences, with about 2.5 times more radioactivity in the hippocampus than in the cerebellum. Pretreatment with 5-HT1A receptor ligands prevented tritium accumulation in the hippocampus but not in the cerebellum. Autoradiograms from brain sections of injected mice confirmed the specific in vivo labeling of 5-HT1A receptors by [3H]S-14506, therefore suggesting further developments with derivatives of this molecule for positron emission tomography in vivo in man.     

Agonist activity of antimigraine drugs at recombinant human 5-HT1A receptors: potential implications for prophylactic and acute therapy

The actions of several serotonergic ligands in use or under development for the treatment of migraine headaches were examined at recombinant human 5-HT1A receptors stably expressed in Chinese Hamster Ovary cells. Affinities (K(i)s) at this site were determined in competition binding experiments with [3H]-8-OH-DPAT ([3H](+/-)8-hydroxy-N,N-dipropylaminotetralin), whilst agonist efficacy was measured by stimulation of [35S]-GTP gamma S (guanylyl-5'-[gamma[35S]thio]-triphosphate) binding. Of the prophylactic antimigraine drugs tested, methysergide and lisuride behaved as efficacious agonists (Emax > or = 90% relative to 5-HT) whereas pitozifen and (-)propranolol acted as a partial agonist (60%) and an antagonist, respectively. This suggests that there is no correlation between agonism at 5-HT1A receptors and prophylactic antimigraine action. In contrast, serotonin, dihydroergotamine, sumatriptan, naratriptan and alniditan, which are effective in acute interruption of migraine attacks, each displayed high efficacy (Emax = 100, 100, 92.6, 79.3, 79.1% respectively) and marked affinity (Ki = 18.7, 0.6, 127, 26.4 and 3.0 nM respectively) at 5-HT1A receptors. EC50 values for agonist stimulation of [35S]-GTP gamma S binding correlated with respective Ki values at 5-HT1A receptors (r = 0.93) and the stimulation of [35S]-GTP gamma S binding by these compounds was antagonised by the selective 5-HT1A antagonist WAY 100,635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclo-hexanecarboxamide; 100 nM). These data suggest that agonism at 5-HT1A receptors may be involved in some actions of drugs used in acute antimigraine therapy. In comparison with the above compounds, novel ligands targeted at 5-HT1B/1D receptors, such as GR125,743 (N-[4-methoxy-3-(4-methyl-piperazin-1-yl)phenyl] -3-methyl-4-(4-pyridyl)benzamide) and GR 127,935 (N-[4-methoxy-3-(4-methylpiperazin-1-yl)-phenyl]-2'-methyl-4'-(5-m ethyl-1, 2,4-oxadiazol-3-yl)-biphenyl-4-carboxamide), only weakly activated [35S]-GTP gamma S binding (32.4 and 32.1% efficacy) and displayed moderate affinity at 5-HT1A receptors (Kis 53.1 and 49.8 nM) suggesting that they constitute useful tools to differentiate 5-HT1A and 5-HT1B/1D receptor-mediated actions. In conclusion, the present data indicates that several antimigraine agents exhibit marked 5-HT1A receptor activity and that although this is unlikely to be important for prophylactic action it may be relevant to the ancilliary properties of drugs used for acute migraine treatment.     

Regulation of hippocampal gonadotropin releasing hormone (GnRH) receptor mRNA and GnRH-stimulated inositol phosphate production by gonadal steroid hormones

Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine3 (5-HT3) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT3 receptors. The affinities of competitive 5-HT3 receptor antagonists were similar regardless of whether the receptors were labeled with [3H]RS-42358, [3H]granisetron, or 1-(m-[3H]chlorophenyl)biguanide ([3H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [3H]mCPG. The dissociation of [3H]mCPG, [3H]RS-42358, and [3H]RS-25259, but not [3H]granisetron, from both cloned and native 5-HT3 receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT3 receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.     

Selective labelling of bradykinin receptor subtypes in WI38 human lung fibroblasts

1. Binding of the B1 bradykinin receptor radioligand, [3H]-des-Arg10-kallidin (-KD) and the B2 receptor radioligand [3H]-bradykinin (-BK) was investigated in membranes prepared from WI38 human foetal lung fibroblasts. 2. One-site analysis of the saturation data for [3H]-des-Arg10-KD gave an equilibrium dissociation constant (KD) value of 0.51 +/- 0.12 nM and a maximum receptor density (Bmax) of 260 +/- 49 fmol mg-1 of protein. [3H]-des-Arg10-KD binding was displaced by ligands in the order: des-Arg10-KD > KD > > des-Arg9[Leu8]-BK > des-Arg9-BK > Hoe 140 > > BK, implying that it was binding selectively to B1 receptors. 3. One-site analysis of the binding of [3H]-BK to W138 membranes indicated that it had a KD value of 0.25 +/- 0.06 nM and a Bmax of 753 +/- 98 fmol mg-1 of protein. The potencies for displacement of [3H]-BK binding were: Hoe 140 > > BK = KD > > > des-Arg10-KD = des-Arg9[Leu8]-BK = des-Arg9-BK, which was consistent with binding to B2 receptors. 4. This is the first characterization of [3H]-des-Arg10-KD binding to include both kinetic and equilibrium data, and demonstrates that [3H]-des-Arg10-KD has a high affinity for human B1 bradykinin receptors and is sufficiently selective to be used as a radioligand for B1 receptors in human cells or tissues expressing an excess of B2 BK receptors.     

Selective in vivo labelling of brain 5-HT1A receptors by [3H]WAY 100635 in the mouse

The novel selective 5-HT1A receptor antagonist radioligand [3H]WAY 100635 ([O-methyl-3H]N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2- pyridyl)cyclohexane-carboxamide) was injected i.v. to mice in an attempt to label in vivo central 5-HT1A receptors. Although 5 min after the i.v. injection of [3H]WAY 100635 (4-7.6 muCi per mouse) the amount of tritium found in the whole brain only accounted for 1.5-1.8% of the injected radioactivity, regional differences in 3H accumulation already corresponded to those of 5-HT1A receptor density. Optimal data were obtained 1 h after [3H]WAY 100635 injection as the distribution of 3H in brain was exactly that of 5-HT1A receptor binding sites in mouse brain sections labelled in vitro with [3H]WAY 100635. In particular, high level of labelling was found in the lateral septum, gyrus dentatus and CA1 area of Ammon's horn in the hippocampus, dorsal raphe nucleus and entorhinal cortex. No labelling was found in he substantia nigra, and 3H accumulated in the cerebellum represented only 12-14% of that found in the hippocampus. Pretreatment with various drugs indicated that only 5-HT1A receptor ligands were able to decrease the accumulation of 3H in all the brain areas examined except in the cerebellum. Assuming that only non-specific binding took place in the latter structure, it was possible to calculate the ID50 values of 5-HT1A receptor agonists (8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), S 14506 (1-[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphthyl+ ++)piperazine) and S 20499 ((+)-4-[N-(5-methoxy-chroman-3-yl)-N-propylamino]butyl-8- azaspiro-(4,5)-decane-7,9-dione)) and antagonists (spiperone, (-)-tertatolol, (+)-WAY 100135 (N-tert-butyl-3,4-(2-methoxyphenyl)piperazin-1-yl-2-phenyl- propanamide)) as inhibitors of 3H accumulation in the hippocampus of [3H]WAY 100635-injected mice. Comparison of these values with the in vitro affinity of the same ligands for hippocampal 5-HT1A receptors revealed marked variations in the capacity of 5-HT1A receptor agonists and antagonists to reach the brain when injected via the subcutaneous route in mice.     

Alniditan, a new 5-hydroxytryptamine1D agonist and migraine-abortive agent: ligand-binding properties of human 5-hydroxytryptamine1D alpha, human 5-hydroxytryptamine1D beta, and calf 5-hydroxytryptamine1D receptors investigated with [3H]5-hydroxytryptamine and [3H]alniditan

