Agonist properties of N,N-dimethyltryptamine at serotonin 5-HT2A and 5-HT2C receptors.

Extensive behavioral and biochemical evidence suggests an agonist role at the 5-HT2A receptor, and perhaps the 5-HT2C receptor, in the mechanism of action of hallucinogenic drugs. However the published in vitro pharmacological properties of N,N-dimethyltryptamine (DMT), an hallucinogenic tryptamine analog, are not consistent with this hypothesis. We, therefore, undertook an extensive investigation into the properties of DMT at 5-HT2A and 5-HT2C receptors. In fibroblasts transfected with the 5-HT2A receptor or the 5-HT2C receptor, DMT activated the major intracellular signaling pathway (phosphoinositide hydrolysis) to an extent comparable to that produced by serotonin. Because drug efficacy changes with receptor density and cellular microenvironment, we also examined the properties of DMT in native preparations using a behavioral and biochemical approach. Rats were trained to discriminate an antagonist ketanserin from an agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in a two-lever choice paradigm. Pharmacological studies showed that responding on the DOI and ketanserin lever reflected agonist and antagonist activity at 5-HT2A receptors, and hence, was a suitable model for evaluating the in vivo functional properties of DMT. Like other 5-HT2A receptor agonists, DMT substituted fully for DOI. Intact choroid plexus was used to evaluate the agonist properties at endogenous 5-HT2C receptors; DMT was a partial agonist at 5-HT2C receptors in this native preparation. Thus, we conclude that DMT behaves as an agonist at both 5-HT2A and 5-HT2A receptors. One difference was evident in that the 5-HT2C, but not the 5-HT2A, receptor showed a profound desensitization to DMT over time. This difference is interesting in light of the recent report that the hallucinogenic activity of DMT does not tolerate in humans and suggests the 5-HT2C receptor plays a less prominent role in the action of DMT.

Regulation of serotonin-2C receptor G-protein coupling by RNA editing.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) elicits a wide array of physiological effects by binding to several receptor subtypes. The 5-HT2 family of receptors belongs to a large group of seven-transmembrane-spanning G-protein-coupled receptors and includes three receptor subtypes (5-HT2A, 5-HT(2B) and 5-HT(2C)) which are linked to phospholipase C, promoting the hydrolysis of membrane phospholipids and a subsequent increase in the intracellular levels of inositol phosphates and diacylglycerol. Here we show that transcripts encoding the 2C subtype of serotonin receptor (5-HT(2C)R) undergo RNA editing events in which genomically encoded adenosine residues are converted to inosines by the action of double-stranded RNA adenosine deaminase(s). Sequence analysis of complementary DNA isolates from dissected brain regions have indicated the tissue-specific expression of seven major 5-HT(2C) receptor isoforms encoded by eleven distinct RNA species. Editing of 5-HT(2C)R messenger RNAs alters the amino-acid coding potential of the predicted second intracellular loop of the receptor and can lead to a 10-15-fold reduction in the efficacy of the interaction between receptors and their G proteins. These observations indicate that RNA editing is a new mechanism for regulating serotonergic signal transduction and suggest that this post-transcriptional modification may be critical for modulating the different cellular functions that are mediated by other members of the G-protein-coupled receptor superfamily.

Identification, molecular cloning, and distribution of a short variant of the 5-hydroxytryptamine2C receptor produced by alternative splicing.

The actions of the neurotransmitter 5-hydroxytryptamine (5-HT) (serotonin) are mediated by multiple receptor subtypes. One of the prominent serotonin receptors in the brain is the 5-HT2C receptor (5-HT2C-R). We report the occurrence of a second 5-HT2C-R transcript, first identified using S1 nuclease protection of total RNA isolated from the choroid plexus. Analyses of the distribution of these two RNAs revealed that the short form is expressed in the same structures as the 5-HT2C-R mRNA, including choroid plexus, striatum, hippocampus, hypothalamus, olfactory tubercles, and spinal cord. Cloning and sequence analyses revealed a second cDNA with a 95-nt deletion in the region coding for the putative second intracellular loop and the fourth transmembrane domain of the 5-HT2C-R. This deletion leads to a frameshift in the coding sequence and the introduction of a premature stop codon. The predicted truncated protein (5-HT2C-tr) contains 172 amino acids, with 153 residues at the amino terminus, identical to the 5-HT2C-R, and 19 carboxyl-terminal amino acids that are unique. Although antibodies specific to the 5-HT2C-tr protein showed that the truncated form is expressed in a transfected fibroblast cell model system, there was no serotonergic ligand binding activity or phosphoinositide hydrolysis. Analyses of the 5-HT2C-R gene revealed that the two transcripts arise from a single gene by differential splicing using alternative donor sites and a common 3'-splice acceptor. Polymerase chain reaction amplification of mouse and human brain cDNAs demonstrated the occurrence of the same splicing patterns in these species. Although this study demonstrates tissue-specific expression of two 5-HT2C mRNA splice variants in rat, mouse, and human, the significance of the truncated form in these three species remains to be established.

