Molecular cloning, pharmacological properties and tissue distribution of the porcine 5-HT(1B) receptor.

Using a combination of RT - PCR and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-HT(1B) receptor cDNA derived from porcine cerebral cortex. Sequence analysis revealed 1170 bp encoding an open reading frame of 390 amino acids showing a 95% similarity with the human 5-HT(1B) receptor. The recombinant porcine 5-HT(1B) cDNA was expressed in monkey Cos-7 cells and its pharmacological profile was determined by radioligand binding assay using [(3)H]-GR125743. The affinities of several agonists (L694247>ergotamine > or =5-carboxamidotryptamine=dihydroergotamine=5-HT>CP122638=zolmitriptan>sumatriptan) and putative antagonists (GR127935>methiothepin>SB224289>ritanserin>ketanserin > or =BRL15572) correlated highly with those described for the recombinant human 5-HT(1B) receptor. In membranes obtained from cells co-expressing the porcine 5-HT(1B) receptor and a mutant G(alphao)Cys(351)Ile protein, 5-HT and zolmitriptan increased, while the 5-HT(1B) receptor antagonist SB224289 decreased basal [(35)S]-GTPgammaS binding, thus showing inverse agonism. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 7.64+/-0.04) agreed with its affinity in displacing the antagonist [(3)H]-GR125743 (pK(i): 7.36+/-0.07). The 5-HT(1B) receptor mRNA was observed by RT-PCR in several blood vessels, cerebral cortex, cerebellum and trigeminal ganglion. In situ hybridization performed in frontal cerebral cortex sections revealed the expression of 5-HT(1B) receptor mRNA in pyramidal cells. In conclusion, we have cloned and established the amino acid sequence, ligand binding profile and location of the porcine 5-HT(1B) receptor. This information may be useful in exploring the role of 5-HT(1B) receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.     

Induction of a high-affinity ketanserin binding site at the 5-Hydroxytryptamine(1B) receptor by modification of its carboxy-terminal intracellular portion

Two chimeric 5-hydroxytryptamine (5-HT) receptors were constructed by exchanging the C-terminal portion of the human (h) 5-HT(1B) receptor with the equivalent domain of the h 5-HT(2A) receptor (5-HT(1B/2A)) or with this domain truncated from its last 44 amino acids (5-HT(1B/2ADelta44)). The equilibrium dissociation constant of the radioligand [(3)H]GR 125743 was similar for both chimera compared to the wild-type (wt) h 5-HT(1B) receptor upon transient expression in COS-7 cells. Ketanserin binding affinity was 21-fold increased from pK(i): 5.79 (wt h 5-HT(1B) receptor) to pK(i): 7.11 at the 5-HT(1B/2A) chimeric receptor, this latter value being close to that of the wt h 5-HT(1D) receptor (pK(i): 7.62). This enhanced ketanserin binding affinity was lost when the last 44 C-terminal amino acids of the 5-HT(2A) receptor were deleted in the chimera 5-HT(1B/2ADelta44) (pK(i): 5.80). The binding affinities of the 5-HT antagonists ritanserin, GR 125743, and SB-224289 were not modified at either chimeric 5-HT receptor. The agonists F 11356, 5-HT, zolmitriptan, and sumatriptan yielded slightly increased (2- to 6-fold) binding affinities at both chimera as compared to the wt h 5-HT(1B) receptor. The present data suggest a role for the C-terminal intracellular receptor domain in modifying ketanserin/5-HT(1B) receptor interactions.     

SB-272183, a selective 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptor antagonist in native tissue.

A novel compound, SB-272183 (5-Chloro-2, 3-dihydro-6-[4-methylpiperazin-1-yl]-1[4-pyridin-4-yl]napth-1-ylaminocarbonyl]-1H-indole), has been shown to have high affinity for human 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptors with pK(i) values of 8.0, 8.1 and 8.7 respectively and is at least 30 fold selective over a range of other receptors. [(35)S]-GTPgammaS binding studies showed that SB-272183 acts as a partial agonist at human recombinant 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptors with intrinsic activities of 0.4, 0.4 and 0.8 respectively, compared to 5-HT. SB-272183 inhibited 5-HT-induced stimulation of [(35)S]-GTPgammaS binding at human 5-HT(1A) and 5-HT(1B) receptors to give pA(2) values of 8.2 and 8.5 respectively. However, from [(35)S]-GTPgammaS autoradiographic studies in rat and human dorsal raphe nucleus, SB-272183 did not display intrinsic activity up to 10 microM but did block 5-HT-induced stimulation of [(35)S]-GTPgammaS binding. From electrophysiological studies in rat raphe slices in vitro, SB-272183 did not effect cell firing rate up to 1 microM but was able to attenuate (+)8-OH-DPAT-induced inhibition of cell firing to give an apparent pK(b) of 7.1. SB-272183 potentiated electrically-stimulated [(3)H]-5-HT release from rat and guinea-pig cortical slices at 100 and 1000 nM, similar to results previously obtained with the 5-HT(1B) and 5-HT(1D) receptor antagonist, GR127935. Fast cyclic voltammetry studies in rat dorsal raphe nucleus showed that SB-272183 could block sumatriptan-induced inhibition of 5-HT efflux, with an apparent pK(b) of 7.2, but did not effect basal efflux up to 1 microM. These studies show that, in vitro, SB-272183 acts as an antagonist at native tissue 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptors.     

(3)H]-SB-269970--A selective antagonist radioligand for 5-HT(7) receptors

Binding of the 5-HT(7) receptor antagonist radioligand [(3)H]-SB-269970 to human 5-HT(7(a)) receptors expressed in HEK293 cell membranes (h5-HT(7(a))/293) and to guinea-pig cerebral cortex membranes, was characterized and compared with [(3)H]-5-CT binding. [(3)H]-SB-269970 (1 nM) showed full association with h5-HT(7(a))/293 membranes after 40 min. Specific binding at equilibrium represented >90% of total binding and was fully reversible by methiothepin (10 microM), full dissociation occurring by 100 min. The association (k(+1)) and dissociation (k(-1)) rate constants were 0.05 nM(-1)min(-1) and 0.05 min(-1) respectively, giving a K(D) (k(-1)/k(+1)) of 1.0 nM. [(3)H]-SB-269970 bound saturably and apparently monophasically to both h5-HT(7(a))/293 and guinea-pig cortex membranes, with K(D) values of 1.25+/-0.05 and 1.7+/-0.3 nM respectively. The B(max) for [(3)H]-SB-269970 to both h5-HT(7(a))/293 and guinea-pig cortex membranes (5780+/-380 and 125+/-8.2 fmoles mg protein(-1) respectively) was similar to that for [(3)H]-5-CT (6190+/-940 and 143+/-19 fmoles mg protein(-1) respectively). These data suggest that, in each tissue, both radioligands labelled the same population of receptors, which appear to be present in an agonist high affinity state. The profile of compound inhibition of [(3)H]-SB-269970 binding to h5-HT(7(a))/293 and guineapig cortex membranes correlated well (corr. coeff. 0.98) with those for [(3)H]-5-CT binding and were consistent with the profiles reported previously for the human 5-HT(7(a)) and guinea-pig cortex 5-HT(7) receptors using [(3)H]-5-CT. Hill slopes for inhibition of [(3)H]-SB-269970 and [(3)H]-5-CT binding were close to 1, consistent with binding to a single receptor population in both tissues. [(3)H]-SB-269970 represents the first selective 5-HT(7) antagonist radioligand, which should aid further characterization of 5-HT(7) receptors in recombinant and native tissues and help establish their role in brain function.     

