Differential radioligand binding properties of [3H]5-hydroxytryptamine and [3H]mesulergine in a clonal 5-hydroxytryptamine1C cell line.
[3H]5-Hydroxytryptamine ([3H]5-HT) and [3H]mesulergine were used to label 5-HT1C 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 less than 0.01). The KD values of [3H]5-HT for the 2 populations are 0.5 +/- 0.1 nM and 31 +/- 15 nM, while the Bmax values are 400 +/- 90 pmol/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 (KD1 = 0.6 +/- 0.06 nM, KD2 = 60 +/- 10 nM; Bmax1 = 740 +/- 90 pmol/g, Bmax2 = 4,000 +/- 700 pmol/g). By contrast, saturation analysis of [3H]mesulergine binding is monophasic (KD = 4.7 +/- 0.7 nM) with a Bmax value (6,800 +/- 1,000 pmol/g protein) that is significantly greater than that obtained using [3H]5-HT (P less than 0.01). Drug competition studies confirm that both [3H]5-HT and [3H]mesulergine label at least 2 subpopulations of expressed 5-HT1C 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-HT1C receptors in NIH 3T3 cells can be differentiated using radioligand binding techniques.
Havlik
S
S
Department of Neurology, Stanford University School of Medicine, CA 94305.
Peroutka
S J
SJ
D000818:Animals; D000978:Antiparkinson Agents / Q000378:metabolism*; D002460:Cell Line; D002498:Centrifugation; D002999:Clone Cells; D003080:Cold Temperature; D004873:Ergolines / Q000378:metabolism*; D005374:Filtration; D006160:Guanosine Triphosphate / Q000378:metabolism; D051379:Mice; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000187:drug effects / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism*
D000978:Antiparkinson Agents; D004873:Ergolines; D011985:Receptors, Serotonin; D012701:Serotonin; D006160:Guanosine Triphosphate; C031649:mesulergine