Specific interaction of 5-HT-moduline with human 5-HT1b as well as 5-HT1d receptors expressed in transfected cultured cells

5-HT_1B receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters. 5-HT-moduline (Leu-Ser-Ala-Leu) has been shown to specifically interact with a very high apparent affinity and in a non-competitive manner with 5-HT_1B receptors (Massot et al. 1996; Rousselle et al. 1996). Using transfected cells expressing either 5-HT_1B or 5-HT_1D receptors, it was shown that 5-HT-moduline prevents the binding of [³H]5-HT to 5-HT_1B as well as to 5-HT_1D receptors with similar biochemical characteristics: the IC₅₀ of the peptide was 1.2×10^–12 M for 5-HT_1B and 9×10^–13 M for 5-HT_1D receptors. The observed effect corresponds to a marked decrease of the maximal binding for [³H]5-HT on 5-HT_1B (–51.2±1%) as well as 5-HT_1D binding (–47.2±7.7% of the control binding) whereas the affinity of 5-HT is increased by a factor close to 3. No effect is observed using the “scrambled” peptide (Ala-Leu-Leu-Ser). Parallel assays using transfected cells expressing 5-HT_1A or 5-ht₆ receptors did not show any significant change induced by the peptide under similar assay conditions. The interaction of the peptide was also studied on the functional activity related to the stimulation of the receptors as measured by the increase in [³⁵S]GTPγS binding reflecting the coupling of the receptor to the G-protein. 5-HT-moduline yields an antagonistic effect on the 5-HT induced coupling with a corresponding IC₅₀=1.2±0.7×10^–12 M for 5-HT_1B and 9.8±4.0×10^–12 M for 5-HT_1D receptors, respectively. The present results demonstrate that 5-HT-moduline interacts with 5-HT_1D as well as 5-HT_1B receptors and possesses a non-competitive antagonistic activity, likely corresponding to its role of endogenous allosteric modulator, specific for both 5-HT_1B and 5-HT_1D receptors. Received: 26 March 1998 / Accepted: 22 May 1998     

Effects of selective h5-HT1B (SB-216641) and h5-HT1D (BRL-15572) receptor ligands on guinea-pig and human 5-HT auto- and heteroreceptors

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT_1B receptor ligand, and of BRL-15572, a preferential h5-HT_1D receptor ligand, on the presynaptic h5-HT_1B and h5-HT_1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT_1D heteroreceptors in the human atrium. The brain preparations, preincubated with [³H]serotonin ([³H]5-HT), and the segments of atrial appendages, preincubated with [³H]noradrenaline, were superfused with modified Krebs’ solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K⁺ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2μM; concentration 154 times higher than its K_i at h5-HT_1D receptors) but was antagonized by SB-216641 (0.1μM; concentration 100 times higher than its K_i at h5-HT_1B receptors; apparent pA₂ 8.45). SB-216641 (0.1μM) by itself facilitated, whereas BRL-15572 (2μM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1μM) also facilitated, and BRL-15572 (2μM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K⁺-evoked tritium overflow. This response was unaffected by BRL-15572 (300nM) but antagonized by SB-216641 (15nM; drug concentrations 23 and 15 times higher than their K_i at h5-HT_1D and h5-HT_1B receptors, respectively). Both drugs, given alone, did not modify the K⁺-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300nM; 23 times K_i at h5-HT_1D receptors) but not by SB-216641 (30nM; 30 times K_i at h5-HT_1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT_1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT_1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT_1B and 5-HT_1D receptors in functional studies. Received: 14 March 1997 / Accepted: 18 May 1997     

SB-216641 and BRL-15572 – compounds to pharmacologically discriminate h5-HT1B and h5-HT1D receptors

Despite only modest homology between h5-HT_1B and h5-HT_1D receptor amino acid sequences, these receptors display a remarkably similar pharmacology. To date there are few compounds which discriminate between these receptor subtypes and those with some degree of selectivity, such as ketanserin, have greater affinity for other 5-HT receptor subtypes. We now report on two compounds, SB-216641 (N-[3-(2-dimethylamino) ethoxy-4-methoxyphenyl]-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1’-biphenyl)-4-carboxamide) and BRL-15572 3-[4-(3-chlorophenyl) piperazin-1-yl]-1,1-diphenyl-2-propanol), which display high affinity and selectivity for h5-HT_1B and h5-HT_1D receptors, respectively. In receptor binding studies on human receptors expressed in CHO cells, SB-216641 has high affinity (pK_i=9.0) for h5-HT_1B receptors and has 25-fold lower affinity at h5-HT_1D receptors. In contrast, BRL-15572 has 60-fold higher affinity for h5-HT_1D (pK_i=7.9) than 5-HT_1B receptors. Similar affinities for these compounds were determined on native tissue 5-HT_1B receptors in guinea-pig striatum. Functional activities of SB-216641 and BRL-15572 were measured in a [³⁵S]GTPγS binding assay and in a cAMP accumulation assay on recombinant h5-HT_1B and h5-HT_1D receptors. Both compounds were partial agonists in these high receptor expression systems, with potencies and selectivities which correlated with their receptor binding affinities. In the cAMP accumulation assay, results from pK_B measurements on the compounds again correlated with receptor binding affinities (SB-216641, pK _B=9.3 and 7.3; BRL-15572, pK_B=<6 and 7.1, for h5-HT_1B and h5-HT_1D receptors respectively). These compounds will be useful pharmacological agents to characterise 5-HT_1B and 5-HT_1D receptor mediated responses. Received: 14 March 1997 / Accepted: 18 May 1997     

