The distribution of 5-HT1D and 5-HT1E binding sites in human brain.

Total 5-HT1, 5-HT1D and 5-HT1E binding sites were measured in homogenates of human frontal cortex, hippocampus, amygdala, globus pallidus, caudate and putamen. Combined 5-HT1D/1E sites were the predominant 5-HT1 subtype (66-95% of total 5-HT1 sites in all regions except hippocampus (38% of total 5-HT1 sites). Globus pallidus contained the highest density and the highest proportion of 5HT1D sites (74% of total 5-HT1 sites). 5HT1D sites in the other brain areas accounted for 19-27% of the total 5-HT1 sites. The highest densities and the highest proportions of 5-HT1E sites were in caudate (72%) and putamen (64%) and the lowest density and lowest proportion in hippocampus (16%).     

Contractile 5-HT1B receptors in human cerebral arteries: pharmacological characterization and localization with immunocytochemistry

1 The cerebrovascular receptor(s) that mediates 5-hydroxytryptamine (5-HT)-induced vasoconstriction in human cerebral arteries (HCA)has proven difficult to characterize, yet these are essential in migraine. We have examined 5-HT receptor subtype distribution in cerebral blood vessels by immunocytochemistry with antibodies selective for human 5-HT1B and human 5-HT1D receptors and also studied the contractile effects of a range of 5-HT receptor agonists and antagonists in HCA. 2 Immunocytochemistry of cerebral arteries showed dense 5-HT1B receptor immunoreactivity (but no 5-HT1D receptor immunoreactivity) within the smooth muscle wall of the HCA. The endothelial cell layer was well preserved and weak 5-HT1B receptor immunoreactivity was present. 3 Pharmacological experiments on HCA with intact endothelium showed that 5-carboxamidotryptamine was significantly more potent than alpha-methyl-5-HT, 2-methyl-5-HT and 5-HT in causing vasoconstriction. The 5-HT1B/1D receptor agonists naratriptan, sumatriptan, zolmitriptan and 181C91 (N-desmethyl zolmitriptan), all induced equally strong contractions and with similar potency as 5-HT. The maximum contractile response was significantly less for avitriptan and dihydroergotamine. There was a significant correlation between vasoconstrictor potency and 5-HT1B- and 5-HT1D-receptor affinity, but not with 5-HT1A-, 5-ht1F or 5-HT2- receptor affinity. 4 The 5-HT1B/1D-receptor antagonist GR 55562 (10-7 - 10-6 M) inhibited the contractile responses to sumatriptan and 5-CT in a competitive manner with a pKB value for GR 55562 of 7.4. Furthermore, ketanserin (10-7 M), prazosin (10-7 M), and sulpiride (10-7 M) were devoid of significant antagonistic activity of 5-HT-induced contraction in the HCA. 5 The results are compatible with the hypothesis that the 5-HT1B receptors play a major role in 5-HT-induced vasoconstriction in HCA.     

Sumatriptan (GR 43175) interacts selectively with 5-HT1B and 5-HT1D binding sites

The ability of sumatriptan (GR 43175; 3-[2-dimethylamino]ethyl-N-methyl-1H-indole-5 methane sulphonamide) to interact with 13 neurotransmitter receptor sites was determined using radioligand binding techniques. Sumatriptan displayed the highest affinity for 5-HT1D (Ki = 17 nM) and 5-HT1B (Ki = 27 nM) binding sites and was slightly less potent at 5-HT1A binding sites (Ki = 100 nM). By contrast, sumatriptan was essentially inactive (Ki greater than 10,000 nM) at each of the 10 other binding sites analyzed. These data indicate that sumatriptan interacts selectively with 5-HT1B and 5-HT1D sites and suggest that these interactions may be the basis of its apparent efficacy in the acute treatment of migraine.     

Autoradiographic mapping of 5-HT1 receptors in the guinea-pig brain with particular reference to the 5-HT1D receptor sites

Summary The anatomical distribution of 5-HT₁ receptors in the guinea-pig brain was studied by means of in vitro quantitative autoradiography using [³H]-5-HT as ligand. The relative presence of the subtypes of the 5-HT₁ binding site was investigated by adding selective concentrations of 8-OH-DPAT, (-)21009, mesulergine and 5-CT In addition, differentiation of 5-HT_1D receptors was achieved by incubation of the tissues with [³H]-5-HT in the presence of 100 nmol/1 8-OH-DPAT together with 100 nmol/1 mesulergine. Areas presenting high densities of 5-HT_1A receptors included the neocortex (internal layers), hippocampal formation (dentate gyrus, CA₁ field), septum and raphe nuclei, while 5-HT_1C sites accounted for most of the [³H]-5-HT binding to the choroid plexus. Non 5-HT_1A-non 5-HT_1C sites (mainly 5-HT_1D and, also probably, 5-HT_1E receptors) were clearly predominant in the guinea-pig brain. These sites were mainly present in the neocortex (external layers), basal ganglia, hypothalamus and midbrain (substantia nigra, superior colliculus). As previously described, sites with the properties of 5-HT_1B receptors could not be clearly identified in the guinea-pig brain. The present results, in addition to providing a detailed map of the 5-HT₁ receptors in the guinea-pig brain, indicate that the guinea-pig is a useful laboratory animal for the study of 5-HT_1D receptors. Correspondence to A. Pazos at the above address     

Autoradiographic characterisation and localisation of 5-HT1D compared to 5-HT1B binding sites in rat brain

Summary The regional distribution and the pharmacology of the binding sites labelled with the novel 5-hydroxytryptamine (serotonin) 5-HT_1B/1D selective radioligand serotonin-O-carboxy-methyl-glycyl-[¹²⁵I]tyrosinamide (abbreviated [¹²⁵I]GTI for the sake of simplicity) was determined using quantitative autoradiography in rat brain. The distribution of [¹²⁵I]GTI binding sites was largely comparable to that of [¹²⁵I] iodocyanopindolol ([¹²⁵I] ICYP) which labels 5-HT_1B binding sites (in the presence of 8-OH-DPAT (8-hydroxy-[2N-dipropylamino]tetralin) and isoprenaline, to prevent binding to 5-HT_1A and -adrenoceptor binding sites), although a detailed analysis revealed differences.The pharmacology of the [¹²⁵I]GTI binding sites was analysed using compounds known to display high affinity for and/or distinguish between 5-HT_1B and 5-HT_1D sites: 5-carboxamidotryptamine (5-CT), sumatriptan, CP 93129 (5-hydroxy-3(4-1,2,5,6-tetrahydropyridyl)-4-azaindole), (–)pindolol, PAPP (4[2-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]ethyl] benzeneamine), rauwolscine, and 8-OH-DPAT. The displacement of [¹²⁵I]GTI by 5-CT was monophasic. By contrast, the selective 5-HT_1B compound CP 93129 and (–)pindolol produced biphasic curves showing a majority of high affinity sites in the globus pallidus and the substantia nigra, whereas PAPP and sumatriptan (which are somewhat 5-HT_1D selective) produced biphasic curves indicating a minority of high affinity sites in these areas. In addition, by blocking the 5-HT_1B sites with 100 nM CP 93129, the remaining population of [¹²⁵I]GTI binding sites could be studied and was found to have high affinity for PAPP, rauwolscine and 8-OH-DPAT. The pharmacological profile of the major binding component was typical of the 5-HT_1B type: 5-CT > CP 93129 (–)pindolol > sumatriptan >/ PAPP > rauwolscine. The profile of the minor component of [¹²⁵I] GTI binding is best characterised as that of a 5-HTID site: 5-CT > PAPP sumatriptan > rauwolscine > (–)pindolol CP 93129.The localisation of the non 5-HT_1B [¹²⁵I]GTI binding sites was characterised by blocking the 5-HT_1B receptors with 100 nM CP 93129. Low densities of the 5-HT_1D recognition sites were found to be present in globus pallidus, ventral pallidum, caudate-putamen, subthalamic nucleus, entopeduncular nucleus, substantia nigra (reticular part), nuclei of the (normal and accessory) optic tract, different nuclei of the geniculate body and frontoparietal cortex, although higher densities of 5-HT_1B sites were always observed in the same structures. Thus, in agreement with the recent cloning of a rat 5-HT_1D receptor cDNA, the presence and the distribution of 5-HT_1D sites could be documented in rat brain. However, when compared to 5-HT_1B sites, 5-HT_1D sites represent only a minor component of the [¹²⁵I]GTI binding in the rat brain structures studied.Correspondence to: D. Hoyer at the above address     

Characterisation of the 5-HT receptor binding profile of eletriptan and kinetics of [3H]eletriptan binding at human 5-HT1B and 5-HT1D receptors

The affinity of eletriptan ((R)-3-(1-methyl-2-pyrrolidinylmethyl)-5-[2-(phenylsulphonyl )ethyl]-1H-indole) for a range of 5-HT receptors was compared to values obtained for other 5-HT1B/1D receptor agonists known to be effective in the treatment of migraine. Eletriptan, like sumatriptan, zolmitriptan, naratriptan and rizatriptan had highest affinity for the human 5-HT1B, 5-HT1D and putative 5-ht1f receptor. Kinetic studies comparing the binding of [3H]eletriptan and [3H]sumatriptan to the human recombinant 5-HT1B and 5-HT1D receptors expressed in HeLa cells revealed that both radioligands bound with high specificity (>90%) and reached equilibrium within 10-15 min. However, [3H]eletriptan had over 6-fold higher affinity than [3H]sumatriptan at the 5-HT1D receptor (K(D)): 0.92 and 6.58 nM, respectively) and over 3-fold higher affinity than [3H]sumatriptan at the 5-HT1B receptor (K(D): 3.14 and 11.07 nM, respectively). Association and dissociation rates for both radioligands could only be accurately determined at the 5-HT1D receptor and then only at 4 degrees C. At this temperature, [3H]eletriptan had a significantly (P<0.05) faster association rate (K(on) 0.249 min(-1) nM(-1)) than [3H]sumatriptan (K(on) 0.024 min(-1) nM(-1)) and a significantly (P<0.05) slower off-rate (K(off) 0.027 min(-1) compared to 0.037 min(-1) for [3H]sumatriptan). These data indicate that eletriptan is a potent ligand at the human 5-HT1B, 5-HT1D, and 5-ht1f receptors and are consistent with its potent vasoconstrictor activity and use as a drug for the acute treatment of migraine headache.     

The 5-HT1-like receptors mediating inhibition of sympathetic vasopressor outflow in the pithed rat: operational correlation with the 5-HT1A, 5-HT1B and 5-HT1D subtypes

1. It has been suggested that the inhibition of sympathetically-induced vasopressor responses produced by 5-hydroxytryptamine (5-HT) in pithed rats is mediated by 5-HT₁-like receptors. The present study has re-analysed this suggestion with regard to the classification schemes recently proposed by the NC-IUPHAR subcommittee on 5-HT receptors.
2. Intravenous (i.v.) continuous infusions of 5-HT and the 5-HT₁ receptor agonists, 8-OH-DPAT (5-HT_1A), indorenate (5-HT_1A), CP 93,129 (5-HT_1B) and sumatriptan (5-HT_1B/1D), resulted in a dose-dependent inhibition of sympathetically-induced vasopressor responses.
3. The sympatho-inhibitory responses induced by 5-HT, 8-OH-DPAT, indorenate, CP 93,129 or sumatriptan were analysed before and after i.v. treatment with blocking doses of the putative 5-HT receptor antagonists, WAY 100635 (5-HT_1A), cyanopindolol (5-HT_1A/1B) or GR 127935 (5-HT_1B/1D). Thus, after WAY 100635, the responses to 5-HT and indorenate, but not to 8-OH-DPAT, CP 93,129 and sumatriptan, were blocked. After cyanopindolol, the responses to 5-HT, indorenate and CP 93,129 were abolished, whilst those to 8-OH-DPAT and sumatriptan (except at the lowest frequency of stimulation) remained unaltered. In contrast, after GR 127935, the responses to 5-HT, CP 93,129 and sumatriptan, but not to 8-OH-DPAT and indorenate, were abolished.
4. In additional experiments, the inhibition induced by 5-HT was not modified after 5-HT₇ receptor blocking doses of mesulergine.
5. The above results suggest that the 5-HT₁-like receptors, which inhibit the sympathetic vasopressor outflow in pithed rats, display the pharmacological profile of the 5-HT_1A, 5-HT_1B and 5-HT_1D, but not that of 5-HT₇, receptors.

     

Brain 5-HT1 binding sites in depressed suicides

5-HT₁ and 5-HT_1A binding sites were measured in brain tissue obtained at postmortem from 19 suicides, with definite evidence of depression, and 19 sex and age-matched controls. Thirteen of the depressed suicides had not been prescribed psychoactive drugs recently (drug-free suicides); six had been receiving antidepressant drugs, alone or in combination with other drugs (antidepressant-treated suicides). No significant differences were found in the number or affinity of 5-HT₁ and 5-HT_1A binding sites in frontal or temporal cortex between drug-free suicides and controls. The number of 5-HT₁ sites was significantly lower (by 20%), affinity unaltered, in hippocampus and the affinity significantly lower (by 33%), number unaltered, in amygdala of drug-free suicides than controls. The number of 5-HT₁ binding sites tended to be higher and the affinity lower in the antidepressant-treated compared to drug-free suicides, and significantly so in hippocampus. The present results, together with our previous studies, provide no evidence of altered cortical 5-HT markers in depressed suicides, but further emphasise abnormalities in the hippocampus.     

5-HT1D binding sites in porcine brain can be sub-divided by GR43175.

We have examined the binding of 5-carboxamidotryptamine (5-CT) and GR43175 (3-(2-dimethylamino)ethyl-N-methyl-1H-indole-5-methane sulphonamide) to 5-HT1D sites labelled with [3H]-5-hydroxytryptamine [( 3H]-5-HT) in neonatal porcine caudate membranes. In competition studies, 5-CT produced shallow inhibition curves (Ki 138 nM, slope 0.31), indicating binding site heterogeneity, while GR43175 interacted with a single population of binding sites (Ki 251 nM, slope 0.98), producing a maximum of only 52% inhibition of [3H]-5-HT binding compared to 100% for 5-HT or 5-CT. In the presence of excess GR43175 (10 microM), 5-CT produced a monophasic inhibition curve with a Ki value of 800 nM for the remaining sites (slope 0.89). These preliminary data suggest that under the conditions employed, GR43175, and to a lesser extent 5-CT, may discriminate between two sub-populations of 5-HT1D binding sites in porcine brain.     

Interaction of the alpha-adrenoceptor agonist oxymetazoline with serotonin 5-HT1A, 5-HT1B, 5-HT1C and 5-HT1D receptors.

Oxymetazoline was recognized with nanomolar affinity by 5-HT1A, 5-HT1B and 5-HT1D binding sites and mimicked the effects of 5-hydroxytryptamine with about the same potency and intrinsic activity as the endogenous amine in the corresponding functional tests. At 5-HT1C receptors, oxymetazoline behaved as a mixed agonist-antagonist. Clonidine had minimal activity. Methiothepin antagonized the effects of oxymetazoline (7.4 less than pKB less than 8.8). Thus, oxymetazoline is a full and potent agonist at 5-HT1A, 5-HT1B and 5-HT1D receptors and a partial agonist at 5-HT1C receptors.     

5-HT1-like receptors mediate 5-hydroxytryptamine-induced contraction of human isolated basilar artery

1. The 5-hydroxytryptamine (5-HT) receptor mediating contraction of endothelium denuded human basilar artery has been characterized in vitro. 2. 5-HT and a variety of 5-HT agonists contracted human isolated basilar artery with a rank order of agonist potency, 5-carboxamidotryptamine (5-CT) greater than 5-HT identical to methysergide greater than GR43175 much greater than 8-OHDPAT much greater than 2-methyl-5-HT. The maximum response produced by these agonists differed. 3. None of the agonists relaxed human basilar artery when tone was elevated with prostaglandin F2 alpha, indeed further contraction was seen. 4. The contractile responses of human basilar artery to 5-HT and the selective 5-HT1-like agonist GR43175 were highly reproducible whilst those to 5-CT were not. 5. The contractile response to both 5-HT and GR43175 was resistant to antagonism by ketanserin and GR38032, thus excluding activation of 5-HT2 and 5-HT3 receptors. The contractile action of 5-HT and GR43175 was also not antagonized by (+/-)-cyanopindolol, excluding the activation of receptors similar to 5-HT1A and 5-HT1B recognition sites identified in ligand binding studies. 6. In marked contrast, methiothepin was a potent antagonist of the contractile actions of both 5-HT and GR43175, with a pA2 value of 8.8 against both agonists. Methiothepin (100 nM) had no effect on the contractile response to the thromboxane A2-mimetic U46619. 7. We conclude that 5-HT and GR43175 contract the human isolated basilar artery by activating the same receptor type.(ABSTRACT TRUNCATED AT 250 WORDS)     

[3H]-5-carboxamidotryptamine labels 5-HT1D binding sites in bovine substantia nigra.

1. [3H]-5-hydroxytryptamine (5-HT) has been shown to radiolabel at least five types of 5-HT binding sites in mammalian brain tissue, 5-HT1A, 5-HT1C, 5-HT1D and 5-HT1D and 5-HT1E (Frazer et al., 1990). Selective masking of 5-HT1A and 5-HT1C receptors, has uncovered binding sites which display both high (5-HT1D) and low (5-HT1E) affinity for 5-carboxamidotryptamine (5-CT). By utilizing [3H]-5-CT we have eliminated a portion of the complex binding (5-HT1E) seen when [3H]-5-HT is used as a radioligand. 2. [3H]-5-CT binding to 5-HT1D sites in bovine substantia nigra was rapid, reversible and saturable, displaying high affinity (Kd = 0.38 +/- 0.04 nM) and low non-specific binding (> 90% specific binding). 3. In bovine substantia nigra, [3H]-5-CT labelled an equivalent number of binding sites to [3H]-5-CT (403 +/- 18 and 362 +/- 20 fmol mg-1 protein, respectively) and binding was sensitive to guanine nucleotides. 4. A linear correlation (r2 = 0.99) existed between the potency of compounds to displace [3H]-5-HT and [3H]-5-CT in bovine substantia nigra. 5. Therefore, [3H]-5-CT is a novel radioligand for the examination of 5-HT1-like binding sites, which under proper experimental conditions can be used to radiolabel selectively 5-HT-1D-like binding sites.     

Localisation of mRNA for h5-HT1B and h5-HT1D receptors in human dorsal raphe

In the mammalian mesencephalon, virtually all serotoninergic neurons are located in the raphe nuclei and the adjacent reticular formation. Pharmacological evidence obtained in rodents suggests that terminal and somatodendritic autoreceptors controlling serotonin (5-hydroxytryptamine, 5-HT) release belong to the 5-HT1B/D subtype of receptors, whereas somatodendritic autoreceptors controlling neuronal cell firing are predominantly of the 5-HT1A subtype. This study investigated the presence of h5-HT1D and h5-HT1B receptor mRNA within the subdivisions of the dorsal raphe of post-mortem human brains by means of in situ hybridisation. Although differences in the labelling intensity, which may be caused by different pre- and/or post-mortem conditions, were obvious among the specimens, all brains expressed both the h5-HT1D and the h5-HT1B mRNA in dorsal raphe neurons. In comparison to h5-HT1D mRNA, expression of h5-HT1B mRNA was slightly more abundant. Information on the existence and localisation of h5-HT1D and h5-HT1B receptors in human dorsal raphe neurons confirms that both subtypes may serve an autoreceptor function in humans. This finding is of pharmacological relevance since these receptors are potential new targets for therapeutic interventions in psychiatric disorders such as depression and anxiety.     

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.

     

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     

Pharmacology of cloned human 5-HT1D receptor-mediated functional responses in stably transfected rat C6-glial cell lines: further evidence differentiating human 5-HT1D and 5-HT1B receptors

