“5-HT1R” or 5-HT1D sites? Evidence for 5-HT 1D binding sites in rabbit brain

Summary Radioligand binding studies were performed in membranes of rabbit whole brain and striatum using the novel iodinated radioligand for 5-hydroxytryptamine 5-HT_1B and 5-HT_1D sites, Serotonin-5-O-Carboxymethyl-Glycyl[¹²⁵I]Tyrosinamide ([¹²⁵I]GTI).[¹²⁵I]GTI labelled a finite number of high affinity sites in rabbit brain membranes, B_max = 191 +- 47 fmol/mg protein, pK_D (-log mol/1) = 8.50 +- 0.13, n = 5. The pharmacological profile of [¹⁵²I]GTI binding was fully comparable to that reported previously in human and other brain preparations known to possess 5-HT_1D sites (using either [³H]5-HT or [¹²⁵I]GTI) and displayed a characteristic rank order of affinity: 5-carboxamidotryptamine > 5-HT = dihydroergotamine _> ergotamine _ sumatriptan >_ CGS 12066 >- metergoline > yohimbine >_ methysergide > ICYP > 8-OH-DPAT >_ CP 93129 > (-)pindolol > ketanserin > isamoltane > mesulergine > corynanthine > buspirone > MDL 72222.Autoradiographic studies were performed on rabbit brain slices using [³H]5-HT in the presence of 100 nmol/1 8-OH-DPAT and mesulergine (in order to mask 5-HT_1A and 5-HT_1c binding sites) and [¹²⁵I]CYP (iodocyanopindolol) in the presence of µmol/I isoprenaline and 100 nmol/l 8-OH-DPAT (in order to mask adrenoceptor and 5-HT_1A binding sites). There was no detectable specific binding of [¹²⁵I]CYP through the brain, thus excluding the presence of 5-HT_1B sites in rabbit brain. By contrast, [³H]5-HT labelled a high density of sites in globus pallidus, substantia nigra and superior colliculus. Other regions displaying labelling include the striatum, the dentate gyrus of the hippocampus and the periaqueductal grey matter. This pattern of distribution is compatible with that reported for 5-HT_1D sites in other species.The present data strongly suggest that rabbit brain has recognition sites with the pharmacological profile and distribution characteristic of the 5-HT_1D recognition site. These findings are in agreement with results obtained by Limberger et al. (1991), suggesting the terminal 5-HT autoreceptor of the rabbit brain to belong to the 5-HT_1D subtype. Except for limited species variations (see Bruinvels et al. 1992), the pharmacology of these sites does not justify an addition to the existing nomenclature (5-HT_1R), in contrast to what has been proposed by Xiong and Nelson (1989). The rabbit represents another laboratory species in which 5-HT_1D receptor mediated effects could be studied. Correspondence to D. Hoyer at the above address     

Differences in ligand binding profiles between cloned rabbit and human 5-HT1Dα and 5-HT1Dβ receptors: ketanserin and methiothepin distinguish rabbit 5-HT1D receptor subtypes

The study of serotonin receptor function has been complicated by the extreme molecular diversity of serotonin receptor subtypes, the lack of selective agonists and antagonists for many of the subtypes, and divergence in the pharmacological properties of a single receptor subtype across different animal species. An example of this pharmacological diversity between species homologues is provided by the 5-HT_1D receptor subfamily. To further advance the ability to characterize and pharmacologically compare functional responses mediated by native 5-HT_1D receptors, we have cloned the 5-HT_1D and 5-HT_1D receptor subtypes from the rabbit and evaluated their pharmacological profiles using radioligand binding assays. The deduced amino acid sequences of the rabbit 5-HT_1D and 5-HT_1D receptor genes displayed 60% overall identity [75% transmembrane (TM) identity] to each other and > 90% overall identity (95% TM identity) to their corresponding human homologues. Two compounds were identified in binding assays which discriminated between the closely-related 5-HT_1D receptors. Ketanserin exhibited high affinity (pK_i = 7.66) and selectivity ( > 20-fold) for the 5-HT_1D receptor while methiothepin displayed high affinity (pK_i = 7.86) and selectivity (16-fold) for the 5-HT_1D receptor subtype. The rabbit and human recombinant 5-HT_1D receptors showed significant intraspecies (rabbit 5-HT_1D vs. 5HT_1D) and interspecies (i.e. rabbit vs. human 5-HT_1D) similarities in their ligand binding profiles. These data suggest that 5-HT_1D-mediated responses in rabbit preparations may provide information relevant to the pharmacology of the 5-HT_1D receptor subtypes in humans.     

