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.     

Characterisation of the contractile activity of eletriptan at the canine vascular 5-HT1B receptor

The functional activity of eletriptan ((R)-3-(1-methyl-2-pyrrolidinylmethyl)-5-[2-(phenylsulphonyl )ethyl]- 1 H-indole) at the contractile serotonin (5-hydroxytryptamine; 5-HT) '1B-like' receptor in dog isolated saphenous vein and basilar artery was investigated. Eletriptan, like 5-HT and sumatriptan potently contracted saphenous vein (pEC50: 6.3, 6.9 and 6.1, respectively) and basilar artery (pEC50 7.2, 7.5 and 6.8, respectively). The maximum responses evoked by eletriptan was, unlike sumatriptan, significantly lower than that to 5-HT (intrinsic activity saphenous vein: eletriptan 0.57, 5-HT 1.0, sumatriptan 0.85; basilar artery: eletriptan 0.77, 5-HT 0.98, sumatriptan 0.89). Contractions evoked by eletriptan were antagonised by the 5-HT1B/1D receptor antagonist GR125743 (N-[4-methoxy-3-(4-methyl piperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide) with pA2 values of 9.1 in saphenous vein and 9.4 in basilar artery. Affinity estimates (pKA) for 5-HT and sumatriptan determined from receptor alkylation studies in saphenous vein were 6.6 and 6.3, respectively, compared to the apparent equilibrium dissociation constant (pKp) for eletriptan of 6.8. The rank order of relative intrinsic efficacies (epsilon) was 5-HT > sumatriptan > eletriptan. Thus, eletriptan required greater receptor occupancy (4.4-fold) to evoke an equivalent contraction to 5-HT and sumatriptan in dog isolated saphenous vein. These data demonstrate that eletriptan is a potent partial agonist at the canine vascular 5-HT1B receptor.     

Pharmacological differences and similarities between the native mouse 5-HT3 receptor in N1E-115 cells and a cloned short splice variant of the mouse 5-HT3 receptor expressed in HEK 293 cells

Human embryonic kidney (HEK) 293 cells were stably transfected with the cDNA encoding the short splice variant of the mouse 5-HT3 receptor (m5-HT3A(b); isolated by RT-PCR from NG108-15 cells) and its pharmacological properties were compared with those of the native 5-HT3 receptor of the mouse neuroblastoma cell line N1E-115. The m5-HT3A(b) receptor of N1E-115 cells differs from that isolated from NG108-15 cells by one amino acid (Val instead of Ile) at position 52 of the amino acid sequence. Both radioligand binding studies with the selective 5-HT3 receptor antagonist [3H]GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone) and functional experiments by measurement of [14C]guanidinium influx evoked by 5-HT in the absence and presence of 10 microM substance P were carried out. Binding of [3H]GR65630 to the recombinant receptor in HEK 293 cells and the native receptor in N1E-115 cells was specific and of high affinity (Kd 4.4 and 3.0 nM, respectively) and characterized by Bmax values of 875 and 1414 fmol/mg protein, respectively. At 10 nM [3H]GR65630, specific binding was inhibited by the selective 5-HT3 receptor antagonist ondansetron (Ki 11 and 42 nM, respectively) and by 5-HT (Ki 294 and 563 nM, respectively). In the transfected HEK 293 cells, 5-HT induced an influx of [14C]guanidinium both in the absence (pEC50 5.7) and presence of substance P (pEC50 6.6,) which was counteracted by 0.3 microM ondansetron; in the N1E-115 cells, 5-HT also evoked [14C]guanidinium influx in the absence (pEC50 6.0) and presence of substance P (pEC50 6.0). Both in transfected HEK 293 cells and in N1E-115 cells, the 5-HT receptor ligand RS-056812-198 ((R)-N-(quinuclidin-3-yl)-2-(1-methyl-1 H-indol-3-yl)-2-oxo-acetamide; in the presence of substance P) induced an influx of [14C]guanidinium (pEC50 9.8 and 8.7, respectively) with a maximum of about 70 and 30% of the maximum response to 5-HT, respectively. 5-HT (in the presence of substance P)-induced [14C]guanidinium influx was inhibited by the imidazoline BDF 6143 (4-chloro-2(2-imidazolin-2-ylamino)-isoindoline; pIC50 4.9 and 5.3, respectively) and by the sigma-site ligand (+/-)-ifenprodil (pIC50 5.0 and 5.2, respectively). In conclusion, most of the drugs exhibited practically identical properties at both the recombinant m5-HT3A(b) receptor in HEK 293 cells and the native m5-HT3 receptor of N1E-115 cells. However, the recombinant receptor had a higher affinity for ondansetron, and the potency of 5-HT in inducing cation influx through the recombinant, but not through the native receptor, was increased by substance P. RS-056812-198 was a 10-fold more potent partial agonist at the recombinant than at the native receptor. These differences may be due to cell-specific post-translational modifications of the 5-HT3 receptor protein in the two cell lines, to the expression of other subunits in addition to the m5-HT3A(b) receptor in N1E-115 cells and/or to the difference in the amino acid sequence at position 52 of the short splice variants of the m5-HT3 receptors expressed in the two cell lines.     

Pharmacological properties of the naturally occurring Phe-124-Cys variant of the human 5-HT1B receptor: changes in ligand binding, G-protein coupling and second messenger formation

The aim of this study was to analyse whether substitution of phenylalanine in position 124 of the human (h) 5-HT1B receptor by cysteine, a naturally occurring variant of this receptor, modifies not only ligand binding, but also G-protein coupling and second messenger formation. Stably transfected rat C6 glioma cells, which express either the h5-HT1B variant receptor (VR) or the wild-type receptor (WTR) were used. In saturation experiments with [3H]5-carboxamidotryptamine ([3H]5-CT), the maximum binding (Bmax) of the VR amounted to only 60% of that to WTR. In competition experiments with 1 nM [3H]5-CT, the following 5-HT receptor ligands exhibited a higher affinity for the mutant receptor than for the WTR: L-694,247, 5-CT, 5-HT, sumatriptan (agonists listed at decreasing order of potency) and SB-224289 (a selective h5-HT1B receptor inverse agonist with competitive antagonistic properties). In contrast, the mixed 5-HT1B/1D receptor antagonist GR-127935 exhibited equal affinity for both isoforms. The efficacy of L-694,247, 5-CT, 5-HT and sumatriptan in stimulating [35S]GTPgammaS binding (a measure of G protein coupling) to membranes of cells expressing the VR was approximately 50-65% lower compared to membranes of cells expressing the WTR, but their potency was 2.8-3.6-fold higher. SB-224289, which decreased [35S]GTPgammaS binding when given alone, but not GR-127935, was more potent in antagonizing the stimulatory effect of 5-CT on [35S]GTPgammaS binding to membranes expressing the VR compared to membranes expressing the WTR. In whole cells expressing the VR, 5-CT and sumatriptan inhibited the forskolin-stimulated cAMP accumulation 3.2-fold more potently than in cells expressing the WTR. In conclusion, our data suggest that the Phe-124-Cys mutation modifies the pharmacological properties of the h5-HT1B receptor and may account for pharmacogenetic differences in the action of h5-HT1B receptor ligands. Thus, the sumatriptan-induced vasospasm which occurs at low incidence as a side-effect in migraine therapy may be related to the expression of the (124-Cys)h5-HT1B receptor in patients with additional pathogenetic factors such as coronary heart disease.     

Evidence for presynaptic location of inhibitory 5-HT1D\-like autoreceptors in the guinea-pig brain cortex

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K⁺ were determined in superfused synaptosomes and slices, preincubated with [³H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [³H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT_1A and 5-HT_2C sites) to guinea-pig cortical synaptosomes and membranes.5-HT receptor agonists inhibited the K⁺-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine (L-694,247) >5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) 5-HT > sumatriptan 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT_1D, and 5-HT_1D receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA₂: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT_1D, but not the 5-HT_1D\, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [³H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [³H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin >5-CT >5-methoxytryptamine >5-HT sumatriptan oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [³H]5-HT binding to synaptosomes and membranes.In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [³H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT_1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT_1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT_1D\-like.     

Functional characterization of the 5-HT terminal autoreceptor in the guinea-pig brain cortex.

1 In guinea-pig cerebral cortical slices in vitro we have shown that the rank order of potency of 5-hydroxytrptamine (5-HT), 5-carboxamidotryptamine and sumatriptan for inhibition of electrically stimulated [3H]-5-HT release correlates well with published data on their 5-HT1D receptor binding affinities. 2 Both the non-selective 5-HT1D receptor antagonist, methiothepin and the selective 5-HT1D receptor antagonist, N-[4-methoxy-3-(4-methyl-1-piperazinyl]phenyl]-2'-methyl-4'- (5-methyl-1,2,4-oxadiazole-3-yl) [1,1-biphenyl]4-carboxamide (GR127935) increased stimulated [3H]-5-HT release per se and also attenuated agonist-induced inhibition of [3H]-5-HT release. GR127935 (10 nM-100 nM) produced a pA2 of 9.0 against 5-HT, which is consistent with its 5-HT1D receptor binding affinity. 3 From these findings we conclude that, in guinea-pig cerebral cortex, the 5-HT terminal autoreceptor is of the 5-HT1D receptor subtype. However, three observations suggest the presence of multiple terminal autoreceptors: shallow inhibition curves to the agonists; a shallow Schild slope of GR127935 antagonism and differences in the maximal responses to 5-HT between whole cortex and frontal cortex.     

L-689,660, a novel cholinomimetic with functional selectivity for M1 and M3 muscarinic receptors.

1. L-689,660, 1-azabicyclo[2.2.2]octane, 3-(6-chloropyrazinyl)maleate, a novel cholinomimetic, demonstrated high affinity binding (pKD (apparent) 7.42) at rat cerebral cortex muscarinic receptors. L-689,660 had a low ratio (34) of pKD (apparent) values for the displacement of binding of the antagonist ([3H]-N-methylscopolamine ([3H]-NMS) compared with the displacement of the agonist [3H]-oxotremorine-M ([3H]-Oxo-M), in rat cerebral cortex. Low NMS/Oxo-M ratios have been shown previously to be a characteristic of compounds that are low efficacy partial agonists with respect to stimulation of phosphatidyl inositol turnover in the cerebral cortex. 2. L-689,660 showed no muscarinic receptor subtype selectivity in radioligand binding assays but showed functional selectivity in pharmacological assays. At M1 muscarinic receptors in the rat superior cervical ganglion, L-689,660 was a potent (pEC50 7.3 +/- 0.2) full agonist in comparison with (+/-)-muscarine. At M3 receptors in the guinea-pig ileum myenteric plexus-longitudinal muscle or in trachea, L-689,660 was again a potent agonist (pEC50 7.5 +/- 0.2 and 7.7 +/- 0.3 respectively) but had a lower maximum response than carbachol. In contrast L-689,660 was an antagonist at M2 receptors in guinea-pig atria (pA2 7.2 (95% confidence limits 7, 7.4)) and at muscarinic autoreceptors in rat hippocampal slices. 3. The putative M1-selective muscarinic agonist, AF102B (cis-2-methylspiro-(1,3-oxathiolane 5,3')-quinuclidine hydrochloride) was found to have a profile similar to L-689,660 but had up to 100 times less affinity in binding and functional assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of a novel 5-HT4 receptor agonist, TS-951, in vitro.

