Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

Journal Article · Sat Jun 01 04:00:00 EDT 1991 · Journal of Pharmacology and Experimental Therapeutics; (United States)

OSTI ID:5595670

Bencherif, M; Lukas, R J ^[1]

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1. Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (USA)

Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate ({sup 3}H-QNB). The rank order potency of selective antagonists that inhibit specific {sup 3}HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2(2-((diethylamino)methyl)-1-(piperidinyl) acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10 microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of {sup 3}HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.

• OSTI ID: 5595670
• Journal Information: Journal of Pharmacology and Experimental Therapeutics; (United States), Journal Name: Journal of Pharmacology and Experimental Therapeutics; (United States) Vol. 257:3; ISSN JPETA; ISSN 0022-3565
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETYLCHOLINE
AFFINITY
AMINES
AMMONIUM COMPOUNDS
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL EFFECTS
CARCINOGENS
CHEMICAL COMPOSITION
DRUGS
ESTERS
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LIGANDS
MAMMALS
MAN
MEMBRANE PROTEINS
NEUROREGULATORS
ORGANIC COMPOUNDS
PARASYMPATHOMIMETICS
PHORBOL ESTERS
PRIMATES
PROTEINS
QUATERNARY COMPOUNDS
REACTION KINETICS
RECEPTORS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES