5-HT3 receptor antagonist effects of DAT-582, (R) enantiomer of AS-5370.

The serotonin 5-HT3 receptor antagonist effects of DAT-582, the (R) enantiomer of AS-5370 ((+/-)-N-[1-methyl-4-(3-methyl-benzyl)hexahydro-1H-1,4-diazepin-6- yl]-1H- indazole-3-carboxamide dihydrochloride), and its antipode were compared with those of AS-5370 and existing 5-HT3 receptor antagonists. In anesthetized rats, DAT-582 antagonized 2-methyl-5-HT-induced bradycardia with an ED50 value of 0.25 microgram/kg i.v., whereas the (S) enantiomer was without effect even at 1000 micrograms/kg i.v. In antagonizing the bradycardia, DAT-582 was as potent as granisetron, slightly more potent than AS-5370, and 2, 5 and 18 times more potent than ondansetron, ICS 205-903 and renzapride, respectively, although it was less potent than zacopride. DAT-582 inhibited cisplatin (10 mg/kg i.v.)-induced emesis in ferrets with an ED50 value of 3.2 micrograms/kg i.v. twice. The antiemetic activity of DAT-582 was more potent than that of the existing 5-HT3 receptor antagonists examined, except zacopride. In contrast, the (S) enantiomer had little effect at 1000 micrograms/kg i.v. twice. In isolated guinea-pig ileum, DAT-582 inhibited 5-HT-induced contractions with an IC50 value of 91 nM, whereas the (S) enantiomer hardly inhibited them even at 1000 nM. These results suggest that DAT-582, the (R) enantiomer of AS-5370, potently and selectively blocks 5-HT3 receptors.     

5-HT3 receptors in NG108-15 neuroblastoma x glioma cells: effect of the novel agonist 1-(m-chlorophenyl)-biguanide.

The effect of the novel agonist, 1-(m-chlorophenyl)-biguanide (mCPBG) was examined on 5-HT3 receptors in NG108-15 mouse neuroblastoma x rat glioma hybrid cells, using whole-cell voltage-clamp and radioligand binding on intact cells. Electrophysiological studies showed that mCPBG is a partial agonist, with an EC50 of 3.1 microM. Displacement of the selective 5-HT3 receptor antagonist [3H]GR65630 by mCPBG revealed a Ki of 14.2 nM. The study suggests that mCPBG may have a high affinity for desensitized 5-HT3 receptors and also revealed some differences between 5-HT3 receptors in NG108-15 and N1E-115 cells.     

Cholecystokinin release mediated by 5-HT3 receptors in rat cerebral cortex and nucleus accumbens.

1. The effects of 5-hydroxytryptamine (5-HT) on the release of cholexystokinin-like immunoreactivity (CCK-LI) were examined in synaptosomes prepared from rat cerebral cortex and nucleus accumbens and depolarized by superfusion with 15 mM KCl. 2. In both areas 5-HT, tested between 0.1 and 100 nM, increased the calcium-dependent, depolarization-evoked CCK-LI release in a concentration-related manner. The concentration-response curves did not differ significantly between the two brain areas (EC50: 0.4 +/- 0.045 nM and 0.48 +/- 0.053 nM, respectively, in cortical and n. accumbens synaptosomes; maximal effect: about 60% at 10 nM 5-HT). 3. The 5-HT1/5-HT2 receptor antagonist methiothepin (300 nM) did not affect the CCK-LI release elicited by 10 nM 5-HT. However, the effects of 10 nM 5-HT were antagonized in a concentration-dependent manner by the 5-HT3 receptor antagonists (3 alpha-tropanyl)-1H-indole-3-carboxylic acid ester (ICS 205-930; 0.1-100 nM; IC50: 3.56 +/- 0.42 nM in the cortex and 3.90 +/- 0.50 nM in the n. accumbens) and ondasetron (IC50: 8.15 +/- 0.73 nM in the cerebral cortex). 5-HT (10 nM) was also strongly antagonized by 100 nM 1 alpha H, 3 alpha 5 alpha H-tropan-3-yl-3,5-dichlorobenzoate (MDL 72222) another blocker of the 5-HT3 receptor. Moreover, the 5-HT3 receptor agonist 1-phenylbiguanide (tested in the cerebral cortex between 0.1 and 100 nM) enhanced CCK-LI release in a manner almost identical to that of 5-HT (EC50 = 0.64 +/- 0.071 nM). 4. It is concluded that 5-HT can act as a potent releaser of CCK-LI in rat cerebrocortex and nucleus accumbens through the activation of receptors of the 5-HT3 type situated on the CCK-releasing terminals. This interaction may provide a rationale for the clinical development of both 5-HT3 and CCK receptor antagonists as novel anxiolytic drugs.     