Identification of rat serotonin 5-HT2C receptors as glycoproteins containing N-linked oligosaccharides.

Antibodies against a portion of the rat 5-HT2C receptor third intracellular loop were generated and used to identify receptors solubilized from cell lines and rat brain. Western blots of CHAPS-soluble proteins were probed with affinity-purified anti-2C antibodies. The specificity of anti-2C was demonstrated with extracts prepared from NIH/3T3 fibroblasts which stably express functional rat 5-HT2C or 5-HT2A receptors. Extracts from the 5-HT2C cell line, but not the 5-HT2A cell line, contained immunoreactive proteins with masses of 51-52 kDa and 58-68 kDa. In the brain, immunoreactive proteins were identified from choroid plexus extracts with masses of 51 kDa and 58-62 kDa. The major 58-62 kDa and minor 51 kDa proteins were not detected in extracts prepared from the hippocampus, striatum, or frontal cortex using the same amount of CHAPS-soluble protein. These results are consistent with previous studies demonstrating that 5-HT2C receptor binding sites and mRNA are most abundant in choroid plexus. The association of asparagine-linked (N-linked) oligosaccharides with the receptors was examined next. The 5-HT2C receptor cell line (3T3/2C) was grown in the presence of tunicamycin to metabolically inhibit N-linked glycosylation. Proteins from the cell extracts were detected with masses of 40 and 41 kDa. Extracts prepared from 3T3/2C cells (grown in the absence of tunicamycin) and from choroid plexus were incubated with N-glycosidase F to enzymatically remove available N-linked sugars. Immunoreactive proteins were detected with masses of 41 and 42 kDa from 3T3/2C cells and 41 kDa from choroid plexus. Neuraminidase, which cleaves sialic acid (N-acetylneuraminic acid) residues from glycoproteins, reduced the mass of the 51 and 58-62 kDa proteins from the choroid plexus to 50 and 54-58 kDa. In contrast, the 51-52 and 58-68 kDa proteins from 3T3/2C cells were not affected by treatment with neuraminidase. These results demonstrate that 5-HT2C receptors contain N-linked sugars and suggest that sialic acid residues associate with 5-HT2C receptors in the choroid plexus. The oligosaccharide moieties, which contribute up to approximately 30% of the relative mass as judged by SDS-polyacrylamide gel electrophoresis, may impart functional properties to 5-HT2C receptors.

Characterization of potent and selective antagonists at postsynaptic 5-HT1A receptors in a series of N4-substituted arylpiperazines.

