John M. Zgombick

A receptor autoradiographic and in situ hybridization analysis of the distribution of the 5-ht7 receptor in rat brain.

1 Receptor autoradiography and in situ hybridization histochemistry have been used to delineate the distribution of the 5‐ht7 receptor and its mRNA in rat brain. Receptor autoradiographic studies were performed using [3H]‐5‐carboxamidotryptamine (5‐CT) as the radioligand. The binding characteristics of the masking compounds were determined in Cos‐7 cells transfected with a panel of 5‐HT receptor subtype cDNAs, including the rat 5‐ht7 cDNA. In situ hybridization studies were carried out with 35S‐labelled oligonucleotide probes to the rat 5‐ht7 mRNA. 2 Specific binding of [3H]‐5‐CT was observed in many areas of the rat brain. Following co‐incubation with 1 μm ergotamine, this binding was completely eliminated. After addition of the masking ligands, [3H]‐5‐CT binding remained in layers 1–3 of cortex, septum, globus pallidus, thalamus, hypothalamus, centromedial amygdala, substantia nigra, periaquaductal gray, and superior colliculus. Addition of the antagonist, methiothepin, to the incubation regimen eliminated most of the remaining [3H]‐5‐CT binding in the brain, with the exception of the globus pallidus and substantia nigra. 3 The 5‐ht7 mRNA was discretely localized in rat brain. The most intense hybridization signals were observed over the thalamus, the anterior hippocampal rudiment, and over the CA3 region of the hippocampus. Other regions containing hybridization signals included the septum, the hypothalamus, the centromedial amygdala and the periaquaductal gray. The regions exhibiting a modest receptor binding signal after methiothepin incubation, the globus pallidus and the substantia nigra, contained no 5‐ht7 hybridization signals, suggesting a non‐5‐ht7 subtype in these two related structures. 4 The distribution of the 5‐ht7 receptor and its mRNA is suggestive of multiple roles for this novel 5‐HT receptor, within several brain systems. The limbic system (centromedial amygdala, anterior hippocampal rudiment, hypothalamus) is particularly well‐represented, indicating a potential role for the 5‐ht7 receptor in affective processes.

5-HT1F receptor agonists inhibit neurogenic dural inflammation in guinea pigs.

THE serotonin (5-HT) receptor subtype mediating inhibition of neurogenic dural inflammation in guinea pigs was investigated using a series of serotonin agonists with differing affinities for the 5-HT1B, 5-HT1D and 5-HT1F receptors. When agonist potencies for inhibiting neurogenic inflammation were compared with affinities for these receptor subtypes, a significant positive correlation was seen only with the 5-HT1F receptor. The potency of agonists in inhibiting adenylate cyclase in cells transfected with human 5-HT1F receptor was also highly correlated with their potency in the animal model of migraine. In situ hybridization demonstrated 5-HT1F receptor mRNA in guinea pig trigeminal ganglion neurons. These data suggest that the 5-HT1F receptor is a rational target for migraine therapeutics.

Characterization of LY344864 as a pharmacological tool to study 5-HT1F receptors: Binding affinities, brain penetration and activity in the neurogenic dural inflammation model of migraine

LY344864 is a selective receptor agonist with an affinity of 6 nM (Ki) at the recently cloned 5-HT1F receptor. It possesses little affinity for the 56 other serotonergic and non-serotonergic neuronal binding sites examined. When examined for its ability to inhibit forskolin-induced cyclic AMP accumulation in cells stably transfected with human 5-HT1F receptors, LY344864 was shown to be a full agonist producing an effect similar in magnitude to serotonin itself. After an intravenous dose of 1 mg/kg, rat plasma LY344864 levels declined with time whereas brain cortex levels remained relatively constant for the first 6 hours after injection. Oral and intravenous LY344864 administration potently inhibited dural protein extravasation caused by electrical stimulation of the trigeminal ganglion in rats. Taken together, these data demonstrate that LY344864 is a selective 5-HT1F receptor agonist that can be used to explore both the in vitro and in vivo functions of this receptor.

Ketanserin and ritanserin discriminate between recombinant human 5-HT1Dα and 5-HT1Dβ receptor subtypes

Abstract Compounds able to discriminate functionally between the closely related cloned human 5-HT1Dα and 5-HT1Dβ receptor subtypes have not been reported previously. In [3H]5-HT competition assays, the classical 5-HT2A receptor antagonists, ritanserin and ketanserin, displayed moderate affinity (pKi = 7.30 and 7.17, respectively) and marked selectivity (22-and 71-fold, respectively) for the recombinant human 5-HT1Dα subtype relative to the 5-HT1Dβ receptor. In contrast, the nonselective 5-HT 1 2 receptor antagonist, methiothepin, exhibited similar binding affinities (pKi=7.64−8.01) for both recombinant 5-HT1D subtypes. The antagonistic properties of these compounds were evaluated for their ability to block 5-HT-induced inhibition of forskolin-stimulated cAMP accumulation in intact cells stably expressing either 5-HT1Dα or 5-HT1Dβ receptors. All three compounds behaved as antagonists devoid of intrinsic activity in the functional assays. The apparent pKb values determined in functional assays closely matched their pKi values obtained in binding assays. Since ketanserin exhibits significant selectivity for the human 5-HT1Dα receptor, this antagonist can be used as a pharmacological tool to discriminate between 5-HT1Dα and 5-HT1Dβ receptor-mediated responses in human tissues.

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-HT1D receptor subfamily. To further advance the ability to characterize and pharmacologically compare functional responses mediated by native 5-HT1D receptors, we have cloned the 5-HT1Dα and 5-HT1Dβ receptor subtypes from the rabbit and evaluated their pharmacological profiles using radioligand binding assays. The deduced amino acid sequences of the rabbit 5-HT1Dα and 5-HT1Dβ 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-HT1D receptors. Ketanserin exhibited high affinity (pKi = 7.66) and selectivity ( > 20-fold) for the 5-HT1Dα receptor while methiothepin displayed high affinity (pKi = 7.86) and selectivity (16-fold) for the 5-HT1Dβ receptor subtype. The rabbit and human recombinant 5-HT1D receptors showed significant intraspecies (rabbit 5-HT1Dα vs. 5HT1Dβ) and interspecies (i.e. rabbit vs. human 5-HT1Dβ) similarities in their ligand binding profiles. These data suggest that 5-HT1D-mediated responses in rabbit preparations may provide information relevant to the pharmacology of the 5-HT1D receptor subtypes in humans.

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-HT1Dα and 5-HT1Dβ receptor subtypes were stably expressed in NIH-3T3 fibroblasts (1Dα cell line) and Y-1 adrenocortical tumor cells (1Dβ cell line), respectively, for pharmacological evaluations of serotonergic compounds to inhibit forskolin-stimulated CAMP accumulation (FSCA). [3H]LSD saturation studies indicated that 5-HT1D receptor expression levels were slightly higher in the 1Dβ cell line (Bmax = 1334 ± 134 fmol/mg protein) than in the (1Dα) cell line (Bmax = 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 Ki/EC50 ratios greater than unity. Methiothepin acted as a silent antagonist at both human 5-HT1Dα and 5-HT1Dβ receptors with apparent dissociation constants (Kb values) of 12 ± 1 nM and 3 ± 1 nM, respectively. Whereas GR 127,935, metergoline, DOI, and quipazine acted as full agonists in the 1Dα cell line, these compounds behaved as partial agonists in the 1Dβ 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 1Dβ cell line whereas nearly linear relationships were observed for these two compounds in the 1Dα cell line. Moreover, KA/EC50 ratios of these compounds were significantly larger in the (1Dα)(10–32) as compared to the 1Dα (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 1Dβ 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 (Ki values) for the closely-related 5-HT1D subtypes, differences in intrinsic activities were observed for a few compounds between the two 5-HT1D 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-HT1D receptors. Higher intrinsic efficacies of compounds at the human 5-HT1Dα receptor relative to the human 5-HT1Dβ subtype may be responsible for the higher intrinsic activities observed in the (1Dα) cell line, even though receptor reserve is apparently lower in this system.

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-HT1B receptors which negatively couple to adenylate cyclase. Since other Gi-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-HT1B receptors could also display dual coupling to these signal transduction mechanisms. Saturation studies using [125I](–)-iodocyanopindolol ([125I]CYP) to radiolabel the 5-HT1B receptor in OK cell membranes (in the presence of 3 µM (–)-isoproterenol to mask β-adrenergic receptors) yielded an equilibrium dissociation constant (pKd) of 10.04 and binding site density (Bmax) of 55 fmol/mg protein. Exposure of intact OK cells to 5-HT, CP 93,129, a selective rodent 5-HT1B receptor agonist, and (±)-cyanopindolol, a mixed 5-HT1A/1B receptor agonist/antagonist, produced concentration-dependent inhibitions of forskolin (3 µM)-stimulated cAMP accumulation (FSCA; Emax=90–95%) and elevations of [Ca2+]i (Emax∼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-HT1B/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 pKb 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 Gi/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-HT1B receptor expression is low in OK cells. The OK cell line continues to serve as a model system to investigate 5-HT1B receptor-mediated signaling events.