title: [3H]dihydroergotamine as a high-affinity, slowly dissociating radioligand for 5-HT1B binding sites in rat brain membranes: evidence for guanine nucleotide regulation of agonist affinity states.
abstract: [3H]Dihydroergotamine (DE) labels a population of binding sites in rat brain membranes with an affinity of approximately 70 pM in both hippocampus (maximal binding at saturation [Bmax] = 340 fmol/mg of protein) and cerebral cortex (Bmax = 250 fmol/mg of protein). Specific binding typically comprises about 97% of total binding at the Kd of the radioligand when nonspecific binding is determined in the presence of 100 nM unlabeled DE. Association kinetics at 37 degrees C are consistent with a uniform association rate constant for all sites labeled. Specific binding is completely reversible with addition of excess unlabeled DE, but dissociation does not proceed with simple first-order kinetics, suggesting the presence of more than one discrete binding site. Competition studies with selective drugs reveal alpha adrenergic, 5-HT1A and 5-HT1B components of [3H]DE specific binding. When phentolamine (500 nM) is included to block alpha receptors and DPAT (100 nM) or spiroxatrine (500 nM) is included to block 5-HT1A receptors, specific binding is exclusively to sites with drug affinities characteristic of 5-HT1B receptors. Under these 5-HT1B-selective conditions, [3H]DE binding is about 90% specific, with a Kd of about 50 to 60 pM and a Bmax of 96 fmol/mg of protein in hippocampus and 77 fmol/mg of protein in cortex. [3H]DE binding to 5-HT1B sites is very slowly dissociable, with a T1/2 of greater than 2 h at 37 degrees C. 5-HT1B antagonists and DE itself yield competition curves at [3H]DE-labeled 5-HT1B sites that are adequately fit assuming a single site in nonlinear regression analysis. Competition by the agonists 5-HT and RU 24969 at 5-HT1B sites are often best described by two site fits. Addition of 100 microM guanylyl 5'-imidodiphosphate appears to convert nearly all 5-HT1B sites to those having low affinity for agonists while having a much smaller effect on the binding of [3H]DE. This suggests that the 5-HT1B site exists in two interconvertable agonist affinity states and is yet another member of the G-protein-linked receptor family. In agreement with previous studies using other radioligands, no 5-HT1B sites can be detected in bovine, porcine or human hippocampus membranes.
authors.affiliation: Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, California.
authors.forename: M W
authors.initials: MW
authors.lastname: Hamblin
authors.forename: K
authors.initials: K
authors.lastname: Ariani
authors.forename: P I
authors.initials: PI
authors.lastname: Adriaenssens
authors.forename: R D
authors.initials: RD
authors.lastname: Ciaranello
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D004087:Dihydroergotamine / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D010646:Phentolamine / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism; D011985:Receptors, Serotonin / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D013552:Swine; D014316:Tritium
chemical_list: D006150:Guanine Nucleotides; D011942:Receptors, Adrenergic, alpha; D011985:Receptors, Serotonin; D014316:Tritium; D006165:Guanylyl Imidodiphosphate; D004087:Dihydroergotamine; D010646:Phentolamine