( 3 H)Rauwolscine: an antagonist radioligand

In previous reports, ( 3 H)5-HT has been used to characterize the pharmacology of the rat and human 5- HT2B receptors. 5-HT, the native agonist for the 5-HT 2B receptor, has a limitation in its usefulness as a radioligand since it is difficult to study the agonist low-affinity state of a G protein-coupled receptor using an agonist radioli- gand. When using ( 3 H)5-HT as a radioligand, rauwolscine was determined to have relatively high affinity for the hu- man receptor (Ki human = 14.3 ± 1.2 nM, compared to Ki rat = 35.8 ± 3.8 nM). Since no known high affinity antag- onist was available as a radioligand, these studies were performed to characterize ( 3 H)rauwolscine as a radioli- gand for the cloned human 5-HT2B receptor expressed in AV12 cells. When ( 3 H)rauwolscine was initially tested for its usefulness as a radioligand, complex competition curves were obtained. After testing several α2-adrenergic ligands, it was determined that there was a component of ( 3 H)rauwolscine binding in the AV12 cell that was due to the presence of an endogenous α2-adrenergic receptor. The α2-adrenergic ligand efaroxan was found to block ( 3 H)rauwolscine binding to the α2-adrenergic receptor without significantly affecting binding to the 5-HT 2B re- ceptor and was therefore included in all subsequent stud- ies. In saturation studies at 37° C, ( 3 H)rauwolscine labeled a single population of binding sites, K d = 3.75 ± 0.23 nM. In simultaneous experiments using identical tissue sam- ples, ( 3 H)rauwolscine labeled 783 ± 10 fmol of 5-HT2B re- ceptors/mg of protein, as compared to 733 ± 14 fmol of 5- HT2B receptors/mg of protein for ( 3 H)5-HT binding. At 0° C, where the conditions for ( 3 H)5-HT binding should la- bel mostly the agonist high affinity state of the human 5- HT2B receptor, ( 3 H)rauwolscine (Bmax = 951 ± 136 fmol/ mg), again labeled significantly more receptors than ( 3 H)5-HT (Bmax = 615 ± 34 fmol/mg). The affinity of ( 3 H)rauwolscine for the human 5-HT2B receptor at 0° C did not change, Kd = 4.93 ± 1.27 nM, while that for ( 3 H)5- HT increased greatly (Kd at 37° C = 7.76 ± 1.06 nM; K d at 0° C = 0.0735 ± 0.0081 nM). When using ( 3 H)rauwolscine as the radioligand, competition curves for antagonist structures modeled to a single binding site, while agonist competition typically resulted in curves that best fit a two site binding model. In addition, many of the compounds with antagonist structures displayed higher affinity for the 5-HT2B receptor when ( 3 H)rauwolscine was the radioli- gand. Typically, ~ 85% of ( 3 H)rauwolscine binding was specific binding. These studies display the usefulness of ( 3 H)rauwolscine as an antagonist radioligand 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 of the human cloned 5-HT2B receptor.