Diagnostic value of otoacoustic emissions. 1

Analogues of the potent and selective 5-HT1A ligand, WAY 100635, were synthesized and examined as potential candidates for imaging 5-HT1A receptors by positron emission tomography (PET). Several of the analogues displayed nanomolar affinity for the 5-HT1A receptor, comparable to WAY 100635. Three of these were examined in a model of human liver metabolism vis-à-vis WAY 100635. All showed a markedly lower propensity for amide hydrolysis than WAY 100635. Radiolabelling of these three potential PET radiotracers with carbon-11 was readily achieved from [11C]-iodomethane, and the newly synthesized radioligands were tested in vivo in rats for binding to 5-HT1A receptors. Whereas two of the ligands failed to bind to 5-HT1A receptors in vivo, one was successful. The latter, [11C]-7 [4-(2'-methoxyphenyl)-1-[2'-[N-(2'-pyridinyl)-2-bicyclo[2.2.2]octanec arboxamido]ethyl]-piperazine], showed good brain penetration, hippocampal:cerebellar ratios of 10:1 at 45 min postinjection. Blocking studies with a variety of drugs demonstrated that the binding of [11C]-7 in vivo was selective for 5-HT1A receptors. [11C]-7 is a promising candidate as a ligand for imaging 5-HT1A receptors by PET.     

Eight inhibitory monoclonal antibodies define the role of individual P-450s in human liver microsomal diazepam, 7-ethoxycoumarin, and imipramine metabolism

Serotonergic 5-hydroxytryptamine-1A (5-HT1A) receptors are of interest in the pathophysiology of several neuropsychiatric disorders such as anxiety, depression and schizophrenia. [Carbonyl-11C]WAY-100635 has recently been shown to be suitable for quantitative determination of 5-HT1A receptors in the human brain using PET. For group comparisons of neuroreceptor distribution on a pixel-by-pixel basis, an anatomic standardization technique is required. In the current study, we have built a database of normal 5-HT1A receptor distribution using [carbonyl-11C]WAY-100635 and an anatomic standardization technique. METHOD: A PET examination lasting 63 min was performed on six subjects after intravenous injection of [carbonyl-11C]WAY-100635. The radioactivity of the PET images were integrated in the interval 12-63 min and normalized by the radioactivity of the cerebellum, providing a measure of the binding potential (BP) in each pixel. Each PET image was transformed into a standard brain anatomy using a computerized brain atlas system. From the standardized PET images, the sample mean and the SD of the BP were calculated in each pixel. RESULT: On the anatomically standardized average image, high BP was observed in the cerebral cortices, hippocampus and raphe nucleus, whereas low BP was observed in the basal ganglia and thalamus. This regional distribution is in good agreement with the distribution of 5-HT1A receptors known from in vitro studies. CONCLUSION: The anatomic standardization technique permits building of a database of the normal 5-HT1A receptor distribution in the living human brain. This technique can be applied for group comparisons of neuroreceptor distribution on a pixel-by-pixel basis.     

The pharmacological characterization of a novel selective 5-hydroxytryptamine1A receptor antagonist, NAD-299

The pharmacological properties of a novel selective 5-hydroxytryptamine1A (5-HT1A) receptor antagonist, NAD-299 [(R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R)-tartrate monohydrate] were examined in vitro and in vivo and compared with the reference 5-HT1A receptor antagonist, WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazin-yl))ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride]. The new compound had high affinity for 5-HT1A receptors in vitro with a Ki value of 0.6 nM. The only other receptors for which NAD-299 had affinity less than 1 microM were alpha-1 and beta adrenoceptors with Ki values of 260 and 340 nM, respectively. Thus, the selectivity of NAD-299 for 5-HT1A receptors was more than 400 times. WAY-100635 had considerably higher affinity than NAD-299 for alpha-1 adrenoceptors (Ki = 45 nM) and dopamine D2 and D3 receptors (Ki = 79 and 67 nM, respectively). Like WAY-100635, NAD-299 competitively blocked 5-HT-induced inhibition of vasoactive intestinal peptide-stimulated cAMP production in GH4ZD10 cells and had no intrinsic activity. Both compounds were therefore 5-HT1A receptor antagonists in vitro and also behaved as such in in vivo experiments. Thus, they competitively antagonized the 8-hydroxy-2-(di-n-propylamino)tetralin-induced 5-HT behavioral effects, hypothermia, corticosterone secretion and inhibition of passive avoidance behavior without causing any actions of their own. The effective dose of NAD-299 varied between 0.03 and 0.35 micromol/kg s.c., depending on the test and the dose of 8-hydroxy-2-(di-n-propylamino)tetralin.     

