Identification of serotonin receptors recognized by anti-idiotypic antibodies.
Anti-idiotypic antibodies were generated by immunizing rabbits with affinity-purified antibodies to serotonin (5-hydroxytryptamine; 5-HT). Anti-5-HT activity was removed from the resulting antisera by chromatography through a 5-HT affinity column. The anti-idiotypic antibodies were demonstrated by enzyme-linked immunosorbent assay to bind to affinity-purified whole anti-5-HT antibodies and their Fab fragments. Anti-idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5-HT were coupled, competed with 5-HT (covalently bound to protein) for the binding sites on anti-5-HT antibodies and serotonin binding protein. The anti-idiotypic antibodies antagonized the binding of [3H]5-HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti-idiotypic antibodies also blocked the binding of the 5-HT1B-selective ligand (-)-[125I]iodocyanopindolol (in the presence of 30 microM isoproterenol) to cortical membranes. In contrast, anti-idiotypic antibodies failed to inhibit binding of the 5-HT1A-selective ligand 8-hydroxy-2-(di-n-[3H]propylamino)-tetralin [( 3H]8-OH-DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti-idiotypic antibodies may recognize some 5-HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti-idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5-HT1C or 5-HT2 receptor or with a genomic clone encoding the 5-HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5-HT1C and 5-HT2 receptors, but not on the surfaces of HeLa cells that expressed 5-HT1A receptors. Immunostaining of cells by the anti-idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5-HT1C receptors was blocked by mesulergine (but not ketanserin, 8-OH-DPAT, or spiperone), whereas that of cells expressing 5-HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8-OH-DPAT). The anti-idiotypic antibodies failed to inhibit the uptake of [3H]5-HT by serotonergic neurons. It is concluded that the anti-idiotypic antibodies generated with anti-5-HT serum recognize the 5-HT1B, 5-HT1C, and 5-HT2 receptor subtypes; however, neither 5-HT1A receptors nor 5-HT uptake sites appear to react with these antibodies.
Tamir
H
H
Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York.
Liu
K P
KP
Hsiung
S C
SC
Yu
P Y
PY
Kirchgessner
A L
AL
Gershon
M D
MD
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000978:Antiparkinson Agents / Q000494:pharmacology; D001921:Brain / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D004873:Ergolines / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D007457:Iodine Radioisotopes; D020120:Iodocyanopindolol; D007650:Ketanserin / Q000494:pharmacology; D000451:Mineralocorticoid Receptor Antagonists / Q000494:pharmacology; D010869:Pindolol / Q000031:analogs & derivatives / Q000378:metabolism; D011817:Rabbits; D011985:Receptors, Serotonin / Q000145:classification / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D012701:Serotonin / Q000276:immunology / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D014316:Tritium
D000730:Androstadienes; D000888:Antibodies, Anti-Idiotypic; D000978:Antiparkinson Agents; D002352:Carrier Proteins; D004873:Ergolines; D007457:Iodine Radioisotopes; D000451:Mineralocorticoid Receptor Antagonists; D011985:Receptors, Serotonin; C025366:serotonin-binding protein; D014316:Tritium; D012701:Serotonin; D020120:Iodocyanopindolol; D007650:Ketanserin; C035143:spirorenone; D010869:Pindolol; C031649:mesulergine