Differential radioligand binding properties of [ 3H]5-hydroxytryptamine and [ 3H]mesulergine in a clonal 5-hydroxytryptamine 1C cell line

Abstract

[ 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.