The pharmacological effect of a novel selective 5-HT4 receptor agonist, TS-951 (N-[endo-8-(3-hydroxypropyl)-8-azabicyclo[3.2.1]oct-3-yl]-1-isopropyl-2-oxo-1,2-dihydro-3-quinolinecarboxamide) was investigated in vitro. TS-951 potently inhibited specific [3H]GR113808 binding both in guinea-pig striatum and in mouse brain. The affinity of TS-951 for the 5-HT4 receptor was higher than those of other agonists, 5-HT, cisapride, mosapride and renzapride. On the longitudinal muscle of the guinea-pig ileum, TS-951 caused a concentration-dependent increase in the amplitude of electrically induced submaximal twitch contractions. On the longitudinal muscle of the guinea-pig distal colon, TS-951 also caused concentration-dependent contractions. TS-951 is a high-affinity, selective and potent 5-HT4 receptor agonist. This compound therefore can be considered as a useful pharmacological tool for investigating 5-HT4 receptor-mediated events.     

Contractile responses to sumatriptan and ergotamine in the rabbit saphenous vein: effect of selective 5-HT(1F) receptor agonists and PGF(2alpha)

Contractile responses to ergotamine, sumatriptan and the novel 5-HT(1F) receptor agonists, LY334370 and LY344864 were examined using the rabbit saphenous vein. Ergotamine (pEC(50)=8.7+/-0.06) was 30 fold more potent than 5-hydroxytryptamine (5-HT) (pEC(50)=7.2+/-0.13) and 300 fold more potent than sumatriptan (pEC(50)=6.0+/-0.08) in contracting the rabbit saphenous vein in vitro. The selective 5-HT(1F) receptor agonists, LY334370 or LY344864 (up to 10(-4) M), did not contract the rabbit saphenous vein. The contractile response to ergotamine in this tissue resulted from activation of both alpha(1) and 5-HT(1B/1D) receptors based on the observation that prazosin (10(-6) M), an alpha-adrenoceptor antagonist, and GR127935 (10(-8) M) a 5-HT(1B/1D) receptor antagonist, dextrally shifted the contractile response to ergotamine. In contrast, prazosin (10(-6) M) did not alter contraction to sumatriptan whereas GR127935 (10(-8) M) was a potent antagonist (-log K(B)=10.0) suggesting that sumatriptan-induced contraction of the rabbit saphenous vein was mediated only by activation of receptors similar or identical to 5-HT(1B/1D) receptors. PGF(2alpha) (3x10(-7) M) produced a modest increase (approximately 5.0 - 10.0% maximum PGF(2alpha) contraction) in saphenous vein force. Precontraction with PGF(2alpha) (3x10(-7) M) dramatically augmented the potency and maximal contractile response to sumatriptan (pEC(50)=7.1) and modestly enhanced the contractile potency of ergotamine (pEC(50)=9.0) in the rabbit saphenous vein. However, PGF(2alpha) (3x10(-7) M) only unmasked a contraction to the 5-HT(1F) receptor agonists when concentrations exceeded 10(-5) M, concentrations considerably higher than their 5-HT(1F) receptor affinities. LY334370 (10(-6) M) pretreatment did not alter contraction to either sumatriptan or ergotamine and a higher concentration (10(-5) M) of LY334370 or LY344864 inhibited contraction to sumatriptan. Thus, activation of 5-HT(1F) receptors will not induce vascular contraction (either alone or following modest tone with PGF(2alpha)) or augment contraction to other contractile agonists in the rabbit saphenous vein.     

