Neurogenic dural protein extravasation induced by meta-chlorophenylpiperazine (mCPP) involves nitric oxide and 5-HT2B receptor activation.

The compound m-chlorophenylpiperazine (mCPP), which is known to trigger migraine-like head pain in some subjects, was evaluated for its ability to induce dural plasma protein extravasation (PPE) in guinea pigs. Intravenous mCPP dose-dependently increased PPE. This effect was inhibited by non-selective 5-HT2 receptor antagonists (methysergide, LY53857, LY215840), by a peripherally restricted 5-HT2 receptor antagonist (xylamidine) and by a 5-HT2B selective receptor antagonist (LY202146). These data suggests that peripheral 5-HT2B receptors mediate mCPP-induced PPE. The nitric oxide synthase inhibitor L-NAME and 5-HT1 agonist sumatriptan also blocked mCPP-induced PPE, suggesting a role for nitric oxide (NO) and the trigeminal system, respectively. NO release has been linked to activation of the 5-HT2B receptor on the vascular endothelium. However, LY202146 did not block PPE induced by electrical stimulation of the trigeminal ganglion. These data are consistent with activation of peripheral 5-HT2B receptors initiating PPE and the theory that selective 5-HT2B antagonists might be effective prophylactic therapies for migraine.

The novel 5-Hydroxytryptamine(1A) antagonist LY426965: effects on nicotine withdrawal and interactions with fluoxetine.

LY426965 [(2S)-(+)-1-cyclohexyl-4-[4-(2-methoxyphenyl)-1-piperazinyl]2-methyl- 2-phenyl-1-butanone monohydrochloride] is a novel compound with high affinity for the cloned human 5-hydroxytryptamine (HT)(1A) receptor (K(i) = 4.66 nM) and 20-fold or greater selectivity over other serotonin and nonserotonin receptor subtypes. Both in vitro and in vivo studies indicate that LY426965 is a full antagonist and has no partial agonist properties. LY426965 did not stimulate [(35)S]guanosine-5'-O-(3-thio) triphosphate (GTPgammaS) binding to homogenates of cells expressing the cloned human 5-HT(1A) receptor in vitro but did inhibit 300 nM 5-HT-stimulated [(35)S]GTPgammaS binding with a K(i) value of 3.07 nM. After both p.o. and s.c. administration, LY426965 blocked the lower lip retraction, flat body posture, hypothermia, and increase in rat serum corticosterone induced by the 5-HT(1A) agonist 8-OH-DPAT (8-hydroxy-2-dipropylaminotetralin). In pigeons, LY426965 dose-dependently blocked the stimulus cue induced by 8-OH-DPAT but had no 8-OH-DPAT-like discriminative properties. LY426965 completely reversed the effects of nicotine withdrawal on the auditory startle reflex in rats. In microdialysis experiments, LY426965 administered together with fluoxetine significantly increased extracellular levels of serotonin above those achievable with fluoxetine alone. In electrophysiological studies, the administration of LY426965 produced a slight elevation of the firing rate of 5-HT neurons in the dorsal raphe nucleus of anesthetized rats and both blocked and reversed the effects of fluoxetine on 5-HT neuronal activity. These preclinical results indicate that LY426965 is a selective, full 5-HT(1A) antagonist that may have clinical use as pharmacotherapy for smoking cessation and depression and related disorders.

Comparisons of hallucinogenic phenylisopropylamine binding affinities at cloned human 5-HT2A, -HT(2B) and 5-HT2C receptors.

