Characterization of muscarinic receptors on intact human neuroblastoma cells: coupling to phosphoinositide hydrolysis and phosphorylation by phorbol esters

Journal Article · Wed Mar 05 00:00:00 EST 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6926843

Serra, M; Watson, M; Roeske, W R; Yamamura, H I

Cloned human neuroblastoma cells (SH-SY5Y) were grown. High affinity binding of (/sup 3/H)(-)quinuclidinyl benzilate ((/sup 3/H)(-)QNB) and its quaternary derivative (/sup 3/H)(-)methyl QNB to muscarinic receptors (MR) on intact SH-SY5Y cells was studied. A 30 min rinse time gave a ratio of specific/total binding of 90% for both ligands. Association rates of (/sup 3/H)(-)QNB and (/sup 3/H)(-)methyl QNB were determined. Both ligands reached steady state by 60 min at 37/sup 0/C. Rates of dissociation for both radioligands were biphasic, although (/sup 3/H)(-)methyl QNB was faster. Saturation studies yielded K/sub d/ (dissociation constant) values of 16 and 260 pM and B/sub max/ (maximal MR density) values of 172 and 134 fmoles/mg prot for (/sup 3/H)(-)QNB and (/sup 3/H)(-)methyl QNB, respectively. Activation of protein kinase C by phorbol esters produced increased phosphorylation of cellular proteins. Pretreatment with 100 nM of 4..beta..-phorbol 12..beta..-myristate 13..cap alpha..-acetate (PMA) induced a decrease in agonist affinity for MR, suggesting a PMA-promoted phosphorylation of the MR protein. Phosphoinositide (PhI) turnover was measured by MR agonist-induced accumulation of inositol-1-phosphate in the presence of Li/sup + +/ (10 mM). Only carbachol and acetylcholine elicited potent responses with oxotremorine (16%) pilocarpine (17%) and McN-A-343 (8%) appearing to be weak partial agonist of low efficacy.

• Research Organization: Univ. of Arizona, Tucson
• OSTI ID: 6926843
• Report Number(s): CONF-8604222-; TRN: 87-009085
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:4; Conference: 70. annual meeting of the Federation of American Society for Experimental Biology, St. Louis, MO, USA, 13 Apr 1986
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
59 BASIC BIOLOGICAL SCIENCES
ACETYLCHOLINE
RECEPTORS
PHORBOL ESTERS
BIOLOGICAL EFFECTS
PHOSPHOLIPIDS
HYDROLYSIS
PHOSPHORYLATION
BIOCHEMICAL REACTION KINETICS
AFFINITY
BENZILIC ACID
LIGANDS
MAN
TRACER TECHNIQUES
TRITIUM COMPOUNDS
TUMOR CELLS
AMINES
AMMONIUM COMPOUNDS
ANIMAL CELLS
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
CARBOXYLIC ACIDS
CARCINOGENS
CHEMICAL REACTIONS
DECOMPOSITION
DRUGS
ESTERS
HYDROXY ACIDS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIPIDS
LYSIS
MAMMALS
MEMBRANE PROTEINS
NEUROREGULATORS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PARASYMPATHOMIMETICS
PRIMATES
PROTEINS
QUATERNARY COMPOUNDS
REACTION KINETICS
SOLVOLYSIS
VERTEBRATES
560300* - Chemicals Metabolism & Toxicology
550201 - Biochemistry- Tracer Techniques