Benzocycloalkyl and benzocycloalkenyl moities linked, directly or via an alkyl chain, to oxygen-bearing heteroarylpiperazines were synthesized, in an attempt to obtain potent and selective antagonists at postsynaptic 5-HT1A receptors. From the numerous arylpiperazines described in the literature, 1-(2,3-dihydro-1,4-benzodioxin-5-yl)piperazine (3a) was chosen as a model of an arylpiperazine in view of its selectivity for 5-HT1A receptors versus alpha 1-, alpha 2-, and beta-adrenergic receptors, as well as dopamine D1 and D2 receptors. Two other closely-related arylpiperazines, 1-(1,5-benzodioxepin-6-yl)piperazine (3b) and 1-(benzofuran-7-yl)piperazine (3c), were also examined in this study. All compounds showed high affinity at 5-HT1A sites (8.10 < or = pKis < or = 9.35), and the majority behaved as antagonists in vivo in blocking the hypothermia induced by the 5-HT1A agonist 8-OH-DPAT in the absence of a marked effect alone at equivalent doses. An in vivo evaluation of dopamine D2 receptor antagonist properties revealed that the majority of compounds was devoid of activity at this site, in marked contrast to BMY 7378 which displayed virtually no selectivity for 5-HT1A versus dopamine D2 receptors. Moreover, six compounds of the present series, 8, 10, 11, 14, 25, and 37, showed > 10-fold selectivity in vitro for 5-HT1A versus alpha 1-adrenergic receptors. Compound 14 displayed an optimal compromise between potency (pKi = 8.75), marked antagonist activity, and selectivity toward alpha 1-adrenergic (81-fold) and dopamine D2 (195-fold) receptors. These characteristics clearly distinguish 14 from previously-reported ligands such as the postsynaptic 5-HT1A antagonist BMY 7378 and the weak partial agonist NAN 190 which, in contrast to the compounds of this series, belong to the well-exemplified class of imido derivatives of (o-methoxyphenyl)piperazines. The availability of 14 (S 15535) should facilitate the further elucidation of the functional role and potential therapeutic significance of 5-HT1A receptors.

Developmental switch in the hippocampal serotonin receptor linked to phosphoinositide hydrolysis.

5-HT2A and 5-HT2C receptors couple to the phosphoinositide hydrolysis signal transduction pathway. The present pharmacological analyses provide evidence for a switch in the functional 5-HT receptor in rat hippocampus (from 5-HT2A to 5-HT2C) between the first and third weeks of life. Spiperone and MDL 100,507, antagonists that bind with 300- to 1000-fold higher affinity to 5-HT2A receptors, blocked 5-HT-induced phosphoinositide hydrolysis in hippocampi of 7-day-old, but not 21-day-old, rats. In contrast, the non-selective 5-HT2A/2C receptor antagonists, mesulergine and mianserin, blocked 5-HT-mediated phosphoinositide hydrolysis in both 7- and 21-day-old rats. These results suggest that the 5-HT-induced phosphoinositide hydrolysis signal in hippocampus of 7-day-old rats is mediated predominantly by 5-HT2A receptors, while in 21-day-old rats the phosphoinositide hydrolysis signal is mediated in large part by 5-HT2C receptors. Neither 5-HT2A or 5-HT2C receptor mRNA nor the binding site densities of the two receptors were altered between the two ages, ruling out developmental changes in receptor density as an explanation for the observed differences. We conclude therefore that the hippocampal 5-HT receptor that links to phosphoinositide hydrolysis switches during postnatal development of rats, perhaps reflecting differences in the coupling of 5-HT2A and 5-HT2C receptors to intracellular effector molecules.

Competitive antagonism of serotonin (5-HT)2C and 5-HT2A receptor-mediated phosphoinositide (PI) turnover by clozapine in the rat: a comparison to other antipsychotics.

The antagonist actions of clozapine and several other antipsychotics at 5-hydroxytryptamine (5-HT)2A and 5-HT2C receptors were studied using the in vitro model of 5-HT-induced phosphoinositide (PI) turnover in rat choroid plexus (5-HT2C) and frontal cortex (5-HT2A). While (-)-sulpiride and raclopride were inactive, clozapine and the other drugs behaved as antagonists both at 5-HT2A and at 5-HT2C receptors. Their order of potency (p Inhibitory Concentration (IC)50) was as follows. 5-HT2A receptors: risperidone (9.07) > spiperone > chlorpromazine > clozapine > thioridazine = fluphenazine > haloperidol (6.03). 5-HT2C receptors: clozapine (7.19) > chlorpromazine > risperidone > thioridazine > fluphenazine > spiperone > haloperidol (< 4.00). In each tissue, clozapine shifted the concentration-effect curve for 5-HT to the right in the absence of an alteration in slope or maximal effect. These findings indicate that clozapine acts as a competitive antagonist at 5-HT2A and 5-HT2C receptors and that its antagonist properties are shared, though less potently at 5-HT2C sites, by several, clinically active antipsychotics.

5-HT1A receptors and the tail-flick response. VI. Intrinsic alpha 1A-adrenoceptor antagonist properties can mask the actions of 5-HT1A receptor agonists in the spontaneous tail-flick paradigm.

In view of the involvement of central alpha 1-adrenoceptors in the expression of 5-HT1A receptor-mediated spontaneous tail-flicks (STFs) in the rat, this study examined whether the putative alpha 1-adrenoceptor antagonist (alpha 1-antagonist) properties of certain 5-HT1A receptor agonists, (+)-flesinoxan and LY 165,163, might modify their behavior in the STF paradigm. Whereas the 5-HT1A receptor agonists 8-OH-DPAT and WY 48,723 dose-dependently elicited STFs, (+)-flesinoxan was only weakly active and LY 165,163 was ineffective. Further, (+)-flesinoxan and LY 165,163 antagonized the induction of STFs by 8-OH-DPAT and WY 48,723. Nevertheless, (+)-flesinoxan and LY 165,163 mimicked 8-OH-DPAT and WY 48,723 in eliciting a pronounced rise in plasma corticosterone and a marked hypothermia: these actions were blocked by the 5-HT1A receptor antagonist, (-)-alprenolol, but they were not affected by the alpha 1-antagonist prazosin. Reflecting its antagonist actions at alpha 1-adrenoceptors, prazosin evoked a pronounced ptosis, an action mimicked by the preferential alpha 1A-antagonists WB 4101, methylurapidil and benoxathian, whereas chlorethylclonidine, which irreversibly inactivates alpha 1B- but not alpha 1A-adrenoceptors, was inactive. Although 8-OH-DPAT and WY 48,723 failed to modify palpebral aperture, (+)-flesinoxan and LY 165,163 provoked a ptosis, suggesting that they possess alpha 1A-antagonist properties. The alpha 1-agonists cirazoline and ST 587 did not elicit STFs alone and failed to modify the induction of STFs by 8-OH-DPAT and WY 48,723. By contrast, they greatly facilitated the ability of both (+)-flesinoxan and LY 165,163 to induce STFs. STFs elicited by (+)-flesinoxan and LY 165,163 in the presence of cirazoline or ST 587 were blocked not only by prazosin but also by (-)-alprenolol, BMY 7378 and S 15535, all of which are antagonists of postsynaptic 5-HT1A receptors. The facilitatory actions of cirazoline and ST 587 were selective in that they did not permit the induction of STFs by agonists at other 5-HT receptor subtypes (5-HT1B, 5-HT1C, 5-HT2 or 5-HT3). In conclusion, in the STF paradigm, the high-efficacy agonist actions of (+)-flesinoxan and LY 165,163 at 5-HT1A receptors are "masked" by their "intrinsic" alpha 1A-antagonist properties, the neutralization of which by alpha 1-agonists reveals the activation of 5-HT1A receptors.

Novel benzodioxopiperazines acting as antagonists at postsynaptic 5-HT1A receptors and as agonists at 5-HT1A autoreceptors: a comparative pharmacological characterization with proposed 5-HT1A antagonists.