Imaging 5-HT1A receptors with positron emission tomography: initial human studies with [11C]CPC-222

The novel radioligand [11C]CPC-222 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-2-bicyclo [2,2,2]octane carboxamide) was evaluated as an in vivo probe of the 5-HT1A receptors using positron emission tomography (PET). Three human volunteers were imaged with PET over a 90 min period following intravenous injection of the radioligand. There was a high accumulation of the radioligand in brain regions with a high density of 5-HT1A receptors. The peak cortical concentration was 1.0-2.5% of the injected dose per litre. The ratio of radioactivity in receptor-rich regions to that of the cerebellum reached a plateau of 2.5-4.0 by 45 min after injection. Analysis of the plasma revealed no detectable amount of the potential metabolite, radiolabelled WAY-100634. This new radioligand has suitable properties to study the 5-HT1A receptors in man with PET.     

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.     

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.     

Quantitative analyses of carbonyl-carbon-11-WAY-100635 binding to central 5-hydroxytryptamine-1A receptors in man

The serotonin 5-hydroxytryptamine-1A (5-HT1A) receptor subtype is of central interest in research on the pathophysiology and treatment of psychiatric disorders. Carbonyl-11 C-WAY-100635 is a new radioligand that, in PET experiments, provides high-contrast delineation of brain regions that are rich in 5-HT1A receptors. The aim of this PET study was to examine the prospects for quantitation of carbonyl-11C-WAY-100635 binding to 5-HT1A receptors in the human brain. METHODS: A PET examination was performed in each of six healthy male subjects after intravenous injection of carbonyl-11C-WAY-100635. Radioactive metabolites in plasma were determined with high-performance liquid chromatography. The metabolite-corrected arterial input function was used in a kinetic three-compartment analysis, and the cerebellum was used as reference region in linear graphical and transient equilibrium analyses. RESULTS: The highest radioactivity concentration was observed in the neocortex and the raphe nuclei, whereas radioactivity was low in the cerebellum. The time-activity curves were well-described by a three-compartment model for all regions. Uptake in the cerebellum could not be described by a two-compartment model. The transient equilibrium and linear graphical analyses, which are both dependent on the cerebellum as the reference region, gave lower binding potential values than did the kinetic analysis. The metabolism was rapid, and the fraction of unchanged carbonyl-11C-WAY-100635 was <10% 10 min after injection in all human subjects. The major radioactive metabolites were unidentified polar components. One metabolite comigrated with reference cyclohexanecarboxylic acid, and another comigrated with reference desmethyl-WAY-100635. CONCLUSION: The suitability of carbonyl-11C-WAY-100635 for research on central 5-HT1A receptors in neuropsychiatric disorders was supported by the observation that the high signals in the neocortex and raphe nuclei can be described using a kinetic analysis with a metabolite-corrected arterial input function. It cannot be excluded that kinetically distinguishable nonspecific binding or the formation of a metabolite that passes the blood-brain barrier may represent measurable components of the low radioactivity in the cerebellum. Simplified quantitative methods, using the relatively low radioactivity in the cerebellum as reference, should accordingly be applied with some caution until the biochemical nature of the radioactivity is better understood and the reliability of these approaches has been confirmed in larger samples.     

