Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line.
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 [( 3H]QNB). The rank order potency of selective antagonists that inhibit specific [3H]QNB 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 [3H]QNB 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Bencherif
M
M
Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona.
Lukas
R J
RJ
D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D007700:Kinetics; D010716:Phosphatidylinositols / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D015290:Second Messenger Systems / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014316:Tritium
D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D014316:Tritium; D011813:Quinuclidinyl Benzilate; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate