Multiple conformations of native and recombinant human 5-hydroxytryptamine(2a) receptors are labeled by agonists and discriminated by antagonists

We have expanded previous studies with the 5-hydroxytryptamine (5-HT)(2) receptor agonist (+/-)-1-(2,5-dimethoxy-4-[(125)I]iodophenyl)-2-aminopropane [(+/-)-[(125)I]DOI] in human brain that had shown biphasic competition curves for several 5-HT(2A) receptor antagonists by using new selective antagonists of 5-HT(2A) (MDL100,907) and 5-HT(2C) (SB242084) receptors together with ketanserin and mesulergine. Autoradiographic competition experiments were performed with these antagonists in human brain regions where (+/-)-[(125)I]DOI labels almost exclusively 5-HT(2A) receptors (frontal cortex and striosomes). Furthermore, the effect of uncoupling receptor/G protein complexes on antagonist competition was studied with guanosine-5'-(beta,gamma-imido)triphosphate [Gpp(NH)p]. Competition experiments with (+/-)-[(3)H]1-(4-bromo-2,5-dimethoxyphenil)-2-aminopropane [(+/-)-[(3)H]DOB] were also performed in membranes from Chinese hamster ovary cells (CHOFA4) expressing cloned human 5-HT(2A) receptors. In both systems, ketanserin and MDL100,907 displayed biphasic competition profiles, whereas SB242084 and mesulergine competed monophasically. In absence of antagonist, 100 microM Gpp(NH)p decreased brain (+/-)-[(125)I]DOI specific binding by 40 to 50% and (+/-)-[(3)H]DOB specific binding to CHOFA4 cells by 30%. The remaining agonist-labeled uncoupled sites were still displaced biphasically by ketanserin and MDL100,907, with unaltered affinities. Saturation experiments were performed in CHOFA4 cells. (+/-)-[(3)H]DOB labeled two sites (K(d(h))= 0.8 nM, K(d(l)) = 31.22 nM). Addition of 100 microM Gpp(NH)p resulted in a single low-affinity (K(d) = 24.44 nM) site with unchanged B(max). [(3)H]5-HT showed no specific binding to 5-HT(2A) receptors. These results conform with the extended ternary complex model of receptor action that postulates the existence of partly activated receptor conformation(s) (R*) in equilibrium with the ground (R) and the activated G protein-coupled (R*G) conformations. Thus, both in human brain and CHOFA4 cells, the agonists possibly label all three conformations and ketanserin and MDL100,907 recognize with different affinities at least two of these conformations.     

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

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

EMD 281014, a new selective serotonin 5-HT2A receptor antagonist

The 5-HT2A receptor ligand 7-[4-[2-(4-fluoro-phenyl)-ethyl]-piperazine-1-carbonyl]-1H-indole-3-carbonitrile HCl (EMD 281014) selectively binds to human (h) and rat 5-HT2A receptors (IC50 values 0.35 and 1 nM, respectively; vs. 1334 nM for h5-HT2C) and inhibited 5-HT-stimulated [35S]guanosine 5'-O-3-thiotriphosphate (GTPgammaS)-accumulation in h5-HT2A transfected Chinese hamster ovary cells (IC50 9.3 nM). EMD 28014 counteracted the N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)-induced decrease of [3H]ketanserin binding in rat frontal cortex (ID50 0.4 mg/kg p.o.) and R-(-)-1-(2,5-dimethoxy-4-iodophenyl)-aminopropane (DOI)-induced head-twitch behaviour in mice (ID50 0.01 mg/kg s.c., 0.06 mg/kg p.o.), demonstrating unique selectivity and efficacy.     

Facilitation of 8-OHDPAT-induced forepaw treading of rats by the 5-HT2 agonist DOI

The potency of the serotonin 1A (5-HT1A) agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT), to induce forepaw treading was increased 20-fold after co-treatment with the 5-HT2 agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). DOI induced head twitches which were inhibited by 8-OHDPAT. The putative 5-HT1B agonist, 1-(3-trifluoromethylphenyl)piperazine (TFMPP), had a weak effect on the responses to DOI or 8-OHDPAT. The forepaw treading induced by 8-OHDPAT plus DOI was inhibited by high doses of (-)-alprenolol, ketanserin or ritanserin, but was not influenced by the beta-adrenoceptor antagonist, ICI 118.551, or the 5-HT3 antagonist, ICS 205-930. A non-effective dose of (-)-alprenolol increased the inhibitory effect of ketanserin and ritanserin. These results indicate a complex and different interaction between 5-HT1A and 5-HT2 receptors in the expression of two behavioural responses mediated by 5-HT.     

Serotonin-glutamate interaction in rat cerebellum: involvement of 5-HT1 and 5-HT2 receptors

The effects of serotonin (5-HT) on the release of endogenous glutamate (GLU) in rat cerebellum were investigated in slices depolarized with 35 mM K+. The Ca2+-dependent release of GLU was potently inhibited by 5-HT in a concentration-dependent way. Release was also inhibited by the 5-HT1 receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and by the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI). The inhibition by 10 nM 5-HT was partly (35-40%) counteracted by the 5-HT2 receptor antagonist ketanserin but was fully blocked by the mixed 5-HT1/5-HT2 receptor antagonist methiothepin. The effect of 8-OH-DPAT was not affected by ketanserin but was totally antagonized by methiothepin, while the effect of DOI was entirely suppressed by ketanserin. Ketanserin or methiothepin themselves increased (by 23 and 55%, respectively, at 10 nM) the K+-evoked release of GLU. In conclusion the release of endogenous GLU in rat cerebellum can be inhibited by 5-HT through receptors of the 5-HT1 and 5-HT2 type. The enhancement of GLU release by ketanserin or methiothepin could suggest a tonic inhibition. The possible localization of the 5-HT receptors involved in the interaction with the GLU systems is discussed.     

Autoradiographic analysis of 5-HT2A binding sites in the brain of Sardinian alcohol-preferring and nonpreferring rats.

The density of 5-HT2A binding sites in the brain of Sardinian alcohol-preferring (sP) and nonpreferring (sNP) rats was evaluated, using [3H]ketanserin for quantitative autoradiography. The highest [3H]ketanserin binding levels were found in the anterior olfactory nucleus, prefrontal cortex, medial prefrontal cortex, post-genual anterior cingulate cortex, insular cortex and claustrum. Statistically significant differences between sP and sNP rats were found in prefrontal cortex, medial prefrontal cortex and post-genual anterior cingulate cortex, where sP rats showed about 20% lower [3H]ketanserin binding levels. No significant difference was found in other areas, although some of them showed slightly lower [3H]ketanserin binding density in sP rats. The 5-HT2A receptor agonist, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane hydrochloride (DOI), microinjected into the medial prefrontal cortex, induced a lower number of wet dog shakes in sP than in sNP rats. These results indicate a different density of 5-HT2A binding sites, and a different functional regulation of 5-HT2A receptor mechanisms in discrete brain areas of sP, in comparison to sNP rats. These findings, and those showing lower levels of 5-HT in the frontal cortex of sP rats, suggest that altered 5-HT function in fronto-cortical areas could be linked to the genetic predisposition to high voluntary ethanol intake in these rats.     

Chronic treatment with (+/-)DOI, a psychotomimetic 5-HT2 agonist, downregulates 5-HT2 receptors in rat brain

(+/-)DOI (2,5-dimethoxy-4-iodo-phenylisopropylamine) is a hallucinogenic phenylalkylamine that has been characterized as a 5-HT2-selective agonist. Chronic treatment with (+/-)DOI [1.0 mg/kg/day (2.8 mumol/kg) for 8 days] significantly reduced the binding of [3H]ketanserin, [125I]LSD, and [125I]R-DOI as measured at single ligand concentrations in rat cortical homogenates. In saturation studies, chronic DOI treatment significantly lowered the Bmax of [3H]ketanserin binding and the high-affinity binding of [125I]R-DOI without altering the Kd values. In rats treated acutely with a single dose of (+/-)DOI, binding of [125I]R-DOI, [125I]LSD, and [3H]ketanserin was not significantly different from controls in membranes preincubated at 37 degrees C for 60 minutes. In all experiments nonspecific binding was determined by incubation with 1 microM ritanserin. This work demonstrates that chronic treatment with a 5-HT2-selective agonist hallucinogen reduces the number of binding sites for 5-HT2 agonists as well as for 5-HT2 antagonists.     

2,5-Dimethoxy-4-iodoamphetamine (DOI) inhibits Delta9-tetrahydrocannabinol-induced catalepsy-like immobilization in mice

The effect of the serotonin 5-HT(2A/2C)-receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on Delta(9)-tetrahydrocannabinol (THC)-induced catalepsy-like immobilization was studied in mice. DOI (0.3 and 1 mg/kg, i.p.) significantly inhibited the catalepsy-like immobilization induced by THC (10 mg/kg, i.p.). In contrast, the selective 5-HT(2C)-receptor agonist 8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one (WAY 161503) had no effect on this catalepsy-like immobilization. Moreover, the 5-HT(2A)-receptor antagonist ketanserin (0.3 mg/kg, i.p.) reversed the inhibition of THC-induced catalepsy-like immobilization caused by DOI (1 mg/kg), whereas the selective 5-HT(2C)-receptor antagonist 6-chloro-2,3-dihydro-5-methyl-N-[6-(2-methyl-3-pyridinyl)oxyl]-3-pyridinyl]-1H-indole-1-carboxyamide (SB 242084) did not affect this inhibitory effect of DOI. On the other hand, ketanserin (0.3 and 1 mg/kg, i.p.) enhanced the catalepsy-like immobilization induced by THC (6 mg/kg, i.p.). Thus, on the basis of these results, it appears that 5-HT(2A)-receptor mechanisms might be responsible for the inhibitory effect of DOI on THC-induced catalepsy-like immobilization.     

Inhibition of amino acid release by 5-HT1B receptor agonist in the rat prefrontal cortex

In vivo microdialysis in conscious rats was used to evaluate the effect of 5-HT1A agonist (+/-)-8-hydroxy-2-(n-dipropylamino)tetralin (8-OH-DPAT), 5-HT2A/2C agonist (+/-)- 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and 5-HT1B receptor agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo-[3,2-b]pyrid-5-one (CP 93129) on the veratridine-evoked glutamate (Glu) and aspartate (Asp) release in the rat prefrontal cortex. CP 93129 at concentrations between 50-500 microM significantly reduced Glu and Asp release. The effect of CP 93129 was attenuated by intraperitoneal (ip) administration of the selective 5-HT1B receptor antagonist N-[3-[3-(dimethylamino)ethoxy]-4-methoxyphenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-[ 1, 1'-biphenyl]-4-carboxamide (SB 216641) at a dose of 2 mg/kg. Neither DOI (100 microM) nor 8-OH-DPAT given locally at concentration of 100 microM or peripherally at doses of 0.1 and 1 mg/kg ip, influenced stimulated Glu and Asp release. We suggest that cortical glutamatergic neurons possess 5-HT1B heteroceptors and their activation may be responsible for the inhibitory effect of 5-HT on Glu and Asp release.     

Development of homogeneous high-affinity agonist binding assays for 5-HT2 receptor subtypes

The serotonin (5-hydroxytryptamine) 5-HT2 receptor subfamily consists of three members, 5-HT2A, 5-HT2B, and 5-HT2C. These receptors share high homology in their amino acid sequence, have similar signaling pathways, and have been indicated to play important roles in feeding, anxiety, aggression, sexual behavior, mood, and pain. Subtype-selective agonists and antagonists have been explored as drugs for hypertension, Parkinson's disease, sleep disorders, anxiety, depression, schizophrenia, and obesity. In this study, we report the development of homogeneous agonist binding assays in a scintillation proximity assay (SPA) format to determine the high-affinity binding state of agonist compounds for the human 5-HT2C, 5-HT2A, and 5-HT2B receptors. The 5-HT2 agonist 1-(4- [125I]iodo-2,5-dimethoxyphenyl)-2-aminopropane ([125I]DOI) was used to label the high-affinity sites for the 5-HT2A and 5-HT2C receptors. The high-affinity sites for the 5-HT2B receptor were labeled with [3H]lysergic acid diethylamide. Total receptor expression was determined with the 5-HT2 antagonist [3H]mesulergine for the 5-HT2B and 5-HT2C receptors, and [3H]ketanserin for the 5-HT2A receptor. The agonist high-affinity binding sites accounted for 2.3% (5-HT(2C) receptor), 4.0% (5-HT2A receptor), and 22% (5-HT2B receptor) of the total receptor population. Competition binding studies using known agonists indicated high Z' values of the agonist binding assays in SPA format (Z' > 0.70). The Ki values of 5-HT, (R)(-)DOI, and VER-3323 for the 5-HT2A, 5-HT2B, and 5-HT2C receptors by SPA format were equivalent to published data determined by filtration binding assays. These results indicate that agonist binding assays in SPA format can be easily adapted to a high throughput assay to screen for selective 5-HT2C receptor agonists, as well as for selectivity profiling of the compounds.     

Cerebral ischaemia reduces the density of 5-HT2 binding sites in the frontal cortex of the gerbil

The 5-HT2 antagonist [3H]ketanserin labels a single population of high affinity sites (Kd 0.48 +/- 0.03 nM; Bmax 206 +/- 20 fmol/mg protein) in the frontal cortex of the gerbil. Specific binding of [3H]ketanserin was displaced by a number of 5-HT2A antagonists ritanserin, cyproheptadine and methysergide) but not by the 5-HT1A agonist, 8-hydroxy-2-(di-n- propylamino)tetralin (8-OH-DPAT) or the 5-HT1A/1B agonists 5-carboxyamidotryptamine (5-CT) or RU 24969, indicating that the labelled site probably represents the 5-HT2 receptor. Cerebral ischaemia induced in either a 3 hr unilateral non-recovery model or a 5 min bilateral, 3-day recovery model, resulted in a significant decrease in the density of 5-HT2 binding sites in the ischaemic frontal cortex without an apparent change in their affinity for the ligand. The decrease in density was not simply related to levels of 5-HT because occlusion of the right carotid artery for 3 hr resulted in bilateral depletion of 5-HT but only in an ipsilateral reduction in the density of binding sites. In addition, a significant decrease in the density of 5-HT2 binding sites occurred in the recovery model at a time when the levels of 5-HT in the cortex were unaltered.     

3H]-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and [3H] ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to [3H]ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding the serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both [3H]DOB and [3H]ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] to this system caused a rightward shift and steepening of agonist competition curves for [3H] ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity [3H]DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that [3H]DOB and [3H]ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein. These observations are consistent with the classical view of interconvertible agonist affinity states of GTP-binding protein-coupled receptors and strongly support the "two state" over the "two receptor" model for DOB binding to the 5-HT2 receptor.     

Effects of the 5-HT2A receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on plasma glucose and glucagon levels of rats

Effects of the 5-hydroxytryptamine (5-HT)2A receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on plasma glucagon levels were studied in rats. Systemic injection of DOI induces significant increases in plasma glucagon levels. Hyperglucagonemia induced by DOI was dose-dependently prevented by the 5-HT2A receptor antagonist ketanserin. Adrenodemedullation abolished hyperglucagonemia elicited by DOI. Previous report demonstrated that the peripheral 5-HT2A receptor agonist induces hyperglycemia in rats but does not increase plasma glucagon levels at doses inducing hyperglycemia. Therefore, our findings suggest that DOI-induced glucagon release was elicited by stimulation of the central 5-HT2A receptor, which in turn increasing adrenaline release.     

Age-related behavioural,neurochemical and radioligand binding changes in the central 5-HT system of Sprague-Dawley rats

Mature (3–4 months) and aged (18–19 months) Sprague-Dawley (SD) rats were treated with 5-HT receptor agonists and drug-induced behaviours monitored. The 5-HT_2/1C agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), induced wet dog shakes and back muscle contractions which were significantly increased in aged, compared to mature, rats, suggesting an age-related enhancement of 5-HT₂ receptor function. In contrast, the selective 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) induced forepaw treading, flat body posture, hypothermia and hyperactivity which were not significantly different in aged compared to mature rats. Levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the hippocampus and frontal cortex were measured using high performance liquid chromatography with electrochemical detection. There were no age-related changes in hippocampal 5-HT or 5-HIAA. However both 5-HT and 5-HIAA were increased in the frontal cortex of aged SD rats. 8-OH-DPAT reduced 5-HIAA in both regions examined in mature rats, an effect which was attenuated in the aged rats, suggesting an age-related reduction in presynaptic 5-HT_1A receptor function. DOI did not induce any changes in 5-HT or 5-HIAA in either of the regions examined. Radioligand binding studies with [³H] ketanserin showed there to be no significant age-related changes in cortical 5-HT₂ receptor density or affinity. In the samples taken from mature rats GTP shifted the competition curve to DOI and reduced the proportion of high affinity agonist binding sites; this effect was not observed in the aged samples, suggesting that there may be age-related changes in G-protein-mediated receptor-effector coupling mechanisms.     

Characterisation of agonist binding on human 5-HT2C receptor isoforms.

The 5-HT2C receptor is expressed in different isoforms as a result of mRNA editing. Both INI (unedited) and VSV (a fully edited version) isoforms are abundant in rat brain. The VSV isoform lacks the high affinity recognition site for 5-HT, which may be caused by low efficiency coupling to G-proteins. In this study we have investigated the pharmacology of the agonist binding site of these two isoforms of the 5-HT2C receptor. The VSV isoform was expressed in Chinese hamster ovary cells (CHO) and the INI isoform in both Chinese hamster ovary cells and human embryonic kidney cells (HEK-293). Saturation analysis using [3H]5-HT revealed high and low affinity recognition sites on the INI isoform in both cell types whilst the VSV isoform did not have the high affinity binding site for [3H]5-HT. Displacement studies were undertaken using [3H]5-HT to label the receptors. In these studies the affinity of agonists (5-HT, Ro600175 ((S)-2-(6-Chloro-5-fluoroindol-1-yl)-1-methylethylamine), MK212 (6-Chloro-2-(piperazinyl) pyrazine), mCPP (1-(m-chlorophenyl)-piperazine), TfMPP (N-(m-trifluoromethylphenyl)piperazine), DOI (1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane), DOB (1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane) and 8OH-DPAT (8-hydroxy-2-(di-N-propylamino)tetralin) was higher at the INI isoform, whilst antagonist affinity (ketanserin and mesulergine) did not change between the two receptor isoforms. There were no differences between the INI isoform expressed in the CHO and HEK-293. This suggests that the INI isoform of the 5-HT2C receptor is pharmacologically similar to the VSV form of the 5-HT2C receptor but that it couples more efficiently to G-proteins.     

Stimulation of 5-HT 1A receptors increases the seizure threshold for picrotoxin in mice

To evaluate the possible role of 5-HT 1A and 5-HT 2A receptors in the anticonvulsant effect of swim stress, mice were pre-treated with agonists and antagonists of these receptors prior to exposure to stress and the intravenous infusion of picrotoxin. 8-OH-DPAT ((+/-)-8-hydroxy-2-(di-n-propylamino) tetralin) and WAY-100635 (a selective agonist and antagonist of 5-HT 1A receptors), DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) and ketanserin (a 5-HT 2A/2C receptor agonist and antagonist) were used. Results demonstrated that 1 and 3 mg/kg of 8-OH-DPAT increased the doses of picrotoxin producing running/bouncing clonus, tonic hindlimb extension and death in stressed and unstressed mice, respectively. Pre-treatment with WAY (0.3 mg/kg) prevented the effect of 8-OH-DPAT (3 mg/kg). DOI (2.5 mg/kg) and ketanserin (1 mg/kg) failed to affect the seizure threshold for picrotoxin. The results show that stimulation of 5-HT 1A receptors exerts anticonvulsant actions in stressed and unstressed mice, while stimulation of 5-HT 2A/2C receptors does not interfere with the effect of stress on picrotoxin-induced convulsions.     

5-HT1 and 5-HT2 binding properties of derivatives of the hallucinugen 1-(2,5-dimethoxyphenyl)-2-aminopropane (2,5-DMA)

The affinities of a series of 1-(2,5-dimethoxyphenyl)-2-aminopropane (2,5-DMA) derivatives, most of which are hallucinogenic in man, and several related agents were determined for rat cortical serotonin (5-HT) binding sites. Competition assays were performed in which these agents were competed for the 5-HT2 binding of [3H]ketanserin, or the 5-HT1 binding of [3H]LSD (in the presence of ketanserin). The R(-)-isomers of DOI, DOM and DON (i.e. the 4-iodo, -methyl and -nitro derivatives of 2,5-DMA) were found to be more potent than their racemates and demonstrated selectivity for 5-HT2 sites. These same agents in competing for [3H]ketanserin binding resulted in Hill coefficients significantly less than unity; computer-assisted analysis indicated a two-state model better fit the data. In the presence of 10(-4) M Gpp(NH)p the competition curve for R(-)-DOI produced a Hill coefficient close to unity. These results are consistent with the hypothesis that certain derivatives of 2,5-DMA, in particular R(-)-DOI, may be potent and selective agonists at 5-HT2 binding sites, sites that may constitute a serotonin receptor that is regulated by a guanine nucleotide regulatory protein. Conversely, the interactions of these agents at 5-HT1 sites was with a lower affinity and a lack of stereoselectivity. Although DOI and DOM are amongst the most potent of these agents as hallucinogens, it is still too premature to draw any conclusions regarding a possible relationship between 5-HT binding and hallucinogenic potency.     

5-HT2 receptors differentially modulate dopamine-mediated auto-inhibition in A9 and A10 midbrain areas of the rat

5-HT (20 microM) enhanced dopamine (DA) D2-like receptor mediated reduction of the firing rate of DA neurons in the substantia nigra pars compacta (A9) and ventral tegmental area (A10) in a rat midbrain slice preparation. Quinpirole (30 nM) induced a mean reduction of the firing rate in A9 and A10 DA neurons to 64 +/- 4%, respectively, 71 +/- 5% of the baseline value. Bath application of 5-HT in the presence of quinpirole further reduced the firing rate to 37 +/- 7% in A9 and 33 +/- 13% in A10. The 5-HT2 receptor agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI, 500 nM) enhanced quinpirole-induced reduction of firing rate of A10 DA neurons, but not of A9 DA neurons, suggesting that different 5-HT receptor subtypes are involved in modulation of dopamine D2-like receptor mediated inhibition in the two regions. The selective 5-HT2A receptor antagonist MDL100907 and the selective 5-HT2C receptor antagonist SB242084 (50 and 500 nM) both abolished the enhancement of quinpirole-induced reduction by either 5-HT or DOI, suggesting the involvement of direct and indirect (possibly via interneurons) modulation pathways in A10. The involvement of 5-HT and specific 5-HT2 receptors in augmentation of auto-inhibition in A10 could have important implications for our understanding of the mechanism of atypical antipsychotic drug action.     

5-HT2 receptor agonists increase spontaneous sympathetic nerve discharge

The selective 5-HT2 agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) produced a marked increase in spontaneous sympathetic nerve discharge recorded from the inferior cardiac nerve in chloralose anesthetized cats. DOI (0.01-1.0 mg/kg i.v.) increased sympathetic nerve discharge to a maximum of 1750% of control values. The increase in sympathetic nerve discharge produced by DOI was reversed by the 5-HT2 antagonists ketanserin and LY 53857. In addition, pretreatment with ketanserin, but not prazosin, completely prevented the increase in sympathetic nerve discharge produced by DOI. These data are discussed in relationship to the role of serotonin in the regulation of activity in central sympathetic pathways.     

Serotonin inhibition of the NMDA receptor/nitric oxide/cyclic GMP pathway in human neocortex slices: involvement of 5-HT(2C) and 5-HT(1A) receptors

The NMDA receptor/nitric oxide (NO)/cyclic GMP pathway and its modulation by 5-hydroxytryptamine (5-HT) was studied in slices of neocortical samples obtained from patients undergoing neurosurgery. The cyclic GMP elevation produced by 100 microM NMDA was blocked by 100 microM of the NO synthase inhibitor N(G)-nitro-L-arginine (L-NOARG) or by 10 microM of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha] quinoxaline-1-one (ODQ). The NMDA effect was prevented by 5-HT or by the 5-HT(2) agonist (+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane ((+/-)-DOI; EC(50)=22 nM). The (+/-)-DOI inhibition was insensitive to the 5-HT(2A) receptor antagonist MDL 100907 or the 5-HT(2B) antagonist rauwolscine; it was largely prevented by 1 microM of the non-selective 5-HT(2C) antagonists mesulergine (5-HT(2A,B,C)), ketanserin (5-HT(2A,C)) or SB 200646A (5-HT(2B,C)); it was completely abolished by 0.1 microM of the selective 5-HT(2C) receptor antagonist SB 242084. The NMDA-induced cyclic GMP elevation also was potently inhibited by the selective 5-HT(2C) agonist RO 60-0175 and by the antidepressant trazodone, both added at 1 microM, in an SB 242084-sensitive manner. Finally, the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 1 microM) inhibited the NMDA-evoked cyclic GMP response, an effect blocked by the selective 5-HT(1A) receptor antagonist WAY 100635. In conclusion, the NMDA receptor/NO/cyclic GMP pathway in human neocortex slices can be potently inhibited by activation of 5-HT(2C) or 5-HT(1A) receptors.