Identification of serotonin 5-HT3 recognition sites in membranes of N1E-115 neuroblastoma cells by radioligand binding.
[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)
Hoyer
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Preclinical Research, Sandoz Ltd, Basel, Switzerland.
Neijt
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HC
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D009447:Neuroblastoma / Q000032:analysis*; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000032:analysis*; D012701:Serotonin / Q000378:metabolism; D012702:Serotonin Antagonists / Q000378:metabolism; D013696:Temperature; D000077526:Tropisetron; D014407:Tumor Cells, Cultured
D007211:Indoles; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D012701:Serotonin; D006165:Guanylyl Imidodiphosphate; D000077526:Tropisetron; D002118:Calcium