CHARACTERIZATION OF HUMAN SEROTONIN IN AND 1B RECEPTORS USING [H-3] GR-125743, A NOVEL RADIOLABELED SEROTONIN 5HT1D 1B RECEPTOR ANTAGONIST/

The study of serotonin (5-HT) receptors from the points of view of the ir anatomical localization and pharmacological characterization has be en linked to the availability of highly selective radioligands exhibit ing high affinity for their targets. This is particularly so in the ca se of serotonin receptors, since many different subtypes with overlapp ing pharmacological profiles have been described. Of these, the seroto nin 5-HT1 receptor family appears to be the most complex in terms of m olecular diversity and pharmacological properties. The lack of appropr iate tools to characterize the different receptor subtypes included in this family has hampered progress in the understanding of biological function. In the case of serotonin 5-HT1D receptors all the radioligan ds used so far in their characterization behave as agonists from the f unctional point of view. This agonistic character is regarded as a dis advantage for radioligands since their interaction with the receptors under study depends on factors other than the abundance of the recepto r, such as the coupling of the receptors with G-proteins. We describe here the binding properties of [H-3]-GR-125743, a new radiolabelled de rivative of a compound that exhibits selective antagonistic properties with respect to the serotonin human (h5-HT1D) and human (h5-HT1B) rec eptors. The compound has been characterized for its ability to label t he cloned h5-HT1D and h5-HT1B receptors. The binding obtained in both cases was specific, saturable and reversible, whereas the percentage o f specific binding depended on the level of expression of the receptor s. Using saturation analysis we have found that, on the specific clone s used in this study, the compound labels a receptor population 5 to 1 0-fold higher that the one revealed using [H-3]-5-carboxamidotryptamin e, a compound with agonist properties for these receptors in functiona l assays. Using [H-3]-GR-125743 as a radioligand we have characterized the pharmacological profile of the same cloned h5-HT1D and h5-HT1B re ceptor preparations for a range of serotonin reference compounds by me ans of displacement assays. The affinities found have been compared, u sing regression analysis, with those obtained for the same radioligand and compounds in membranes obtained from human substantia nigra, a ti ssue known to be rich in h5-HT1B/1D receptors. We have found a better correlation, both in terms of correlation coefficient and of slope, be tween the substantia nigra data and the h5-HT1B data compared with the h5-HT1D data (0.94 and 1.05 vs. 0.86 and 0.64 respectively). Finally, the addition of 100 mu M GTP reduced the binding of [H-3]-GR-125743 t o h5-HT1D and h5-HT1B receptor subtypes by approximately 20% without a ffecting the affinities obtained for different displacers. Therefore, [H-3]-GR-125743 appears to be a suitable radioligand for the character ization of h5-HT1D and h5-HT1B receptor subtypes, being potentially mo re useful than previously existing compounds.