(/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
(/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), Vol. 243:3
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ERGOTAMINE
BIOCHEMICAL REACTION KINETICS
RECEPTORS
SEROTONIN
AFFINITY
BRAIN
CATTLE
CELL MEMBRANES
GUANINE
IN VITRO
LIGANDS
RATS
REGRESSION ANALYSIS
SWINE
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ALKALOIDS
AMINES
ANIMALS
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
AZAARENES
AZOLES
BODY
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM
DOMESTIC ANIMALS
DRUGS
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
INDOLES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MAMMALS
MATHEMATICS
MEMBRANE PROTEINS
MEMBRANES
NERVOUS SYSTEM
NEUROREGULATORS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PROTEINS
PURINES
PYRROLES
RADIOPROTECTIVE SUBSTANCES
REACTION KINETICS
RODENTS
RUMINANTS
STATISTICS
SYMPATHOLYTICS
SYMPATHOMIMETICS
TRYPTAMINES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques