(/sup 3/H)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

Journal Article · Tue Dec 01 04:00:00 EST 1987 · J. Pharmacol. Exp. Ther.; (United States)

OSTI ID:5363074

Hamblin, M W; Ariani, K; Adriaenssens, P I; Ciaranello, R D

(/sup 3/H)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 (/sup 3/H)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, (/sup 3/H)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. (/sup 3/H)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 (/sup 3/H)DE-labeled 5-HT1B sites that are adequately fit assuming a single site in nonlinear regression analysis. 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 (/sup 3/H)DE.

• Research Organization: Stanford Univ. School of Medicine, CA
• OSTI ID: 5363074
• Journal Information: J. Pharmacol. Exp. Ther.; (United States), Journal Name: J. Pharmacol. Exp. Ther.; (United States) Vol. 243:3; ISSN JPETA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ALKALOIDS
AMINES
ANIMALS
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
AZAARENES
AZOLES
BIOCHEMICAL REACTION KINETICS
BODY
BRAIN
CATTLE
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
DOMESTIC ANIMALS
DRUGS
ERGOTAMINE
GUANINE
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
IN VITRO
INDOLES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIGANDS
MAMMALS
MATHEMATICS
MEMBRANE PROTEINS
MEMBRANES
NERVOUS SYSTEM
NEUROREGULATORS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PROTEINS
PURINES
PYRROLES
RADIOPROTECTIVE SUBSTANCES
RATS
REACTION KINETICS
RECEPTORS
REGRESSION ANALYSIS
RODENTS
RUMINANTS
SEROTONIN
STATISTICS
SWINE
SYMPATHOLYTICS
SYMPATHOMIMETICS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
TRYPTAMINES
VERTEBRATES