Characterization of SB-269970-A, a selective 5-HT(7) receptor antagonist

The novel 5-HT(7) receptor antagonist, SB-269970-A, potently displaced [(3)H]-5-CT from human 5-HT(7(a)) (pK(i) 8.9+/-0.1) and 5-HT(7) receptors in guinea-pig cortex (pK(i) 8.3+/-0.2). 5-CT stimulated adenylyl cyclase activity in 5-HT(7(a))/HEK293 membranes (pEC(50) 7.5+/-0.1) and SB-269970-A (0.03 - 1 microM) inhibited the 5-CT concentration-response with no significant alteration in the maximal response. The pA(2) (8.5+/-0.2) for SB-269970-A agreed well with the pK(i) determined from [(3)H]-5-CT binding studies. 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes (pEC(50) of 8.4+/-0.2) was inhibited by SB-269970-A (0.3 microM) with a pK(B) (8.3+/-0.1) in good agreement with its antagonist potency at the human cloned 5-HT(7(a)) receptor and its binding affinity at guinea-pig cortical membranes. 5-HT(7) receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. SB-269970-A was CNS penetrant (steady-state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min(-1) kg(-1)). Following a single dose (3 mg kg(-1)) SB-269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea-pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested. 5-CT (0.3 mg kg(-1) i.p.) induced hypothermia in guinea-pigs was blocked by SB-269970-A (ED(50) 2.96 mg kg(-1) i.p.) and the non-selective 5-HT(7) receptor antagonist metergoline (0.3 - 3 mg kg(-1) s.c.), suggesting a role for 5-HT(7) receptor stimulation in 5-CT induced hypothermia in guinea-pigs. SB-269970-A (30 mg kg(-1)) administered at the start of the sleep period, significantly reduced time spent in Paradoxical Sleep (PS) during the first 3 h of EEG recording in conscious rats.     

5-HT(1A) receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems

1. It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5-HT(1A) receptors. 2. Saturation studies using [(3)H]-8-OH-DPAT and [(3)H]-MPPF revealed a single, high affinity site (K(D)approximately 1 nM) in HEK293 cells expressing human 5-HT(1A) receptors and rat cortex. In recombinant cells, [(3)H]-MPPF labelled 3 - 4 fold more sites than [(3)H]-8-OH-DPAT suggesting the presence of more than one affinity state of the receptor. [(3)H]-Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5-HT(1A) receptors but did not bind to native tissue 5-HT(1A) receptors. These data suggest that, in transfected HEK293 cells, human 5-HT(1A) receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state. 3. Receptor agonists inhibited [(3)H]-MPPF binding from recombinant 5-HT(1A) receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [(3)H]-8-OH-DPAT and [(3)H]-spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [(3)H]-MPPF and [(3)H]-8-OH-DPAT in a monophasic manner. 4. Functional evaluation of compounds, using [(35)S]-GTPgammaS binding, produced a range of intrinsic activities from full agonism, displayed by 5-HT and 5-CT to inverse agonism displayed by spiperone. 5. [(3)H]-8-OH-DPAT : [(3)H]-MPPF pK(i) difference correlated well with functional intrinsic activity (r=0.86) as did [(3)H]-8-OH-DPAT : [(3)H]-spiperone pK(i) difference with functional intrinsic activity (r=0.96). 6. Thus agonist : antagonist binding affinity differences may be used to predict functional efficacy at human 5-HT(1A) receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5-HT(1A) receptors in which only the high agonist affinity state was detectable.     

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.     

The effect of SB-269970, a 5-HT(7) receptor antagonist, on 5-HT release from serotonergic terminals and cell bodies

1. The presence of 5-HT(7) receptor mRNA and protein in 5-HT neurons suggests that this receptor may act as a 5-HT autoreceptor. In this study, the effect of the 5-HT(7) receptor antagonist, SB-269970 ((R)-1-[3-hydroxy phenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine), was investigated on 5-HT release in the guinea-pig and rat cortex and the rat dorsal raphe nucleus (DRN), using the techniques of in vitro [(3)H]-5-HT release or fast cyclic voltammetry, respectively. 2. Cortical slices were loaded with [(3)H]-5-HT and release was evoked by electrical stimulation. 5-CT inhibited the evoked release of [(3)H]-5-HT in a concentration-dependent manner. SB-269970 had no significant effect on [(3)H]-5-HT release while the 5-HT(1B) receptor antagonist, SB-224289 significantly potentiated [(3)H]-5-HT release. In addition, SB-269970 was unable to attenuate the 5-CT-induced inhibition of release while SB-224289 produced a rightward shift of the 5-CT response, generating estimated pK(B) values of 7.8 and 7.6 at the guinea-pig and rat terminal 5-HT autoreceptors respectively. 3. Rat DRN slices were electrically stimulated and the evoked 5-HT efflux detected by voltammetric analysis. 8-OH-DPAT inhibited evoked 5-HT efflux and was fully reversed by WAY 100635. SB-269970 had no effect on either 5-HT efflux per se or 8-OH-DPAT-induced inhibition of 5-HT efflux. In addition, 5-CT inhibited 5-HT efflux in a concentration-dependent manner. SB-269970 was unable to attenuate the 5-CT-induced inhibition of 5-HT efflux. 4. In conclusion, we were unable to provide evidence to suggest a 5-HT autoreceptor role for 5-HT(7) receptors. However, investigations with more selective 5-HT(7) receptor agonists are needed to confirm the data reported here.     

Exploration of the ligand binding site of the human 5-HT(4) receptor by site-directed mutagenesis and molecular modeling

Among the five human 5-HT(4) (h5-HT(4)) receptor isoforms, the h5-HT(4(a)) receptor was studied with a particular emphasis on the molecular interactions involved in ligand binding. For this purpose, we used site-directed mutagenesis of the transmembrane domain. Twelve mutants were constructed with a special focus on the residue P4.53 of helix IV which substitutes in h5-HT(4) receptors the highly conserved S residue among the rhodopsin family receptors. The mutated receptors were transiently expressed in COS-7 cells. Ligand binding or competition studies with two h5-HT(4) receptor agonists, serotonin and ML10302 and two h5-HT(4) receptor antagonists, [(3)H]-GR113808 and ML10375 were performed on wild type and mutant receptors. Functional activity of the receptors was evaluated by measuring the ability of serotonin to stimulate adenylyl cyclase. Ligand binding experiments revealed that [(3)H]-GR113808 did not bind to mutants P4.53A, S5.43A, F6.51A, Y7.43A and to double mutant F6.52V/N6.55L. On the other hand mutations D3.32N, S5.43A and Y7.43A appeared to promote a dramatic decrease of h5-HT(4(a)) receptor functional activity. From these studies, S5.43 and Y7.43 clearly emerged as common anchoring sites to antagonist [(3)H]-GR113808 and to serotonin. According to these results, we propose ligand-receptor complex models with serotonin and [(3)H]-GR113808. For serotonin, three interaction points were selected including ionic interaction with D3.32, a stabilizing interaction of this ion pair by Y7.43 and a hydrogen bond with S5.43. [(3)H]-GR113808 was also docked, based on the same type of interactions with S5.43 and D3.32: the proposed model suggested a possible role of P4.53 in helix IV structure allowing the involvement of a close hydrophobic residue, W4.50, in a hydrophobic pocket for hydrophobic interactions with the indole ring of [(3)H]-GR113808.     

[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.     

Agonist interactions with 5-HT3 receptor recognition sites in the rat entorhinal cortex labelled by structurally diverse radioligands.

1. The pharmacological properties of 5-HT3 receptor recognition sites labelled with [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 in membranes prepared from the rat entorhinal cortex were investigated to assess the presence of cooperativity within the 5-HT3 receptor complex. 2. In rat entorhinal cortex homogenates, [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 labelled homogeneous densities of recognition sites (defined by granisetron, 10 microM) with high affinity (Bmax = 75 +/- 5, 53 +/- 5, 92 +/- 6 and 79 +/- 6 fmol mg-1 protein, respectively; pKd = 9.41 +/- 0.04, 8.69 +/- 0.14, 8.81 +/- 0.06 and 10.14 +/- 0.04 for [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330, respectively, n = 3-8). 3. Quipazine and granisetron competed for the binding of each of the radioligands in the rat entorhinal cortex preparation at low nanomolar concentrations (pIC50; quipazine 9.38-8.51, granisetron 8.62-8.03), whilst the agonists, 5-hydroxytryptamine (5-HT), phenylbiguanide (PBG) and 2-methyl-5-HT competed at sub-micromolar concentrations (pIC50; 5-HT 7.16-6.42, PBG 7.52-6.40, 2-methyl-5-HT 7.38-6.09). 4. Competition curves generated with increasing concentrations of quipazine, PBG, 5-HT and 2-methyl-5-HT displayed Hill coefficients greater than unity when the 5-HT3 receptor recognition sites in the entorhinal cortex preparation were labelled with [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330. These competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]-(S)-zacopride. Competition for the binding of [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 by granisetron generated Hill coefficients around unity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of 5-HT(1A) receptor-stimulated [35S]-GtpgammaS binding as measured by quantitative autoradiography following chronic agonist administration

1. Because changes 5-HT(1A) receptor number do not occur following repeated agonist treatment, we hypothesized that the basis for 5-HT(1A) receptor desensitization involves changes in receptor-G protein coupling. We measured the effect of repeated agonist administration on 5-HT(1A) receptor-stimulated [(35)S]-GTPgammaS binding in forebrain areas, (i.e. anterior cingulate cortex, lateral septum, hippocampus, entorhinal cortex), and serotonergic cell body areas, the dorsal and median raphe nuclei. 2. Following treatment of rats with (+/-)8-OH-DPAT (1 mg kg(-1), s.c.) for 7 or 14 days, 5-HT(1A) receptor-stimulated [(35)S]-GTPgammaS binding was significantly attenuated in both the dorsal and median raphe nuclei. 3. 5-HT(1A) receptor-stimulated [(35)S]-GTPgammaS binding was significantly attenuated in the CA(1) region of the hippocampus after 7, but not 14 days of 8-OH-DPAT administration. 5-HT(1A) receptor-stimulated [(35)S]-GTPgammaS binding was not altered in other forebrain areas examined. 4. The binding of [(3)H]-MPPF to 5-HT(1A) receptor sites was not altered in any brain region examined following repeated agonist administration, suggesting that the observed changes in (+/-)8-OH-DPAT-stimulated [(35)S]-GTPgammaS binding were not due to changes in 5-HT(1A) receptor number. 5. Our data indicate that in serotonergic cell body areas the regulation of presynaptic 5-HT(1A) receptor function following repeated agonist administration occurs at the level of receptor-G protein interaction. In forebrain areas, however, the regulation of postsynaptic 5-HT(1A) receptor sensitivity appears not to be at the level of receptor-G protein coupling.     

(3)H]-8-OH-DPAT binding in the rat brain raphe area: involvement of 5-HT(1A) and non-5-HT(1A) receptors

1. The 5-HT(1A) agonist 8-OH-DPAT has been shown to have additional 5-HT uptake inhibiting properties. The present work was undertaken to examine further the binding of [(3)H]-8-OH-DPAT in the raphe area of the rat brain, a region rich in 5-HT(1A) receptors and 5-HT uptake sites. 2. 5-HT inhibited [(3)H]-8-OH-DPAT binding in a biphasic manner (pK(i1): 8.82+/-0.01, pK(i2): 6.07+/-0.05, n=4) with the low affinity site representing 36+/-4% of the total population. A biphasic inhibition curve was found also with the 5-HT(1A) antagonist, WAY 100635 (pK(i1): 8.65+/-0.17, pK(i2): 4.26+/-0.38, n=3). In the presence of 1 microM WAY 100635 to mask 5-HT(1A) receptors, 5-HT inhibited [(3)H]-8-OH-DPAT binding in a monophasic manner (pK(i): 6.04+/-0.07, n=3). 3. The affinities of various compounds for sites labelled by [(3)H]-8-OH-DPAT in the presence of 1 microM WAY 100635 and for sites labelled by [(3)H]-citalopram (a selective 5-HT uptake inhibitor) were determined. There was a significant correlation between pK(i) values at 5-HT uptake sites and at non-5HT(1A) sites labelled by [(3)H]-8-OH-DPAT (r=0.80, P<0. 001, n=17), suggesting these latter sites to be 5-HT uptake sites. 4. Whereas the affinities of R(+) and S(-) enantiomers of 8-OH-DPAT for the 5-HT uptake site are similar, R(+)8-OH-DPAT has 10 times higher affinity for the non-5-HT(1A) site than S(-)8-OH-DPAT and was considered as an outlier in the correlation. It is suggested that [(3)H]-8-OH-DPAT labels other, as yet unknown binding sites in the raphe.     

Characterisation of a 5-HT(7) binding site in mouse ileum

The aim of the present study was to identify 5-hydroxytryptamine(7) (5-HT(7)) binding sites in the mouse ileum, where the presence of mRNA for the receptor has been reported. Studies were performed using [3H]mesulergine, an antagonist with high affinity at 5-HT(7) receptors. In the presence of a combination of masking drugs to inhibit the binding of the radioligand to other receptors at which it has affinity, such as 5-HT(2A), 5-HT(2C) and dopamine D(2) receptors as well as alpha(1)/alpha(2)-adrenoceptors, [3H]mesulergine labelled two sites with pK(D) values of 9.7+/-0.7 and 7.4+/-0.4 and B(max) values of 37.2+/-21.4 and 247.8+/-62.1 fmol mg protein(-1), respectively. Displacement studies also indicated the presence of non-homogenous binding sites, which showed a significant correlation (Pearson correlation factors of 0.91 and 0. 85) with the 5-HT(2C) and 5-HT(7) receptors, respectively. Total binding to the 5-HT(2C) receptor was minimal; <30% of the total specific receptor binding. The antagonist order of affinity at the greater proportion of receptors was: risperidone (pK(i)pindolol (5. 6). This receptor also showed a high affinity for 5-carboxamidotryptamine (5-CT; 10.6) and moderate affinity for (+/-)-2-dipropyl-amino-8-hydroxy-1,2,3,4,-tetrahydronaphthalene (8-OH-DPAT; 7.2), which is typical of the 5-HT(7) receptor profile.     

An investigation of the 5-HT1D receptor binding affinity of 5-hydroxytryptamine, 5-carboxyamidotryptamine and sumatriptan in the central nervous system of seven species.

The ability of three 5-HT1 receptor agonists, 5-HT (5-hydroxytryptamine), 5-CT (5-carboxyamidotryptamine) and sumatriptan to inhibit the binding of [3H]5-HT, in the presence of cyanopindolol and mesulergine, from cerebral cortical and/or caudate membranes in seven species (dog, guinea-pig, rabbit, pig, human, hamster and calf) has been investigated. Under the experimental conditions used, 5-CT and sumatriptan consistently yielded displacement curves best fit to a two-site model whereas 5-HT always gave a monophasic displacement curve. The pIC50 values obtained with 5-HT displacement gave a mean of 8.1 +/- 0.1 (mean +/- S.E.M.). In contrast the biphasic displacement curves for 5-CT and sumatriptan yielded high and low affinity pIC50 values of 8.3 +/- 0.1, 5.5 +/- 0.1 and 7.6 +/- 0.1, 5.0 +/- 0.1, respectively. These data indicate that under these experimental conditions the high affinity component labelled by [3H]5-HT is the same receptor subtype, previously denoted the 5-HT1D receptor, in all seven species.     

Characterization of [(125)I]-SB-258585 binding to human recombinant and native 5-HT(6) receptors in rat, pig and human brain tissue

SB-258585 (4-Iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzen esulphonamide) is a high affinity ligand at 5-HT(6) receptors. It displays over 100 fold selectivity for the 5-HT(6) receptor over all other 5-HT receptors tested so far. SB-258585 has been radiolabelled, to high specific activity, for its characterization as a 5-HT(6) receptor selective radioligand. [(125)I]-SB-258585 bound, with high affinity, to a single population of receptors in a cell line expressing human recombinant 5-HT(6) receptors. Kinetic and saturation binding experiments gave pK(D) values of 9.01+/-0.09 and 9.09+/-0.02, respectively. In membranes derived from rat or pig striatum and human caudate putamen, [(125)I]-SB-258585 labelled a single site with high levels (>60%) of specific binding. Saturation analysis revealed pK(D) values of 8.56+/-0.07 for rat, 8.60+/-0.10 for pig and 8.90+/-0.02 for human. B(max) values for the tissues ranged from 173+/-23 and 181+/-25 fmol mg(-1) protein in rat and pig striatum, respectively, to 215+/-41 fmol mg(-1) protein in human caudate putamen. The pK(i) rank order of potency for a number of compounds, determined in competition binding assays with [(125)I]-SB-258585, at human caudate putamen membranes was: SB-271046>SB-258585>SB-214111>methiothepin>clozapine>5-Me-OT>5-HT>Ro 04-6790>mianserin>ritanserin=amitriptyline>5-CT>mesulergine. Similar profiles were obtained from pig and rat striatal membranes and recombinant 5-HT(6) receptors; data from the latter correlated well with [(3)H]-LSD binding. Thus, [(125)I]-SB-258585 is a high affinity, selective radioligand which can be used to label both recombinant and native 5-HT(6) receptors and will facilitate further characterization of this receptor subtype in animal and human tissues.     