Peptide leukotriene antagonistic activity of AS-35, a new antiallergic drug.

The effects of 9-[(4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (AS-35), a newly synthesized compound, on leukotrienes (LTs) antagonistic activities were investigated in vitro and in vivo. In isolated guinea pig preparations, AS-35 antagonized LTC4-, LTD4- and LTE4-induced contractions of the ileum with IC50 values of 8 nM, 4 nM and 3 nM, respectively. In the trachea, the agent also antagonized LTD4- and LTE4-induced contractions with IC50 values of 10 nM and 20 nM, respectively. However, LTC4-induced tracheal contraction in the presence of L-serine borate was not antagonized by AS-35. Histamine-, acetylcholine-, serotonin- and bradykinin-induced contractions of the ileum, carbachol-, prostaglandin D2-, prostaglandin F2 alpha-induced contractions of the trachea and LTB4-induced chemotaxis of rat polymorphonuclear leukocytes were not inhibited by AS-35. As to the in vivo models, AS-35 (i.v.) dose-dependently antagonized bronchoconstriction induced by i.v.-injection of LTC4 and LTD4 in anesthetized guinea pigs, but did not inhibit histamine-induced bronchoconstriction. Oral administration of AS-35 also antagonized LTD4- as well as antigen-induced LT-mediated bronchoconstriction. In addition, LTD4-induced increase in the cutaneous vascular permeability of guinea pig was inhibited by the drug (p.o.). These results indicate that AS-35 is an orally effective, potent and selective peptide LT antagonist.     

Comparison of [125I]beta-endorphin binding to rat brain and NG108-15 cells using a monoclonal antibody directed against the opioid receptor

The properties of [125I]beta h-endorphin-binding sites from rat brain membranes and membranes from the NG108-15 cell line were compared using a monoclonal antibody directed against the opioid receptor and opioid peptides as probes. The binding of [125I]beta h-endorphin to both rat brain and NG108-15 membranes yielded linear Scatchard plots with Kd values of 1.2 nM and 1.5 nM, respectively, and Bmax values of 865 fmol/mg rat brain membrane protein and 1077 fmol/mg NG108-15 membrane protein. A monoclonal antibody, OR-689.2.4, capable of inhibiting mu and delta binding but not kappa binding to rat brain membranes, noncompetitively inhibited the binding of 1 nM [125I]beta h-endorphin to rat brain and NG108-15 membranes with an IC50 value of 405 nM for rat brain membranes and 543 nM for NG108-15 membranes. The monoclonal antibody also inhibited the binding of 3 nM [3H] [D-penicillamine2, D-penicillamine5] enkephalin to NG108-15 membranes with an IC50 value of 370 nM. In addition to blocking the binding of [125I]beta h-endorphin to brain membranes, the antibody also displaced [125I]beta h-endorphin from membranes. Site-specific opioid peptides had large variations in their IC50 values depending on whether they were inhibiting [125I]beta h-endorphin binding to rat brain or the NG108-15 membranes. When the peptides were tested with the monoclonal antibody for their combined ability to inhibit [125I]beta h-endorphin binding to both membrane preparations, the peptides and antibody blocked binding as though they were acting at allosterically coupled sites, not two totally independent sites. These studies suggest that mu-, delta-, and beta-endorphin-binding sites share some sequence homology with the 35,000-dalton protein that the antibody is directed against.     

Characteristics of 5-HT3 binding sites in NG108-15, NCB-20 neuroblastoma cells and rat cerebral cortex using [3H]-quipazine and [3H]-GR65630 binding.

1. The biochemical and pharmacological properties of 5-HT3 receptors in homogenates of NG108-15 and NCB-20 neuroblastoma cells and rat cerebral cortex have been ascertained by the use of [3H]-quipazine and [3H]-GR65630 binding. 2. In NG108-15 and NCB-20 cell homogenates, [3H]-quipazine bound to a single class of high affinity (NG108-15: Kd = 6.2 +/- 1.1 nM, n = 4; NCB-20: Kd = 3.0 +/- 0.9 nM, n = 4; means +/- s.e.means) saturable (NG108-15: Bmax = 1340 +/- 220 fmol mg-1 protein; NCB-20: Bmax = 2300 +/- 200 fmol mg-1 protein) binding sites. In rat cortical homogenates, [3H]-quipazine bound to two populations of binding sites in the absence of the 5-hydroxytryptamine (5-HT) uptake inhibitor, paroxetine (Kd1 = 1.6 +/- 0.5 nM, Bmax1 = 75 +/- 14 fmol mg-1 protein; Kd2 = 500 +/- 300 nM, Bmax2 = 1840 +/- 1040 fmol mg-1 protein, n = 3), and to a single class of high affinity binding sites (Kd = 2.0 +/- 0.5 nM, n = 3; Bmax = 73 +/- 6 fmol mg-1 protein) in the presence of paroxetine. The high affinity (nanomolar) component probably represented 5-HT3 binding sites and the low affinity component represented 5-HT uptake sites. 3. [3H]-paroxetine bound with high affinity (Kd = 0.02 +/- 0.003 nM, n = 3) to a site in rat cortical homogenates in a saturable (Bmax = 323 +/- 45 fmol mg-1 protein, n = 3) and reversible manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Common pharmacological and physico-chemical properties of 5-HT3 binding sites in the rat cerebral cortex and NG 108-15 clonal cells

On account of the postulated existence of 5-HT3 receptor subtypes, the respective physico-chemical and pharmacological properties of specific binding sites for the potent 5-HT3 antagonist [3H]zacopride were compared using membranes from the rat posterior cortex or neuroblastoma-glioma NG 108-15 clonal cells. In both membrane preparations, [3H]zacopride bound to a single class of specific sites with a Kd close to 0.5 nM. However, the Bmax value in NG 108-15 cell membranes (970 +/- 194 fmol/mg protein) was approximately 50 times larger than that in cortical membranes (19 +/- 2 fmol/mg protein). The specific binding of [3H]zacopride was equally affected by temperature, pH and molarity of the assay medium, and equally insensitive to thiol- and disulfide-reagents (N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, dithiothreitol) and GTP in cortical as well as NG 108-15 cell membranes. Determination of the molecular size of [3H]zacopride specific binding sites by radiation inactivation yielded values close to 35 kDa for both membrane preparations. Finally, a highly significant positive correlation (r = 0.979) was found between the respective pKi values of 34 different drugs for their inhibition of [3H]zacopride specific binding to cortical or NG 108-15 cell membranes. Among them, the most potent was S(-)zacopride (pKi = 9.55), followed by BRL 43964, ICS 205-930, quipazine, R(+)zacopride, GR 38032F and MDL 72222. Atypical antidepressants (mianserin, amoxapine) and neuroleptics (clotiapine, loxapine and clozapine) were active in rather low concentrations (pKi less than 6.5), suggesting that recognition of 5-HT3 sites might be relevant to part of the in vivo effects of these drugs. Such identical physico-chemical and pharmacological properties of [3H]zacopride specific binding in cortical and NG 108-15 cell membranes strongly suggest that the same 5-HT3 receptor (subtype?) exists in these two preparations.     

Distinct functional profiles of aripiprazole and olanzapine at RNA edited human 5-HT2C receptor isoforms

