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.     

Antagonism of the effects of the hallucinogen DOM and the purported 5-HT agonist quipazine by 5-HT2 antagonists

Rats trained to discriminate 1.0 mg/kg of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) from saline in a two-lever operant choice task were administered doses of mescaline, LSD, 5-methoxy-N,N-dimethyltryptamine (5-OMe DMT), quipazine, TFMPP and RU-24969. The DOM-stimulus generalized to the three hallucinogenic agents and to quipazine, but not to the purported serotonin agonists TFMPP or RU-24969. Pretreatment of the animals with the 5-HT2 antagonists ketanserin and pirenperone antagonized the effect produced by DOM. Pirenperone also blocked DOM-stimulus generalization to mescaline, LSD, 5-OMe DMT and quipazine. The results of this study suggest that the discriminative stimulus effects of DOM, the three hallucinogenic agents to which DOM-stimulus generalization occurred, and quipazine, may involve those sub-populations of serotonin receptors that are labeled by tritiated ketanserin (i.e. 5-HT2 sites).     

Regulation of 5-HT2 receptors in rat cortex. Studies with a putative selective agonist and an antagonist

The phenylisopropylamine derivative 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) has been suggested recently as a selective serotonin2 (5-HT2) receptor agonist. Because of the potential importance of such a tool for investigations of 5-HT2 receptor regulation, receptor binding studies were performed in rats after acute and chronic treatment with DOI, the selective 5-HT2 antagonist ketanserin, or vehicle. Single injections of 5 or 10 mg/kg DOI reduced the Bmax of cortical sites labeled with [3H]1-(2,5-dimethoxy-4-bromo-phenyl)-2-aminopropane and [3H]ketanserin (9-32 or 32-46%, respectively). Chronic daily treatment with DOI (3-9 mg/kg) further down-regulated 5-HT2 sites in cortex identified with either [3H]ketanserin (-60%) or with [3H]DOB (-75%), without altering Kd values or affecting 5-HT1 sites. In vitro addition to the [3H]ketanserin or [3H]DOB binding assay of 10 nM to 1 microM DOI resulted in competitive inhibition, suggesting that down-regulation found in vivo was not secondary to residual drug. Chronic treatment with ketanserin (10 mg/kg) also down-regulated both [3H]ketanserin (-38%) and [3H]DOB (-58%) sites in cortex without charges in 5-HT1 sites. In naive cortex, competition experiments revealed a Ki (nM) for ( +/- )-DOI of 1.7 +/- 0.02 at sites labeled by [3H]DOB, and a KH and KL of 4.8 +/- 1.5 and 53 +/- 2 nM at sites labeled by [3H]ketanserin. These data indicate that in chronic treatment, DOI, like ketanserin, is highly selective for 5-HT2 vs 5-HT1 sites at behaviorally useful doses. However, a representative putative 5-HT2 selective agonist and antagonist have similar effects on 5-HT2 receptors labeled by agonist or antagonist radioligands.     

A structure-affinity study of the binding of 4-substituted analogues of 1-(2,5-dimethoxyphenyl)-2-aminopropane at 5-HT2 serotonin receptors

With [3H]ketanserin as the radioligand, structure-affinity relationships (SAFIRs) for binding at central 5-HT2 serotonin receptors (rat frontal cortex) were examined for a series of 27 4-substituted 1-(2,5-dimethoxyphenyl)-2-aminopropane derivatives (2,5-DMAs). The affinity (Ki values) ranged over a span of several orders of magnitude. It appears that the lipophilic character of the 4-position substituent plays a major role in determining the affinity of these agents for 5-HT2 receptors, 2,5-DMAs with polar 4-substituents (e.g. OH, NH2, COOH) display a very low affinity (Ki greater than 25,000 nM) for these receptors, whereas those with lipophilic functions display a significantly higher affinity. The results of these studies prompted us to synthesize and evaluate examples of newer lipophilic derivatives and several of these (e.g. n-hexyl, n-octyl) bind with very high (Ki values = 2.5 and 3 nM, respectively) affinities at central 5-HT2 sites. Although, 2,5-DMAs are generally considered to be 5-HT2 agonists, preliminary studies with isolated rat thoracic aorta suggest that some of the more lipophilic derivatives (e.g. the n-hexyl and n-octyl derivatives) are 5-HT2 antagonists.     

5-HT1 and 5-HT2 binding characteristics of 1-(2,5-dimethoxy-4-bromophenyl)-2-aminopropane analogues

1-(2,5-Dimethoxy-4-bromophenyl)-2-aminopropane (DOB; 1a) is a purported serotonin (5-HT) agonist that binds selectively to central 5-HT2 binding sites. Systematic removal of any or all of the aromatic substituents had relatively little effect on 5-HT1 binding but reduced 5-HT2 binding by approximately 2 or more orders of magnitude. Demethylation of the 2-methoxy group of 1a, or introduction of an N-n-propyl group, doubled 5-HT1-site affinity but decreased 5-HT2-site affinity by 3- and 30-fold, respectively. In tests of stimulus generalization, using rats trained to discriminate DOM from saline, the 2-demethyl and N-propyl derivatives were found to produce stimulus effects similar to those of DOB. In addition, the S-(+) isomer of the iodo analogue of 1a was found to possess one-third the affinity of its R-(-) enantiomer at 5-HT2 sites and also resulted in DOM-stimulus generalization. Of the DOB analogues examined, DOB (1a) possesses optimal selectivity for 5-HT2 binding.     

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

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

Membrane stabilising properties of the selective 5-HT2 receptor agonist, (+/-)-DOI.

The effects of (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a selective 5-HT2 receptor agonist, on nerve conduction in the isolated frog sciatic nerve trunk were examined. Compound action potential amplitude was concentration dependently decreased by (+/-)-DOI (EC50 = 1.2 mM), an effect that was not altered by the presence of ketanserin and with a similar potency to lignocaine. (+/-)-propranolol and disopyramide. The results indicate that (+/-)-DOI has membrane stabilising properties, in addition to its reported 5-HT2 agonist actions.     

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.     

(+/-)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane(DOI) and alpha-methyl-5-HT: 5-HT2 receptor agonistic action on phosphatidylinositol metabolism in the rat fronto-cingulate and entorhinal cortex.

In the present study, the effects of 5-HT and two 5-HT1c/5-HT2 receptor agonists, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and alpha-methyl-serotonin (alpha-Me-5-HT) on phosphoinositide hydrolysis were compared, to determine whether DOI and alpha-Me-5-HT were full agonists. Consistent with the results obtained from previous studies, both (+/-)-DOI and alpha-Me-5-HT stimulated turnover of phosphoinositide in a concentration-dependent manner. However, the response obtained with these 5-HT1c/5-HT2 receptor agonists was only 30-40% of that of 5-HT. The stimulation of hydrolysis of phosphoinositide, produced by both 5-HT2 receptor agonists, was potently antagonized by ritanserin (a 5-HT1c/5-HT2 receptor antagonist) and alpha-phenyl-1-(2-phenylethyl)-4-piperine methanol [(+)-MDL 11,939, a 5-HT2 receptor antagonist] but not by granisetron (BRL a 5-HT3 receptor antagonist), suggesting that the action of DOI and alpha-Me-5-HT was primarily mediated by 5-HT2 receptors. When the effect of increasing the concentration of 5-HT on turnover of phosphoinositide was measured in the presence of a 1 microM concentration of the 5-HT3 receptor antagonist granisetron, the response obtained was similar to the response produced by the 5-HT2 receptor agonists, DOI and alpha-Me-5-HT. These results confirm the previous finding that 5-HT stimulates hydrolysis of phosphoinositide by interacting with 5-HT1c/5-HT2 and 5-HT3 receptors. Moreover, they suggest that DOI and alpha-Me-5-HT are full agonists at the 5-HT2 receptor, coupled to hydrolysis of phosphoinositide in the cortex of the rat.     

Differential interactions of indolealkylamines with 5-hydroxytryptamine receptor subtypes

Affinities of drugs for 21 indolealkylamine derivatives, some with putative hallucinogenic activity, were determined at 5-HT1A, 5-HT2A and 5-HT2B recognition sites, using radioligand competition studies. Nearly all of the derivatives displayed greatest potency for the 5-HT2A receptor, labelled by [125I]R-(-)DOI in the cortex of the rat. Most derivatives displayed 2-10 times lower affinity at the HT2B receptor labelled by [3H]ketanserin in bovine cortex. Derivatives lacking ring substituents displayed lower affinities for all of the recognition sites, compared to derivatives substituted in the 4- or 5-position of the indole ring. The 4-hydroxylated derivatives displayed 25-380-fold selectivity for the 5-HT2A site, vs the 5-HT1A site, while the 5-substituted derivatives displayed approximately equal potency at the 5-HT1A and 5-HT2A sites. Affinity of all the compounds at the 5-HT2B site was greater than 300 nM. The 6-substituted derivatives displayed greater than micromolar affinities for all of the 5-HT recognition sites examined. The size of the N,N-dialkyl substituent was a secondary determinant of affinity, with groups larger than N,N-diisopropyl resulting in a marked reduction in affinity at both the 5-HT2A and 5-HT1A recognition sites. This study demonstrated that hallucinogenic 4-hydroxy-indolealkylamines, like psychotomimetic phenylisopropylamines, bind potently and selectively to the 5-HT2A recognition site, labelled by [125I]R-(-)DOI. This provides further evidence indicating that this recently described subtype of the 5-HT2 receptor may partially mediate the action of hallucinogenic agents.     

N-methyl derivatives of the 5-HT2 agonist 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane

1-(4-Bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB) is a serotonin (5-HT) agonist that displays a high affinity and selectivity for a certain population of central 5-HT binding sites (i.e., 5-HT2 sites). In the present study, (a) an enantiomeric potency comparison was made for the optical isomers of DOB and (b) the activity of N-monomethyl-,N,N-dimethyl-, and N,N,N-trimethyl-DOB was examined. (R)-(-)-DOB (Ki = 0.39 nM) was found to have 6 times greater affinity than its S-(+) enantiomer at [3H]DOB-labeled (rat cortical homogenates) 5-HT2 sites; N-methylation of racemic DOB resulted in a decrease in affinity that was at least 1 order of magnitude per methyl group. Similar results were obtained in an in vivo drug discrimination paradigm with rats as subjects and (R)-(-)-DOB (0.2 mg/kg) as the training drug. Thus, the R-(-) isomer of DOB is more active than its S-(+) enantiomer and than any of the possible N-methyl derivatives of DOB, both with respect to affinity at central 5-HT2 binding sites and with respect to potency in the behavioral (i.e., stimulus generalization) studies.     

The selective 5-HT2 receptor antagonist amperozide attenuates 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane-induced inhibition of male rat sexual behavior.

This study was aimed at exploring the role of 5-HT2/5-HT1C neurotransmission in male rat sexual behavior. The administration of the 5-HT2/5-HT1C agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (1 mg/kg), suppressed sexual activity in most of the animals. The suppressive effect of DOI was antagonized by treatment with amperozide, a selective 5-HT2 receptor antagonist, in doses which did not by themselves affect sexual activity. In addition, several other serotonin antagonists were tested with varying affinity profiles for 5-HT2/5-HT1C receptors, including ketanserin, ritanserin, and mesulergine. All these compounds antagonized the suppressive action of DOI. In contrast, no antagonizing effect was obtained by treatment with (-)-alprenolol, a 5-HT1A antagonist. The present findings suggest that 5-HT2/5-HT1C receptors might be involved in the neural control of male rat sexual behavior, presumably by exerting an inhibitory influence on the behavior.     

Do functional relationships exist between 5-HT1A and 5-HT2 receptors?

To investigate the possible functional relationship between 5-HT1 and 5-HT2 receptors, we studied the effects of a nonselective 5-HT agonist (5-MeO DMT), a 5-HT1A-selective (8-OH-DPAT) and a 5-HT1B/5-HT1C-selective (TFMPP) agonist on the head-twitch behavior induced by the putative 5-HT2-selective receptor agonist (+/-)-DOI. In the mouse (+/-)-DOI produced the head-twitch response in a dose-dependent manner and (-)-DOI was twice as potent as the (+) isomer. Selective 5-HT2 antagonists, ketanserin and spiperone, dose-dependently inhibited the (+/-)-DOI-induced head-twitch response. The nonselective and the 5-HT1A-selective agonists also dose-dependently reduced the behavior, whereas 5-HT1B/5-HT1C-selective agonist (TFMPP) failed to affect the (+/-)-DOI-induced response. Taken together with previously published literature data, we propose a 5-HT1A inhibitory action on the 5-HT2 receptor-mediated response when induced by its selective agonist (+/-)-DOI.     

Comparison of behavioral properties of di- and tri-methoxyphenylisopropylamines

Prominent among the class of hallucinogenic phenylisopropylamines is the 2,5-dimethoxy substitution pattern; this pattern has long been recognized as being an important feature of the more potent agents within this class. The purpose of this present study was to explore the behavioral properties of a series of methoxylated phenylisopropylamines in order to determine the effect of other substitution patterns and the relative importance of individual methoxy groups. Rats, trained to discriminate the hallucinogenic agent 2,5-dimethoxy-4-methyl-phenylisopropylamine (DOM) from saline in a two-lever drug discrimination task, were challenged with a series of di- and trimethoxyphenylisopropylamines (i.e., DMA and TMA derivatives). DOM-stimulus generalization was found to occur with 2,4-DMA but not with 2,3-DMA, 2.6-DMA, or 3,5-DMA; generalization also occurred with 2,3,4-TMA, 2,3,5-TMA, 2,4,6-TMA and 3,4,5-TMA. The 2,4-dimethoxy pattern also emerges as an important feature among the more active agents.     

5-HT(2A) and 5-HT(2C) receptor stimulation are differentially involved in the cortical dopamine efflux-Studied in 5-HT(2A) and 5-HT(2C) genetic mutant mice

Both 5-HT(2A) and 5-HT(2C) receptors modulate cortical dopamine efflux, but in opposite directions. We have now compared the ability of the three 5-HT(2A/2C) receptor agonists, DOI (R(-)-2,5-dimethoxy-4-iodoamphetamine), mCPP (meta-chlorophenylpiperazine) and MK-212 (6-Chloro-2-(piperazinyl) pyrazine), to modulate cortical dopamine efflux in 5-HT(2A) and 5-HT(2C) genetic mutant mice. In the 5-HT(2A) mice, the preferential 5-HT(2A) receptor agonist DOI (2.5mg/kg, s.c.) induced a slight but significant increase in cortical dopamine efflux only in the wild type (WT) mice; MK-212 (2.5mg/kg) reduced dopamine efflux in both WT and receptor knockout (KO) mice; moreover, MCPP, 2.5mg/kg, had no effect in either types. In 5-HT(2C) mice, DOI increased dopamine efflux in both types; while MK-212 decreased dopamine efflux in the WT, but not the receptor KO mice. These results provide new evidence that 5-HT(2A) receptor stimulation enhances and 5-HT(2C) receptor stimulation inhibits cortical dopamine efflux, and suggest the effects of DOI, MK-212 and mCPP on the cortical dopamine efflux are due to their different abilities on 5-HT(2A) and 5-HT(2C) receptors stimulation. Of these three agents, only DOI, the more selective 5-HT(2A) receptor agonist, is hallucinogenic. The absence of hallucinations with mCPP may be due to its relatively more potent 5-HT(2C) receptor agonist effect, inhibiting the ability of mCPP to enhance dopamine efflux in cortical and perhaps limbic regions as well. The present data provide additional evidence that hallucinations are due, in part, to 5-HT(2A) rather than 5-HT(2C) receptor stimulation. These findings suggest that 5-HT(2C) receptor agonists may be useful as antipsychotics, consistent with previous suggestions.     

Stereoselective LSD-like activity in d-lysergic acid amides of (R)- and (S)-2-aminobutane.

The (R)- and (S)-2-butylamides of d-lysergic acid were prepared and evaluated in behavioral and biochemical assays of 5-HT2 agonist activity. In rats trained to discriminate 0.08 mg/kg LSD tartrate from saline, both isomers completely substituted for the training stimulus. Similarly, both isomers were found to possess very high affinity in displacing [125I]-(R)-DOI ([125I]-(R)-1-(2,5-dimethoxy-4-iodophenyl)-2- aminopropane) from rat cortical homogenate 5-HT2 receptors and in displacing [3H]-8-OH-DPAT ([3H]-8-hydroxy-2-(di-n-propylamino)tetralin) from rat hippocampal 5-HT1A receptors. The difference in activity between the two isomeric amides was significant in both the behavioral and binding assays, with the R isomer possessing greater potency. Molecular mechanics were used to predict the active geometries of the subject compounds. It was found that the (R)-2-butylamide has a conformation quite similar to LSD, while the (S)-2-butylamide does not. These results suggest that stereochemical properties of the amide substituent of hallucinogenic lysergamides may exert a critical influence on activity. It is concluded that the conformation of the amide function may directly affect binding through stereoselective interactions with a hydrophobic region on the receptor, indirectly by inducing conformational changes elsewhere in the molecule, or by a combination of these two mechanisms.     

Evidence for the involvement of 5-HT2A receptors in mild mesenteric ischemia/reperfusion dysfunctions in mice

In this study, the involvement of 5-HT2A receptors on mesenteric ischemia-reperfusion injury was examined in mice. Intestinal ischemia produced by 45 min occlusion of superior mesenteric artery was followed by 24h reperfusion (I/R). The 5-HT2A selective antagonist, ketanserin (0.5 mgkg(-1)) or the 5-HT2A agonist DOI (0.25 mgkg(-1)) was intravenously administered before ischemia and 8h after the beginning of reperfusion. The effects were compared with those obtained in sham operated animals (S). Ketanserin prevented the upper gastrointestinal transit delay induced by I/R (P<0.01), protected intestine from leukocyte recruitment as indicated by jejunal myeloperoxidase activity (P<0.05) and reverted Evans Blue extravasation elicited by I/R in lung, colon and jejunum (P<0.05). On the other hand, 5-HT2A activation by DOI mimicked the effects of I/R in S mice prolonging small intestine transit (P<0.05) and enhancing neutrophil accumulation in jejunal tissues (P<0.05). Furthermore, the reduction of ADP-induced platelet aggregation in plasma of I/R mice was prevented by ketanserin treatment. All together, these findings support the critical involvement of 5-HT2A receptor subtype in mediating the damage induced by mesenteric I/R in mice.     

Role of 5-HT(2) receptors in the tryptamine-induced 5-HT syndrome in rats

We distinguished the functions of the different 5-hydroxytryptamine-2 (5-HT(2)) receptor (5-HT(2)R) subtypes in the tryptamine-induced 5-HT syndrome in rats using (1) the 5-HT(2A)R antagonist R93274 (N-[(3-p-fluorophenyl-1-propyl)-4-methyl-4-piperidinyl]-4-amino-5-iodo-2-methoxybenzamide), the 5-HT(2A/C)R antagonist R99647 (2-(dimethylaminomethyl)2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine), the 5-HT(2B/C)R antagonist SB-242084 (6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline), and several 5-HT(2)R antagonists (ketanserin, risperidone, pipamperone and mianserin); and (2) chronic 5-HT(2)R activation by 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). In contrast to SB-242084, the selective 5-HT(2A)R antagonist R93274 as well as the non-selective 5-HT(2A)R antagonists (R99647, ketanserin, risperidone, pipamperone and mianserin) significantly inhibited tryptamine-induced forepaw treading and tremors, and reversed peripherally mediated cyanosis into hyperaemia; only the 5-HT(2A/C)R antagonists R99647 and mianserin inhibited the tryptamine-induced hunched back. Intermittent DOM administration (intravenously every 48 h for 12 days) did not change the centrally mediated tryptamine-induced forepaw treading, tremors and hunched back at 1, 4 or 7 days after the last DOM pretreatment. The DOM-induced head twitch response, measured immediately after every DOM injection, was not affected. In contrast, peripherally mediated cyanosis was reversed into hyperaemia in 75, 11 and 20% of all pretreated rats at 1, 4 and 7 days, respectively, after the last DOM administration. Taken together, these finding suggest that central 5-HT(2A)Rs mediate tryptamine-induced forepaw treading and tremors, that peripheral 5-HT Rs mediate tryptamine-induced cyanosis, and that 5-HT(2A)Rs mediate tryptamine-induced hunched back. Peripheral 5-HT(2C)Rs are more sensitive to desensitization after intermittent treatment with an agonist than central 5-HT(2A)Rs.     

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.     

Evidence that 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced hyperthermia in rats is mediated by stimulation of 5-HT2A receptors

The effects of various 5-HT receptor subtype-selective antagonists were studied on phenylisopropylamine hallucinogen1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced hyperthermia in Wistar rats, in an attempt to characterize the 5-HT receptor subtype mediating DOI-induced hyperthermia. Intraperitoneal administration of DOI to rats produced hyperthermia with a peak effect at 60 min. Pretreatment with propranolol (-adrenoceptor antagonist that also has binding affinity for 5-HT_1A, 5-HT_1B and 5-HT_2C sites), MDL-72222 or ondansetron (5-HT₃ antagonists) did not attenuate DOI-induced hyperthermia. In contrast, pretreatment with metergoline (5-HT₁/5-HT₂ antagonist), ketanserin, LY53857, mesulergine, mianserin and ritanserin (5-HT_2C/5-HT_2A antagonists), as well as spiperone (5-HT_1A/5-HT_2A/D₂ antagonist), significantly attenuated DOI-induced hyperthermia. Furthermore, daily administration of DOI (2.5 mg/kg per day) for 17 days did not produce either tolerance to its hyperthermic effect or modifym-CPP-induced hyperthermia in rats. These findings suggest that DOI-induced hyperthermia in rats is mediated by stimulation of 5-HT_2A receptors.