Characterisation of 5-HT₃ recognition sites in membranes of NG 108-15 neuroblastoma-glioma cells with [³H]ICS 205-930

Summary

• 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: B _max = 58 ± 3 fmol/mg protein, pK _D = 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 > ICS 205-930 > SDZ 206-792 > BRL 43694 > quipazine > BRL 24924 > SDZ 210-204 > MDL 72222 > SDZ 210-205. Metoclopramide, mCPP and mianserin showed submicromolar affinity. The rank order of potency of agonists was: 5-HT = 2-methyl-5-HT > phenylbiguanide ≫ 8-OH-DPAT > 5-carboxamidotryptamine. Drugs acting at 5-HT₁, 5-HT₂, dopamine receptors, α- and β-adrenoceptors, (methysergide, ketanserin, pindolol, spiperone, SCH 23390) showed very low affinities for [³H]ICS 205-930 recognition sites.

• 4. The binding of [³H]ICS 205-930 was not affected by quanine or adenine nucleotides (GTP, GppNHp and ATP) at 1 mmol/l. Moreover, these nucleotides did not affect the binding of agonists suggesting that 5-HT₃ recognition sites are not coupled to guanine nucleotide regulatory proteins.

• 5. 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 unlabelled compounds: apparent B _max values were not reduced whereas apparent K _D values were increased in the presence of competing ligands. There was a good agreement between apparent K _B values determined in saturation experiments with agonists and antagonists and their K _D values determined in competition experiments.

• 6. These findings are consistent with [³H]ICS 205-930 labelling 5-HT₃ receptors on NG 108-15 cells. The pharmacological profile of the sites labelled by [³H]ICS 205-930 on NG 108-15 cells is very similar to that of the 5-HT₃ sites identified on neuroblastoma NlE-115 cells (Hoyer and Neijt 1988a, b).

• 7. The present data demonstrate that [³H]ICS 205-930 is a suitable ligand for the identification of 5-HT₃ recognition sites in membrane preparations.