Since the classical hallucinogens were initially reported to produce their behavioral effects via a 5-HT2 agonist mechanism (i.e., the 5-HT2 hypothesis of hallucinogen action), 5-HT2 receptors have been demonstrated to represent a family of receptors that consists of three distinct subpopulations: 5-HT2A, 5-HT2B, and 5-HT2C receptors. Today, there is greater support for 5-HT2A than for 5-HT2C receptor involvement in the behavioral effects evoked by these agents. However, with the recent discovery of 5-HT2B receptors, a new question arises: do classical hallucinogens bind at 5-HT2B receptors? In the present study we examined and compared the binding of 17 phenylisopropylamines at human 5-HT2A, 5-HT2B, and 5-HT2C receptors. Although there was a notable positive correlation (r>0.9) between the affinities of the agents at all three populations of 5-HT2 receptors, structural modification resulted only in small differences in 5-HT2B receptor affinity such that the range of affinities was only about 50-fold. As with 5-HT2A and 5-HT2C receptor affinity, there is a significant correlation (r>0.9, n=8) between 5-HT2B receptor affinity and human hallucinogenic potency. Nevertheless, given that 5-HT2A and 5-HT2A/2C antagonists - antagonists with low affinity for 5-HT2B receptors - have been previously shown to block the stimulus effects of phenylisopropylamine hallucinogens, it is likely that 5-HT2A receptors play a more prominent role than 5-HT2B and 5-HT2C receptors in mediating such effects despite the affinity of these agents for all three 5-HT2 receptor subpopulations.

Human 5-HT1F receptor-stimulated [35S]GTPgammaS binding: correlation with inhibition of guinea pig dural plasma protein extravasation.

To determine the potency and efficacy of 5-HT1F receptor ligands, a [35S]GTPgammaS binding assay was developed and optimized for the human 5-HT1F receptor. Compounds which are known to be effective in the abortive treatment of migraine were tested for efficacy and potency in this assay. Naratriptan, sumatriptan, zolmitriptan, and rizatriptan all had agonist activity. The 5-HT1F receptor ligand LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]-benzamide) was the most potent compound tested with an EC50 of 2.13 +/- 0.15 nM. LY302148 (5-fluoro-3-[1-[2-(1-methyl-1H-pyrazol-4-yl)ethyl]-4-piperidinyl]-1H-ind ole), methysergide, LY306258 (3-dimethylamino-2,3,4,9-tetrahydro-1H-carbazol-6-ol), dihydroergotamine (DHE), L-694,247 and CP-122,288 were also investigated for potency and efficacy. There was a statistically significant correlation between the pEC50 for the stimulation of [35S]GTPgammaS binding and the pID50 for the inhibition of trigeminal nerve-stimulated dural plasma protein extravasation in the guinea pig. In the course of these studies, it was found that the purportedly selective 5-HT1D receptor antagonist GR127935 inhibited 5-HT1F receptor-stimulated [35S]GTPgammaS binding with a Ki of 39.6 +/- 9.5 nM. These studies demonstrate that 5-HT1F receptor-mediated stimulation of [35S]GTPgammaS binding in a clonal cell system is a reproducible, high throughput assay that is predictive of an in vivo model of 5-HT1F receptor activation.

Substituted naphthofurans as hallucinogenic phenethylamine-ergoline hybrid molecules with unexpected muscarinic antagonist activity.

