Characteristics of 5‐HT3 binding sites in NG108‐15, NCB‐20 neuroblastoma cells and rat cerebral cortex using [3H]‐quipazine and [3H]‐GR65630 binding
Authors: Sharif N.A. , Wong E.H.F. , Loury D.N. , Stefanich E. , Michel A.D. , Eglen R.M. , Whiting R.L. 
Published: July 2012

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.

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.

[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 (pKi < 7). No detectable specific [3H]‐paroxetine binding was observed in NG108‐15 or NCB‐20 cell homogenates.

[3H]‐quipazine binding to homogenates of NG108‐15, NCB‐20 cells and rat cortex (in the presence of 0.1 μm 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 ≥ granisetron > ondansetron > ICS 205–209 ≥ (R)‐zacopride > quipazine > renzapride > MDL‐72222 > butanopride > metoclopramide.

[3H]‐GR65630 labelled a site in NCB‐20 cell homogenates with an affinity (Kd = 0.7 ± 0.1 nm, n = 4) and density (Bmax = 1800 ± 1000 fmol mg−1 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.

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 μm 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.