Structure Activity Relationship of Homochiral 7-Substituted 1-Aminoindans as 5-HT1A Receptor Ligands

A series of homochiral 7-substituted 1-aminoindans was prepared by means of asymmetric reductive amination from racemic 7-substituted 1-indanones via E-imines and E-imine/ enamine mixtures, respectively, and subjected to 5-hydroxytryptamine (5-HT) receptor subtype binding studies. The ee’s of the title compounds were determined by HPLC of the corresponding Mosher’s amides and range from 96 to 99.9%. The absolute configuration was elucidated by means of correlation CD spectroscopy. On the basis of the pK_I values obtained from various test systems, structure activity relationships have been established with respect to the absolute configuration, degree of N-alkylation, and the type of 7-substituents. The tested 1-aminoindans show the highest affinity to the 5-HT_1A receptor, with decreasing magnitude for the 5-HT_2A, 5-HT_1D, and 5-HT_2C receptors. The highest affinities for the 5-HT_1A receptor were observed for the R-(–)-7-propoxy-1-(di-n-propylamino)indan hydrochloride (R-(–)-30), S,S′-(+)-7-benzylamido-1-(1-phenylethylamino)indan hydrochloride [S,S′-(+)- 19 ] and R-(–)-7-methoxy-1-(di-n-propylamino)indan hydrogenembonate (R-(–)- 29 ) with pK_I = 7.07 ± 0.19, 6.40 ± 0.09, and 6.22 ± 0.10, respectively, in comparison to 8-OH-DPAT with pK_I = 8.70 ± 0.30. Nearly all other compounds showed low affinity at all 5-HT receptors tested. The three above mentioned ligands were tested on HeLa cells (cell line HA6) expressing recombinant human 5-HT_1A receptors for their effects on forskolin-stimulated cAMP accumulation in intact cells. Both the di-n-propylamino substituent bearing R-(–)- 30 and R-(–)- 29 acted as efficacious agonists with a pEC₅₀ value of 5.89 ± 0.20 for R-(–)- 30 compared to 5-HT with a pEC₅₀ value of 8.06 ± 0.14. S,S′-(+)- 19 with the 1-phenylethylamino substituent was devoid of intrinsic activity up to the highest tested concentration (10 μM).     

Actions of roxindole at recombinant human dopamine D2, D3 and D4 and serotonin 5-HT1A, 5-HT1B and 5-HT1D receptors

Roxindole is a potential antidepressant agent. The present study determined its affinity and agonist efficacy at recombinant human (h) dopamine hD₂, hD₃ and hD₄ and serotonin (5-HT) h5-HT_1A, h5-HT_1B and h5-HT_1D receptors. Roxindole exhibited high affinity at hD₃ as well as at hD₂ (short isoform) and hD₄ (4-repeat isoform) receptors (pK _i values 8.93, 8.55 and 8.23, respectively). Further, it displayed high affinity at h5-HT_1A receptors (pK _i = 9.42) but modest affinity at 5-HT_1B and 5-HT_1D receptors (pK _i values 6.00 and 7.05, respectively). In [³⁵S]GTPγS binding experiments, roxindole was >20-fold more potent in stimulating [³⁵S]GTPγS binding at hD₃ than at hD₂ or hD₄ receptors (pEC₅₀ = 9.23 vs. 7.88 and 7.69). However, whereas roxindole exhibited partial agonist activity at hD₃ and hD₄ sites (E _max = 30.0% and 35.1%, respectively, relative to dopamine = 100%), it only weakly activated hD₂ receptors (E _max = 10.5%). Roxindole potently blocked dopamine-stimulated [³⁵S]GTPγS binding at hD₂ receptors (pK _B = 9.05). In comparison, the dopamine receptor agonist, (-)quinpirole, acted as a partial agonist at hD₃ and hD₄ sites (E _max = 67.4% and 66.3%, respectively) but surpassed the efficacy of dopamine at hD₂ receptors (E _max = 132%). At h5-HT_1A receptors, roxindole behaved as a high affinity (pK _i = 9.42) partial agonist (E _max = 59.6%, relative to 5-HT = 100%), whereas (-)quinpirole had negligible activity. The selective 5-HT_1A antagonist, WAY 100,635, blocked roxindole (100 nM)-stimulated [³⁵S]GTPγS binding at h5-HT_1A receptors in a concentration-dependent manner (pK _B = 9.28). Roxindole only weakly stimulated [³⁵S]GTPγS binding at 5-HT_1B and 5-HT_1D receptors (E _max = 27.1% and 13.7%). The present data suggest that roxindole activates mainly D₃ vs. D₂ or D₄ receptors and 5-HT_1A vs. 5-HT_1B or 5-HT_1D receptors. Activation of D₃ and/or 5-HT_1A receptors may thus contribute to its potential antidepressant properties. Received: 4 February 1999 / Accepted: 29 March 1999     

Native 5-HT1B receptors expressed in OK cells display dual coupling to elevation of intracellular calcium concentrations and inhibition of adenylate cyclase

The opossum kidney (OK) cell line has been shown previously to express endogenous 5-HT_1B receptors which negatively couple to adenylate cyclase. Since other G_i-linked receptors have been shown to inhibit adenylate cyclase and to elevate intracellular calcium concentrations ([Ca²⁺]_i), studies were initiated to determine whether native opossum 5-HT_1B receptors could also display dual coupling to these signal transduction mechanisms. Saturation studies using [¹²⁵I](–)-iodocyanopindolol ([¹²⁵I]CYP) to radiolabel the 5-HT_1B receptor in OK cell membranes (in the presence of 3 μM (–)-isoproterenol to mask β-adrenergic receptors) yielded an equilibrium dissociation constant (pK _d) of 10.04 and binding site density (B _max) of 55 fmol/mg protein. Exposure of intact OK cells to 5-HT, CP 93,129, a selective rodent 5-HT_1B receptor agonist, and (±)-cyanopindolol, a mixed 5-HT_1A/1B receptor agonist/antagonist, produced concentration-dependent inhibitions of forskolin (3 μM)-stimulated cAMP accumulation (FSCA; E _max=90–95%) and elevations of [Ca²⁺]_i (E _max∼200 nM increase above basal levels). Agonist potencies (pEC₅₀) ranged from 9.7 to 8.1 and were comparable between the two second messenger assays, although slightly higher agonist potencies (∼three-fold) were observed in the cAMP assay. GR 127,935, a selective 5-HT_1B/1D receptor antagonist, behaved as a strong partial agonist in both the cAMP and calcium assays, with an intrinsic activity of 0.7 relative to 5-HT. Methiothepin, a nonselective 5-HT receptor antagonist, competitively antagonized the inhibitory cAMP response elicited by CP 93,129, yielding an apparent pK _b value of 7.3. Methiothepin (10 μM) completely antagonized the stimulatory calcium response evoked by a saturating concentration of CP 93,129 (100 nM). Pertussis toxin pretreatment blocked the CP 93,129-induced inhibition of FSCA and elevation of [Ca²⁺]_i in OK cells, indicating the involvement of G_i/o proteins in transducing these second messenger responses. The agonist properties of (±)-cyanopindolol and GR 127,935 observed in both second messenger assays suggests that a large degree of receptor reserve may be present, even though 5-HT_1B receptor expression is low in OK cells. The OK cell line continues to serve as a model system to investigate 5-HT_1B receptor-mediated signaling events. Received: 31 March 1998 / Accepted: 21 July 1998     

5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes

The effect of the selective r5-HT_1B agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo [3,2-b] pyril-5-one (CP93,129) on the K⁺-evoked overflow of [³H]dopamine was studied in rat striatal synaptosomes loaded with [³H]dopamine. The aim of the study was to investigate the participation of 5-HT_1B receptors in the serotonergic modulation of striatal dopaminergic transmission. The Ca²⁺-dependent, tetrodotoxin-resistant K⁺-evoked overflow of [³H]dopamine was inhibited by CP93,129 (0.01–100 μM) in a concentration-dependent manner (IC₅₀=1.8 μM; maximal inhibition by 35.5% of control). [±]8-OH-DPAT, a 5-HT_1A receptor agonist, [+/–]DOI, a 5-HT₂ receptor agonist, and 2-methyl-5-hydroxytryptamine, a 5-HT₃ receptor agonist, at concentrations ranging from 0.01 μM to 100 μM did not show any significant effect. Neither ketanserin (1 μM and 5 μM), a selective 5-HT₂/5-HT_1D receptor antagonist, nor ondansetron (1 μM), a 5-HT₃ receptor antagonist, changed the inhibitory effect of CP93,129. SB224289, GR55562, GR127935, isamoltane and metergoline, selective and non-selective 5-HT_1B receptor antagonists, in contrast, used at a concentration of 1 μM, antagonized the inhibitory effect of CP93,129 (3 μM and 10 μM). SB224289, a selective 5-HT_1B receptor antagonist, inhibited the effect of CP93,129 in a concentration-dependent manner; the calculated K _i value was 1.8 nM. Our results indicate that in rat striatal axon terminals the K⁺-evoked release of dopamine is regulated by the presynaptic 5-HT_1B heteroreceptors. Received: 7 September 1998 / Accepted: 2 November 1998     

5-Bicycloindoles as 5-HT1D agonists

In this patent, novel 3-(2-aminoethyl)-, 3-(piperidin-4-yl)- and (R)-3- (pyrrolidin-2-ylmethyl) 5-bicycloindole derivatives are claimed. They are claimed to be agonists at the 5-HT_1D receptors useful for the treatment of 5-HT_1D-mediated disorders such as migraine. The preparation of the compounds is illustrated by six schemes and exemplified by eight examples.The compounds were tested for their inhibition of [³H]-5-HT binding at 5-HT_1D and 5-HT_1B receptors. The compounds are stated to bind selectively to the 5-HT_1D receptor, relative particularly to the 5-HT_1B receptors.     

