1 The biochemical and pharmacological properties of 5-HT3 receptor in homogenates of NG108-15 and NCB-20 neuroblastoma cells and rat cerebral cortex have been ascertained by the use of [H-3]-quipazine and [H-3]-GR65630 binding. 2 In NG108-15 and NCB-20 cell homogenates, [H-3]-quipazine bound to a single class of high affinity (NG108-15: K(d) = 6.2 +/- 1.1 nM, n = 4; NCB-20: K(d) = 3.0 +/- 0.9 nm, n = 4; means +/- s.e.means +/- s.e. means) saturable (NG108-15: B(max) 1340 +/- 220 fmol mg-1 protein; NCB-20: B(max) = 2300 +/- 200 fmol mg-1 protein) binding sites. In rat cortical homogenates, [H-3]-quipazine bound to two populations of binding sites in the absence of the 5-hydroxytryptamine (5-HT) uptake inhibitor, paroxetine (K(dl) = 1.6 +/- 0.5 nM, B(max1) = 75 +/- 14 fmol mg-1 protein; K(d2) = 500 +/- 300 nM, B(max2) = 1040 fmol mg-1 protein, n = 3), and to a single class of high affinity binding sites (K(d) = 2.0 +/- 0.5 nM, n = 3; B(max) 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 [H-3]-paroxetine bound with high affinity (K(d) = 0.002 +/- 0.00 nM, n = 3) to a site in rat cortical homogenates in a saturable (B(max) = 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 (pK(i)s = 8.6 - 9.9), while 5-HT3 receptor ligands exhibited only low affinity (pK(i) < 7). No detectable specific [H-3]-paroxetine binding was observed in NG108-15 or NCB-20 cell homogenates. 4 [H-3]-quipazine binding to homogenates of NG108-15, NCB-20 cells and rat cortex (in the presence of 0.1-mu-m paroxetine) exhibited similar pharmacological characteristics. 5-HT3 receptor antagonists competed for [H-3]-quipazine binding with high nanomolar affinities in the three preparations and the rank order of affinity was: (S)-zacopride > quarternized ICS 205-930 greater-than-or-equal-to granisetron > ondansetron > ICS 205-209 greater-than-or-equal-to (R)-zacopride > quipazine > renzapride > MDL-72222 > butanopride > metoclopramide. 5 [H-3]-GR65630 labelled a site in NCB-20 cell homogenates with an affinity (K(d) = 0.7 +/- 0.1 nM, n = 4) and density (B(max) = 1800 +/- 1000 fmol mg-1 protein) comparable to that observed with [H-3]-quipazine. Competition studies also indicated a good correlation between the pharmacology of 5-HT3 binding sites when [H-3]-GR65630 and [H-3]-quipazine were used in these cells. 6 In conclusion, [H-3]-quipazine labelled 5-HT3 receptor sites in homogenates of NG108-15 cells, NCB-20 cells and rat cerebral cortex. In rat cortical homogenates, [H-3]-quipazine also bound to 5-HT uptake sites, which could be blocked by 0.1-mu-M paroxetine. The pharmacological specificity of the 5-HT3 receptor labelled by [H-3]-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, [H-3]-GR65630.
