[H-3]RAUWOLSCINE - AN ANTAGONIST RADIOLIGAND FOR THE CLONED HUMAN 5-HYDROXYTRYPTAMINE(2B) (5-HT2B) RECEPTOR

In previous reports, [H-3]5-HT has been used to characterize the pharm acology of the rat and human 5-HT2B receptors. 5-HT, the native agonis t for the 5-HT2B receptor, has a limitation in its usefulness as a rad ioligand since it is difficult to study the agonist low-affinity state of a G protein-coupled receptor using an agonist radioligand. When us ing [H-3]5-HT as a radioligand, rauwolscine was determined to have rel atively high affinity for the human receptor (K-i human = 14.3 +/- 1.2 nM, compared to K-i rat = 35.8 +/- 3.8 nM). Since no known high affin ity antagonist was available as a radioligand, these studies were perf ormed to characterize [H-3]rauwolscine as a radioligand for the cloned human 5-HT2B receptor expressed in AV12 cells. When [H-3]rauwolscine was initially tested for its usefulness as a radioligand, complex comp etition curves were obtained. After testing several alpha(2)-adrenergi c ligands, it was determined that there was a component of [H-3]rauwol scine binding in the AV12 cell that was due to the presence of an endo genous alpha(2)-adrenergic receptor. The alpha(2)-adrenergic ligand ef aroxan was found to block [H-3]rauwolscine binding to the alpha(2)-adr energic receptor without significantly affecting binding to the 5-HT2B receptor and was therefore included in all subsequent studies. In sat uration studies at 37 degrees C, [H-3]rauwolscine labeled a single pop ulation of binding sites, K-d = 3.75 +/- 0.23 nM. In simultaneous expe riments using identical tissue samples, [H-3]rauwolscine labeled 783 /- 10 fmol of 5-HT2B receptors/mg of protein, as compared to 733 +/- 1 4 fmol of 5-HT2B receptors/mg of protein for [H-3]5-HT binding. At 0 d egrees C, where the conditions for [H-3]5-HT binding should label most ly the agonist high affinity state of the human 5-HT2B receptor, [H-3] rauwolscine (B-max = 951 +/- 136 fmol/mg), again labeled significantly more receptors than [H-3]5-HT (B-max = 615 +/- 34 fmol/mg). The affin ity of [H-3]rauwolscine for the human 5-HT2B receptor at 0 degrees C d id not change, K-d = 4.93 +/- 1.27 nM, while that for [H-3]5-HT increa sed greatly (K-d at 37 degrees C = 7.76 +/- 1.06 nM; K-d at 0 degrees C = 0.0735 +/- 0.0081 nM). When using [H-3]rauwolscine as the radiolig and, competition curves for antagonist structures modeled to a single binding site, while agonist competition typically resulted in curves t hat best fit a two site binding model. In addition, many of the compou nds with antagonist structures displayed higher affinity for the 5-HT2 B receptor when [H-3]rauwotscine was the radioligand. Typically, simil ar to 85% of [H-3]rauwolscine binding was specific binding. These stud ies display the usefulness of [H-3]rauwolscine as an antagonist radiol igand for the cloned human 5-HT2B receptor. This should provide a good tool for the study of both the agonist high-and low-affinity states o f the human cloned 5-HT2B, receptor.