Differences in the sensitivity to purinergic stimulation of myelinating and non-myelinating Schwann cells in peripheral human and rat nerve

The administration of the 5-hydroxytryptamine (5-HT) precursor 5-hydroxytryptophan (5-HTP) (25 mg/kg i.p.), in combination with an inhibitor of peripheral 5-HTP decarboxylase, produced a dose-dependent increase in the ejaculation latency of male rats, and this effect was enhanced by additional treatment with the 5-HT1 receptor antagonist (-)-pindolol (2 mg/kg s.c.). The 5-HT2A/C receptor agonist (+/-) 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.125-0.5 mg/kg s.c.) did not by itself affect male ejaculatory behavior, but additional treatment with (-)-pindolol (2 mg/kg s.c.) produced a dose-dependent decrease in number of ejaculating animals. The increased ejaculation latency produced by 5-HTP was fully antagonized by treatment with the 5-HT1B receptor antagonist isamoltane (4 mg/kg s.c.), but not by ritanserin (2 mg/kg s.c.) treatment. The selective 5-HT1A receptor antagonist WAY-100635 (0.15 mg/kg s.c.) enhanced the inhibitory actions of 5-HTP on the male rat ejaculatory behavior, and this dose of WAY-100635 fully antagonized 8-OH-DPAT-induced facilitation (0.25 mg/kg s.c.) of the ejaculatory behavior. WAY-100635 (0.04-0.60 mg/kg s.c.) did not, by itself, significantly affect male rat sexual behavior. Taken together, the results suggest an inhibitory role for postsynaptic 5-HT1B receptors in the effects produced by 5-HTP on male rat ejaculatory behavior. Furthermore, 5-HTP-induced inhibition of male rat ejaculatory behavior is partially controlled by stimulation of inhibitory 5-HT1A autoreceptors, since the effects of 5-HTP were accentuated by treatment with (-)-pindolol, as well as by the more selective 5-HT1A receptor antagonist WAY-100635.     

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.     

The discriminative stimulus effects of KA 672, a putative cognitive enhancer: evidence for a 5-HT1A component

Stimulus control was established in a group of seven rats using a dose of KA 672 [7-methoxy-6-[3-[4-(2-methoxyphenyl)-1-piperazinyl] propoxy]3,4-dimethyl-2H-1-benzopyran-2-one HCl] of 1.0 mg/kg, administered i.p., 15 min before training. A two-lever operant task using a fixed-ratio 10 schedule of sweetened milk reinforcement was used. Based upon a criterion for the presence of stimulus control of five consecutive sessions during which 83% or more of all responses were on the appropriate lever, a mean of 23 sessions was required to reach criterion performance. Subsequently, it was observed that KA 672-induced stimulus control is partially but significantly antagonized by the selective 5-HT1A antagonist, WAY-100635. Furthermore, KA 672 generalized to the selective 5-HT1A agonist, 8-hydroxy-dipropylaminotetralin [8-OH-DPAT], and this generalization was blocked by WAY-100635. Other tests of generalization were conducted with the structural analogs, scoparone, CD-127, and OMPP, as well as with the receptor-selective ligands ketamine, PCP, dizocilpine, prazosin, urapidil, apomorphine, and DTG. Of these drugs only dizocilpine met the criteria for full substitution while an intermediate level of generalization was observed to ketamine, PCP, urapidil, and apomorphine. The present results indicate that KA 672-induced stimulus control is mediated in part by activity at the 5-HT1A receptor and that behaviorally significant interactions occur as well at PCP/NMDA, dopaminergic, and adrenergic receptors.     

18F]fluoroalkyl analogues of the potent 5-HT1A antagonist WAY 100635: radiosyntheses and in vivo evaluation

Two analogues of the potent 5-HT1A antagonist WAY 100635 have been synthesized and radiolabelled with 18F, namely N-[2-[4-(2-2'-[18F] fluoroethoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohe xan e carboxamide ([18F]FEC) and N-[2-[4-(2-3'-[18F] fluoropropoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cycloh exa ne carboxamide ([18F]FPC). Biodistribution studies in rats showed selective uptake of both radiotracers in regions known to be rich in 5-HT1A receptors following i.v. injection. The ratio of radioactivity in hippocampus to that in the cerebellum was 5.5 (for [18F]FEC) and 7.5 (for [18F]FPC) at 60 min postinjection. Regional brain heterogeneity of radioactivity could be abolished by pretreatment with WAY 100635 and FPC but was unaffected by pretreatment with a variety of drugs including ketanserin, sulpiride, and SCH 23390. These results are compared vis-a-vis with those obtained using [11C]WAY 100635 to evaluate [18F]FEC and [18F]FPC as potential radiotracers for imaging 5-HT1A receptors by positron emission tomography.     