The novel benzodioxopiperazines [4-(benzodioxan-5-yl)1-[2- (benzocyclobutane-1-yl)ethyl]piperazine] (S 14489), [4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine)] (S 15535) and [4-(benzodioxan-5-yl)1-[2(indan-1-yl)ethyl]piperazine (S15931) competitively displaced the binding of [3H]-8-OH-DPAT at serotonin (5-HT)1A receptors with affinities (pKis) of 9.2, 8.8 and 8.9, respectively. These values compared favorably with those of the structurally related eltoprazine (8.0) and the proposed 5-HT1A antagonists NAN-190 (9.2), MDL 73005 EF (8.9), SDZ 216-525 (8.8), BMY 7378 (8.7), (-)-tertatolol (8.1), (-)-alprenolol (7.7), WAY 100,135 (7.5) and spiperone (6.9). The affinities of S 14489, S 15535 and S 15931 for other 5-HT receptor types (5-HT1B, 5-HT1C, 5-HT1D, 5-HT2 and 5-HT3) were about 50 to 1000-fold lower. The spontaneous tail-flicks, flat-body posture and hypothermia mediated by an action of the 5-HT1A agonist 8-OH-DPAT at postsynaptic 5-HT1A receptors were dose-dependently and completely antagonized by S 14489, S 15535 and S15931 at doses of 0.63 to 10.0 and 2.5 to 40.0 mg/kg for s.c. and oral administration, respectively. They did not induce these responses alone, and in their presence, dose-response curves for 8-OH-DPAT were shifted in parallel to the right without loss of maximal effect. By contrast, eltoprazine, MDL 73005 EF, BMY 7378 and NAN-190 behaved as "partial" agonists and only incompletely antagonized the actions of 8-OH-DPAT in these tests. At 5-HT1A autoreceptors, S 14489, S 15535 and S 15931 acted as agonists in inhibiting striatal 5-hydroxytryptophan accumulation (0.16-2.5 mg/kg, s.c.) and in abolishing the electrical activity of the dorsal raphe nucleus (0.005-0.100 mg/kg, i.v.). Eltoprazine, BMY 7378, NAN-190 and MDL 73005 EF also behaved as agonists at these 5-HT1A autoreceptors, whereas WAY 100,135, spiperone, (-)-tertatolol, (-)-alprenolol and SDZ 216-525 inhibited neither accumulation nor firing. WAY 100,135 and spiperone antagonized the inhibition of DRN firing induced by S 14489, S 15535 and S 15931. The affinity of 15535 for dopamine D1 and D2 receptors, as well as for beta-, alpha 1- and alpha 2-adrenoceptors, was > 100-fold lower than its affinity for 5-HT1A receptors. Further, in vivo, at doses of 10.0 to 40.0 mg/kg, s.c., it showed minimal activity in tests of dopamine D2 (and D1) receptor-mediated activity. Similarly, in vivo, S 15535 was weakly active in a test of alpha 1-adrenoceptor-mediated activity.(ABSTRACT TRUNCATED AT 400 WORDS)

WAY 100,135 and (-)-tertatolol act as antagonists at both 5-HT1A autoreceptors and postsynaptic 5-HT1A receptors in vivo.

In binding studies, WAY 100,135 (N-tertiobutyl-3-[4-(2-methoxyphenyl)-piperazinyl]-2-phenylpropana mide) and (-)-tertatolol showed affinities (Ki) of 29 nM and 10 nM, respectively, at 5-HT1A receptors. In vivo, they both dose dependently blocked the flat-body posture and corticosterone secretion provoked by an action of the 5-HT1A receptor agonist, S 14671 (1-[2-(2-thenoyl-amino)ethyl]-4-[1-(7- methoxynaphtyl)]piperazine), at postsynaptic 5-HT1A receptors. Alone, they exerted little effect. The firing rate of dorsal raphe neurones, which bear inhibitory 5-HT1A autoreceptors, was reduced by S 14671 whereas it was not affected by WAY 100,135 and was increased by (-)-tertatolol. Both WAY 100,135 and (-)-tertatolol blocked the ability of S 14671 to inhibit raphe firing. In conclusion, these data demonstrate that WAY 100,135 and (-)-tertatolol behave as antagonists at both 5-HT1A autoreceptors and postsynaptic 5-HT1A receptors in vivo.

Induction of hypothermia as a model of 5-hydroxytryptamine1A receptor-mediated activity in the rat: a pharmacological characterization of the actions of novel agonists and antagonists.

