Differential radioligand binding properties of [H]5-hydroxytryptamine and [H]mesulergine in a clonal 5-hydroxytryptamine1C cell line

[³H]5-Hydroxytryptamine ([³H]5-HT) and [³H]mesulergine were used to label 5-HT_1C receptors expressed in NIH 3T3 mouse fibroblast cells. Using a rapid filtration assay, saturation analysis of the [³H]5-HT radioligand data indicate that the binding is biphasic. Based on computerized analysis of the data, a 2-site model of radioligand binding is significantly more consistent with the data than a one-site model (P < 0.01). The K_D values of [³H]5-HT for the 2 populations are0.5±0.1nM and31±15nM, while the B_max values are400±90pmol/g protein and 3,000±600 pmol/g protein, respectively. A biphasic binding pattern is also observed with [³H]5-HT using a centrifugation assay (K_D1 = 0.6±0.06nM, K_D2 = 60±10nM;B_max1 = 740±90pmol/g, B_max2 = 4,000±700pmol/g). By contrast, saturation analysis of [³H]mesulergine binding is monophasic (K_D = 4.7±0.7nM) with a B_max value (6,800±1,000pmol/g protein) that is significantly greater than that obtained using [³H]5-HT (P < 0.01). Drug competition studies confirm that both [³H]5-HT and [³H]mesulergine label at least 2 subpopulations of expressed 5-HT_1C receptors in NIH 3T3 cells. 10⁻⁴ M GTP eliminates the high affinity [³H]5-HT-labeled binding sites with minimal effect on the low affinity [³H]5-HT-labeled sites and no effect on [³H]mesulergine-labeled sites. These data demonstrate that at least 2 distinct subpopulations of 5-HT_1C receptors in NIH 3T3 cells can be differentiated using radioligand binding techniques.