Labelling of recombinant human and native rat serotonin 5-HT1A receptors by a novel, selective radioligand, [3H]-S 15535: definition of its binding profile using agonists, antagonists and inverse agonists.

The novel benzodioxopiperazine, 5-HT1A receptor weak partial agonist, S 15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine) bound with high affinity and selectivity to membranes of Chinese Hamster Ovary cells stably expressing the human (h) 5-HT1A receptor (Ki = 0.6 nM versus [3H]-8-hydroxy-dipropylamino-tetralin, [3H]-8-OH-DPAT): its affinity at h5-HT1A receptors was more than 70-fold higher than its affinity at > 50 other binding sites. S 15535 was tritiated to high specific activity (50 Ci/mmol) and its binding profile characterised. At 22 degrees C, [3H]-S 15535 associated and dissociated from h5-HT1A receptors with half-times of 2.9 and 5.0 min, respectively, yielding a Kd estimate of 3.6 nM. In saturation binding experiments, [3H]-S 15535 displayed a Bmax value for h5-HT1A receptors (1630 fmol/mg), higher than that obtained with the agonist [3H]-8-OH-DPAT (1023 pmol/mg). Guanylyl imidodiphosphate (GppNHp, 100 microM) reduced the binding of [3H]-S 15535 by only 25% compared with 79% for [3H]-8-OH-DPAT at h5-HT1A receptors. [3H]-S 15535 also showed high affinity, saturable binding to rat hippocampal membranes (Bmax = 820 fmol/mg versus 647 fmol/mg for [3H]-8-OH-DPAT). For both h5-HT1A and rat 5-HT1A receptors, the Ki values for competition binding of 15 serotonergic ligands with [3H]-S 15535 was highly correlated with that of [3H]-8-OH-DPAT. However, important differences were also observed. The agonist, 5-hydroxytryptamine (5-HT), displayed biphasic competition curves with [3H]-S 15535 but not with [3H]-8-OH-DPAT at h5-HT1A receptors. Similarly, the 'antagonists', spiperone, methiothepin and (+)butaclamol, showed biphasic competition isotherms versus [3H]-S 15535 but not [3H]-8-OH-DPAT. When [3H]-S 15535 competition binding experiments were carried out in the presence of GppNHp (100 microM) the 5-HT and 8-OH-DPAT competition curves shifted to the right, whereas the spiperone and methiothepin competition curves shifted to the left. In contrast, in the presence of GppNHp, the competition isotherms for N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo-h exanecarboxamide (WAY 100,635) were not altered. Taken together, these data show that (i) [3H]-S 15535 is a highly selective 5-HT1A receptor ligand which labels both G-protein-coupled and uncoupled 5-HT1A receptors, (ii) antagonists, such as WAY 100,635, which yield monophasic isotherms in competition with both [3H]-agonists and [3H]-antagonists, are not sensitive to the G-protein coupling state of the receptor, but (iii) spiperone and methiothepin behaved as inverse agonists, their competition isotherms with [3H]-S 15535 being modulated in an opposite manner to those of agonists.