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.     

Differential regulation of somatodendritic serotonin 5-HT1A receptors by 2-week treatments with the selective agonists alnespirone (S-20499) and 8-hydroxy-2-(Di-n-propylamino)tetralin: microdialysis and autoradiographic studies in rat brain

Single treatment with the serotonin (5-hydroxytryptamine) 5-HT1A receptor agonists 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and alnespirone (S-20499) reduces the extracellular 5-HT concentration (5-HText) in the rat midbrain and forebrain. Given the therapeutic potential of selective 5-HT1A agonists in the treatment of affective disorders, we have examined the changes in 5-HT1A receptors induced by 2-week minipump administration of alnespirone (0.3 and 3 mg/kg/day) and 8-OH-DPAT (0.1 and 0.3 mg/kg/day). The treatment with alnespirone did not modify baseline 5-HText but significantly attenuated the ability of 0.3 mg/kg s.c. alnespirone to reduce 5-HText in the dorsal raphe nucleus (DRN) and frontal cortex. In contrast, the ability of 8-OH-DPAT (0.025 and 0.1 mg/kg s.c.) to reduce 5-HText in both areas was unchanged by 8-OH-DPAT pretreatment. Autoradiographic analysis revealed a significant reduction of [3H]8-OH-DPAT and [3H]WAY-100635 [3H-labeled N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexa necarboxamide x 3HCl] binding to somatodendritic 5-HT1A receptors (but not to postsynaptic 5-HT1A receptors) of rats pretreated with alnespirone but not with 8-OH-DPAT. In situ hybridization analysis revealed no change of the density of the mRNA encoding the 5-HT1A receptors in the DRN after either treatment. These data indicate that continuous treatment for 2 weeks with alnespirone, but not with 8-OH-DPAT, causes a functional desensitization of somatodendritic 5-HT1A receptors controlling 5-HT release in the DRN and frontal cortex.     

Cell-specific transcription of the smooth muscle gamma-actin gene requires both positive- and negative-acting cis elements

We recently labeled with carbon-11, a high affinity, selective, 5-HT3 receptor (5-HT3R) ligand, S21007, for potential positron emission tomography (PET) applications. To evaluate the in vivo binding properties of [11C]S21007, its brain regional distribution, tissue and plasma pharmacokinetics and plasma metabolisation were characterized. To circumvent the problem of highly discrete brain localization of the 5-HT3R (area postrema, hippocampus), we designed an original approach combining high-resolution imaging techniques (ex vivo phosphor plate autoradiography and MRI-guided coronal PET in the rat and baboon, respectively). After i.v. injection of trace amounts of [11C]S21007 to rats, phosphorimager autoradiography failed to reveal in vivo specific binding to, nor selectivity for 5-HT3R-rich areas. PET studies in the baboon showed consistent results, i.e., there was no selective accumulation of [11C]S21007 in the area postrema or hippocampus, and neither displacement nor presaturation with cold S21007 resulted in significant changes in tissue distribution or kinetics of [11C]S21007.     

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.     

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.     

Altered postreceptor signal transduction of formyl-Met-Leu-Phe receptors in polymorphonuclear leukocytes of patients with non-insulin-dependent diabetes mellitus

The 5-HT1A receptor agonist (-)-(R)-2-[4-[[(3,4-dihydro-2H-1-benzopyran-2-yl)methyl]amino]butyl]-1,2 -benzisothiazol-3(2H)-one1,1-dioxide monohydrochloride (BAY x 3702) was recently shown to have pronounced neuroprotective effects in rat models of cerebral ischemia and traumatic brain injury. In the present study we investigated the neuroprotective effects of BAY x 3702 in primary cultures of hippocampal and cortical neurons. Cell death was induced by 25 nM of the apoptosis inducing agent staurosporine and analyzed 24 h later by release of lactate dehydrogenase, formation of apoptotic bodies and DNA fragmentation. A significant neuroprotection was seen after pretreatment of the affected neurons with 50 pM to 1 microM BAY x 3702. The effects of BAY x 3702 were completely blocked by the selective 5-HT1A receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride) (WAY-100635). These results indicate that low concentrations of BAY x 3702 protect cortical as well as hippocampal neurons from apoptotic cell death via a 5-HT1A receptor mediated pathway.     

