title: Characteristics of 5-HT3 binding sites in NG108-15, NCB-20 neuroblastoma cells and rat cerebral cortex using [3H]-quipazine and [3H]-GR65630 binding.
abstract: 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. Binding to this site was potently inhibited by 5-HT uptake blockers such as paroxetine and fluoxetine (pKi s = 8.6-9.9), while 5-HT3 receptor ligands exhibited only low affinity (pK; < 7). No detectable specific [3H]-paroxetine binding was observed in NG108-15 or NCB-20 cell homogenates. 4. [3H]-quipazine binding to homogenates of NG108-15, NCB-20 cells and rat cortex (in the presence of 0.1 microM paroxetine) exhibited similar pharmacological characteristics. 5-HT3 receptor antagonists competed for [3H]-quipazine binding with high nanomolar affinities in the three preparations and the rank order of affinity was: (S)-zacopride > quarternized ICS 205-930 2 granisetron > ondansetron > ICS 205-209 (R)-zacopride > quipazine > renzapride > MDL-72222 > butanopride > metoclopramide. 5. [3H]-GR65630 labelled a site in NCB-20 cell homogenates with an affinity (Kd = 0.7 + 0.1 nms n = 4) and density (B__ = 1800 + 1000 fmol mg- protein) comparable to that observed with [3H]-quipazine. Competition studies also indicated a good correlation between the pharmacology of 5-HT3 binding sites when [3H]-GR65630 and [3H]-quipazine were used in these cells. 6. In conclusion, [3H]-quipazine labelled 5-HT3 receptor sites in homogenates of NG108-15 cells, NCB-20 cells and rat cerebral cortex. In rat cortical homogenates, [3H]-quipazine also bound to 5-HT uptake sites, which could be blocked by 0.1 microM paroxetine. The pharmacological specificity of the 5-HT3 receptor labelled by [3H]-quipazine was similar in the neuroblastoma cells and rat cortex and was substantiated in NCB-20 cells by the binding profile of the selective 5-HT3 receptor antagonist, [3H]-GR65630.
authors.affiliation: Department of Neurosciences, Institute of Pharmacology (R7-101), Syntex Research, Palo Alto, CA 94304.
authors.forename: N A
authors.initials: NA
authors.lastname: Sharif
authors.forename: E H
authors.initials: EH
authors.lastname: Wong
authors.forename: D N
authors.initials: DN
authors.lastname: Loury
authors.forename: E
authors.initials: E
authors.lastname: Stefanich
authors.forename: A D
authors.initials: AD
authors.lastname: Michel
authors.forename: R M
authors.initials: RM
authors.lastname: Eglen
authors.forename: R L
authors.initials: RL
authors.lastname: Whiting
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D002540:Cerebral Cortex / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism*; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D051379:Mice; D009423:Nervous System Neoplasms / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism*; D017374:Paroxetine; D010880:Piperidines / Q000378:metabolism; D011814:Quipazine / Q000378:metabolism*; D051381:Rats; D011985:Receptors, Serotonin / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000378:metabolism*
chemical_list: D007093:Imidazoles; D007211:Indoles; D008024:Ligands; D010880:Piperidines; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; C059068:GR 65630; D017374:Paroxetine; D011814:Quipazine