Constitutive activity at serotonin 5-HT(1D) receptors: detection by homologous GTPgammaS versus [(35)S]-GTPgammaS binding isotherms

Although many G-protein-coupled receptors (GPCRs) may display constitutive activity, their detection has, to date, depended on the use of inverse agonists. The present study exploited a novel procedure to investigate constitutive activity at recombinant human (h) serotonin (5-HT) 5-HT(1D) receptors stably expressed in Chinese hamster ovary (CHO) cells. 5-HT modestly stimulated guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]-GTPgammaS) binding to CHO-h5-HT(1D) membranes whereas methiothepin and the 5-HT(1B/1D)-selective ligand, SB224,289, exerted robust inhibition of basal [(35)S]-GTPgammaS binding (inverse agonism). These actions were specific inasmuch as they were reversed by the novel, selective 5-HT(1B/1D) ligand, S18127. Constitutive activity was investigated by homologous inhibition of [(35)S]-GTPgammaS binding to CHO-h5-HT(1D) membranes with unlabelled GTPgammaS. Under 'basal' conditions (absence of receptor ligand), biphasic isotherms were observed. Most (80%) [(35)S]-GTPgammaS binding sites were in the high affinity (HA) versus low affinity (LA) component of the isotherms. HA binding was augmented by 5-HT (to 155%; relative to basal values=100%), but decreased by methiothepin (to 23%) and by SB224,289 (to 67%). In contrast, LA binding was not altered. Further, membranes of untransfected CHO cells exhibited only LA binding sites, indicating that the latter are not related to h5-HT(1D) receptor-G-protein coupling. Thus, at 5-HT(1D) receptors expressed in this CHO cell line, HA binding detected in homologous inhibition experiments (GTPgammaS versus [(35)S]-GTPgammaS) under basal conditions provides a measure of constitutive G-protein activation. Thus, it is suggested that for h5-HT(1D) receptors and, possibly, other GPCRs, inverse agonists will be detectable by [(35)S]-GTPgammaS binding if a HA component is present under basal conditions.     

SB-656104-A,a novel selective 5-HT7 receptor antagonist,modulates REM sleep in rats

1 (6-((R)-2-[2-[4-(4-Chloro-phenoxy)-piperidin-1-yl]-ethyl]-pyrrolidine-1-sulphonyl)-1H-indole hydrochloride) (SB-656104-A), a novel 5-hydroxytryptamine (5-HT(7)) receptor antagonist, potently inhibited [(3)H]-SB-269970 binding to the human cloned 5-HT(7(a)) (pK(i) 8.7+/-0.1) and 5-HT(7(b)) (pK(i) 8.5+/-0.2) receptor variants and the rat native receptor (pK(i) 8.8+/-0.2). The compound displayed at least 30-fold selectivity for the human 5-HT(7(a)) receptor versus other human cloned 5-HT receptors apart from the 5-HT(1D) receptor ( approximately 10-fold selective). 2 SB-656104-A antagonised competitively the 5-carboxamidotryptamine (5-CT)-induced accumulation of cyclic AMP in h5-HT(7(a))/HEK293 cells with a pA(2) of 8.5. 3 Following a constant rate iv infusion to steady state in rats, SB-656104 had a blood clearance (CL(b)) of 58+/-6 ml min(-1) kg(-1) and was CNS penetrant with a steady-state brain : blood ratio of 0.9 : 1. Following i.p. administration to rats (10 mg kg(-1)), the compound displayed a t(1/2) of 1.4 h with mean brain and blood concentrations (at 1 h after dosing) of 0.80 and 1.0 micro M, respectively. 4 SB-656104-A produced a significant reversal of the 5-CT-induced hypothermic effect in guinea pigs, a pharmacodynamic model of 5-HT(7) receptor interaction in vivo (ED(50) 2 mg kg(-1)). 5 SB-656104-A, administered to rats at the beginning of the sleep period (CT 0), significantly increased the latency to onset of rapid eye movement (REM) sleep at 30 mg kg(-1) i.p. (+93%) and reduced the total amount of REM sleep at 10 and 30 mg kg(-1) i.p. with no significant effect on the latency to, or amount of, non-REM sleep. SB-269970-A produced qualitatively similar effects in the same study. 6 In summary, SB-656104-A is a novel 5-HT(7) receptor antagonist which has been utilised in the present study to provide further evidence for a role for 5-HT(7) receptors in the modulation of REM sleep.     