Interaction of 5-HT1B/D ligands with recombinant h 5-HT1A receptors:Intrinsic activity and modulation by G-protein activation state

Many 5-HT_1B/D receptor ligands have affinity for 5-HT_1A receptors. In the present study, the intrinsic activity of a series of 5-HT_1B/D ligands was investigated at human 5-HT_1A (h 5-HT_1A) receptors by measuring G-protein activation in recombinant C6-glial and HeLa membranes, using agonist-stimulated [³⁵S]GTPγS binding. In these two membrane preparations, the density of h 5-HT_1A receptors (i.e., 246 to 320 fmol mg^–1 protein) and of their G-proteins, and the receptor: G-protein density ratio (0.08 to 0.18) appeared to be similar. It was found that: (i) the maximal [³⁵S]GTPγS binding responses induced by the 5-HT_1B/D receptor ligands in the HeLa preparation at 30 μM GDP were comparable to that of the native agonist 5-HT; (ii) as compared to 5-HT (1.00), similar potencies but lower maximal responses were observed in the C6-glial preparation at 0.3 μM GDP for zolmitriptan (0.89), dihydroergotamine (0.81), rizatriptan (0.71), CP122638 (0.69), naratriptan (0.60) and sumatriptan (0.53); and that (iii) maximal [³⁵S]GTPγS binding responses induced by 5-HT_1B/D ligands in the C6-glial preparation were either unaffected or significantly enhanced by increasing the GDP concentration from 0.3 to 30 μM and higher concentrations. These features differ from those observed with 5-HT_1A receptor agonists; the latter display the same rank order of potency and efficacy in both membrane preparations, and increasing the amount of GDP with C6-glial membranes results in an attenuation of both the agonist’s maximal effect and the apparent potency of partial agonists. The differential regulation of 5-HT_1A and 5-HT_1B/D agonist responses by GDP suggests that different G-protein subtypes are involved upon 5-HT_1A receptor activation by 5-HT_1A and 5-HT_1B/D agonists. Received: 28 October 1997 / Accepted: 14 February 1998     

Agonistic properties of alniditan,sumatriptan and dihydroergotamine on human 5-HT1B and 5-HT1D receptors expressed in various mammalian cell lines

1. Alniditan, a novel migraine abortive agent, is a potent 5-HT_1B/5-HT_1D receptor agonist of nM affinity. We compared the agonistic properties of alniditan, sumatriptan and dihydroergotamine on the cloned human 5-HT_1B receptor expressed at 200 fmol mg⁻¹ protein (B_max) in non-induced L929sA cells, at 740 fmol mg⁻¹ protein in HEK 293 and at 2300 fmol mg⁻¹ protein in mIFNβ-induced L929sA cells, and on the human cloned 5-HT_1D receptor expressed in C6 glioma cells (B_max 780 fmol mg⁻¹ protein).
2. Sodium butyrate treatment increased the expression level of human (h)5-HT_1B receptors in HEK 293 cells and h5-HT_1D receptors in C6 glioma cells approximately 3 fold, the binding affinities of [³H]-5-HT and [³H]-alniditan were unaffected.
3. Agonistic properties were evaluated based on inhibition of cyclic AMP accumulation in the cells after stimulation of adenylyl cyclase by forskolin or isoproterenol. Alniditan, sumatriptan and dihydroergotamine were full agonists at the h5-HT_1B receptor (IC₅₀ values were 1.7, 20 and 2 nM, respectively in HEK 293 cells) and h5-HT_1D receptors (IC₅₀ values of 1.3, 2.6 and 2.2 nM, respectively). At the h5-HT_1B receptor the agonist potency of the compounds slightly increased with higher receptor density. The opposite was seen for antagonists (ocaperidone, risperidone and ritanserin).
4. This comparative study demonstrated that alniditan was 10 times more potent than sumatriptan at the h5-HT_1B receptor, and twice as potent at the h5-HT_1D receptor. Dihydroergotamine was more potent an agonist at the h5-HT_1B receptor when expressed at high and low level in L929sA cells (but not in HEK 293 cells), and was less potent at the h5-HT_1D receptor.

     

5-Hydroxytryptamine 5-HT1D receptors mediating inhibition of cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells

5-Hydroxytryptamine 5-HT_1B/5-HT_1D receptors are members of the same receptor subfamily, but display a different pharmacology (Hartig et al. (1992) Trends Pharmacol Set 13:152–159). Whereas several cell lines have been reported to contain 5-HT_1B receptors, none has been described, however, that endogenously expresses well-characterized 5-HT_1D receptors. The present study deals with the identification of 5-HT_1D receptors inhibiting cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells. 5-HT (1 nM– 10 M) induced a concentration-dependent inhibition of the cyclic AMP accumulation stimulated by prostaglandin E₁ (1 M) in MDCK cells. The maximal effect of 5-HT averaged 50% inhibition and was abolished after a pre-treatment of the cells with pertussis toxin. Other agonists mimicked the effects of 5-HT, with the following rank order of potency (pEC₅₀ ± SEM, n 3): 5-carboxamidotryptamine (8.36 ± 0.48) > PAPP (p-aminophenylethyl-m-trifluoromethylphenyl piperazine, 7.89 ± 0.23) > 5-HT (7.35 ± 0.05) > sumatriptan (6.65 ± 0.27). PAPP behaved as a partial agonist. 8-OH-DPAT (8-hydroxy-2(di-n-propylamino)tetralin) was less potent, its maximal effect being not reached at 0.1 mM. Methiothepin, GR127935, (–)propranolol, rauwolscine and ketanserin were all devoid of intrinsic activity (up to 10 M or 0.1 mM). Methiothepin (10 nM, 0.1 M and 1 M) antagonized 5-HT effect (pA₂ 8.57 ± 0.44, Schild slope 1.17 ± 0.21, n = 3). GR127935 (1 nM, 10 nM and 0.1 M) shifted the curve of 5-HT to the right, but the antagonism was not fully surmountable (apparent pK_B value, 9.80 ± 0.16, n = 9). From the shifts obtained with rauwolscine (1 M) and (–)propranolol (10 M), respective pK_B values were estimated 6.68 ± 0.30 and 5.4 (n = 3 each). PAPP, when tested as an antagonist at 1 M, also shifted the curve of 5-HT to the right, with a pK_B of 8.27 ± 0.16 (n = 3). Finally, ketanserin (10 M) also antagonized the effects of 5-HT, the pK_B being 6.54 ± 0.16 (n = 9). The rank orders of agonist and antagonist potencies strongly suggest 5-HT receptors mediating inhibition of cyclic AMP accumulation in MDCK cells to be 5-HT_1D receptors. This is the first report of a cell line expressing endogenous, well-characterized, 5-HT_1D receptors. With regard to the 5-HT_1D receptor subtype involved, the relatively high potency of ketanserin would suggest it to be a 5-HT_1D subtype or a mixture of 5-HT_1D/5-HT_1D\ subtypes. However, caution must be exercised here, owing to the poor knowledge of canine 5-HT_1D receptor subtypes.     

[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-HT_2B receptors. 5-HT, the native agonist for the 5-HT_2B 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 (K_i human = 14.3 ± 1.2 nM, compared to K_i 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-HT_2B 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 α₂-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 α₂-adrenergic receptor. The α₂-adrenergic ligand efaroxan was found to block [³H]rauwolscine binding to the α₂-adrenergic receptor without significantly affecting binding to the 5-HT_2B receptor and was therefore included in all subsequent studies. In saturation studies at 37° C, [³H]rauwolscine labeled a single population of binding sites, K_d = 3.75 ± 0.23 nM. In simultaneous experiments using identical tissue samples, [³H]rauwolscine labeled 783 ± 10 fmol of 5-HT_2B receptors/mg of protein, as compared to 733 ± 14 fmol of 5-HT_2B receptors/mg of protein for [³H]5-HT binding. At 0° C, where the conditions for [³H]5-HT binding should label mostly the agonist high affinity state of the human 5-HT_2B receptor, [³H]rauwolscine (B_max = 951 ± 136 fmol/ mg), again labeled significantly more receptors than [³H]5-HT (B_max = 615 ± 34 fmol/mg). The affinity of [³H]rauwolscine for the human 5-HT_2B receptor at 0° C did not change, K_d = 4.93 ± 1.27 nM, while that for [³H]5-HT increased greatly (K_d at 37° C = 7.76 ± 1.06 nM; K_d at 0° 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-HT_2B receptor when [³H]rauwolscine was the radioligand. Typically, ∼ 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-HT_2B receptor. This should provide a good tool for the study of both the agonist high- and low-affinity states of the human cloned 5-HT_2B receptor. Received: 26 June 1997 / Accepted: 30 August 1997     

Interactions of GR127935, a 5-HT1B/D receptor ligand, with functional 5-HT receptors

GR127935 (N-[methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl) [1, 1-biphenyl]-4-carboxamide hydrochloride) has been recently introduced as an experimental tool to antagonize 5-HT_1B/D receptor-mediated functional responses. The compound indeed exhibits a very high affinity and selectivity for 5-HT_1B/D binding sites and it antagonizes a number of 5-HT_1B/D receptor-mediated responses. The present experiments were performed to investigate the selectivity of GR127935 against functional responses mediated by 5-HT₁-like, ‘orphan’ 5-HT₁-like (5-ht₇?), 5-HT₂, 5-HT₃ or 5-HT₄ receptors in several invivo preparations. Intravenous (i.v.) treatment with GR127935 (300μg?kg^-1) potently antagonized decreases in total carotid blood flow as well as hypotensive responses induced by the 5-HT₁-like receptor agonist sumatriptan in rabbits. I.v. bolus injections of GR127935 (up to 500 and/or 1500μg?kg^-1) did not significantly modify 5-HT-induced: (i) tachycardia in the pig (5-HT₄ receptor-mediated) and cat (‘orphan’ 5-HT₁-like or, perhaps, 5-ht₇ receptor-mediated); (ii) depressor effects in the rat and cat (‘orphan’ 5-HT₁-like or 5-ht₇ receptor-mediated); (iii) vonBezold-Jarisch reflex in the rat or the early phase of the urinary bladder contraction in the cat (both 5-HT₃ receptor-mediated). In contrast, high doses (500-1500μg?kg^-1) of GR127935 suppressed 5-HT-induced pressor responses in the rat and cat and urinary bladder contractions (secondary phase) in the cat as well as the DOI ((±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride)-induced pressor responses in the rat, which are all mediated by 5-HT_2A receptors. In conclusion, the present study demonstrates that GR127935 is a selective 5-HT_1B/D receptor antagonist devoid of interactions at ‘orphan’ 5-HT₁-like (5-ht₇?), 5-HT₃ and 5-HT₄ receptors. However, GR127935 possesses a moderate 5-HT_2A receptor blocking property, which is consistent with its binding profile (pK_i: 7.4). Lastly, in view of the potent antagonist action of GR127935, the sumatriptan-induced hypotension in rabbits seems to be mediated by 5-HT_1B/D receptors.     