How selective is GR 43175? Interactions with functional 5-HT1A, 5-HT1B, 5-HT1C and 5-HT1D receptors

Summary GR 43175 (3-[2-dimethylamino]ethyl-N-methyl-1H-indole-5 methane sulphonamide) is a novel 5-HT₁-like receptor-selective agonist which was reported to be active in the treatment of migraine attacks. The effects of the compound were investigated in radioligand binding studies and in functional 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 phosphate production in pig choroid plexus).GR 43175 displayed the following order of affinity for 5-HT recognition sites (pK_D values, -log mol/l, in parentheses): 5-HT_1D (7.54) > 5-HT_1B (6.35) > 5-HT_1A (6.13) 5-HT_1C (4.13) > 5-HT₂ (3.67). The same order of potency was observed at functional 5-HT₁ receptors, at which GR 43175 acted as a full agonist, with the exception of the 5-HT_1C receptor, where the compound was a weak antagonist (pEC₅₀ or pK_B values, -log mol/l, in parentheses): 5-HT_1D (6.28) > 5-HT_1B (6.03) > 5-HT_1A (5.57) > 5-HT_1C (4.25).The present data show that GR 43175 interacts preferentially as an agonist with 5-HT_1B and 5-HT_1D receptors. Since 5-HT_1B receptors have not yet been identified in human brain, it seems possible that it is the 5-HT_1D receptor which is relevant to the reported antimigraine effects of this compound.Send offprint requests to D. Hoyer at the above address     

Selection process for investigators and study sites

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The recovery of α-adrenoceptor function and binding sites after phenoxybenzamine

Summary The recovery of peripheral -adrenoceptor function and binding sites was studied in male New Zealand white rabbits after treatment with the irreversible adrenoceptor antagonist phenoxybenzamine. Phenoxybenzamine (5 mg/kg) was administered intravenously and the animals studied 30 min to 12 days later. Pressor dose response curves to intravenous phenylephrine, noradrenaline and guanabenz were constructed in vivo in conscious animals. The contractile response of abdominal aorta and renal artery to phenylephrine and noradrenaline was examined in vitro and the recovery of specific prazosin and clonidine binding to spleen membranes investigated in radioligand binding studies.The half life (t _1/2) for recovery of maximum pressor response in vivo ranged from 0.9±0.2 days for phenylephrine to 1.4±0.1 days for guanabenz. The t _1/2 for recovery of ED₅₀ was not significantly different to t _1/2 for recovery of maximum pressor response and ranged from 0.8±0.2 days for noradrenaline to 1.3±0.3 days for phenylephrine.Half life for recovery of maximum response and EC₅₀ in the isolated tissues was similar to that obtained in vivo for recovery of pressor responses and ranged from 0.4±0.1 days for the EC₅₀ of noradrenaline in the renal artery to 1.2±0.6 days for maximum response to phenylephrine in the abdominal aorta.The rate of recovery of specific clonidine binding did not differ significantly from the rate of recovery of pressor responses to the ₂-selective agonist guanabenz. t _1/2 for maximum number of specific clonidine binding sites, B _max was 1.6±0.9 days. However t _1/2 for recovery of specific prazosin binding was significantly longer than recovery of responses to phenylephrine and noradrenaline, t _1/2 for B _max was 3.6 ±0.1 day.     

Inhibition of 5-HT3 receptor function by imidazolines in mouse neuroblastoma cells: potential involvement of σ2 binding sites

The influence of several imidazolines and -site ligands on cation influx through the 5-HT₃ receptor channel in NIE-115 mouse neuroblastoma cells was studied by measuring the 2-min influx of the organic cation [¹⁴C] guanidinium induced by 1 M 5-HT (in the presence of 10 M substance P in all experiments). In addition, we determined specific binding of [³H]DTG (1,3-di(2-tolyl)-guanidine), a selective -site radioligand, and [³H] GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone), a selective 5-HT₃ receptor antagonist, to membranes prepared from NIE-115 cells.The 5-HT-induced [¹⁴C]guanidinium influx was inhibited by the imidazolines, ondansetron, antazoline, idazoxan, BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), cirazoline, naphazoline, clonidine and by the guanidine agmatine, but not by the catecholamine adrenaline. The inhibitory effect of the imidazolines on cation influx through the 5-HT₃ receptor channel was mimicked by the -site ligands, (±)-ifenprodil, (+)-3-PPP ((R)-3-(3-hydroxyphenyl)-N-propylpiperidine), DTG (1,3-di-tolyl-guanidine), haloperidol, dizocilpine, and ketamine as well as by the polyamines, arcane and spermidine. — Ondansetron inhibited [³H]GR65630 binding with high affinity, whereas inhibition of binding of this radioligand to the 5-HT₃ receptor by antazoline, BDF 6143, idazoxan, cirazoline, (±)-ifenprodil, (+)-3-PPP, DTG and haloperidol occurred in the high micromolar range. In the competition experiments with [³H]DTG, (±)-ifenprodil, haloperidol, unlabelled DTG, BDF 6143 and (+)-3-PPP inhibited binding of the radioligand at moderate affinity (K_i values in the range of 1 M or lower), whereas ondansetron, amazoline, idazoxan, cirazoline, naphazoline, clonidine, tolazoline, efaroxan, RX821002 (2-[2-(2-methoxy-1,4-benzodioxanyl)]imidazoline), ketamine and spermidine exhibited affinity in the high micromolar or millimolar range only. Comparison of the potencies of the ligands (pIC_50% values) in inhibiting 5-HT-induced [¹⁴C]guanidinium influx with their affinities (pK_i values) at the 5-HT recognition sites of the 5-HT₃ receptor and at the ₂-sites of the N1E-115 cells by means of multiple regression analysis revealed a significant correlation with the affinities at both sites.In conclusion, our data suggest that imidazolines and -ligands, which as a rule possess low affinity for the 5-HT recognition site of the 5-HT₃ receptor, may be assumed to exert their inhibitory effect on cation influx through the 5-HT₃ receptor channels, at least in part, by interacting with ₂-binding sites.     

Effects of acute selective 5-HT1, 5-HT2, 5-HT3 receptor andα 2 adrenoceptor blockade on naloxone-induced antinociception

Several studies have demonstrated a paradoxical form of antinociception induced by the repeated administration of opioid antagonists accompanied by exposure to a painful stimulus. The underlying mechanism of this naloxone-induced antinociception (NIA) is still unknown, but the results of several studies suggest that it is a non-opioid response. This study was designed to investigate serotonergic and noradrenergic involvement in NIA. Rats were treated daily with systemic injections of 5 mg/kg naloxone, followed by a 45-s hot plate test of nociception (temperature=51.5 ± 0.5°C). After rats reached plateau levels of NIA, they received a test trial in which they were treated with various doses of different selective 5-HT or ₂ adrenoceptor antagonists in addition to naloxone before the hot plate test. Rats treated with 0.16, 0.32 and 0.63 mg/kg pirenperone or 2.5 mg/kg ritanserin showed significant reductions in paw lick latency with respect to rats treated with vehicle. In addition, high doses of yohimbine (7.5–10 mg/kg) also effectively reversed NIA. In contrast, NIA was not affected by acute blockade of 5-HT₁ or 5-HT₃ receptors by methiothepin or MDL 72222, respectively, or by the ₂ adrenoceptor blocker idazoxan. None of the 5-HT or ₂ adrenoceptor antagonists had any effect on the paw lick latencies of saline-treated rats. A possible role of 5-HT₂ receptors in the antinociception induced by opioid receptor blockade is discussed.     

Structural analysis by the comparative molecular field analysis method of the affinity of β-adrenoreceptor blocking agents for 5-HT1A and 5-HT1B receptors

The affinities of 17β-adrenoreceptor antagonists for 5-HT_1A and 5-HT_1B receptors were evaluated in binding assays. A large range of K_i values (2–10,000 nM) was observed and ortho or meta substitution of the aromatic ring carrying the amino chain was implicated in the high affinity K_i values, whereas para substitution elicited a dramatic drop in activity. These variations were analyzed with two molecular design tools: the active analogue approach (AAA) and the new 3D-QSAR (quantitative structure activity relationship) method, comparative molecular field analysis (CoMFA). The AAA method emphasized, by superimposition of selected conformations of the molecules, the favorable and unfavorable volumes implicated in the receptor recognition. CoMFA generated a linear expression between the biological data and the different values of electrostatic and steric fields surrounding the molecules. It predicted the values of selected molecules but also those of new molecules not included in the study. The excellent accuracy of the prediction revealed the potential of the method for the design of new compounds. CoMFA demonstrated the important contribution of steric parameters, evaluated at 92%, compared to the electrostatic field (evaluated at 8%) to explain the affinity for 5-HT_1A and 5-HT_1B receptors. This study emphasizes also the importance of the occupancy of a hydrophobic pocket in the receptor site located near the area interacting with the aromatic moiety, and subsequently its use for the design of new, potent, specific antagonists of 5-HT_1A and 5-HT_1B receptors.     

κ1-Opioid binding sites are the dominant opioid binding sites in surgical specimens of human pheochromocytomas and in a human pheochromocytoma (KAT45) cell line

The adrenal medulla produces opioids which exert paracrine effects on adrenal cortical and chromaffin cells and on adrenal splanchnic nerves, via specific binding sites. The opioid binding sites in the adrenals are detectable mainly in the medullary part of it and differ in type between species. Thus, the bovine adrenal medulla contains mostly κ-opioid binding sites and fewer δ- and μ-opioid binding sites while primate adrenals contain mainly δ sites and few κ-opioid binding sites. Most chromaffin cell tumors, the pheochromocytomas, produce opioids which suppress catecholamine production by the tumor. The aim of the present work was to identify the types of opioid binding sites in human pheochromocytomas. For this purpose, we characterized the opioid binding sites on crude membrane fractions prepared from 14 surgically excised pheohromocytomas and on whole KAT45 cells, a recently characterized human pheochromocytoma cell line. Our data showed that human pheohromocytomas are heterogeneous, as expected, with regard to the production of catecholamines and the distribution and profile of their opioid binding sites. Indeed, only one out of the 14 pheochromocytomas expressed exclusively δ and μ opioid sites, while in the remaining 13 tumors κ-type binding sites were dominant. The KAT45 cell line possessed a significant number of κ₁ binding sites, fewer κ₂-opioid binding sites and κ₃-opioid binding sites, and minimal binding capacity for δ- and μ-opioid receptor agonists sites. More specifically, the κ₁ sites/cell were approximately 18,000, the κ₂ 4500/cell and the κ₃ sites 2000/cell. Our findings for the surgical specimens and the cell line combined with previously published pharmacological data obtained from KAT45 cells suggest that κ sites appear to be the most prevalent opioid binding sites in pheochromocytomas. Finally, in normal bovine adrenals the profile of opioid binding sites differs in adrenaline and noradrenaline producing chromaffin cells. To test the hypothesis that the type of catecholamine produced by a pheochromocytoma depends on its cell of origin, we compared our binding data with the catecholamine content of each pheochromocytoma examined. We found no correlation between the type of the predominant catecholamine produced and the opioid binding profile of each tumor suggesting that this hypothesis may not be valid.     