Alniditan is a new migraine-abortive agent. It is a benzopyran derivative and therefore structurally unrelated to sumatriptan and other indole-derivatives and to ergoline derivatives. The action of sumatriptan is thought to be mediated by 5-hydroxytryptamine (5-HT)1D-type receptors. We investigated the receptor-binding profile in vitro of alniditan compared with sumatriptan and dihydroergotamine for 28 neurotransmitter receptor subtypes, several receptors for peptides and lipid-derived factors, ion channel-binding sites, and monoamine transporters. Alniditan revealed nanomolar affinity for calf substantia nigra 5-HT1D and for cloned h5-HT1D alpha, h5-HT1D beta and h5-HT1A receptors (Ki = 0.8, 0.4, 1.1, and 3.8 nM, respectively). Alniditan was more potent than sumatriptan at 5-HT1D-type and 5-HT1A receptors. Alniditan showed moderate-to-low or no affinity for other investigated receptors; sumatriptan showed additional binding to 5-HT1F receptors. Dihydroergotamine had a much broader profile with high affinity for several 5-HT, adrenergic and dopaminergic receptors. In signal transduction assays using cells expressing recombinant h5-HT1D alpha, h5-HT1D beta, or h5-HT1A receptors, alniditan (like 5-HT) was a full agonist for inhibition of stimulated adenylyl cyclase (IC50 = 1.1, 1.3, and 74 nM, respectively, for alniditan). Therefore, in functional assays, the potency of alniditan was much higher at 5-HT1D receptors than at 5-HT1A receptors. We further compared the properties of [3H]alniditan, as a new radioligand for 5-HT1D-type receptors, with those of [3H]5-HT in membrane preparations of calf substantia nigra, C6 glioma cells expressing h5-HT1D alpha, and L929 cells expressing h5-HT1D beta receptors. [3H]Alniditan revealed very rapid association and dissociation binding kinetics and showed slightly higher affinity (Kd = 1-2 nM) than [3H]5-HT. We investigated 25 compounds for inhibition of [3H]alniditan and [3H]5-HT binding in the three membrane preparations; Ki values of the radioligands were largely similar, although some subtle differences appeared. Most compounds did not differentiate between 5-HT1D alpha and 5-HT1D beta receptors, except methysergide, ritanserin, ocaperidone, risperidone, and ketanserin, which showed 10-60-fold higher affinity for the 5-HT1D alpha receptor. The Ki values of the compounds obtained with 5-HT1D receptors in calf substantia nigra indicated that these receptors are of the 5-HT1D beta-type. We demonstrated that alniditan is a potent agonist at h5-HT1D alpha and h5-HT1D beta receptors; its properties probably underlie its cranial vasoconstrictive and antimigraine properties.     

Characterization of desamino-5-[125I]iodo-3-methoxy-zacopride ([125I]MIZAC) binding to 5-HT3 receptors in the rat brain

1. Antagonists at 5-HT3 receptors have shown activity in animal models of mental illness, however, few radiolabeled 5-HT3 ligands are available for preclinical studies. MIZAC, an analogue of the selective 5-HT3 antagonist, zacopride, binds with high affinity (1.3-1.5 nM) to CNS 5-HT3 sites. The authors report here the selectivity of MIZAC for these sites in rat brain homogenates. 2. Ninety-seven percent of total specific binding of [125I]MIZAC (0.1 nM) of was displaced by bemesetron (3 microM), a selective 5-HT3 antagonist. Competition studies using ligands with known affinities for 5-HT3 sites give a high correlation with reported pKi values (r2 0.98). Bemesetron displaceable binding has a regional distribution consistent with that of the 5-HT3 receptor, i.e. highest in cortex and hippocampus, and lowest in striatum and cerebellum. 3. Potent antagonists present at concentrations sufficient to occupy 95% of other 5-HT receptor populations (1A, 1B, 1D, 2A, 2B, 2C, 5A, 5B, 6, and 7) showed minimal ability to displace [125I]MIZAC binding (3 nM). Specificity studies using radioligand binding assays selective for 5-HT4, 5-HT6, and 5-HT7 receptors, and for binding sites of other neurotransmitters indicate a high degree of selectivity of [125I]MIZAC for the 5-HT3 receptor. 4. [125I]MIZAC binds to an apparent low affinity (benzac) site having a unique pharmacology. Low affinity binding was displaceable by benztropine, but not by other muscarinic agents nor inhibitors of dopamine uptake. The regional distribution of the low affinity site differed markedly from that of the high affinity site. The apparent affinity of [125I]MIZAC for the benzac site is two orders of magnitude lower than for the 5-HT3 receptor. Given its high selectivity for 5-HT3 binding sites, [125I]MIZAC appears to be a promising ligand for labeling 5-HT3 receptors in vitro and in vivo.     