This study was undertaken to investigate the pharmacology of human serotonin (5-HT)_1D receptor sites by measuring two functional cellular responses, inhibition of forskolin-stimulated cAMP formation and promotion of cell growth, using transfected rat C6-glial cell lines and a broad series of 5-HT receptor agonists. Stable and separate transfection of a pcDNA3 or pRcRSV plasmid, each containing a cloned human 5-HT_1D receptor gene, in rat C6-glial cells was confirmed with RT PCR of 5-HT_1D receptor mRNA and radioligand binding with [³H] 5-carboxamidotryptamine (5-CT) and [³H] sumatriptan. The 5-HT_1D receptor density was 350 and 1050 fmol/mg protein for the C6-glial/pcDNA3/5-HT_1D and C6-glial/pRcRSV/5-HT_1D cell line, and forskolin (100 M)-induced cAMP formation was inhibited by 45 and 78% in the presence of 1 M 5-HT, respectively. A comparison of the intrinsic agonist activities for sixteen 5-HT receptor ligands with their corresponding binding affinities for the human 5-HT_1D receptor site showed similar results for both cell lines with the exception of the partial agonist m-trifluoro-phenyl-piperazine (TFMPP). Three classes of compounds were observed: 1. efficacious agonists, such as 5-CT, 5-methoxytryptamine, 5-HT, sumatriptan, bufotenine, 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)1H-indole (RU 24,969), tryptamine and 8-hydroxy-2(di-n-propilamino)tetralin (8-OH-DPAT), with agonist potency close to their binding affinity; 2. the partial agonists metergoline, 7-trifluoromethyl-4(4-methyl-l-piperazinyl)-pyrolo-(1,2-a) quinoxaline (CGS 12066B), 1-naphthylpiperazine and 2-methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)-phenyl]-amide (GR 127,935) with marked intrinsic agonist activity but at concentrations higher than their binding affinity; and 3. the silent antagonists ritanserin, ketanserin and methiothepin, apparently free of intrinsic agonist activity, with antagonist potency close to their binding affinity. The cAMP data were further supported by the observed promotion of cell growth by stimulation of both transfected cell lines with sumatriptan under serum-free conditions; half-maximal stimulation was obtained at 4.4 nM (C6-glial/pcDNA3/5-HT_1D) fully in agreement with its EC₅₀-value (5.7 nM) for inhibition of cAMP formation. This growth promoting effect was antagonised by 1 M methiothepin and not observed in pcDNA3-plasmid-transfected and non-transfected C6-glial cells. A comparative study with a C6-glial/pcDNA3/5-HT_1B cell line expressing a similar amount of cloned human 5-HT_1B receptors (B _max: 360 fmol/mg protein) showed almost no intrinsic agonist activity for metergoline, 1-naphtylpiperazine and GR 127,935. Together with the 5-HT_1D receptor binding selectivity and antagonist activity of ketanserin and ritanserin, the findings define important pharmacological differences between cloned human 5-HT_1D and 5-HT_1B receptor sites.     

Molecular pharmacology of 5-HT1D recognition sites: Radioligand binding studies in human,pig and calf brain membranes

1) The binding characteristics of [3H]5-HT (5-hydroxytryptamine, serotonin) were investigated in membrane preparations of several regions from calf, pig and human brain in the presence of 100 nmol/l 8-OH-DPAT (8-hydroxy-2[di-n-dipropylamino]tetralin) and 100 nmol/l mesulergine in order to mask 5-HT1A and 5-HT1C sites. 2) [3H]5-HT bound rapidly, reversibly and stereo-selectively to a population of high affinity recognition sites in membranes from pig caudate, calf caudate and human cortex, caudate and substantia nigra. 3) Saturation experiments carried out with [3H]5-HT in the presence of 100 nmol/l 8-OH-DPAT and 100 nmol/l mesulergine revealed that non-5-HT1A non-5-HT1C sites represented from 50 to more than 90% of the total 5-HT1 sites (determined with [3H]5-HT in the absence of 8-OH-DPAT and mesulergine), depending on the tissue source. 4) The pharmacological profile of these sites was characterized in competition experiments performed with a variety of ligands in membranes of calf, pig and human caudate membranes. Under these conditions, [3H]5-HT labelled a population of "5-HT1-like" sites which display nanomolar affinity for tryptamines (5-carboxamidotryptamine greater than 5-HT greater than or equal to 5-methoxytryptamine greater than tryptamine) and some ergolines (metergoline greater than methysergide). In contrast, these sites showed low affinity for drugs with high affinity and/or selectivity for 5-HT1A (8-OH-DPAT, buspirone), 5-HT1B (21-009, RU 24969), 5-HT1C (mesulergine, mianserin) and 5-HT2 sites (ketanserin, cinanserin).(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

Homogeneous 5-HT1D recognition sites in the human substantia nigra identified with a new iodinated radioligand.

The novel iodinated radioligand, serotonin-5-O-carboxymethyl-glycyl[125I] tyrosinamide, was used for binding studies with membranes of human substantia nigra. Evidence is provided for the existence in this tissue of a homogeneous population of recognition sites with the pharmacological profile of the 5-HT1D site.