Pindolol-insensitive [3H]-5-hydroxytryptamine binding in the rat hypothalamus;: identity with 5-hydroxytryptamine7 receptors

1 Pindolol-insensitive [H-3]-5-hydroxytryptamine ([H-3]-5-HT) binding to rat hypothalamic membranes was pharmacologically and functionally characterized to resolve whether this procedure selectively labels 5-HT7 receptors. 2 Consistent with a previous report, 3 mu M and not 100 nM pindolol was required to occupy fully 5-HT1A and 5-HT1B receptors. Remaining [H-3]-5-HT binding was saturable (K-D, 1.59 +/- 0.21 nM; B-max, 53.8 +/- 3.1 fmol.mg protein(-1)). 3 Displacement of [H-3]-5-HT with metergoline and 5-CT revealed shallow Hill slopes (<0.5) but seven other compounds had slopes >0.8 and pK(1) values and the rank order of affinity were significantly correlated (r = 0.81 and 0.93, respectively) with published [H-3]-5-HT binding to rat recombinant 5-HT7 receptors. 4 In the presence of pindolol. 5-HT-enhanced accumulation of [P-32]-cyclic AMP was unaffected by the 5-HT4 antagonist RS39604 (0.1 mu M) or the 5-ht(6) antagonist Ro 04-6790 (1 mu M) but significantly attenuated by mesulergine (250 nM), ritanserin (450 nM) or methiothepin (200 nM) which have high affinity for the 5-HT7 receptor. 5 Intracerebroventricular pretreatment with the serotonergic neurotoxin 5,7-dihydroxytryptamine, 5,7-DHT, elevated the [H-3]-5-HT B-max 2 fold, indicating that the hypothalamic 5-HT7 receptor is post-synaptic to 5-HT nerve terminals and regulated by synaptic 5-HT levels. 6 These results suggest that, in the presence of 3 mu M pindolol, [H-3]-5-HT selectively labels hypothalamic binding sites consistent with functional 5-HT7 receptors.