A series of substituted racemic naphthofurans were synthesized as "hybrid" molecules of the two major prototypical hallucinogenic drug classes, the phenethylamines and the tryptamines/ergolines. Although it was hypothesized that these new agents might possess high affinity for the serotonin 5-HT2A/2C receptor subtypes, unexpected affinity for muscarinic receptors was observed. The compounds initially synthesized for this study were (+/-)-anti- and syn-4-amino-6-methoxy-2a,3,4,5-tetrahydro-2H-naphtho[1,8-bc]furan (4a,b), respectively, and their 8-bromo derivatives 4c,d, respectively. The brominated primary amines 4c,d were assayed initially for activity in the two-lever drug discrimination (DD) paradigm in rats trained to discriminate saline from LSD tartrate (0. 08 mg/kg). Also, 4c,d were evaluated for their ability to compete against agonist and antagonist radioligands at cloned human 5-HT2A, 5-HT2B, and 5-HT2C receptors. After the syn diastereomers were found to have the highest activity in these preliminary assays, the N-alkylated analogues syn-N,N-dimethyl-4-amino-6-methoxy-2a,3,4, 5-tetrahydro-2H-naphtho[1,8-bc]furan (4e) and syn-N, N-dipropyl-4-amino-6-methoxy-2a,3,4,5-tetrahydro-2H-naphtho[1, 8-bc]furan (4f) were prepared and assayed for their affinities at [3H]ketanserin-labeled 5-HT2A and [3H]-8-OH-DPAT-labeled 5-HT1A sites. All of the molecules tested had relatively low affinity for serotonin receptors, yet a preliminary screen indicated that compound 4d had affinity for muscarinic receptors. Thus, 4b,d,e were evaluated for their affinity at muscarinic M1-M5 receptors and also assessed for their functional characteristics at the M1 and M2 isoforms. Compound 4d had affinities of 12-33 nM at all of the muscarinic sites, with 4b,e having much lower affinity. All three compounds fully antagonized the effects of carbachol at the M1 receptor, while only 4d completely antagonized carbachol at the M2 receptor. The fact that the naphthofurans lack LSD-like activity suggests that they do not bind to the serotonin receptor in a way such that the tricyclic naphthofuran nucleus is bioisosteric with, and directly superimposable upon, the A, B, and C rings of LSD. This also implies, therefore, that the hallucinogenic phenethylamines cannot be directly superimposed on LSD in a common binding orientation for these two chemical classes, contrary to previous hypotheses.

[3H]Rauwolscine: an antagonist radioligand for the cloned human 5-hydroxytryptamine2b (5-HT2B) receptor.

In previous reports, [3H]5-HT has been used to characterize the pharmacology of the rat and human 5-HT2B receptors. 5-HT, the native agonist for the 5-HT2B receptor, has a limitation in its usefulness as a radioligand since it is difficult to study the agonist low-affinity state of a G protein-coupled receptor using an agonist radioligand. When using [3H]5-HT as a radioligand, rauwolscine was determined to have relatively high affinity for the human receptor (Ki human = 14.3+/-1.2 nM, compared to Ki rat = 35.8+/-3.8 nM). Since no known high affinity antagonist was available as a radioligand, these studies were performed to characterize [3H]rauwolscine as a radioligand for the cloned human 5-HT2B receptor expressed in AV12 cells. When [3H]rauwolscine was initially tested for its usefulness as a radioligand, complex competition curves were obtained. After testing several alpha2-adrenergic ligands, it was determined that there was a component of [3H]rauwolscine binding in the AV12 cell that was due to the presence of an endogenous alpha2-adrenergic receptor. The alpha2-adrenergic ligand efaroxan was found to block [3H]rauwolscine binding to the alpha2-adrenergic receptor without significantly affecting binding to the 5-HT2B receptor and was therefore included in all subsequent studies. In saturation studies at 37 degrees C, [3H]rauwolscine labeled a single population of binding sites, Kd = 3.75+/-0.23 nM. In simultaneous experiments using identical tissue samples, [3H]rauwolscine labeled 783+/-10 fmol of 5-HT2B receptors/mg of protein, as compared to 733+/-14 fmol of 5-HT2B receptors/mg of protein for [3H]5-HT binding. At 0 degrees C, where the conditions for [3H]5-HT binding should label mostly the agonist high affinity state of the human 5-HT2B receptor, [3H]rauwolscine (Bmax = 951+/-136 fmol/mg), again labeled significantly more receptors than [3H]5-HT (Bmax = 615+/-34 fmol/mg). The affinity of [3H]rauwolscine for the human 5-HT2B receptor at 0 degrees C did not change, Kd = 4.93+/-1.27 nM, while that for [3H]5-HT increased greatly (Kd at 37 degrees C = 7.76+/-1.06 nM; Kd at 0 degrees C = 0.0735+/-0.0081 nM). When using [3H]rauwolscine as the radioligand, competition curves for antagonist structures modeled to a single binding site, while agonist competition typically resulted in curves that best fit a two site binding model. In addition, many of the compounds with antagonist structures displayed higher affinity for the 5-HT2B receptor when [3H]rauwolscine was the radioligand. Typically, approximately 85% of [3H]rauwolscine binding was specific binding. These studies display the usefulness of [3H]rauwolscine as an antagonist radioligand for the cloned human 5-HT2B receptor. This should provide a good tool for the study of both the agonist high- and low-affinity states of the human cloned 5-HT2B receptor.

Mutations of transmembrane IV and V serines indicate that all tryptamines do not bind to the rat 5-HT2A receptor in the same manner.

Two mutations of the rat serotonin 5-HT2A receptor were made, expressed and examined for their ability to bind and be stimulated by certain tryptamines as well as their ability to bind antagonists. Mutation of Ser207 to an Ala (S207A) resulted in no substantial changes in binding of either 5-HT2A antagonists or agonists. In contrast, mutation of Ser239 to an Ala (S239A) resulted in significant changes in the 5-HT2A receptor with some but not all agonists and antagonists examined. Specifically, 5-HT had decreased affinity for the S239A mutated 5-HT2A receptor, showing over a 10-fold decrease in receptor-binding displacement, while still being capable of stimulating IP3 formation. However, the agonists tryptamine, 5-methoxytryptamine (5-MeOT), and N-1-isopropyl-5-methoxytryptamine; and the antagonists ketanserin, LY 86057, and LY 53857 were significantly less affected by a S239A mutation. These results suggest that while 5-HT might have a direct interaction with the Ser239 of the 5-HT2A receptor, tryptamine and 5-MeOT interact with this receptor in a different manner.

Dihydrobenzofuran analogues of hallucinogens. 4. Mescaline derivatives.

Dihydrobenzofuran and tetrahydrobenzodifuran functionalities were employed as conformationally restricted bioisosteres of the aromatic methoxy groups in the prototypical hallucinogen, mescaline (1). Thus, 4-(2-aminoethyl)-6,7-dimethoxy-2,3-dihydrobenzofuran hydrochloride (8) and 1-(8-methoxy-2,3,5,6-tetrahydrobenzo[1,2-b:5,4-b']difuran-4-yl)-2- aminoethane hydrochloride (9) were prepared and evaluated along with 1 for activity in the two-lever drug discrimination (DD) paradigm in rats trained to discriminate saline from LSD tartrate (0.08 mg/kg). Also, 1, 8, and 9 were assayed for their ability to displace [3H]ketanserin from rat cortical homogenate 5-HT2A receptors and [3H]8-OH-DPAT from rat hippocampal homogenate 5-HT1A receptors. In addition, these compounds were evaluated for their ability to compete for agonist and antagonist binding to cells expressing cloned human 5-HT2A, 5-HT2B, and 5-HT2C receptors. Finally, agonist efficacy was assessed by measurement of phosphoinositide hydrolysis in NIH 3T3 cells expressing the rat 5-HT2A or 5-HT2C receptors. Although 1 fully substituted for LSD in the DD assays (ED50 = 33.5 mumol/kg), neither 8 nor 9 substituted for LSD, with just 50% of the rats administered 8 selecting the drug lever, and only 29% of the rats administered 9 selecting the drug lever. All of the test compounds had micromolar affinity for the 5-HT1A and 5-HT2A receptors in rat brain homogenate. Curiously, the rank order of affinities of the compounds at 5-HT2A sites was opposite their order of potency in the behavioral assay. An evaluation for ability to stimulate phosphoinositide turnover as a measure of functional efficacy revealed that all the compounds were of approximately equal efficacy to serotonin in 5-HT2C receptors. At 5-HT2A receptors, however, 8 and 9 were significantly less efficacious, eliciting only 61 and 45%, respectively, of the maximal response. These results are consistent with the proposed mechanism of action for phenethylamine hallucinogens, that such compounds must be full agonists at the 5-HT2A receptor subtype. In contrast to the 2,5-dimethoxy-substituted phenethylamines, where rigidification of the methoxy groups had no deleterious effect on activity, the loss of activity in the 3,4,5-trioxygenated mescaline analogues may suggest that the 3 and 5 methoxy groups must remain conformationally mobile to enable receptor activation.

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

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

Pharmacologic characterization of the human 5-hydroxytryptamine2B receptor: evidence for species differences.

The 5-Hydroxytryptamine2B (5-HT2B) receptor was cloned originally from rat stomach fundus and its pharmacology was determined to be consistent with that of the receptor responsible for contraction of rat fundal tissue in response to 5-HT. Recently, the cloning of the human homolog of the 5-HT2B receptor has been reported and, in this study, we report a detailed pharmacological characterization of this human receptor. The cloned human 5-HT2B receptor has high affinity for [3H]5-HT (Kd = 10.6 +/- 1.5 nM), and the pharmacology of this receptor matches closely the rat 5-HT2B receptor, consistent with the structural relatedness of these two proteins. Most compounds tested show no difference in affinity for the human or rat receptors. There were, however, groups of compounds that discriminated between the human and rat 5-HT2B receptors. Examples include certain ergolines such as methysergide and mesulergine, which have higher affinity for the human than for the rat receptor. Similarly, certain benzoylpiperidines, e.g., ketanserin, pirenperone and pipamperone, and the antipsychotics clozapine and olanzapine have higher affinity for the human 5-HT2B receptor. These pharmacological findings reinforce the desirability of having the human forms of receptors when considering drug actions.

Receptor subtype and density determine the coupling repertoire of the 5-HT2 receptor subfamily.

The 5-Hydroxytryptamine (5-HT)2C receptor (originally known as the 5-HT1C receptor) is a member of the 5-HT2 subfamily of G protein coupled receptors, which is known to couple to phospholipase C. Within the 5-HT2 subfamily, only the 5-HT2C receptor also coupled to inhibition of forskolin-stimulated cAMP production when expressed at high density (12 pmol/mg membrane protein) in stably transformed AV12 cells. The 5-HT2C receptor coupled with high efficacy to both phospholipase C as measured by IP3 (inositol 1,4,5-trisphosphate) production and to inhibition of forskolin-stimulated cAMP production (EC50 = 2.98 nM +/- 0.9 and IC50 = 47.99 nM +/- 10.25 respectively). The 5-HT2A and 5-HT2B receptors, while coupling to phospholipase C with high affinity (EC50s of 19.24 nM +/- 6.44 and 1.24 nM +/- 0.136 respectively), did not decrease adenylyl cyclase activity. The 5-HT2C receptor actions in both systems showed the expected pharmacology for the 5-HT2C receptor, e.g., mesulergine antagonized the effects of 5-HT and spiperone did not. Preincubation of cells with PTX showed that the G protein coupling of the 5-HT2C receptor to phospholipase C is PTX insensitive, while the G protein coupling to inhibition of adenylyl cyclase is PTX sensitive, even to concentrations as low as 20 ng/ml of PTX. PTX pretreatment of the 5-HT2C bearing cells also unmasked a small stimulatory effect on adenylyl cyclase. When expressed at low density the 5-HT2C receptor potentiated forskolin-stimulated cAMP production by 2 fold while still maintaining its ability to enhance PI hydrolysis. A more modest potentiation of cAMP production was noted with low density expression of the 5-HT2B receptor. Thus the ability of the 5-HT2C receptor to interact with several effectors through at least two different G proteins is, in part, receptor subtype specific but also influenced by receptor density.

A novel class of 5-HT2A receptor antagonists: aryl aminoguanidines.

Local delivery of serotonin (5-HT) produces a rapid edematous response in soft tissues via increased fluid extravasation which is prevented by 5-HT2 antagonists such as ketanserin or mianserin. Here we report the effects of a new class of aminoguanidine 5-HT2 antagonists, with relative selectivity for 5-HT2A receptors which are potent inhibitors of 5-HT-induced paw edema in the rat. Radioligand binding studies with 125I DOI on human 5-HT2A and 5-HT2C receptors and with 3H-5-HT on human 5-HT2B receptors demonstrated that, LY314228, and LY320954 displayed some selectivity for the 5-HT2A receptor. When compared to binding at other 5-HT2 receptor subtypes, LY314228 had an 18.6-fold greater affinity for the 5-HT2A site over the 5-HT2B site, and 2.6 fold greater at the 5-HT2C site. LY320954 displayed similar preference for 5-HT2A sites. Both compounds also inhibited 5-HT-induced paw swelling in rats, with ED50's of 6.4 and 4.8 mg/kg (for LY314228 and LY320954, respectively). These studies offer evidence for a novel class of pharmacophores for the 5-HT2 receptor family which show greater relative affinities for the 5-HT2A receptor subclass.

Molecular biology of serotonin receptors.

Over the last several years the use of molecular cloning technology has revealed a vast diversity among serotonin (5-HT) receptors, whereby what was previously thought to be a family of three pharmacologically defined classes of 5-HT receptors is actually composed of seven distinct subfamilies designated 5-HT1-7. The 5-HT1, 5-HT2, and 5-HT5 subfamilies currently consist of five, three and two subtypes respectively while the 5-HT3, 5-HT4, 5-HT6, and 5-HT7 "subfamilies" have at present one subtype each. Fourteen separate genes encode 13 receptors which fall in the superfamily of G protein-coupled receptors and one ligand-gated ion channel receptor. Our lab has contributed to the elucidation of this subtype diversity by cloning the cDNAs from both rat and human encoding the 5-HT2B receptor. This receptor subtype is equally homologous (approximately 70%) to the 5-HT2A and 5-HT2C receptors when amino acids comprising the transmembrane domains are compared and is clearly the third member of the 5-HT2 subfamily. The 5-HT2B receptor has been shown to couple to phosphoinositide hydrolysis as do the other two members of this subfamily when expressed in AV12-664 cells. Limited pharmacological analyses indicated that both rat and human 5-HT2B receptors are similar but distinguishable. With one tantalizing exception, the mRNAs for these receptors appear to be similarly distributed within rat and human. The 5-HT2B receptor mRNA is not found in rat brain, whereas in human brain it has been identified in multiple regions. This later finding suggests that the 5-HT2B receptor may be serving a unique CNS function in man that is absent in rat.

Molecular cloning, functional expression, and mRNA tissue distribution of the human 5-hydroxytryptamine2B receptor.

Clones encoding a portion of the human 5-hydroxytryptamine (5-HT)2B receptor gene were isolated from a human placental genomic library. Based on distribution studies of 5-HT2B receptor mRNA, human uterus cDNA libraries were constructed and screened, resulting in the isolation of several full-length cDNA clones. These clones harbored a common single open reading frame encoding a protein of 481 amino acids. The deduced amino acid sequence of the human 5-HT2B receptor displayed 91.5% identity within the transmembrane domains and 82% identity overall with the rat 5-HT2B receptor. The human 5-HT2B receptor stably expressed in AV12-664 cells demonstrated high affinity (Kd = 10.18 +/- 1.60 nM), saturable [3H]serotonin binding, similar to that previously described for the rat 5-HT2B receptor. The pharmacological profile of the human 5-HT2B receptor was virtually identical to that of the rat 5-HT2B receptor, with the exceptions of the 5-HT2A receptor antagonists ketanserin and spiperone. Both compounds exhibited higher affinity at the human 5-HT2B receptor (ketanserin, Ki = 376 +/- 58 nM; spiperone, Ki = 697 +/- 54 nM) than at the rat 5-HT2B receptor (ketanserin, Ki = 3559 +/- 175 nM; spiperone, Ki = 3278 +/- 92 nM). Functional coupling of the human 5-HT2B receptor was also demonstrated in AV12-664 cells, where 5-HT produced a dose-dependent increase in phosphatidylinositol hydrolysis (EC50 = 27 +/- 12 nM) analogous to that seen with the rat 5-HT2B receptor. Reverse transcription-polymerase chain reaction studies revealed human 5-HT2B receptor mRNA to be expressed in many tissues, including the central nervous system. The presence of 5-HT2B receptor mRNA in human brain and not in rat brain raises the possibility that the 5-HT2B receptor may be of significance in higher brain function.

DOI and alpha-methylserotonin: comparative vascular and nonvascular smooth muscle effects and central 5-hydroxytryptamine2 receptor affinities.

Both alpha-methylserotonin and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) are agonists at 5-hydroxytryptamine2 (5-HT2) receptors. The present study compared these agonists for their binding affinities at the high- and low-affinity states of the 5-HT2 receptor and for their contractile activities in certain smooth muscle preparations. Both agonists contracted the rat aorta and rat jugular vein, tissues possessing 5-HT2 receptors, and contraction was blocked by ketanserin. However, alpha-methylserotonin produced greater maximal response (80-90% maximum response to serotonin) than DOI. In the rat jugular vein, the calculated dissociation constant of DOI (-log Kb = 7.7) corresponded well with its affinity for [3H]ketanserin- (pKi = 7.5) but not [125I]DOI- (pKi = 8.6) radiolabeled sites. This might suggest that binding to the agonist low-affinity state of the 5-HT2 receptor is more relevant to vascular agonist activity. alpha-Methylserotonin was slightly more potent than serotonin in contracting the rat aorta but not the jugular vein, whereas DOI was more potent in the jugular vein than in the aorta. In the jugular vein but not the aorta, the relative potency of these agents corresponded well with their relative affinities for either the [3H]ketanserin- or the [125I]DOI-labeled 5-HT2 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Species differences in the pharmacology of the 5-hydroxytryptamine2 receptor: structurally specific differentiation by ergolines and tryptamines.

Species differences in the recognition of a series of ergolines by the 5-hydroxytryptamine2 (5-HT2, serotonin2) receptor were investigated in four species, the rat, pig, squirrel monkey and human. In pig frontal cortical membranes the initial studies showed that the ergolines gave shallow displacement curves against [3H]ketanserin binding. The component of [3H]ketanserin binding having low affinity for the ergolines was determined to be the result of [3H]ketanserin binding to alpha-1 adrenergic receptors. Thus, in all subsequent assays prazosin was used to mask [3H]ketanserin binding to alpha-1 adrenergic receptors. Examination of a series of ergolines revealed a distinct pattern in the species selectivity. Compounds that were unsubstituted at the N1 position of the ergoline nucleus showed higher affinity for the pig, squirrel monkey and human 5-HT2 receptors than for the rat. Conversely, compounds that had an N1-isopropyl substituent showed higher affinity for the rat receptor compared to the pig, squirrel monkey and human 5-HT2 receptors. For example, LY53857, a widely used 5-HT2 antagonist, has an isopropyl substituent at position N1 of the ergoline nucleus and exhibited a 4- to 5-fold higher affinity for the rat 5-HT2 receptor, whereas its N1-unsubstituted homologue, LY86057, had more than 10-fold higher affinity for the pig, squirrel monkey and human 5-HT2 receptors. Similar results were seen with three additional ergoline pairs, each having different substituents at the C8 position compared to LY53857. Even an N1-substitution on LY53857 as small as a methyl group, LY108742, resulted in the compound having higher affinity for the rat 5-HT2 receptor compared to the other species.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacological characteristics of the newly cloned rat 5-hydroxytryptamine2F receptor.

The rat 5-hydroxytryptamine (5-HT)2F (serotonin2F) receptor is a newly cloned member of the 5-HT2/1C receptor family. The pharmacology of the 5-HT2F receptor was explored using a variety of structurally different compounds in a radioligand binding assay. In addition, the 5-HT2F receptor was shown to stimulate production of inositol 1,4,5-trisphosphate in the transformed cells. Based on the affinities of the compounds tested, their known affinities for certain of the other 5-HT receptors, and the fact that activation of the cloned 5-HT2F receptor stimulates inositol 1,4,5-trisphosphate production, the 5-HT2F receptor was determined to be a novel receptor and a member of the 5-HT2/1C receptor family. In addition, several agonists and partial agonists were evaluated for contractile activity in the rat stomach fundus, and these activities were correlated with their binding affinities at the 5-HT2F receptor. A highly significant correlation was found, providing additional evidence that is consistent with the 5-HT2F receptor being the stomach fundal contractile receptor. [3H]5-HT had high affinity for this receptor both at 37 degrees and at 0 degree (Kd = 7.87 +/- 0.55 and 0.12 +/- 0.02 nM, respectively). The difference in affinity for [3H]5-HT at the two temperatures prompted an investigation of potential temperature-dependent differences in the binding affinities of agonists versus antagonists. Agonists such as 5-HT, 5-methoxytryptamine, etc., showed higher affinity for the 5-HT2F receptor at 0 degree than at 37 degrees, whereas antagonists such as methysergide, 1-naphthylpiperazine, etc., showed no difference in affinity for this receptor at the two different temperatures. Therefore, the affinity of a compound for the 5-HT2F receptor at 37 degrees versus 0 degree was shown to be useful for predicting agonist or antagonist activity. Additionally, information is provided about some of the structural requirements for the affinity of certain tryptamines at the 5-HT2F receptor.

Molecular cloning, functional expression, and pharmacological characterization of a novel serotonin receptor (5-hydroxytryptamine2F) from rat stomach fundus.

Using the polymerase chain reaction amplification technique in conjunction with conventional cloning techniques, we have isolated a novel member of the serotonin [5-hydroxytryptamine (5-HT)] 1C/2 receptor subfamily (designated 5-HT2F) from rat stomach fundus. Two DNA fragments were amplified from cDNA synthesized from rat stomach fundus poly(A)+ RNA using the polymerase chain reaction technique with degenerate oligonucleotide primers derived from sequence comparisons of the second, third, and sixth putative transmembrane domains of known 5-HT receptors. These fragments were used as hybridization probes to isolate full length cDNA clones from rat stomach fundus cDNA libraries. Full length cDNA clones contained one open reading frame encoding a 479-amino acid protein with seven hydrophobic domains, characteristic of members of the guanine nucleotide-binding protein-coupled receptor superfamily. Within these seven putative transmembrane domains, the 5-HT2F receptor shared greatest homology with the rat 5-HT1C and 5-HT2 receptor subtypes (70% and 68%, respectively). Cell lines stably expressing the 5-HT2F receptor were established and demonstrated functional coupling to phosphatidylinositol hydrolysis upon 5-HT stimulation analagous to that observed for the 5-HT1C and 5-HT2 receptors. Membranes from the stably transfected cell lines (but not the untransfected parental lines) exhibited high affinity (Kd = 7.9 nM), saturable binding of [3H]5-HT. Maximum binding ranged from 0.1 to 2.4 pmol/mg of protein, depending on the clonal isolate. Using [3H]5-HT as the basis for a radioligand binding assay, the relative affinities of several tryptamine and piperazine derivatives for the cloned 5-HT2F receptor correlated with their relative potencies to contract the rat stomach fundus. These data suggest a probable relationship between this novel 5-HT2F receptor and the serotonin contractile receptor of the rat stomach fundus.