Lymphocyte proliferative responses to recombinant bovine herpes virus type 1 (BHV-1) glycoprotein gD (gIV) in immune cattle: identification of a T cell epitope

Effects of the antipsychotics risperidone and clozapine on 5-HT2 and D2-dopamine receptor binding were examined using [3H]N-methylspiperone ([3H]NMSP) and in vitro receptor autoradiography on human whole hemisphere cryosections. The 5-HT2 receptor antagonist ketanserin and the D2-dopamine receptor antagonist raclopride were used as references. [3H]NMSP binding was observed in caudate nucleus, putamen, and cerebral cortex indicating binding to D2-dopamine and 5-HT2 receptors. Risperidone and clozapine counteracted the binding to both receptor types. This was in contrast to raclopride, which selectively blocked the D2-dopamine receptors in the basal ganglia, and ketanserin, which selectively blocked the 5-HT2 receptors in the cerebral cortex. Risperidone (100 nM and 10 microM) blocked up to 90% of [3H]NMSP binding to both receptor types, whereas the blocking capacity of clozapine (10 microM) was lower (approximately 60%). The lack of total blockade of D2-dopamine receptors is in line with results obtained in with [11C]raclopride and positron emission tomography studies of clozapine treated human subjects. However, autoradiographic studies of clozapine competition of [3H]raclopride binding show total displacement of the binding at high clozapine concentrations, thus contradicting the PET results with [11C]raclopride, as well as the autoradiographic results obtained with [3H]NMSP. In conclusion it can be stated that pharmacological concentrations of the two drugs clozapine and risperidone block a large proportion of D2-dopamine receptors and 5-HT2 receptors in the human brain. Moreover, the study shows the usefulness of human whole hemisphere autoradiography for the study of interaction of drugs with different central neurotransmitter receptors.     

Phenylpiperazine derivatives with strong affinity for 5HT1A, D2A and D3 receptors

Four 7-[3-(4-phenyl-1-piperazinyl)propoxy]coumarins were synthesized. The affinities of these compounds for DA (D2A, D3) and 5HT1A receptors were evaluated for their ability to displace [3H]-raclopride and [3H]-8-OH-DPAT respectively from their specific binding sites. The affinities of the target compounds were all in the nanomolar range and followed the order 5-HT1A > D2 > D3.     

Selective labelling of 5-HT7 receptor recognition sites in rat brain using [3H]5-carboxamidotryptamine

The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT7 receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, (+/-)-pindolol (10 microM)-insensitive [3H]5-CT ([3H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 microM) displayed a pharmacological profile similar to the recombinant 5-HT7 receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, (+/-)-pindolol (10 microM)-insensitive [3H]5-CT recognition sites also resembled, pharmacologically, the 5-HT7 receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [3H]5-CT binding to residual, possibly, 5-HT1A sites. Competition for this [3H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT7 receptor. Saturation studies also indicated that (+/-)-pindolol (10 microM)/WAY 100635 (100 nM)-insensitive [3H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity (Bmax=33.2+/-0.7 fmol mg(-1) protein, pKd=8.78+/-0.05, mean+/-S.E.M., n=3). The development of this 5-HT7 receptor binding assay will aid investigation of the rat native 5-HT7 receptor.     

Dihydrobenzofuran analogues of hallucinogens. 3. Models of 4-substituted (2,5-dimethoxyphenyl)alkylamine derivatives with rigidified methoxy groups

Tetrahydrobenzodifuran functionalities were employed as conformationally restricted bioisosteres of the aromatic methoxy groups in prototypical hallucinogenic phenylalkylamines 1 and 2. Thus, a series of 8-substituted 1-(2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b']difuran-4-yl)-2-aminoal kanes (7a-e) were prepared and evaluated for activity in the two-lever drug discrimination paradigm in rats trained to discriminate saline from LSD tartrate (0.08 mg/kg) and for the ability to displace [3H]ketanserin from rat cortical homogenate 5-HT2A receptors and [3H]-8-OH-DPAT from rat hippocampal homogenate 5-HT1A receptors. In addition, 1-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b']difuran-4-yl)-2-am inopropane (7b), which was found to be extremely potent in the rat in vivo assays, was evaluated for its ability to compete with [125I]DOI and [3H]ketanserin binding to cells expressing cloned human 5-HT2A, 5-HT2B, and 5-HT2C receptors. All of the dihydrofuranyl compounds having a hydrophobic substituent para to the alkylamine side chain had activities in both the in vitro and in vivo assays that equaled or surpassed the activity of the analogous conformationally flexible parent compounds. For example, 7b substituted for LSD in the drug discrimination assay with an ED50 of 61 nmol/kg and had Kj values in the nanomolar to subnanomolar range for the displacement of radioligand from rat and human 5-HT2 receptors, making it one of the most potent hallucinogen-like phenylalkylamine derivatives reported to date. The results suggest that the dihydrofuran rings in these new analogues effectively model the active binding conformations of the methoxy groups of the parent compounds 1 and 2. In addition, the results provide information about the topography and relative orientation of residues involved in agonist binding in the serotonin 5-HT2 receptors.