Pharmacological analysis of contractile effects of eletriptan and sumatriptan on human isolated blood vessels

Eletriptan, a second-generation triptan with high affinity for 5-HT(1B/1D) receptors, is highly effective in migraine, with or without aura. We compared the effects of eletriptan and sumatriptan on the human isolated middle meningeal and coronary arteries and saphenous vein, used as models for therapeutic efficacy and potential side effects, and have investigated the role of 5-HT(1B/1D) receptors in contractions induced by these triptans. Concentration-response curves to eletriptan and sumatriptan were constructed in the absence or presence of a selective 5-HT(1B/1D) receptor antagonist, N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-py rid yl) benzamide (GR125743). All three blood vessels constricted in response to eletriptan and sumatriptan, but the middle meningeal artery relaxed following the highest concentration (100 microM) of eletriptan. In the middle meningeal artery, GR125743 antagonised the contractions induced by both eletriptan (pEC(50): 7.34+/-0.13) and sumatriptan (pEC(50): 6.91+/-0.17) to a similar degree (pA(2): 8. 81+/-0.17 and 8.64+/-0.21, respectively). In the human coronary artery and saphenous vein, sumatriptan-induced contractions (pEC(50): 6.24+/-0.14 and 6.19+/-0.12, respectively) were also potently antagonised by GR125743 (pA(2): 8.18+/-0.27 and 8.34+/-0.12, respectively). The eletriptan-induced contractions of the human saphenous vein (pEC(50): 6.09+/-0.13) were antagonised less effectively by GR125743 (pK(B): 7.73+/-0.18), and those of the human coronary artery (pEC(50): 5.54+/-0.22) remained unaffected by GR125743 up to a concentration of 100 nM. These results suggest that (i) based on the differences in pEC(50) values, the cranioselectivity of eletriptan (63-fold) is higher than that of sumatriptan (5-fold) in coronary artery, (ii) the contractile effects of sumatriptan and eletriptan (lower concentrations) in the three blood vessels are mediated via the 5-HT(1B) receptor, and (iii) additional mechanisms seem to be involved in coronary artery and saphenous vein contractions and middle meningeal artery relaxation following high concentrations of eletriptan.     

Chronic fluoxetine treatment selectively uncouples raphe 5-HT(1A) receptors as measured by [(35)S]-GTP gamma S autoradiography

1. Selective Serotonin Reuptake Inhibitors (SSRIs) are thought to have a delay in therapeutic efficacy because of the need to overcome the inhibitory influence of raphe 5-HT(1A) autoreceptors. Prolonged SSRI administration has been reported to desensitize these autoreceptors. We have used [(35)S]-GTP gamma S autoradiography to determine whether this desensitization occurs at the level of receptor/G protein coupling. 2. Male mice were injected intraperitoneally once a day with saline or 20 mg kg(-1) fluoxetine for either 2 days or 14 days. 5-HT(1A) receptor binding and coupling to G proteins were assessed using [(3)H]-8-OH-DPAT and [(35)S]-GTP gamma S autoradiography, respectively. 3. The 5-HT receptor agonist 5-carboxamidotryptamine (5-CT) stimulated [(35)S]-GTP gamma S binding in the substantia nigra, as well as in hippocampus and dorsal raphe nucleus. The 5-HT(1A) receptor antagonist p-MPPF (4-fluoro-N-(2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl)-N-(2-pyridinyl)benzamide) blocked this effect in the latter regions, whereas the 5-HT(1B/D) antagonist GR-127,935 (2'-methyl-4'-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-l-yl)-phenyl]-amide) only decreased labelling in substantia nigra. 4. Fourteen-day fluoxetine treatment decreased 5-CT-stimulated [(35)S]-GTP gamma S binding in dorsal raphe (saline: 112 +/- 12% stimulation; fluoxetine: 66 +/- 13%), but not in substantia nigra (99 +/- 14% vs 103 +/- 7%) or hippocampus (157 +/- 3% vs 148 +/- 18%). Two-day fluoxetine treatment did not alter 5-CT-stimulated [(35)S]-GTP gamma S binding in any of the brain areas investigated. 5. Decreased [(35)S]-GTP gamma S binding was not due to receptor down-regulation, since the density of raphe [(3)H]-8-OH-DPAT binding sites was unaffected by fluoxetine treatment. 6. These results suggest that the desensitization of presynaptic 5-HT(1A) receptor function occurs at the level of receptor-G protein interaction on dorsal raphe neurons, and may underlie the therapeutic efficacy of long-term SSRI treatment.