5-HT3- and 5-HT2C-antagonist properties of cyamemazine: significance for its clinical anxiolytic activity

Rationale: Cyamemazine is a neuroleptic compound which possesses anxiolytic properties in humans. On the other hand, 5-HT₃- and 5-HT_2C-receptors have been implicated in anxiety disorders and a previous binding study has shown that cyamemazine possesses high affinity for both serotonin receptor types. Objective: The present study was undertaken to establish whether cyamemazine antagonizes 5-HT₃- and/or 5-HT_2C-mediated responses, and whether it compares with reference compounds. Methods: Cyamemazine was tested for its ability to antagonize: (i) 5-HT₃-dependent contraction of the isolated guinea-pig ileum and bradycardic responses in the rat and (ii) 5-HT_2C-dependent phospholipase C (PLC) stimulation in rat brain membranes. Results: In isolated guinea-pig ileum, cyamemazine potently and competitively antagonized 5-HT-dependent contractions (pA₂=7.52±0.08; n=5). In this test, cyamemazine was 5–7 times more potent (pIC₅₀=6.75±0.13) than tropisetron (pIC₅₀=6.02±0.04). In rats, cyamemazine i.v. antagonized 5-HT-dependent bradycardic responses with ID_50%=3.2±1.5 mg/kg (n=4). Finally, in rat brain membranes cyamemazine antagonized 5-HT_2C-dependent PLC stimulation with K_i=424 nM (mianserin exhibits a K_i=113 nM). Conclusions: Cyamemazine behaves as an antagonist at both 5-HT₃- and 5-HT_2C-receptors, which compares well with reference compounds. These 5-HT₃- and 5-HT_2C-antagonistic actions of cyamemazine can be involved, at least in part, in its beneficial therapeutic actions in anxiety disorders. Received: 19 May 1999 / Final version: 3 August 1999     

Interaction of arylpiperazines with 5-HT1A, 5-HT1B, 5-HT1C and 5-HT1D receptors: do discriminatory 5-HT1B receptor ligands exist?

Summary The effects of several putative 5-HT₁ receptorsubtype selective ligands were investigated in biochemical models for 5-HT_1A, 5-HT_1B, and 5-HT_1D receptors (inhibition of forskolin-stimulated adenylate cyclase activity in calf hippocampus, rat and calf substantia nigra, respectively) and 5-HT_1C receptors (stimulation of inositol phosphates production in pig choroid plexus). Following compounds were studied: 5-HT (5-hydroxytryptamine), TFMPP (1-(mtrifluoromethylphenyl)piperazine), mCPP (1-m-chlorophe-nyl)piperazine, 1 CGS 12066 (7-trifluoromethyl-4-(4-methyl1-piperazinyl)-pyrrolo[1,2-a]quinoxaline 1), isamoltane (CGP 361A, 1-(2-(1-pyrrolyl)-phenoxy)-3-isopropylamino-2-propranol), quipazine, 1-NP (1-(1-naphthyl)piperazine), and PAPP (LY165163, 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)-piperazine). Among reported 5-HT_1B receptor selective drugs, TFMPP had similar potency at 5HT_1A, 5-HT_1B and 5-HT_1C receptors, mCPP did not separate between 5-HT_1B and 5-HT_1C receptors, CGS 12066 was equipotent at 5-HT_1B and 5-HT_1D receptors, and isamoltane was only slightly 5-HTIB versus 5-HT_1A selective. Quipazine showed equal potency at 5-HTIB and 5-HT_1C receptors and 1-NP did not discriminate between the four receptor subtypes. PAPP described as 5-HT_1A receptor selective, was equally potent at 5-HT_1A and 5-HT_1D receptors. The potencies determined in second messenger studies were in good agreement with the affinity values determined in radioligand binding studies. Thus 5-HT_1A, 5-HT_1B, 5-HT_1C and 5-HT_1D receptors have different pharmacological profiles as predicted from radioligand binding studies. Despite claims to the contrary, none of the tested compounds had actual selectivity for a given 5-HT₁ receptor subtype. Of interest were the properties of several of these drugs, which behaved as agonists at some receptors and as antagonists at others (e. g. quipazine, 1-NP, PAPP and isamoltane). Send offprint requests to D. Hoyer at the above address     

