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

The biochemical and pharmacological properties of 5‐HT 3 receptors in homogenates of NG108‐15 and NCB‐20 neuroblastoma cells and rat cerebral cortex have been ascertained by the use of [ 3 H]‐quipazine and [ 3 H]‐GR65630 binding. In NG108‐15 and NCB‐20 cell homogenates, [ 3 H]‐quipazine bound to a single class of high affinity (NG108‐15: K d = 6.2 ± 1.1 n m , n = 4; NCB‐20: K d = 3.0 ± 0.9 n m , n = 4; 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, [ 3 H]‐quipazine bound to two populations of binding sites in the absence of the 5‐hydroxytryptamine (5‐HT) uptake inhibitor, paroxetine ( K d1 = 1.6 ± 0.5 n m , B max1 = 75 ± 14 fmol mg −1 protein; K d2 = 500 ± 300 n m , B max2 = 1840 ± 1040 fmol mg −1 protein, n = 3), and to a single class of high affinity binding sites ( K d = 2.0 ± 0.5 n m , n = 3; B max = 73 ± 6 fmol mg −1 protein) in the presence of paroxetine. The high affinity (nanomolar) component probably represented 5‐HT 3 binding sites and the low affinity component represented 5‐HT uptake sites. [ 3 H]‐paroxetine bound with high affinity ( K d = 0.02 ± 0.003 n m , 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‐HT 3 receptor ligands exhibited only low affinity (pK i < 7). No detectable specific [ 3 H]‐paroxetine binding was observed in NG108‐15 or NCB‐20 cell homogenates. [ 3 H]‐quipazine binding to homogenates of NG108‐15, NCB‐20 cells and rat cortex (in the presence of 0.1 μ m paroxetine) exhibited similar pharmacological characteristics. 5‐HT 3 receptor antagonists competed for [ 3 H]‐quipazine binding with high nanomolar affinities in the three preparations and the rank order of affinity was: ( S )‐zacopride > quarternized ICS 205–930 ≥ granisetron > ondansetron > ICS 205–209 ≥ ( R )‐zacopride > quipazine > renzapride > MDL‐72222 > butanopride > metoclopramide. [ 3 H]‐GR65630 labelled a site in NCB‐20 cell homogenates with an affinity ( K d = 0.7 ± 0.1 n m , n = 4) and density ( B max = 1800 ± 1000 fmol mg −1 protein) comparable to that observed with [ 3 H]‐quipazine. Competition studies also indicated a good correlation between the pharmacology of 5‐HT 3 binding sites when [ 3 H]‐GR65630 and [ 3 H]‐quipazine were used in these cells. In conclusion, [ 3 H]‐quipazine labelled 5‐HT 3 receptor sites in homogenates of NG108‐15 cells, NCB‐20 cells and rat cerebral cortex. In rat cortical homogenates, [ 3 H]‐quipazine also bound to 5‐HT uptake sites, which could be blocked by 0.1 μ m paroxetine. The pharmacological specificity of the 5‐HT 3 receptor labelled by [ 3 H]‐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‐HT 3 receptor antagonist, [ 3 H]‐GR65630.