Comparison of the vasoconstrictor effects of the selective 5-HT1D-receptor agonist L-775,606 with the mixed 5-HT1B/1D-receptor agonist sumatriptan and 5-HT in human isolated coronary artery

AIMS: Vasoconstriction in human coronary artery can be mediated via activation of both 5-HT2 and 5-HT1B-receptors. Coronary vasoconstriction is a rare, but potential adverse effect of the antimigraine drug sumatriptan. In order to investigate the receptor population involved we compared the vasoconstrictor effects of sumatriptan (a mixed 5-HT1B/1D-receptor agonist) with those of L-775, 606 (a selective 5-HT1D-receptor agonist) and 5-HT (the endogenous ligand) in human isolated coronary arteries. METHODS: Coronary arteries were obtained from human hearts removed prior to transplant surgery. Several endothelium denuded ring segments (4 mm in length) were obtained from each artery and mounted for isometric tension recording. Each segment was first exposed to 45 mm KCl and then to 5-HT (1 nm-100 microm ). Concentration-effect curves to L-775,606 (1-(3-(5-(1,2, 4-triazol-4-yl)-1H-indol-3-yl)propyl)-4-(2-(3-fluorophenyl)ethyl)p ipe razine) and sumatriptan were then performed in a consecutive and random manner. The response to repeated application of 5-HT was obtained in separate segments. RESULTS: Twenty-five segments from seven different coronary arteries were studied. Concentration-effect curves were fitted to the data using nonlinear regression analysis. The maximum contraction for L-775,606 was significantly less than that for sumatriptan with Emax values (% relative to 45 mm KCl=100%) of 30.1+/-4.22 and 41.5+/-2.7, respectively. L-775,606 was significantly (30-fold) less potent than sumatriptan in causing contraction compared with sumatriptan (EC50 values were 6.0 microm and 0.2 microm, respectively). For comparison the Emax value for 5-HT was 77.2% and the EC50 value was 0.2 microm. CONCLUSIONS: The selective 5-HT1D-receptor agonist L-775,606 has less propensity towards vasoconstriction in human isolated coronary artery (endothelium-denuded) than was mixed 5-HT1B/1D-receptor agonist sumatriptan. The contractions produced were at concentrations where L-775,606 would be expected to occupy 5-HT1B-receptors.     

Vasodilator and vasoconstrictor responses induced by 5-hydroxytryptamine in the in situ blood autoperfused hindquarters of the anaesthetized rat

In the present study we attempted to characterise the responses and receptors involved in the effects of 5-hydroxytryptamine (5-HT, serotonin) in in situ autoperfused rat hindquarters. Intra-arterial administration of the lowest doses of 5-HT used (0.12-12.5 ng/kg) induced vasodilator responses, whereas the highest doses (25-1000 ng/kg) produced vasoconstriction. The vasodilator effect was inhibited by methiothepin (a non-specific 5-HT(1,2,5,6,7) receptor antagonist) and by a 5-HT(1D/1B) receptor antagonist, i.e., 3-[4-(4-chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanolol (BRL 15572), but not by ritanserin (a selective 5-HT(2) receptor antagonist), 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f] indole (SB 206553, a selective 5-HT(2B/2C) receptor antagonist) or mesulergine (a non-specific serotonergic antagonist that shows affinity to the 5-HT(7) receptor). This vasodilator effect was mimicked by administration of a selective 5-HT(1) receptor agonist - 5-carboxamidotryptamine (5-CT) - and by 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-1 H-indol-3-yl]ethanamine (L-694,247, a selective 5-HT(1D/1B) receptor agonist). Methiothepin, but not mesulergine, inhibited 5-CT-induced vasodilatation and the selective 5-HT(1D/1B) receptor antagonist (BRL 15572) inhibited the vasodilator action induced by L-694,247.The vasoconstrictor effect of 5-HT was significantly decreased by methiothepin, ritanserin and SB 206553, and was mimicked by alpha-methyl-5-HT (a selective 5-HT(2) receptor agonist) but not by administration of BW 723C86, a selective 5HT(2B) receptor agonist. Ritanserin, SB 206553 and spiperone (a non-specific 5-HT(1/2A) receptor antagonist) inhibited the alpha-methyl-5HT-induced vasoconstriction.Our data suggest that the vasodilator response induced by 5-HT in autoperfused rat hindquarters is mainly mediated by 5-HT(1D/1B) receptors, whereas the vasoconstrictor effect is mainly due to the activation of 5-HT(2A) receptors.     

MDL 72832: a potent and stereoselective ligand at central and peripheral 5-HT1A receptors

In receptor binding assays (+/-)MDL 72832, 8-[4-(1,4-benzodioxan-2-ylmethylamino)butyl]-8-azaspiro++ +[4,5] decane-7,9-dione, was a potent (pIC50 9.1), selective and stereospecific ligand for central 5-HT1A recognition sites. In functional tests, (+/-)MDL 72832 and its S(-) and R(+) enantiomers blocked stereoselectively the 8-OH-DPAT-induced neuronal inhibition of the transmurally stimulated guinea-pig ileum and the cardiovascular effects of 8-OH-DPAT in anaesthetized rats. In contrast, (+/-)MDL 72832 and its enantiomers were exclusively '8-OH-DPAT-like' in their ability to fully and stereoselectively generalize to the 8-OH-DPAT discriminative stimulus and, in reserpinised rats, to induce forepaw treading and flat body posture. These results characterize (+/-)MDL 72832 as a potent, stereoselective ligand with mixed agonist and antagonist properties at central and peripheral 5-HT1A receptors. The similar stereoselective requirements for the recognition site and functional effects provides compelling evidence that the 5-HT1A recognition site is indeed a functional receptor.     

Mode of action of gingerols and shogaols on 5-HT3 receptors: binding studies, cation uptake by the receptor channel and contraction of isolated guinea-pig ileum

Ginger (rhizomes of Zingiber officinale) has been shown to exert potent anti-emetic properties, but its mode of action has not yet been elucidated. Among its active constituents, [6]-, [8]- and [10]-gingerol as well as [6]-shogaol were shown in different in vivo studies to be at least partly responsible for the drug's anti-emetic properties. In an attempt to gain more insight into the mode of action of these compounds, three different in vitro models were used to investigate their effects on 5-HT(3) receptors (serotonin receptor subtype) in more detail: [(14)C]guanidinium influx into N1E-115 cells which express 5-HT(3) receptors, isotonic contractions of the isolated guinea-pig ileum and equilibrium competition binding studies using a radioactively labeled 5-HT(3) receptor antagonist ([(3)H]GR65630) (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone). All four compounds inhibited the [(14)C]guanidinium influx through 5-HT(3) receptor channels as well as contractions of the guinea-pig ileum induced by SR57227A ((4-amino)-(6-chloro-2-pyridyl)l-piperidine hydrochloride), a highly selective 5-HT(3) receptor agonist. Both effects were concentration-dependent, with the following order of potency for both models: [6]-shogaol> or =[8]-gingerol>[10]-gingerol> or =[6]-gingerol. All compounds showed also weak anticholinergic and antineurokininergic activities in the guinea-pig ileum (acetylcholine and substance P are mediators of the 5-HT(3) receptor effect). The vanilloid receptor did not seem to be involved derived from experiments using capsazepine. None of the tested ginger substances, however, was able to displace [(3)H]GR65630 from its binding site (5-HT(3) receptor) neither on intact N1E-115 cells nor on the purified membranes of HEK-293 cells over-expressing the h5-HT(3) receptor. It may be concluded that [6]-, [8]-, [10]-gingerol and [6]-shogaol exert their anti-emetic effect at least partly by acting on the 5-HT(3) receptor ion-channel complex, probably by binding to a modulatory site distinct from the serotonin binding site. This may include indirect effects via receptors in the signal cascade behind the 5-HT(3) receptor channel complex such as substance P receptors and muscarinic receptors; this needs further investigation since ginger is effective against motion sickness which is cured by some vanilloids and by anticholinergics such as scopolamine.     

Characterization of 5-HT receptor subtypes mediating contraction in human umbilical vein. 2. Evidence of involvement of 5-HT1B receptors using functional studies

Previous studies have shown that a heterogeneous 5-HT receptor population may be involved in vasoconstrictor actions of 5-HT in human umbilical vein (HUV). The aim of the present study was to evaluate whether the 5-HT(1B/1D) receptor subtype mediates contraction in this tissue. 5-HT(1B/1D)-mediated responses can be enhanced or unmasked after exposure to threshold or sub-threshold KCl concentrations. In HUV rings, when 5-HT, alpha-Me-5HT or bradykinin concentration-response curves (CRC) were generated in the presence or absence of sub-threshold concentrations of KCl, there were not significant differences between the control and the treated rings. On the other hand, sumatriptan, the classic selective 5-HT(1B/1D) receptor agonist, produced a concentration-related contraction that was potentiated in the presence of sub-threshold KCl concentration. In addition, L-694,247, the novel selective 5-HT(1B/1D) receptor agonist, displayed a concentration-dependent contraction with high potency in HUV. The presence of sub-threshold concentrations of KCl produced a marked leftward shift of its CRCs.GR-55562, a 5-HT(1B/1D)-selective antagonist, competitively blocked sumatriptan CRCs with an estimated p A(2) of 8.00 and a slope not different from unity. Likewise, SB-216641, a selective 5-HT(1B) antagonist, produced a parallel rightward shift of sumatriptan CRCs in HUV. The Schild analysis yielded a p A(2) of 9.29, with a slope not different from unity. In addition, L-694,247 contractile responses were competitively blocked by SB-216641 with an estimated p A(2) value of 9.12 and a Schild slope not different from unity. On the other hand, ketanserin behaved as a weak antagonist of L-694,247-induced responses, yielding a calculated p A(2) value of 6.40.In summary, the results obtained in this study support that the 5-HT(1B) receptor subtype is involved in vasoconstrictor responses in HUV.     

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.     

D,L-(tetrazol-5-yl) glycine: a novel and highly potent NMDA receptor agonist.

This paper describes the pharmacological activity of D,L-(tetrazol-5-yl)glycine, a structurally novel and highly potent agonist at the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor. D,L-(Tetrazol-5-yl)glycine potently displaced NMDA receptor binding to rat brain membranes as measured using [3H]CGS19755 (IC50 = 98 +/- 7 nM) and [3H]glutamate (IC50 = 36 +/- 18 nM) as ligands. D,L-(Tetrazol-5-yl)glycine did not appreciably inhibit the binding of D,L-alpha-[5-methyl-3H] amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, or [3H]glycine (IC50s greater than 30,000 nM). D,L-(Tetrazol-5-yl)glycine was more potent than NMDA or cis-methanoglutamate as a depolarizing agent in the rat cortical slice, and unlike these other agents induced rapid receptor-mediated neurotoxicity. Depolarization by D,L-(tetrazol-5-yl)glycine was antagonized by LY233053, a selective NMDA receptor antagonist. D,L-(Tetrazol-5-yl)glycine was a highly potent convulsant when given to neonatal rats (ED50 = 0.071 mg/kg i.p.). Convulsions in neonatal rats or lethality in mice induced by D,L-(tetrazol-5-yl)glycine were selectively antagonized by competitive and non-competitive NMDA receptor antagonists. D,L-(Tetrazol-5-yl)glycine is a structurally novel (tetrazole-substituted) compound that is a highly potent and selective NMDA receptor agonist. D,L-(Tetrazol-5-yl)glycine could be used to probe further NMDA receptor function in vitro and in vivo.     

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.     