5-HT重摄取抑制/5-HT1A受体拮抗双重作用抗抑郁药研究进展

5-HT重摄取抑制/5-HT1A受体拮抗双重作用抗抑郁药是在单胺递质学说的基础上研发的新一代抗抑郁药,此类药物研发的策略主要是通过分子拼合原理,在单一分子中引入2种药效团在体内共同发挥抗抑郁作用.文中对该类药物中几种典型的结构构效关系及生物活性评价结果进行综述,以期为抗抑郁药物的研发提供参考.新型化合物在有效性、安全耐受性及起效时间等方面表现出很多优势,但是目前还没有相关的临床报道数据.     

Melatonin receptors,heterodimerization, signal transduction and binding sites: what's new?

Melatonin is a neurohormone that has been claimed to be involved in a wide range of physiological functions. Nevertheless, for most of its effects, the mechanism of action is not really known. In mammals, two melatonin receptors, MT1 and MT2, have been cloned. They belong to the G-protein-coupled receptor (GPCR) superfamily. They share some specific short amino-acid sequences, which suggest that they represent a specific subfamily. Another receptor from the same subfamily, the melatonin-related receptor has been cloned in different species including humans. This orphan receptor also named GPR50 does not bind melatonin and its endogenous ligand is still unknown. Nevertheless, this receptor has been shown to behave as an antagonist of the MT1 receptor, which opens new pharmacological perspectives for GPR50 despite the lack of endogenous or synthetic ligands. Moreover, MT1 and MT2 interact together through the formation of heterodimers at least in cells transfected with the cDNA of these two receptors. Lastly, signalling complexes associated with MT1 and MT2 receptors are starting to be deciphered. A third melatonin-binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). Inhibition of QR2 by melatonin may explain melatonin's protective effect that has been reported in different animal models and that is generally associated with its well-documented antioxidant properties.     

Vertebrate cyclodiene insecticide resistance: Role of γ-aminobutyric acid and diazepam binding sites

Certain populations of the mosquitofish (Gambusia affinis) are highly resistant to cyclodiene and cyclodiene-type insecticides that competitively interact with the picrotoxinin binding site of the -aminobutyric acid (GABA) receptor-ionophore complex in the central nervous system. Resistance involves a reduction in affinity of the picrotoxinin binding site. The present study reports that GABA receptor binding is increased in resistant brain membranes compared to membranes from susceptible fish at concentrations of free radioligand above 0.2 M. The increase appears to be due to a greater number of binding sites (B_max) in the resistant population. Diazepam binding affinity (K_d) and B_max were not different in membranes from resistant fish compared to those from susceptible fish. Up-regulation of GABA binding sites in the resistant fish population may compensate for a possible reduction of GABAergic transmission caused by chronic environmental exposure to cyclodiene insecticides. However, a lack of cross-resistance to bicuculline (a competitive GABA antagonist) indicates that an increase in GABA sites is not a mechanism of cyclodiene resistance.This work was supported by National Institute of Environmental Health Sciences (NIEHS) Grant ES03069     

5-HT家族中5-HT2亚家族研究

Elaine Sanders-Bush教授，现任Vanderbilt大学脑科学研究所所长，兼任《分子药理学》以及《神经精神药理学》等多家学术杂志的编委，担任美国药理和实验治疗学会和美国精神分裂症抑郁症研究学会等众多学术团体的委员。主要研究内容包括5-羟色胺5-HT2受体的分子基因组学多态性研究。     

Dorsal-striatal 5-HT₂A and 5-HT₂C receptors control impulsivity and perseverative responding in the 5-choice serial reaction time task

Rational  

Prefrontal cortex (PFC) and dorsal striatum are part of the neural circuit critical for executive attention. The relationship between 5-HT and aspects of attention and executive control is complex depending on experimental conditions and the level of activation of different 5-HT receptors within the nuclei of corticostriatal circuitry.     

Search for correlations between serotonin 5-HT1A receptor expression and cognitive functions—a strategy in translational psychopharmacology

Rationale Animal studies and studies of human aging have suggested that the serotonin 5-HT_1A receptor may serve as a biomarker for cognitive functioning and a target for pharmacological treatment of cognitive deficits.Objectives The purpose of this positron emission tomography (PET) study was to search for relationships between interindividual variability in serotonin 5-HT_1A receptor binding potential (BP) and cognitive functioning.Materials and methods Twenty-four male control subjects, age 20–55 years, were examined with [¹¹C]WAY100635 PET and a battery of cognitive tests. 5-HT_1A receptor binding potential were calculated for the raphe nuclei, the hippocampus and the neocortex. Correlation coefficients between BP and cognitive performance were obtained for each region.Results There was a severalfold of variability in 5-HT_1A BP between individuals. We found no significant correlation between regional [¹¹C]WAY100635 binding and cognitive performance.Conclusions The results do not provide support for involvement of the 5-HT_1A receptor in cognitive functioning in man and question the predictive validity of some currently used animal models in translational neuroscience.     

5-HT1-like receptor mediated changes in porcine carotid haemodynamics: are 5-HT1D receptors involved?

Summary 5-Hydroxytryptamine (5-HT) reduces porcine arteriovenous shunting in the carotid vascular bed by stimulation of both 5-HT₁-like and 5-HT₂ receptors and increases capillary flow to some tissues, like the skin and ears, by different 5-HT₁-like receptors. In view of the heterogeneous nature of the 5-HT₁-like receptors and the relative selectivity for the 5-HT_1D binding sites of sumatriptan, which also reduces porcine arteriovenous shunting and slightly increases capillary blood flow towards skin and ears by 5-HT₁-like receptors, we have attempted to determine whether one or both of these carotid 5-HT₁-like receptors belong to the 5-HTID subtype.Pentobarbitone anaesthetized pigs, subjected to bilateral cervical vagosympathectomy, received either 5-HT (2 g · kg^–1 · min^–1) in the carotid artery or cumulative i.v. doses of sumatriptan (10, 30, 100 and 300 g · kg^–1). Their effect on the total carotid blood flow and its distribution into capillary and arteriovenous anastomotic parts was determined with radioactive microspheres. The effect of metergoline (1 mg·kg^–1), a substance with a very high affinity for the 5-HT_1D receptor as well as for the 5-HT_1A, 5-HT_1B, 5-HT_1C and 5-HT₂ receptors, was studied on the responses to 5-HT and sumatriptan.Both 5-HT and sumatriptan reduced carotid arteriovenous anastomotic blood flow. 5-HT and, to a lesser extent, sumatriptan also increased capillary blood flow towards some tissues. Metergoline by itself did not affect the distribution of porcine carotid blood flow. It attenuated the constrictor response, but increased the vasodilator response to 5-HT, in a manner similar to the 5-HT₂ receptor antagonists cyproheptadine, ketanserin and WAL 1307 in our former experiments. These effects seem, therefore, to be related to the blockade of 5-HT₂ receptors by metergoline. On the other hand, metergoline had no significant effect against the responses to sumatriptan.It is concluded that neither the constrictor nor the dilator carotid 5-HT₁-like receptors seem to be related to the known 5-HT₁ binding subtypes, including the 5-HT_1D subtype. Send offprint requests to M. O. Den Boer at the above address     

Are 5-HT(1B/1D) and 5-HT(2A/2B/2C) receptors involved in learning and memory processes?

Pharmacological data provide clear evidence that 5-HT(1B/1D) and 5-HT(2A/2B/2C )receptors are involved in the consolidation of learning.     