In this study, we examined the localization of the 5-hydroxytryptamine (5-HT)1A receptors mediating hypothermia in the rat, evaluated the pharmacological specificity of this response and examined the influence of a series of novel 5-HT1A receptor ligands upon core temperature. Administered s.c., 8-hydroxy-(2-di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), an agonist at both pre- and postsynaptic 5-HT1A receptors, elicited pronounced hypothermia. In contrast, BMY 7378, which shows low efficacy at postsynaptic 5-HT1A receptors but high efficacy at presynaptic 5-HT1A receptors, elicited only mild hypothermia. Similarly, 8-OH-DPAT was more efficacious than BMY 7378 in eliciting corticosterone secretion, a response mediated by postsynaptic 5-HT1A receptors, whereas BMY 7378 was as efficacious as 8-OH-DPAT in inhibiting striatal accumulation of 5-hydroxytryptophan, a response mediated by presynaptic 5-HT1A receptors. These data suggest, by analogy, that postsynaptic 5-HT1A receptors mediate hypothermia, an interpretation supported by the observation that destruction of central 5-HT neurons with 5,7-dihydroxytryptamine failed to reduce 8-OH-DPAT-induced hypothermia (DIH). Agonists at 5-HT1B, 5-HT1C, 5-HT2 and/or 5-HT3 receptors did not elicit hypothermia, and drugs releasing 5-HT elicited hyperthermia. In contrast, DIH was fully mimicked by the novel 5-HT1A receptors agonists, eltoprazine, WY 48,723, MDL 72832, tandospirone, S 14671, S 14506 and WY 50,324, whereas the novel partial agonist, zalospirone, was less efficacious. DIH was blocked by (-)-alprenolol, (+/-)-pindolol and the novel beta-blocker, (-)-tertatolol, which also has high affinity for 5-HT1A receptors; in distinction, betaxolol and ICI 118,551, antagonists at beta-1 and beta-2 adrenoceptors, respectively, were inactive. Spiperone, NAN-190 and BMY 7378 also inhibited DIH whereas ritanserin, SCH 39166, raclopride and prazosin, antagonists at 5-HT2 receptors, D1 and D2 dopamine receptors and alpha-1 adrenoceptors, respectively, were inactive. The novel 5-HT1A antagonists, WAY 100,135, MDL 73005 EF and (very potently) SDZ 216-525 all blocked DIH. Potency for induction of hypothermia and inhibition of DIH correlated well with affinity for 5-HT1A binding sites. In conclusion, hypothermia is a highly specific and sensitive response to activation of postsynaptic 5-HT1A receptors. Furthermore, DIH is inhibited by their selective blockade. At postsynaptic 5-HT1A receptors mediating hypothermia, eltoprazine, WY 48,723, MDL 72832 and tandospirone are agonists, zalospirone is a partial agonist and (-)-tertatolol, WAY 100,135, MDL 73005 EF and SDZ 216-525 are antagonists.

S 15535: a highly selective benzodioxopiperazine 5-HT1A receptor ligand which acts as an agonist and an antagonist at presynaptic and postsynaptic sites respectively.

The novel benzodioxopiperazine, S 15535 (4-(benzodioxan-5-yl)1-(indan-2- yl)piperazine), displayed high affinity for 5-HT1A binding sites (1.8 nM) whereas its affinity was 100-fold lower at other 5-HT receptor types, at alpha 1, alpha 2- and beta-adrenoceptors and at dopamine D1 and D2 receptors. In vivo, S 15535 (0.16-10 mg/kg s.c.) acted as an antagonist at postsynaptic 5-HT1A receptors in completely blocking the flat-body posture and hypothermia elicited by the 5-HT1A receptor agonist, 8-OH-DPAT. It had no effect when applied alone. At presynaptic 5-HT1A receptors, S 15535 acted as an agonist in inhibiting striatal accumulation of 5-hydroxytryptophan (0.04-0.63 mg/kg s.c.) and in spiperone reversibly reducing electrical activity of the dorsal raphe nucleus (0.004-0.031 mg/kg i.v.). At doses up to 40.0 mg/kg s.c., S 15535 neither inhibited methylphenidate-induced gnawing nor elicited ptosis suggesting a lack of antagonist properties at, respectively, dopamine D2 receptors and alpha 1-adrenoceptors. In conclusion, S 15535 is a potent 5-HT1A ligand which acts, in vivo, as a highly selective agonist and antagonist at presynaptic and postsynaptic 5-HT1A receptors, respectively.

S 14671: a naphtylpiperazine 5-hydroxytryptamine1A agonist of exceptional potency and high efficacy possessing antagonist activity at 5-hydroxytryptamine1C/2 receptors.