Stimulated [35S]GTP gamma S binding by 5-HT1A receptor agonists in recombinant cell lines. Modulation of apparent efficacy by G-protein activation state

G-protein activation by different 5-HT receptor ligands was investigated in h5-HT1A receptor-transfected C6-glial and HeLa cells using agonist-stimulated [35S]-GTP gamma S binding to membranes in the presence of excess GDP. 5-HT (10 microM) stimulated [35S]GTP gamma S binding in the C6-glial membrane preparation to a larger extent than in the HeLa preparation; maximal responses with 30 microM GDP were 490 +/- 99 and 68 +/- 12%, respectively. With the 5-HT receptor agonists that were being investigated, the two preparations displayed the same rank order of potency for stimulation of [35S]GTP gamma S binding. In the C6-glial preparation at 0.3 microM GDP, the rank order of maximal effects was: 5-HT (1.00) > 8-OH-DPAT (0.90) = R(+)-8-OH-DPAT (0.87) = 5-CT (0.86) = L694247 (0.84) > S(-)8-OH-DPAT (0.68) = buspirone (0.67) = spiroxatrine (0.67) = flesinoxan (0.64) > ipsapirone (0.53) = (-)-pindolol (0.50) > SDZ216525 (0.25). However, differences in maximal response in the C6-glial preparation were magnified by increasing the GDP concentrations, indicating that the activity state of G-proteins can affect the maximal response. With the exception of 5-CT and L694247, increasing the amount of GDP to 30 microM and higher concentrations resulted in an attenuation of both the ligand's maximal effect (24 to 56%) and apparent potency (6 to 24-fold). Each of the [35S]GTP gamma S binding responses was mediated by a 5-HT1A receptor as indicated by the competitive blockade by WAY100635 and spiperone. Only 5-CT and L694247 in some conditions displayed an efficacy similar to that of 5-HT at the h5-HT1A receptor; the other agents with intrinsic activity are partial agonists at this receptor. The data also suggest that the activity state of the G-proteins is involved in the maximal effects that can be produced by activating the h5-HT1A receptor.     

Preferential potentiation of the effects of serotonin uptake inhibitors by 5-HT1A receptor antagonists in the dorsal raphe pathway: role of somatodendritic autoreceptors

5-HT1A autoreceptor antagonists enhance the effects of antidepressants by preventing a negative feedback of serotonin (5-HT) at somatodendritic level. The maximal elevations of extracellular concentration of 5-HT (5-HT(ext)) induced by the 5-HT uptake inhibitor paroxetine in forebrain were potentiated by the 5-HT1A antagonist WAY-100635 (1 mg/kg s.c.) in a regionally dependent manner (striatum > frontal cortex > dorsal hippocampus). Paroxetine (3 mg/kg s.c.) decreased forebrain 5-HT(ext) during local blockade of uptake. This reduction was greater in striatum and frontal cortex than in dorsal hippocampus and was counteracted by the local and systemic administration of WAY-100635. The perfusion of 50 micromol/L citalopram in the dorsal or median raphe nucleus reduced 5-HT(ext) in frontal cortex or dorsal hippocampus to 40 and 65% of baseline, respectively. The reduction of cortical 5-HT(ext) induced by perfusion of citalopram in midbrain raphe was fully reversed by WAY-100635 (1 mg/kg s.c.). Together, these data suggest that dorsal raphe neurons projecting to striatum and frontal cortex are more sensitive to self-inhibition mediated by 5-HT1A autoreceptors than median raphe neurons projecting to the hippocampus. Therefore, potentiation by 5-HT1A antagonists occurs preferentially in forebrain areas innervated by serotonergic neurons of the dorsal raphe nucleus.     

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.