tropisetron and Neuroblastoma

The serotonin 5-HT3 receptor, a ligand-gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5-HT3 receptors induce Ca2+ influx. Whereas postsynaptic 5-HT3 receptors induce depolarization, being permeant to Na+ and K+, the basis of presynaptic 5-HT3 receptor-induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 nM 1-(m-chlorophenyl)biguanide (mCPBG), a highly specific 5-HT3 receptor agonist, induced increases in internal free Ca2+ concentration ([Ca2+]i) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG-induced increases in [Ca2+]i ranged from 1.3 to 1.6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5-HT3 receptor antagonist, but were insensitive to the removal of external free Na+ (substituted with N-methyl-D-glucamine), to prior depolarization induced on addition of 20 mM K+, or to voltage-gated Ca2+ channel blockade by 10 microM Co2+/Cd2+ or by 1 microM omega-conotoxin MVIIC/1 microM oemga-conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca2+ influx induced by 5-HT3 receptor activation in NG108-15 cells by 1 microM mCPBG was substantially reduced by 10 microM Co2+/Cd2+ and was completely blocked by 1 microM nitrendipine, an L-type Ca2+ channel blocker. We conclude that in contrast to the perikaryal 5-HT3 receptors, presynaptic 5-HT3 receptors appear to be uniquely calcium-permeant.

Topics: Animals; Biguanides; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Membrane Permeability; Corpus Striatum; Glioma; Hybrid Cells; Immunohistochemistry; Indoles; Ion Channel Gating; Mice; Microscopy, Confocal; Neuroblastoma; Nitrendipine; Potassium; Presynaptic Terminals; Rats; Receptors, Serotonin; Receptors, Serotonin, 5-HT3; Serotonin Antagonists; Serotonin Receptor Agonists; Synaptosomes; Tropisetron

In the present study, we investigated the effects of chronic in vitro administration of amitriptyline, a tricyclic antidepressant, on cyclic GMP formation stimulated by 5-hydroxytryptamine (5-HT) in the neuroblastoma x glioma hybrid cell line, NG 108-15, 5-HT (0.01-100 microM)-stimulated cyclic GMP formation was concentration-dependent and was sensitive to ICS 205-930, a 5-HT3 receptor antagonist. Exposure of NG 108-15 cells to 5 microM amitriptyline for 3 days significantly reduced 5-HT-stimulated cyclic GMP formation. Acute treatment with amitriptyline had no effect on 5-HT-stimulated cyclic GMP formation. The reduction by chronic amitriptyline exposure of 10 microM 5-HT-stimulated cyclic GMP formation was concentration-dependent over the concentration range examined (0.5 to 10 microM). The IC50 of amitriptyline was 1.9 microM. In contrast, amitriptyline exposure, even at a concentration of 8 microM, failed to modify cyclic GMP formation stimulated by bradykinin, sodium nitroprusside, or atrial natriuretic peptide. Increases in intracellular Ca2+ concentration ([Ca2+]i) evoked by 10 microM 5-HT were attenuated in amitriptyline-exposed cells, while 100 nM bradykinin-induced [Ca2+]i increases were not affected. In addition, chronic exposure to 5 microM amitriptyline caused a decrease in affinity (Kd) of [3H]zacopride specific binding to 5-HT3 recognition sites. The Bmax for the labelled ligand remained unchanged. These results suggest that chronic amitriptyline exposure reduces 5-HT-stimulated cyclic GMP formation and [Ca2+]i increases, and this may reflect the functional changes of 5-HT3 receptors.

Topics: Amitriptyline; Antidepressive Agents, Tricyclic; Benzamides; Bradykinin; Brain Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cyclic GMP; Fluorescent Dyes; Fura-2; Glioma; Hybrid Cells; Indoles; Kinetics; Neuroblastoma; Receptors, Serotonin; Serotonin Antagonists; Tropisetron; Tumor Cells, Cultured

The 5-HT3-receptor antagonist, ondansetron, has been shown to have positive effects in selected in-vivo models of memory impairment and anxiety. The exact mechanisms underlying such bioactivities are unknown. In the present work, an 86Rb efflux bioassay was used to show that ondansetron has a unique ability to block voltage-gated potassium channels in TE671 human neuroblastoma cells. This intrinsic potassium-channel-blocking (KCB) property is relatively weak (IC50 20 microM), but is not shared by other 5-HT3-receptor ligands including zatosetron, MDL 72222, LY 278, 584, zacopride, 1-phenylbiguanide, and ICS 205-930 (tropisetron). Pre-incubation of the target neuroblastoma cells with several 5-HT-receptor ligands including 5-hydroxytryptamine, 8-OH-DPAT, ketanserin, 2-methyl-5-HT, as well as a number of potent 5-HT3 agonists and antagonists and two selective neurotoxins, failed to abolish the KCB action of ondansetron. A preliminary structure-activity relationship analysis indicates that the KCB activity of ondansetron is almost entirely attributable to its structural nucleus, 2,3-dihyro-9-methyl-4(1H)-carbazolone. It is hypothesized that the KCB action of ondansetron is mediated through receptors other than 5-HT3 receptors. The KCB activity of ondansetron may be a significant factor in the in-vivo cognition-enhancing activities of this compound, conceivably due to depolarization of the hippocampal synaptic membranes and a consequent augmentation of neurotransmission.

Topics: Anti-Anxiety Agents; Benzamides; Benzofurans; Biguanides; Bridged Bicyclo Compounds, Heterocyclic; Humans; Hypoglycemic Agents; Indazoles; Indoles; Neuroblastoma; Neurotoxins; Ondansetron; Potassium Channels; Receptors, Serotonin; Receptors, Serotonin, 5-HT3; Rubidium Radioisotopes; Serotonin Antagonists; Serotonin Receptor Agonists; Structure-Activity Relationship; Tropanes; Tropisetron; Tumor Cells, Cultured

The effects of the novel antagonist S 11978 (Endo-7-[(8-methyl-8-azabicyclo[3,2,1]-3-octyl)oxycarbonyl] benzo[b] thiophene) on 5HT3 receptors were examined in N1E-115 mouse neuroblastoma x rat glioma hybrid cells, with radioligand binding and whole cell patch clamp techniques. The 5HT3 receptor ligand [3H] quipazine was displaced by ICS 205-930, GR 38032F and S 11978 with KI values of 2.25 nM, 36.5 nM and 1.75 nM respectively. Electrophysiological studies showed that S 11978 is a potent 5HT3 antagonist: IC50 values for inhibition of 5HT-induced inward current by ICS 205-930, GR 38032F and S 11978 were 0.22 nM, 0.63 nM and 0.43 nM respectively at a holding potential of -65 mV. It is concluded that S 11978 is a potent, high affinity 5HT3 receptor antagonist.

Topics: Animals; Binding, Competitive; Electrophysiology; Indoles; Kinetics; Mice; Neuroblastoma; Ondansetron; Quipazine; Serotonin Antagonists; Thiophenes; Tritium; Tropisetron; Tumor Cells, Cultured

The effect of a novel 5-HT3 receptor antagonist, BRL 46470, has been studied on two electrophysiological models for 5-HT3 receptors: grease-gap recordings from rat isolated vagus nerve and whole-cell patch-clamp recordings from mouse neuroblastoma-rat glioma NG108-15 cells. Its action on the rat vagus nerve was compared to that of four other 5-HT3 receptor antagonists. On the rat vagus, BRL 46470 reduced the maximum depolarizing response to 5-HT in a concentration-dependent manner with an IC50 of 0.3-1.0 nM, but the EC50 for 5-HT was not appreciably affected. This action was similar to that of granisetron and ICS 205-930, but differed from that of GR38032F and (+)-tubocurarine which produced clear rightward shifts of the concentration-response curve to 5-HT. The 5-HT-induced fast inward current of voltage-clamped NG108-15 cells was also antagonized by 1 nM BRL 46470 in an insurmountable manner. In contrast to (+)-tubocurarine, the action of BRL 46470 on the rat vagus nerve and NG108-15 cells did not readily reverse on washing with antagonist-free medium. It is concluded that BRL 46470 is a potent, insurmountable 5-HT3 receptor antagonist on the rat vagus and NG108-15 cells.

Topics: Animals; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Electrophysiology; Glioma; Granisetron; In Vitro Techniques; Indoles; Kinetics; Male; Neuroblastoma; Neurons; Ondansetron; Rats; Rats, Sprague-Dawley; Serotonin Antagonists; Tropisetron; Tubocurarine; Tumor Cells, Cultured; Vagus Nerve

4-(2-Methoxyphenyl)-2-[4(5)-methyl-5(4)-imidazolylmethyl]thiazole (5) is a highly potent member of a structurally novel series of selective serotonin-3 receptor antagonists. The synthesis of tritiated 5 and its binding profile in neuroblastoma-glioma 108-15 cells are described. Furthermore, in vivo studies in rat with this radioligand indicate that it effectively penetrates the blood-brain barrier upon peripheral administration. Thus, 5 should be a useful pharmacological tool for both in vitro and in vivo studies of this class of compounds.

Topics: Animals; Blood-Brain Barrier; Brain; Chemical Phenomena; Chemistry; Glioma; Imidazoles; Mice; Molecular Structure; Neuroblastoma; Serotonin Antagonists; Thiazoles; Tumor Cells, Cultured

Both substance P and, to a lesser degree, serotonin activate cation permeability in neuroblastoma x glioma hybrid cells, as determined by measurement of [14C]guanidinium uptake. Serotonin potentiates the action of substance P by shifting the concentration-effect curve of substance P to the left. The EC50 value for the synergistic effect of serotonin was around 0.3 microM. Dopamine and noradrenaline displayed comparable activity, albeit only at 50 and 130 times higher concentrations, respectively. The order of potency of various substance P-analogues was not changed by serotonin, indicating that the specificity of the substance P site on the hybrid cells was not affected by serotonin. Various other neurotransmitters and peptides had no effect on the response of the hybrid cells to substance P. The serotonin receptor interacting with the substance P receptor may be classified as a 5-HT3-receptor since methysergide, cimetidine, and ketanserin were ineffective, but two inhibitors specific for 5-HT3-receptors, ICS 205-930 (3 alpha-tropanyl-1H-indole-3-carboxylic acid ester) and MDL 72222 (1 alpha H,3 alpha,5 alpha H-tropan-3-yl-3,5-dichlorobenzoate), blocked the effect of serotonin at nanomolar concentrations. However, the two serotonin antagonists might also be blocking the ion permeability, since at higher concentrations they fully inhibited the stimulation of guanidinium uptake by substance P or by substance P plus serotonin. The synergism between substance P and serotonin on the hybrid cells offers the opportunity to study the mechanism of interaction of neurotransmitter receptors on a permanent neuronal cell line.

Topics: Catecholamines; Cations; Cell Line; Cell Membrane Permeability; Drug Interactions; Glioma; Guanidine; Guanidines; Indoles; Neuroblastoma; Neurons; Serotonin; Substance P; Tropanes; Tropisetron; Tumor Cells, Cultured

Topics: Animals; Cerebral Cortex; Indoles; Neuroblastoma; Psychotropic Drugs; Rats; Receptors, Serotonin; Serotonin Antagonists; Tropisetron; Tumor Cells, Cultured

[3H]ICS 205-930 labelled 5-HT3 recognition sites in membranes prepared from murine neuroblastoma N1E-115 cells. Binding was rapid, reversible, saturable and stereoselective to an apparently homogeneous population of sites. Kinetic studies revealed that agonists and antagonists produced a monophasic dissociation reaction of [3H]ICS 205-930 from its recognition sites. The dissociation rate constant of the radioligand was similar whether the dissociation was induced by an agonist or an antagonist. Competition studies carried out with agonists and antagonists also suggested the presence of a homogeneous population of [3H]ICS 205-930 recognition sites. Competition curves were best fit for a 1 site model. [3H]ICS 205-930 binding sites displayed the pharmacological profile of a 5-HT3 receptor. The interactions of agonists and antagonists with [3H]ICS 205-930 recognition sites were apparently competitive in nature, as demonstrated in kinetic and equilibrium experiments. In saturation experiments carried out with [3H]ICS 205-930 in the presence and the absence of unlabelled agonists and antagonists, apparent Bmax values were not reduced whereas apparent Kd values were increased in the presence of competing ligands. There was a good agreement between apparent pKB values calculated for the competing ligands in saturation experiments and pKd values calculated from competition experiments. The present data demonstrate that [3H]ICS 205-930 labels a homogeneous population of sites at which agonists and antagonists interact competitively.

Topics: Animals; Binding, Competitive; Indoles; Kinetics; Mice; Neuroblastoma; Receptors, Serotonin; Serotonin Antagonists; Tritium; Tropisetron; Tumor Cells, Cultured

[3H]ICS 205-930 recognition sites were analyzed in membranes prepared from murine neuroblastoma N1E-115 cells. [3H]ICS 205-930 bound rapidly, reversibly, and stereoselectively to a homogeneous population of high affinity recognition sites: Bmax = 40 +/- 5 fmol/mg of protein, pKD = 9.20 +/- 0.05 (n = 11). Nonlinear regression and Scatchard analysis of saturation data suggested the existence of a single class of [3H]ICS 205-930 recognition sites on N1E-115 cells. The affinity of [3H]ICS 205-930 determined in kinetic studies was in agreement with that obtained under equilibrium conditions. Competition studies carried out with a large variety of agonists and antagonists also suggested the presence of a homogeneous population of [3H]ICS 205-930 recognition sites. [3H]ICS 205-930-binding sites displayed the pharmacological profile of a 5-HT3 receptor. Potent 5-HT3 receptor antagonists showed nM affinities for [3H]ICS 205-930-binding sites with the following rank order of potency: SDZ 206-830 greater than SDZ 206-792 greater than ICS 205-930 greater than BRL 43694 greater than quipazine greater than BRL 24924 greater than MDL 72222 greater than GR 38032F. Methiothepine, mCPP, and metoclopramide showed sub-microM affinity. The rank order of potency of agonists was: 5-HT greater than phenylbiguanide = 2-methyl-5-HT much greater than 5-methoxytryptamine = 5-carboxamidotryptamine. All antagonist competition curves were steep (pseudo-Hill coefficients not lower than 1), monophasic, and best fit for a one-site model; 5-HT and 2-methyl-5-HT produced pseudo-Hill coefficients of 1.2-1.4. Drugs acting at 5-HT1, 5-HT2, alpha- and beta-adrenergic, dopaminergic, and histaminergic receptors (methysergide, ketanserin, propranolol, phentolamine, sulpiride, SCH 23390, cimetidine) were essentially inactive at 10 mumol/liter. The binding of [3H]ICS 205-930 was not affected by guanine and adenine nucleotides (GTP, GppNHp, and ATP) at 1 mmol/liter. These nucleotides did not affect the binding of agonists, suggesting that 5-HT3 recognition sites are not coupled to G-proteins. The interactions of agonists and antagonists with [3H]ICS 205-930 recognition sites were competitive in nature, as demonstrated by saturation experiments carried out with [3H]ICS 205-930 in the presence and the absence of unlabeled compounds: apparent Bmax values were not reduced, whereas apparent KD values were increased in the presence of competing ligands.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Binding, Competitive; Calcium; Guanylyl Imidodiphosphate; Indoles; Kinetics; Mice; Neuroblastoma; Radioligand Assay; Receptors, Serotonin; Serotonin; Serotonin Antagonists; Temperature; Tropisetron; Tumor Cells, Cultured

1. The binding characteristics of [3H]ICS 205-930, a potent and selective 5-hydroxytryptamine 5-HT3 receptor antagonist, were investigated in membranes prepared from murine neuroblastoma-glioma NG 108-15 cells. 2. [3H]ICS 205-930 bound rapidly, reversibly and stereoselectively to a homogeneous population of high affinity recognition sites: Bmax = 58 +/- 3 fmol/mg protein, pKD = 9.01 +/- 0.08 (n = 11). Non linear regression and Scatchard analysis of saturation data suggested the existence of a single class of [3H]ICS 205-930 recognition sites on NG 108-15 cells. The binding was rapid, stable and reversible. The affinity of [3H]ICS 205-930 determined in kinetic studies was in agreement with that obtained under equilibrium conditions. 3. Competition studies performed with a variety of agonists and antagonists also suggested the presence of a homogeneous population of [3H]ICS 205-930 recognition sites. All competition curves were steep and monophasic and were best fit by a 1 receptor site model. [3H]ICS 205-930 binding sites displayed the pharmacological profile of a 5-HT3 receptor. Potent 5-HT3 receptor antagonists showed nanomolar affinities for [3H]ICS 205-930 binding sites with the following rank order of potency: SDZ 206-830 greater than ICS 205-930 greater than SDZ 206-792 greater than BRL 43694 greater than quipazine greater than BRL 24924 greater than SDZ 210-204 greater than MDL 72222 greater than SDZ 210-205. Metoclopramide, mCP and mianserin showed submicromolar affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding, Competitive; Cell Membrane; Glioma; Indoles; Kinetics; Mice; Neuroblastoma; Nucleotides; Radioligand Assay; Receptors, Serotonin; Serotonin Antagonists; Stereoisomerism; Temperature; Tropisetron; Tumor Cells, Cultured

Topics: Cell Membrane; Cells, Cultured; Glioma; Humans; Indoles; Neuroblastoma; Receptors, Serotonin; Serotonin Antagonists; Tropisetron