5-HT1B receptor-mediated contractions in human temporal artery: evidence from selective antagonists and 5-HT receptor mRNA expression

1. In the human temporal artery both 5-HT_1-like and 5-HT_2A receptors mediate the contractile effects of 5-hydroxytryptamine (5-HT) and we have suggested that the 5-HT_1-like receptors resemble more closely recombinant 5-HT_1B than 5-HT_1D receptors. To investigate further which subtype is involved, we investigated the blockade of 5-HT-induced contractions by the 5-HT_1B-selective antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2-methyl-4′[(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-yl] carbonyl} furo[2,3-f]indole-3-spiro-4′-piperidine oxalate) and the 5-HT_1D-selective antagonist BRL-15572 (1-phenyl-3[4-3-chlorophenyl piperazin-1-yl] phenylpropan-2-ol). We also used RT-PCR to search for the mRNA of 5-HT_1B, 5-HT_1D and other 5-HT receptors.
2. The contractile effects of 5-HT in temporal artery rings were partially antagonized by SB-224289 (20, 200 nM) (apparent K_B=1 nM) and ketanserin (1 μM) but not by BRL-15572 (500 nM).
3. Sumatriptan evoked contractions (EC₅₀, 170 nM) that were resistant to blockade by BRL-15572 (500 nM) but antagonized by SB-224289 (20, 200 nM).
4. The potency of 5-HT (EC₅₀) was estimated to be 94 nM for the ketanserin-sensitive receptor and 34 nM for the SB-224289-sensitive receptor. The fraction of maximal 5-HT response mediated through SB-224289-sensitive receptors was 0.20–0.67, the remainder being mediated through ketanserin-sensitive receptors.
5. We detected arterial receptor mRNA for the following receptors (incidence): 5-HT_1B (8/8), 5-HT_1D (2/8), 5-HT_1F (0/4), 5-HT_2A (0/8), 5-HT_2B (0/8), 5-HT_2C (0/8), 5-HT₄ (4/8) and 5-HT₇ (4/8).
6. We conclude that the ketanserin-resistant fraction of the 5-HT effects and the effects of sumatriptan are mediated by 5-HT_1B receptors. The lack of antagonism by BRL-15572 rules out 5-HT_1D receptors as mediators of the contractile effects of 5-HT and sumatriptan.

     

5-Hydroxytryptamine 5-HT1B and 5-HT1D receptors mediating inhibition of adenylate cyclase activity

Summary 5-Hydroxytryptamine_1B (5-HT_1B) receptor mediated-inhibition of forskolin-stimulated adenylate cyclase activity in rat substantia nigra was characterized pharmacologically and compared to 5-HT_1D receptor mediated-inhibition of forskolin-stimulated adenylate cyclase activity in calf substantia nigra. Special attention was paid to the effects of drugs known to bind with high affinity to 5-HT_1B (pindolol, propranolol, cyanopindolol, SDZ 21-009, isamoltane) or 5-HT_1D recognition sites (yohimbine, rauwolscine).PEC₅₀ or pK _B values of a variety of 5-HT-receptor ligands (6 agonists including 5-HT, and 12 antagonists) for the inhibition of adenylate cyclase activity in rat substantia nigra, correlated significantly to the corresponding pK _D values at 5-HT_1B binding sites (r = 0.90, P = 0.0001). Amongst the ₂- and -adrenoceptor antagonists tested, none of the drugs expressed more than 35% of the intrinsic activity of 5-HT at 5-HT_1B receptors. When tested as antagonists, their pK _B values were in good agreement with their pK _D values for 5-HT_1B sites. By contrast, these drugs displayed marked intrinsic activity at 5-HT_1D receptors: their pEC₅₀ values were close to their pK _D values for 5-HT_1D sites and their effects could be potently antagonized by methiothepin. The rank orders of potency of the tested compounds at 5-HT_1B and 5-HT_1D were markedly different.The results strengthen the identity between 5-HT receptors mediating inhibition of adenylate cyclase activity in rat and calf substantia nigra and 5-HT_1B and 5-HT_1D binding sites, respectively. They underline the differences between these receptors in terms of intrinsic activities and potencies of drugs. Send offprint requests to: D. Hoyer at the above address     

A Search for New 5-HT1A/5-HT2A Receptor Ligands. In Vitro and in vivo Studies of 1-[ω-(4-Aryl-1-piperazinyl)alkyl]indolin-2(1H)-ones

A series of 1-[ω-(4-aryl-1-piperazinyl)alkyl]indolin-2(1H)-one derivatives 2–14 was synthesized in order to obtain ligands with a dual 5-HT_1A/5-HT_2A activity. The majority of those compounds ( 2–5, 7, 10–13 ) exhibited a high 5-HT_1A (K_i = 2 – 44 nM) and/or 5-HT_2A affinity (K_i = 51 and 39 for 5 and 7 , respectively). Induction of lower lip retraction (LLR) and behavioral syndrome and inhibition of these efects evoked by 8-hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT) were used for determination the agonistic and antagonistic activity, respectively, at 5-HT_1A receptors. The 5-HT_2A antagonistic activity was assessed by the blocking effect on the head twitches induced by (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in mice. Two of the tested compounds, 1-{3-[4-(3-chlorophenyl)-1-piperazinyl]propyl}-6-fluoroindolin-2(1H)-one ( 5 ) and 1-{3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl}indolin-2(1H)-one ( 7 ), demonstrated a high 5-HT_1A/5-HT_2A affinity and an in vivo antagonistic activity towards both receptor subtypes.     