Studies on 1-arylpiperazine derivatives with affinity for rat 5-HT7 and 5-HT1A receptors

Several 1-aryl-4-(2-arylethyl)piperazine derivatives were synthesized and tested in-vitro for their binding affinity for 5-HT(7) and 5-HT(1A) receptors. These compounds displayed 5-HT(7 )receptor affinity ranging between K(i) = 474 nM and K(i) = 8.2 nM, besides high affinity for the 5-HT(1A) receptor. Intrinsic activity of the most potent compounds was assessed. 4-[2-(3-Methoxyphenyl)ethyl]-1-(2-methoxyphenyl)piperazine (16) and 1-(1,2-benzisoxazol-3-yl)-4-[2-(3-methoxyphenyl)ethyl]piperazine (20) (K(i) = 24.5 and 8.2 nM, respectively) behaved as partial agonist and full agonist, respectively, when tested for 5-HT(7) receptor-mediated relaxation of substance P-induced guinea-pig ileum contraction.     

Agonist properties of pindolol at h5-HT1A receptors coupled to mitogen-activated protein kinase

At h5-HT1A receptors, stably transfected into Chinese Hamster Ovary Cells (CHO-h5-HT1A), the selective 5-HT1A receptor agonist, (+)8-hydroxy-dipropyl-amino-tetralin, ((+)8-OH-DPAT), transiently activated mitogen-activated protein kinase (MAPK) with a pEC50 of 8.5. The arylalkylamine, (-)-pindolol, also behaved as an agonist with a maximal effect of 57% relative to (+)8-OH-DPAT (100%), and with a pEC50 of 7.2. The selective 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo-hexane carboxamide (WAY100,635), blocked (+)8-OH-DPAT- and (-)-pindolol-induced MAPK activation with pK(B)s of 9.7 and 9.9, respectively, whereas the selective 5-HT(1B) receptor antagonist, 1'-Methyl-5-[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-ylcarbonyl]-2,3,6,7-tetrahydro-5H-spiro[furo[2,3-f]indole-3,4'-piperidine] (SB224,289) was inactive. Pertussis toxin blocked the actions of (+)8-OH-DPAT and (-)-pindolol demonstrating implication of G(i)/G(o) proteins. Thus, stimulation of MAPK provides an intracellular marker and signal for expression of the agonist actions of (-)-pindolol at h5-HT1A receptors.     

The antimigraine drugs ergotamine and dihydroergotamine are potent 5-HT1C receptor agonists in piglet choroid plexus.

1. Fozard & Gray (1989) proposed that migraine is mediated by stimulation of 5-HT1C receptors. We have examined the interaction of two effective anti-migraine agents, ergotamine and dihydroergotamine (DHE), with these receptors. Binding (inhibition of labelling by [3H]-mesulergine) and agonist activity (phosphoinositide hydrolysis) were measured in piglet choroid plexus, a tissue rich in 5-HT1C receptors. 2. The pKD for [3H]-mesulergine binding was 8.4. Ergotamine and DHE both inhibited [3H]-mesulergine binding with a pKD of 7.1. This was similar to the potency of m-chlorophenylpiperazine (m-CPP) (pKD 7.4) and rather less than that of 5-hydroxytryptamine (5-HT) (pKD 8.1). 3. Both ergotamine and DHE were full agonists (pEC50S 7.5 and 7.6 respectively) with potencies similar to that of 5-HT (pEC50 7.7) and greater than that of m-CPP (pEC50 7.1). Mesulergine 10(-7) M produced near-parallel rightward shifts of the concentration-response curves for all these agents of 1.8-2.2 log units, consistent with an action of the agonists at the same receptor. 4. There was no effect of prazosin, spiperone, mepyramine or atropine on the phosphoinositide hydrolysis induced by ergotamine, ruling out an action via alpha 1-adrenoceptors, 5-HT2, histamine H1, or muscarinic receptors. 5. It is concluded that, together with 5-HT, ergotamine and DHE are the most potent 5-HT1C agonists reported so far. These findings do not support the theory that 5-HT1C receptor activation causes migraine.