Interaction of a radiolabeled agonist with cardiac muscarinic cholinergic receptors

Journal Article · Thu Dec 01 04:00:00 EST 1983 · J. Pharmacol. Exp. Ther.; (United States)

OSTI ID:6540232

Harden, T K; Meeker, R B; Martin, M W

The interaction of a radiolabeled muscarinic cholinergic receptor agonist, (methyl-/sup 3/H)oxotremorine acetate ((/sup 3/H)OXO), with a washed membrane preparation derived from rat heart, has been studied. In binding assays at 4 degrees C, the rate constants for association and dissociation of (/sup 3/H)OXO were 2 X 10(7) M-1 min-1 and 5 X 10(-3) min-1, respectively, Saturation binding isotherms indicated that binding was to a single population of sites with a Kd of approximately 300 pM. The density of (/sup 3/H)OXO binding sites (90-100 fmol/mg of protein) was approximately 75% of that determined for the radiolabeled receptor antagonist (/sup 3/H)quinuclidinyl benzilate. Both muscarinic receptor agonists and antagonists inhibited the binding of (/sup 3/H)OXO with high affinity and Hill slopes of approximately one. Guanine nucleotides completely inhibited the binding of (/sup 3/H)OXO. This effect was on the maximum binding (Bmax) of (/sup 3/H)OXO with no change occurring in the Kd; the order of potency for five nucleotides was guanosine 5'-O-(3-thio-triphosphate) greater than 5'-guanylylimidodiphosphate greater than GTP greater than or equal to guanosine/diphosphate greater than GMP. The (/sup 3/H)OXO-induced interaction of muscarinic receptors with a guanine nucleotide binding protein was stable to solubilization. That is, membrane receptors that were prelabeled with (/sup 3/H)OXO could be solubilized with digitonin, and the addition of guanine nucleotides to the soluble, (/sup 3/H)OXO-labeled complex resulted in dissociation of (/sup 3/H)OXO from the receptor. Pretreatment of membranes with relatively low concentrations of N-ethylmaleimide inhibited (/sup 3/H)OXO binding by 85% with no change in the Kd of (/sup 3/H)OXO, and with no effect on (/sup 3/H)quinuclidinyl benzilate binding.

• Research Organization: Univ. of North Carolina, Chapel Hill
• OSTI ID: 6540232
• Journal Information: J. Pharmacol. Exp. Ther.; (United States), Journal Name: J. Pharmacol. Exp. Ther.; (United States) Vol. 227:3; ISSN JPETA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL PATHWAYS
BODY
CARDIOVASCULAR SYSTEM
DRUGS
HEART
IN VITRO
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MAMMALS
MEASURING METHODS
MEMBRANES
MOLECULAR BIOLOGY
ORGANS
RATS
REACTION KINETICS
RECEPTORS
RODENTS
SYMPATHOMIMETICS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES