Characterization of [3H]5-hydroxytryptamine and [3H]spiperone binding sites in clathrin-coated vesicles from bovine brain.
Coated vesicles prepared from bovine brain cerebral cortex exhibited [3H]5-hydroxytryptamine (5-HT, serotonin) and [3H]spiperone binding activities. The binding activities were localized in the inner core vesicles. Binding reached an equilibrium level by 30-45 min at 30 degreesC, and was reversed by the addition of 100 microM 5-HT for [3H]5-HT binding or 10 microM ketanserin for [3H]spiperone binding. The saturation binding experiments indicated a single class of binding sites for [3H]5-HT and [3H]spiperone with apparent Kd values of 2.4 and 1.75 nM, respectively. The binding of [3H]5-HT was displaced by 5-HT and 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), but not by ketanserin. The binding of [3H]spiperone was displaced by spiperone and ketanserin but not by 5-HT or 8-OH-DPAT even at 1 mM. The coated vesicles were shown by immunoblotting assay to contain alpha-subunits of GTP-binding proteins, Galphas, Galphai2, Galphai3, Galphao and Galphaq/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 bovine brain coated vesicles and that the 5-HT1A receptors are coupled to adenylate cyclase activity via GTP binding proteins.
Moroi
K
K
Division of Cardiovascular Biology, Center for Biomedical Science, Chiba University School of Medicine, Chiba, 260, Japan.
Ozaki
N
N
Kadota
T
T
Kadota
K
K
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D002966:Clathrin; D018699:Coated Vesicles / Q000378:metabolism*; D016015:Logistic Models; D011869:Radioligand Assay; D011954:Receptors, Dopamine / Q000378:metabolism*; D011985:Receptors, Serotonin / Q000378:metabolism*; D013134:Spiperone / Q000378:metabolism*; D014316:Tritium
D002966:Clathrin; D011954:Receptors, Dopamine; D011985:Receptors, Serotonin; C031611:spiroperidol receptor; D014316:Tritium; D013134:Spiperone; D000262:Adenylyl Cyclases