Characterization of (/sup 3/H)pirenzepine binding to muscarinic cholinergic receptors solubilized from rat brain
Membranes prepared from rat cerebral cortex were solubilized in buffer containing 1% digitonin. Material present in the supernatant after centrifugation at 147,000 X g was shown to contain binding sites for both (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) and (/sup 3/H)pirenzepine ((/sup 3/H)PZ). Recovery of binding sites was approximately 25% of the initial membrane-bound (/sup 3/H)QNB binding sites. The Kd values for (/sup 3/H)QNB and (/sup 3/H)PZ binding to solubilized receptors were 0.3 nM and 0.1 microM, respectively. As has been observed previously in membrane preparations, (/sup 3/H)PZ appeared to label fewer solubilized binding sites than did (/sup 3/H)QNB. Maximum binding values for (/sup 3/H)PZ and (/sup 3/H)QNB binding to solubilized receptors were approximately 400 and 950 fmol/mg of protein, respectively. Competition curves for PZ inhibiting the binding of (/sup 3/H)QNB, however, had Hill slopes of 1, with a Ki value of 0.24 microM. The k1 and k-1 for (/sup 3/H)PZ binding were 3.5 X 10(6) M-1 min-1 and 0.13 min-1, respectively. The muscarinic receptor antagonists atropine, scopolamine and PZ inhibited the binding of (/sup 3/H)QNB and (/sup 3/H)PZ to solubilized receptors with Hill slopes of 1, as did the muscarinic receptor agonist oxotremorine. The muscarinic receptor agonist carbachol competed for (/sup 3/H)QNB and (/sup 3/H)PZ binding with a Hill slope of less than 1 in cerebral cortex, but not in cerebellum. GTP did not alter the interactions of carbachol or oxotremorine with the solubilized receptor. Together, these data suggest that muscarinic receptor sites solubilized from rat brain retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.
- Research Organization:
- Univ. of Pennsylvania, Philadelphia
- OSTI ID:
- 5221138
- Journal Information:
- J. Pharmacol. Exp. Ther.; (United States), Vol. 1
- Country of Publication:
- United States
- Language:
- English
Similar Records
Comparison of (/sup 3/H)pirenzepine and (/sup 3/H)quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain
/sup 3/H)pirenzepine and (-)-(/sup 3/H)quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites. II. Characterization and regulation of antagonist binding to putative muscarinic subtypes
Related Subjects
ACETYLCHOLINE
RECEPTORS
PARASYMPATHOLYTICS
CHEMICAL BONDS
BIOCHEMICAL REACTION KINETICS
BRAIN
CELL MEMBRANES
DETERGENTS
IN VITRO
RATS
SOLUBILITY
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ADDITIVES
AMINES
AMMONIUM COMPOUNDS
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
BODY
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM
DRUGS
EMULSIFIERS
ESTERS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MAMMALS
MEMBRANES
NERVOUS SYSTEM
NEUROREGULATORS
ORGANIC COMPOUNDS
ORGANS
PARASYMPATHOMIMETICS
QUATERNARY COMPOUNDS
REACTION KINETICS
RODENTS
SURFACTANTS
VERTEBRATES
WETTING AGENTS
550201* - Biochemistry- Tracer Techniques