IDENTIFICATION OF SEROTONIN 5-HT3 RECOGNITION SITES IN MEMBRANES OF N1E-115 NEURO-BLASTOMA CELLS BY RADIOLIGAND BINDING

Abstract

[3H]ICS 205-930 recognition sites were analyzed in membranes prepared from murine neuroblastoma N1E-115 cells. [4H]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 > SDZ 206-792 > ICS 205-930 > BRL 43694 > quipazine > BRL 24924 > MDL 72222 > GR 38032F. Methiothepine, mCPP, and metoclopramide showed sub-.mu.M affinity. The rank order of potency of agonists was: 5-HT > phenylbiguanide = 2-methyl-5-HT .mchgt. 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 .mu.mol/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. The present data demonstrate that [3H]ICS 205-930 is a suitable ligand for the identification of 5-HT3 recognition sites in membrane preparations. These findings are consistent with previous electrophysiological experiments where 5-HT3 receptors were demonstrated in N1R-115 cells.