Pharmacological characterizations of recombinant human 5-HT1D1Dα and 5-HT1Dβ receptor subtypes coupled to adenylate cyclase inhibition in clonal cell lines: apparent differences in drug intrinsic efficacies between human 5-HT1D subtypes

Recombinant human 5-HT_1D and 5-HT_1D receptor subtypes were stably expressed in NIH-3T3 fibroblasts (1D cell line) and Y-1 adrenocortical tumor cells (1D cell line), respectively, for pharmacological evaluations of serotonergic compounds to inhibit forskolin-stimulated CAMP accumulation (FSCA). [³H]LSD saturation studies indicated that 5-HT_1D receptor expression levels were slightly higher in the 1D cell line (B _max = 1334 ± 134 fmol/mg protein) than in the (1D) cell line (B _max = 900 ± 900 fmol/mg protein). 5-HT inhibited FSCA with similar potencies (EC₅₀ 2 nM) in both assay systems. The rank order of agonist potencies in both clonal cell lines matched their pharmacological profiles previously determined in binding studies: dihydroergotamine >- 5-carboxamidotryptamine (5-CT) > LSD >- 5-HT > sumatriptan > 1-naphthylpiperazine (1-NP) > yohimbine > 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) > 1-(2,5-dimethoxy4-iodophenyl)-2-aminopropane (DOI), with K_i/EC₅₀ ratios greater than unity. Methiothepin acted as a silent antagonist at both human 5-HT_1D and 5-HT_1D receptors with apparent dissociation constants (K_b values) of 12 ± 1 nM and 3 ± 1 nM, respectively. Whereas GR 127,935, metergoline, DOI, and quipazine acted as full agonists in the 1D cell line, these compounds behaved as partial agonists in the 1D cell line.To determine whether high levels of receptor reserve might mask partial agonist activity in the two second messenger assay systems, studies were performed using the irreversible receptor alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The relationships between receptor occupancy and inhibition of FSCA were determined for 5-HT, sumatriptan, and 1-NP in both clonal cell lines after partial receptor inactivation using Furchgott analysis. Hyperbolic relationships between receptor occupancy and second messenger response were determined for 5-HT in both transfected cell lines. Steep hyperbolic relationships were also found for sumatriptan and 1-NP in the 1D cell line whereas nearly linear relationships were observed for these two compounds in the 1D cell line. Moreover, K_A/EC₅₀ ratios of these compounds were significantly larger in the (1D)(10–32) as compared to the 1D (0.9–2.5) cell line. These data are consistent with the hypothesis that the two heterologous expression systems contain a differential amount of receptor reserve. Despite the presence of an apparently larger receptor reserve in the 1D cell line, GR 127,935, metergoline, DOI, and quipazine behaved as partial agonists.Although the potencies (EC₅₀ values) of compounds matched their respective affinity constants (K_i values) for the closely-related 5-HT_1D subtypes, differences in intrinsic activities were observed for a few compounds between the two 5-HT_1D receptor expression systems. Since receptor reserve is dependent on the properties of both the assay system and drug, the observed variations in intrinsic activity, although influenced by the variable amounts of receptor reserve in the two transfected cell lines, reflect primarily system-independent differences in the intrinsic efficacy of the tested compounds at the two human 5-HT_1D receptors. Higher intrinsic efficacies of compounds at the human 5-HT_1D receptor relative to the human 5-HT_1D subtype may be responsible for the higher intrinsic activities observed in the (1D) cell line, even though receptor reserve is apparently lower in this system.Abbreviations CRC Concentration-response curve - FSCA forskolin-stimulated cAMP accumulation - KA pseudo-dissociation constants - 5-CT 5-carboxamidotryptamine - 5-HT 5-hydroxytryptamine - 5MeOT 5-methoxytryptamine - PAPP 1-[2-(4-aminophenyl) ethyl] -4-(3-trifluoromethylphenyl)-piperazine - 1-NP 1-(1-naphthyl) piperazine - 8-OH-DPAT 8-hydroxy-2-(di-n-propylamino) tetralin - DOI 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane - MK-462 (N,N-dimethyl-2-[5-(1, 2, 4-triazol-l-yl methyl)-1H-indole3-yl]ethylamine - GR 127,935 (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 46611 5-[4-methoxybenzyl ethylene]-1H-indole3-yl]ethyl amine) - L-694,247 (2-[5-[3-(4-methylsulfonylamino)benzyl-1, 2, 4-oxadiazol-5-yl]-1H-indole3-yl]ethylamine)