The interaction at 5-hydroxytryptamine (5-HT) receptors of the novel naphtylpiperazine, S 14671 (1-[2-(2-thenoylamino)ethyl]-4[1-(7- methoxynaphtyl)]piperazine), was compared to that of the 5-HT1A ligands, 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), WY 50,324 [N-(29(4-(2-pyrimidinyl)-1-piperazinyl)ethyl)tricyclo(3.3.1.1(3,7) )- decane-1-carboxamide], (+)-flesinoxan, buspirone and BMY 7378 [(8-[2-[4-(2-methoxyphenyl)- 1-piperazinyl]ethyl]-8-azaspirol[-4-]-decane-7,9-dione 2HCl]. S 14671 showed a very high affinity for 5-HT1A sites (pKi, 9.3) as compared to the reference ligands (pKi values, 9.2, 8.7, 8.7, 7.9 and 8.7, respectively). S 14671 bound in an apparently competitive manner and, in distinction to the reference compounds, possessed a Hill Coefficient (1.4) significantly superior to 1. Although showing low affinity at 5-HT1B and 5-HT3 sites, S 14671 displayed significant affinity at both 5-HT1C and 5-HT2 sites; pKi, 7.8 in each case. Furthermore, S 14671 acted as an antagonist of 5-HT-stimulated phosphoinositide turnover in rat choroid plexus (5-HT1C) and cortex (5-HT2). In vivo, upon s.c. administration, S 14671 acted as a high efficacy agonist in models of 5-HT1A receptor-mediated activity: induction of flat-body posture, spontaneous tail-flicks, hypothermia and corticosterone secretion and inhibition of morphine-induced antinociception. In every test, S 14671 was the most potent compound: it was active at doses as low as 5 micrograms/kg s.c. Relative potency across all tests was S 14671 greater than 8-OH-DPAT greater than WY 50,324 greater than (+)-flesinoxan greater than buspirone with BMY 7378 too weak for comparison to be meaningful. The action of S 14671 in 5-HT1A tests was blocked by BMY 7378 and the 5-HT1A antagonist, (-)-alprenolol, but unaffected by the 5-HT1C/2 antagonist, ritanserin, and the 5-HT3 antagonist, ondansetron. Activation of postsynaptic 5-HT1A receptors was confirmed in 5,7-dihydroxytryptamine-lesioned rats, in which the potency of S 14671 to elicit spontaneous tail-flicks was potentiated. Activation of presynaptic receptors was demonstrated by inhibition of the electrical activity of the dorsal raphe nucleus with the following order of relative potency: S 14671 greater than 8-OH-DPAT greater than WY 50,324 greater than BMY 7378 greater than buspirone. Spiperone, which acts as a pure 5-HT1A antagonist at raphe 5-HT1A receptors, blocked the action of S 14671. In conclusion, S 14671 is a structurally novel ligand manifesting high efficacy and exceptional potency at both pre- and postsynaptic 5-HT1A receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Histopathological effects in Poecilia reticulata (guppy) exposed to methyl mercury chloride.

To evaluate the usefulness of histopathology in aquatic toxicity testing, studies were carried out on the small freshwater fish Poecilia reticulata (guppy) following aqueous methyl mercury chloride exposure. Fish were exposed to concentrations of 0, 1.0, 1.8, 3.2, 5.6, or 10 micrograms/L for 1 and 3 months. Histopathological changes included the occurrence of multiple granulomas in various tissues, in particular, the integument and orbit. These changes were accompanied by hyperplasia of monocytopoietic interrenal tissue, and hepatocellular change which was confirmed by morphometry. The latter findings were probably a result of monocyte "consumption" by granulomas, and hepatic synthesis of ("stress") proteins, respectively. The bile duct and, focally, the proximal intestine, showed hyperplasia of the epithelium. In the testis of sexually mature fish (3-month study), degeneration and necrosis of sperm occurred, with severe cases exhibiting Sertoli cell hypertrophy, interstitial inflammation, and absence of mature sperm. Epidermal mucous cells disappeared in the highest concentration used, and, after 3 months, clusters of undifferentiated basophilic cells were seen in the gas gland, which occasionally were suggestive of malignant growth. The changes in the kidney tubules were characterized by degeneration and necrosis of single cells which also showed mitotic figures. This is considered a result of the mitosis-disturbing activity of methyl mercury (MeHg). It is concluded that MeHg has effects on various target organs in guppies with the occurrence of granulomas as the most sensitive indicator, yielding a no-observed-effect concentration (NOEC) of 1.0 micrograms/L. In contrast to mammalian species, no morphologic evidence for neurotoxicity was found.

S 14671: a novel naphthylpiperazine 5-HT1A agonist of high efficacy and exceptional in vivo potency.

The novel, naphthylpiperazine 5-HT1A agonist, S 14671 (4-[(thenoyl-2)aminoethyl]-1-(7-methoxynaphtylpiperazine], displayed very high affinity for 5-HT1A binding sites (pKi = 9.3) as compared to the serotonin (5-HT)1A agonists, 8-OH-DPAT (9.2) and (+)-flesinoxan (8.7) and the 5-HT1A partial agonists, buspirone (7.9) and BMY 7378 (8.8). In vivo, S 14671 induced the typical 5-HT1A agonist-induced responses of hypothermia and spontaneous tail-flicks at doses as low as greater than or equal to 5 micrograms/kg s.c. and greater than or equal to 40 micrograms/kg s.c., respectively. In each test, it was about 10-fold more potent than 8-OH-DPAT and 100-fold more potent than (+)-flesinoxan and buspirone. The actions of S 14671 could be blocked by BMY 7378 and the 5-HT1A receptor antagonist, (-)-alprenolol, but not by the 5-HT1C/2 receptor antagonist, ritanserin, nor the 5-HT3 receptor antagonist, ICS 205930. Thus, S 14671 is a novel 5-HT1A ligand of high efficacy and exceptional in vivo potency.

Glutamate and glycine co-activate while polyamines merely modulate the NMDA receptor complex.

1. Agonists may act at any one of three sites on the N-methyl-D-aspartate (NMDA) receptor-effector complex to promote opening of the associated ion channel. The three sites are activated by i) NMDA, L-glutamate, aspartate, and other dicarboxylic amino acids; ii) glycine, D-serine, D-cycloserine, and others; iii) the polyamines spermine or spermidine, but not cadaverine or putrescine. 2. This opening by exogenous agonists is reflected by an enhanced binding of the phencyclidine-like dissociative anesthetic [3H]MK-801 to rat cortical membranes (well washed to remove endogenous agonists, e.g., L-glutamate, glycine). 3. The effects of adding combinations of agonists yielded stimulation approximately equal to the sum of each agonist's effect, suggesting that in the first approximation the three classes act at independent sites. 4. When the glutamate (E) site was antagonized with D-2-amino-5-phosphonopentanoate (D-AP5), no stimulation in binding could be elicited by agonists at the two other sites. Activation of the E site is therefore necessary but not sufficient for channel opening. 5. When the glycine (G) site was antagonized with 7-chlorokynurenate, no stimulation in binding could be elicited by agonists at the other two sites. Activation of the G site is therefore necessary but not sufficient for channel opening. 6. Of the two putative antagonists for the polyamine (PA) site, ifenprodil fails to completely inhibit the binding of [3H]MK-801, whereas arcaine inhibited [3H]MK-801 binding completely. We present data which question the selectivity of arcaine for the polyamine site, and propose that the polyamine site is merely modulatory, but neither necessary nor sufficient, for channel opening.

Binding of typical and atypical antipsychotics to 5-HT1C and 5-HT2 sites: clozapine potently interacts with 5-HT1C sites.

We determined the affinity of several typical and atypical antipsychotics for the 5-HT1C and 5-HT2 sites using radioligand binding assays. Most of the antipsychotics tested appeared to bind to 5-HT2 sites with affinities that were fairly high (i.e. pKi values between 7 and 9) and significantly higher than for 5-HT1C sites. In contrast, clozapine was found to have a significantly higher affinity for 5-HT1C than for 5-HT2 sites. Clozapine had the highest affinity for 5-HT1C sites of all the compounds tested. These findings are consistent with the hypothesis that an interaction with 5-HT2 receptors may be relevant to the clinical activity of typical antipsychotics. The findings also suggest, however, that an interaction with 5-HT1C sites may be relevant to the mechanism of clinical action of clozapine and, perhaps, of other atypical antipsychotics.

Peritoneal lavage in the treatment of acute pancreatic ascites.

A case of intraperitoneal rupture of a pseudocyst associated with ascites is presented. Peritoneal lavage was successful in the initial management and allowed definitive surgery to be performed at a more opportune time. When faced with a case of intraperitoneal rupture of a pseudocyst, peritoneal lavage should be considered as an alternative to emergency surgery.