Recombinant saphenous vein 5-HT1B receptors of the rabbit: comparative pharmacology with human 5-HT1B receptors

1. The rabbit recombinant saphenous vein 5-hydroxytryptamine_1B (rb 5-HT_1B) receptor stably transfected in rat C6-glial cells was characterized by measuring adenosine 3′:5′-cyclic monophosphate (cyclic AMP) formation upon exposure to various 5-HT receptor ligands. The effects of agonists and antagonists were compared with their effects determined previously at the human cloned 5-HT_1B (h 5-HT_1B) receptor under similar experimental conditions.
2. Intact C6-glial cells expressing rb 5-HT_1B receptors exhibited [³H]-5-carboxamidotryptamine (5-CT) binding sites with a K_d of 0.80±0.13 nM and a B_max between 225 to 570 fmol mg⁻¹ protein. The binding affinities of a series of 5-HT receptor ligands determined in a membrane preparation with [³H]-5-CT or [³H]-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR 125,743) were similar. With the exception of ketanserin, ligand affinities were comparable to those determined at the cloned h 5-HT_1B receptor site.
3. rb 5-HT_1B receptors were negatively coupled to cyclic AMP formation upon stimulation with 5-HT agonists. Of the several 5-HT agonists tested, 5-CT was the most potent, the potency rank order being: 5-CT>5-HT>zolmitriptan>naratriptan>rizatriptan>sumatriptan>R(+)-8-(hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The maximal responses of these agonists were similar to those induced by 5-HT. The potency of these agonists showed a positive correlation (r²=0.87; P<0.002) with their potency at the cloned h 5-HT_1B receptor subtype.
4. 2′-Methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR 127,935), methiothepin and ketanserin each behaved as silent, competitive antagonists at rb 5-HT_1B receptors; pK_B values were 8.41, 8.32 and 7.05, respectively when naratriptan was used as an agonist. These estimates accorded with their binding affinities and the potencies found on 5-HT and/or sumatriptan-mediated contraction of isolated rabbit saphenous vein segments.
5. In conclusion, the recombinant saphenous vein 5-HT_1B receptor of the rabbit shares important pharmacological similarities with the cloned h 5-HT_1B receptor. However, ketanserin is a more potent antagonist of rb 5-HT_1B receptors.

     

Evidence that hypophagia induced bymCPP and TFMPP requires 5-HT1C and 5-HT1B receptors; hypophagia induced by RU 24969 only requires 5-HT1B receptors

Male Sprague-Dawley rats deprived of food for 18 h were injected with the 5-HT agonists RU 24969, 1-(3-chlorophenyl)piperazine (mCPP) or 1-[3-(trifluoromethyl)phenyl)]piperazine (TFMPP) and 20 min later presented with their normal diet. Food intake was determined 1, 2 and 4 h later. All three drugs reduced intake over 1 and 2 h. Three out of four drugs with high affinity for 5-HT_1C receptors (metergoline, mianserin, and mesulergine but not cyproheptadine) opposed hypophagia caused bymCPP. Another drug reported to have high affinity for the 5-HT_1C site, 1-naphthyl-piperazine (1-NP), also blocked the hypophagic response tomCPP at doses which attenuatedmCPP-induced hypolocomotion. Only one of the above drugs (metergoline) which also has high affinity for other 5-HT sites opposed hypophagia caused by RU 24969. Two out of three 5-HT_1B receptor antagonists [(±) cyanopindolol, (−) propranolol, but not (−) pindolol)] which oppose hypophagia caused by RU 24969 (Kennett et al. 1987) also opposed hypophagia caused bymCPP. The 5-HT₂ antagonists ketanserin and ritanserin, the 5-HT₃ antagonist ICS 205-930 and the α₂ adrenoceptor antagonist idazoxan did not oppose the hypophagic effect ofmCPP. In agreement with results formCPP, hypophagia caused by TFMPP was opposed by both, mianserin and (±) cyanopindolol. Given alone, mianserin 1-NP and cyproheptadine but not ICS 205-930 increased food consumption of normally fed rats. The results suggest that RU 24969-induced hypophagia depends on 5-HT_1B receptors but not on 5-HT_1C receptors, whilemCPP (and TFMPP)-induced hypophagia may depend on both receptors. Thus, 5-HT_1C and 5-HT_1B receptors may evoke hypophagia via a common pathway but the effect of antagonists implies that at the doses usedmCPP and TFMPP act predominantly at 5-HT_1C receptors. Since only the hypophagic response tomCPP is blocked by cyanopindolol and (−) propranolol (Kennett and Curzon 1988) it is unlikely to be secondary to hypoactivity induced by the drug.