CHARACTERIZATION OF [H-3]5-HYDROXYTRYPTAMINE AND [H-3]SPIPERONE BINDING-SITES IN CLATHRIN-COATED VESICLES FROM BOVINE BRAIN

Coated vesicles prepared from bovine brain cerebral cortex exhibited [ H-3]5-hydroxytryptamine (5-MT, serotonin) and [H-3]spiperone binding a ctivities. The binding activities were localized in the inner core ves icles. Binding reached an equilibrium level by 30-45 min at 30 degrees C, and was reversed by the addition of 100 mu M 5-HT for [H-3]5-HT bi nding or 10 mu M ketanserin for [H-3]spiperone binding. The saturation binding experiments indicated a single class of binding sites for [H- 3]5-HT and [H-3]spiperone with apparent K-d values of 2.4 and 1.75 nM, respectively. The binding of [H-3]5-HT was displaced by 5-HT and 8-hy droxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), but not by ketanserin . The binding of [H-3]spiperone was displaced by spiperone and ketanse rin but not by 5-HT or 8-OH-DPAT even at 1 mM. The coated vesicles wer e shown by immunoblotting assay to contain alpha-subunits of GTP-bindi ng proteins, G alpha s, G alpha i2, G alpha i3, G alpha o and Gaq/11. Forskolin-stimulated adenylate cyclase activity in the coated vesicles was inhibited to 80% of the control level by 5-HT or 8-OH-DPAT. These results suggested that 5-HT1A and 5-HT2A receptors are present in bov ine brain coated vesicles and that the 5-HT1A receptors are coupled to adenylate cyclase activity via CTP binding proteins. (C) 1998 Elsevie r Science B.V. All rights reserved.