In this study we have functionally characterized aripiprazole (OPC-14597; 7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyloxy-3,4-dihydro-2-(1H)-quinolinone), the prototype of a new generation antipsychotic drug termed dopamine-serotonin-system stabilizer, in cells expressing 5-hydroxytryptamine2 (5-HT2) receptor subtypes in comparison with olanzapine. In Chinese hamster ovary (CHO) cells stably expressing 5-HT2 receptors, aripiprazole displayed a dual agonist/antagonist profile for 5-HT2C receptor (VNI isoform) mediated calcium signaling (EC50 1070 nM, IC50 281 nM). It exhibited no appreciable 5-HT2A or 5-HT2B agonism, whereas it antagonized 5-HT-stimulated calcium increase at either 5-HT2A or 5-HT2B receptor expressed in CHO cells (IC50s of 369 and 0.46 nM, respectively). In comparison, olanzapine was devoid of agonism but was an antagonist at all three subtypes, with a potency rank order of 5-HT2A (IC50, 2.5 nM)>5-HT2B (47 nM)>5-HT2C (69 nM). In human embryonic kidney (HEK) cells transiently expressing 5-HT2C receptor isoforms, aripiprazole exhibited full agonism at the unedited INI, but partial agonism at the partially edited VNI and fully edited VSV isoforms (EC50s of 571, 1086 and 2099 nM, respectively). A partial antagonism was also observed for aripiprazole at the two edited isoforms (IC50s of 1138 and 1000 nM, respectively). In contrast, while lacking agonist activity at the VNI and VSV, olanzapine showed inverse agonism at the INI isoform (IC50 594 nM), reaching a maximal attenuation of 20%. In addition, olanzapine was a full antagonist at all three isoforms, with a rank order of potency of VNI (IC50, 79 nM)>VSV (101 nM)>INI (3856 nM). The modest 5-HT2A antagonism and 5-HT2C partial agonism, along with reported D2 and 5-HT1A partial agonism, may allow aripiprazole to stabilize the disturbed dopamine-serotonin interplay in schizophrenia with a moderate yet adequate pharmacological intervention. 5-HT2C agonism may also underlie the minimal weight gain seen with aripiprazole.     

Evidence that the atypical 5-HT3 receptor ligand, [3H]-BRL46470, labels additional 5-HT3 binding sites compared to [3H]-granisetron.

1. The radioligand binding characteristics of the 3H-derivative of the novel 5-HT3 receptor antagonist BRL46470 were investigated and directly compared to the well characterized 5-HT3 receptor radioligand [3H]-granisetron, in tissue homogenates prepared from rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen. 2. In rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, [3H]-BRL46470 bound with high affinity (Kd (nM): 1.57 +/- 0.18, 2.49 +/- 0.30, 1.84 +/- 0.27, 3.46 +/- 0.36, respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 102 +/- 16, 44 +/- 4, 968 +/- 32 and 2055 +/- 105, respectively; mean +/- s.e. mean, n = 3-4) but failed to display specific binding in human putamen homogenates. 3. In the same homogenates of rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen as used for the [3H]-BRL46470 studies, [3H]-granisetron also bound with high affinity (Kd (nM): 1.55 +/- 0.61, 2.31 +/- 0.44, 1.89 +/- 0.36, 2.03 +/- 0.42 and 6.46 +/- 2.58 respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 39 +/- 4, 20 +/- 2, 521 +/- 47, 870 +/- 69 and 18 +/- 2, respectively; mean +/- s.e. mean, n = 3-4).(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction of antidepressant drugs with central and peripheral (enteric) 5-HT3 and 5-HT4 receptors.

1. A combined study of receptor binding in central neuronal cell membranes and functional responses in isolated segments of guinea-pig small intestine allowed characterization of the interaction of four antidepressant drugs with central and peripheral 5-HT3 and 5-HT4 receptors. 2. Clomipramine, paroxetine and fluoxetine inhibited [3H]-DAU 6215 binding to 5-HT3 recognition sites in NG 108-15 cells with IC50 values in the range 1.3-4 microM. Litoxetine had an IC50 of 0.3 microM. The specific binding of [3H]-GR 113808 to 5-HT4 recognition sites in pig striatal membranes was inhibited by all four antidepressants with negligible potency (IC50 values > or = 20 microM). 3. In whole ileal segments, concentration-response curves to 5-HT were biphasic, with the high- and low-potency phases involving 5-HT4 and 5-HT3 receptors, respectively. Curves to 2-methyl-5-hydroxytryptamine (2-methyl-5-HT: a 5-HT3 receptor agonist) and 5-methoxytryptamine (5-MeOT: a 5-HT4 receptor agonist) were monophasic. All antidepressants were used at concentrations lacking anticholinoceptor properties, as demonstrated in both electrically stimulated longitudinal muscle-myenteric plexus preparations (LMMPs) and in unstimulated LMMPs following addition of acetylcholine (100 nM). 4. Fluoxetine (0.1-1 microM) and litoxetine (0.3-3 microM) antagonized both the high- and low-potency phases of the 5-HT curve. Schild analysis for the low-potency phase yielded pA2 estimates of 6.6 +/- 0.3 (Schild slope of 1.1) and of 6.6 +/- 0.1 (Schild slope of 1.1), respectively. At higher concentrations (3 microM), fluoxetine markedly inhibited the 5-HT response maximum. Clomipramine (10-300 nM) inhibited, by a mechanism independent of concentration, both phases of the 5-HT curve with a reduction of the maximum response. Paroxetine (1 microM) was ineffective on the high-potency phase, but caused a rightward shift of the low-potency phase (pKB: 6.1 +/- 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic serotonin receptors regulate [3H]serotonin release from rat spinal cord synaptosomes

Superfused rat spinal cord synaptosomes were studied to determine if inhibitory serotonin (5-HT) receptors (autoreceptors) exist on spinal serotonergic nerve terminals. Exogenous 5-HT (1-50 nM) produced a concentration-dependent inhibition of K+-induced [3H]5-HT release but did not affect basal [3H]5-HT release. A 32-44% inhibition was produced by 30 nM 5-HT. The inhibitory effect of 30 nM 5-HT was effectively antagonized by 100 nM metitepine, a 5-HT autoreceptor antagonist. The results provide evidence for the existence of 5-HT autoreceptors in rat spinal cord tissue.     

The novel 5-HT1A receptor antagonist (S)-UH-301 antagonizes 8-OH-DPAT-induced effects on male as well as female rat copulatory behaviour.

The 5-HT1A receptor agonist 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) facilitates male rat copulatory behaviour but inhibits female rat copulatory behaviour. The effect of the novel 5-HT1A receptor antagonist (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin [S)-UH-301) on these 8-OH-DPAT-induced responses was tested. 8-OH-DPAT was given s.c. in a dose of 0.176 mumol/kg (50 micrograms/kg). The doses of (S)-UH-301 given s.c. were 1.76 mumol/kg (0.53 mg/kg) and 5.28 mumol/kg (1.60 mg/kg). The administration of (S)-UH-301 10 min before 8-OH-DPAT antagonized the 8-OH-DPAT-induced effects on both male and female rat copulatory behaviour. The results presented strongly support the classification of (S)-UH-301 as a 5-HT1A receptor antagonist. In addition, the effect of the enantiomer of (S)-UH-301, (R)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin [R)-UH-301), on male rat copulatory behaviour was tested. This enantiomer was found to facilitate male rat copulatory behaviour in a 8-OH-DPAT-like manner, supporting a 5-HT1A agonistic action of (R)-UH-301.     

An electrophysiological investigation of the properties of a murine recombinant 5-HT3 receptor stably expressed in HEK 293 cells.

1. The pharmacological and biophysical properties of a recombinant 5-HT3 receptor have been studied by use of patch-clamp techniques applied to HEK 293 cells stably transfected with the murine 5-HT3 R-A cDNA. 2. At a holding potential of -60 mV, 77% of cells investigated responded to ionophoretically applied 5-HT with an inward current. Such currents were unaffected by methysergide (1 microM), or ketanserin (1 microM), but were antagonized in a concentration-dependent and reversible manner by the selective 5-HT3 receptor antagonist, ondansetron (IC50 = 440 pM) and the non-selective antagonists (+)-tubocurarine (IC50 = 1.8 nM) and metoclopramide (IC50 50 nM). 3. The 5-HT-induced current reversed in sign (E5-HT) at approximately -2mV and exhibited inward rectification. The influence of extra- and intracellular ion substitutions upon E5-HT indicates the 5-HT-evoked current to be mainly mediated by a mixed monovalent cation conductance. 4. Calcium and magnesium (0.1-10 nM) produced a concentration-dependent, voltage-independent, inhibition of the 5-HT-induced response. Zinc (0.3-300 microM) exerted a biphasic effect with low concentrations enhancing, and high concentrations depressing, the 5-HT-evoked current. 5. Fluctuation analysis of inward currents evoked by a low (1 microM) concentration of 5-HT suggests the current to be mediated by the opening of channels with a conductance of 420 fS. 6. The pharmacological and biophysical properties of the 5-HT3 R-A are similar to those previously described for 5-HT3 receptors native to murine neuroblastoma cell lines, with the exception that the function of the recombinant receptor was enhanced by low concentrations of zinc. This observation suggests that the properties of the native receptor are not completely represented by the 5-HT3 R-A subunit alone.     

1-(m-chlorophenyl)-biguanide, a potent high affinity 5-HT3 receptor agonist

1-(m-Chlorophenyl)-biguanide (mCPBG) was examined and compared with three 5-HT3 receptor agonists in three 5-HT3 receptor models. mCPBG inhibited [3H]GR67330 binding to 5-HT3 receptors with high affinity (IC50 1.5 nM). mCPBG depolarized the rat vagus nerve with an EC50 one tenth of that for 5-HT (0.05 vs. 0.46 microM); the maximum depolarization was approximately half that for 5-HT. The mCPBG depolarization was potently blocked by the selective 5-HT3 antagonist, ondansetron (pKB 8.6 +/- 0.1). In anaesthetised cats, mCPBG potently evoked the Bezold-Jarisch reflex which was blocked by low doses of ondansetron (10 micrograms/kg i.v.). It is concluded that mCPBG is a potent, high affinity 5-HT3 receptor agonist.     

Characteristics of ATP-induced current through P2X7 receptor in NG108-15 cells: unique antagonist sensitivity and lack of pore formation

ATP activates the mouse P2X7 receptor and induces a nonselective-cation current in NG108-15 cells. We investigated the effects of five receptor antagonists on the ATP-induced nonselective-cation current through P2X7 receptor (I(NS.P2X7)) in NG108-15 cells. Nonselective P2 receptor antagonists, RB-2, PPADS and suramin inhibited the I(NS.P2X7) with IC50 values of 4.3, 53 and 40 microM, respectively. However, KN-04, which is a potent antagonist of human P2X7 receptors but is not that of rat P2X7 receptors, had only a weak blocking effect. Furthermore, oxidized-ATP (300 microM), an antagonist of the P2X7 receptor-mediated pore-formation, did not affect the I(NS.P2X7). Prolonged ATP application did not increase the membrane permeability to large molecules, N-methyl-D-glucamine or Yo-Pro-1, indicating that pore-formation was not promoted by the P2X7 receptor activation in NG108-15 cells. These results suggest that antagonist sensitivities and pore-forming properties of the P2X7 receptors in NG108-15 cells are different from those of other cells types.     

Anti-thrombotic and vascular effects of AR246686, a novel 5-HT2A receptor antagonist

We have evaluated the anti-platelet and vascular pharmacology of AR246686, a novel 5-hydroxytryptamine2A (5-HT2A) receptor antagonist. AR246686 displayed high affinity binding to membranes of HEK cells stably expressing recombinant human and rat 5-HT2A receptors (Ki=0.2 nM and 0.4 nM, respectively). Functional antagonism (IC50=1.9 nM) with AR246686 was determined by inhibition of ligand-independent inositol phosphate accumulation in the 5-HT2A stable cell line. We observed 8.7-fold and 1360-fold higher affinity of AR246686 for the 5-HT2A receptor vs. 5-HT2C and 5-HT2B receptors, respectively. AR246686 inhibited 5-HT-induced amplification of ADP-stimulated human platelet aggregation (IC50=21 nM). Similar potency was observed for inhibition of 5-HT stimulated DNA synthesis in rat aortic smooth muscle cells (IC(50)=10 nM) and 5-HT-mediated contraction in rat aortic rings. Effects of AR246686 on arterial thrombosis and bleeding time were studied in a rat model of femoral artery occlusion. Oral dosing of AR246686 to rats resulted in prolongation of time to occlusion at 1 mg/kg, whereas increased bleeding time was observed at a dose of 20 mg/kg. In contrast, both bleeding time and time to occlusion were increased at the same dose (10 mg/kg) of clopidogrel. These results demonstrate that AR246686 is a high affinity 5-HT2A receptor antagonist with potent activity on platelets and vascular smooth muscle. Further, oral administration results in anti-thrombotic effects at doses that are free of significant effects on traumatic bleeding time.     

Bradyzide, a potent non-peptide B(2) bradykinin receptor antagonist with long-lasting oral activity in animal models of inflammatory hyperalgesia

Bradyzide is from a novel class of rodent-selective non-peptide B(2) bradykinin antagonists (1-(2-Nitrophenyl)thiosemicarbazides). Bradyzide has high affinity for the rodent B(2) receptor, displacing [(3)H]-bradykinin binding in NG108-15 cells and in Cos-7 cells expressing the rat receptor with K(I) values of 0.51+/-0.18 nM (n=3) and 0.89+/-0.27 nM (n=3), respectively. Bradyzide is a competitive antagonist, inhibiting B(2) receptor-induced (45)Ca efflux from NG108-15 cells with a pK(B) of 8.0+/-0.16 (n=5) and a Schild slope of 1.05. In the rat spinal cord and tail preparation, bradyzide inhibits bradykinin-induced ventral root depolarizations (IC(50) value; 1.6+/-0.05 nM (n=3)). Bradyzide is much less potent at the human than at the rodent B(2) receptor, displacing [(3)H]-bradykinin binding in human fibroblasts and in Cos-7 cells expressing the human B(2) receptor with K(I) values of 393+/-90 nM (n=3) and 772+/-144 nM (n=3), respectively. Bradyzide inhibits bradykinin-induced [(3)H]-inositol trisphosphate (IP(3)) formation with IC(50) values of 11.6+/-1.4 nM (n=3) at the rat and 2.4+/-0.3 microM (n=3) at the human receptor. Bradyzide does not interact with a range of other receptors, including human and rat B(1) bradykinin receptors. Bradyzide is orally available and blocks bradykinin-induced hypotension and plasma extravasation. Bradyzide shows long-lasting oral activity in rodent models of inflammatory hyperalgesia, reversing Freund's complete adjuvant (FCA)-induced mechanical hyperalgesia in the rat knee joint (ED(50), 0.84 micromol kg(-1); duration of action >4 h). It is equipotent with morphine and diclofenac, and 1000 times more potent than paracetamol, its maximal effect exceeding that of the non-steroidal anti-inflammatory drugs (NSAIDs). Bradyzide does not exhibit tolerance when administered over 6 days. In summary, bradyzide is a potent, orally active, antagonist of the B(2) bradykinin receptor, with selectivity for the rodent over the human receptor. British Journal of Pharmacology (2000) 129, 77 - 86     

Chronic treatment with imipramine inhibits cell growth and enhances serotonin 2C receptor mRNA expression in NG 108-15 cells

We previously reported that NG108-15 cells contain intrinsic serotonin 2C receptor (5-HT2CR). The effects of imipramine, a 5-HT2CR antagonist, on cell growth, cell viability, and the 5-HT2CR mRNA level were investigated in this study. Repeated treatment with imipramine at concentrations of 1 - 10 microM for 5 days inhibited cell growth in a concentration-dependent manner without affecting cell viability. In addition, the level of 5-HT2CR mRNA was elevated. At 30 microM, imipramine significantly reduced cell viability. Our findings suggest that the effect of imipramine on neuronal growth may be related to its effects on 5-HT2CR.     

Importance of phenylalanine 107 in agonist recognition by the 5-hydroxytryptamine(3A) receptor

The 5-hydroxytryptamine (5-HT)(3) receptor is a member of the ligand-gated ion channel receptor family with significant homology to the nicotinic acetylcholine, gamma-aminobutyric acid(A), and glycine receptors. In this receptor class, the agonist binding site is formed by parts of the extracellular amino-terminal region. This study examines the effects of altering phenylalanine 107 (F107) of the 5-HT(3AL) subunit, obtained from NG108-15 cells, using site-directed mutagenesis. The wild-type (WT) and mutant receptors were expressed in HEK 293 cells and characterized using both whole-cell patch-clamp and radioligand binding. The tyrosine mutant F107Y exhibits a significantly lower affinity for the agonist 5-HT (K(i) = 203 versus 15.6 nM) and an increase of similar magnitude in the EC(50) value (10.6 versus 1.2 microM) compared with WT. The activation kinetics of the maximal currents generated by 5-HT with this mutant were markedly slower than those of the WT receptor, but application of supramaximal concentrations of the agonist markedly decreased the time to half-peak. The asparagine mutant F107N displayed a significantly higher affinity for 5-HT than the WT receptor (1.62 versus 15.6 nM), which was mirrored in direction and magnitude by changes in the EC(50) value for this agonist (0.2 versus 1.2 microM). In contrast to the WT receptor, the mutant F107N was activated by acetylcholine (EC(50) = 260 microM). The response to acetylcholine was blocked by the 5-HT(3) receptor antagonist renzapride with a similar IC(50) value as that determined against currents generated by 5-HT in the WT receptor. These data suggest that F107 is an important determinant of agonist recognition at the 5-HT(3) 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.