title: Methoctramine binding sites sensitive to alkylation on muscarinic receptors from tracheal smooth muscle.
abstract: The binding of L-[benzilic-4,4'-3H]quinuclidinyl benzilate was studied in the plasma membrane fraction of bovine tracheal smooth muscle treated with the alkylating agent N-ethylmaleimide (NEM). It was found that NEM (2.5 mM) reduced significantly the Bmax from 1116 to 853 fmol/mg protein and increased the KD values of the muscarinic acetylcholine receptor (mAchR) activity from 36 to 61 pM. The mAchR subtypes in these plasma membranes were studied using competition experiments with selective antagonists. Pirenzepine displayed low competitive activity, having a pKi of 6.91 +/- 0.03, which was similar to that of AF-DX 116 (11[[2-[(diethylamino)methyl]- 1-piperidinyl]-acetyl]-5,11-dihydro-6H-pyrido[2,3- b][1,4]benzodiazepine-6-one); (pKi = 6.90 +/- 0.04), whereas hexahydrodifenidol (HDD) and its p-fluoro-derivative (p-FHHSiD) showed higher affinities than pirenzepine, having pKi values of 7.45 +/- 0.05 and 7.17 +/- 0.06, respectively. The antagonist 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) showed a pKi of 8.25 +/- 0.03, which did not differ significantly from the affinity shown by methoctramine (pKi = 8.00 +/- 0.04). These data indicate that the mAchR associated with the plasma membrane fraction isolated from bovine airway smooth muscle can be classified as an M2 subtype muscarinic receptor. NEM treatment altered the affinities of the mAchR towards specific antagonists, such as methoctramine (Ki increased 3 times), and the results indicated that the alkylated mAchR behaves as a chemically modified M2 subtype. This suggests the presence of thiol groups controlling the antagonist binding activity of this muscarinic receptor subtype.
authors.affiliation: Sección de Biomembranas, Instituto de Medicina Experimental (IME), Universidad Central de Venezuela (UCV), Caracas.
authors.forename: A J
authors.initials: AJ
authors.lastname: Misle
authors.forename: I
authors.initials: I
authors.lastname: Lippo de Bécemberg
authors.forename: R
authors.initials: R
authors.lastname: González de Alfonzo
authors.forename: M J
authors.initials: MJ
authors.lastname: Alfonzo
mesh_terms: D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003959:Diamines / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D009130:Muscle, Smooth / Q000378:metabolism*; D010277:Parasympathomimetics / Q000378:metabolism*; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism*; D014132:Trachea / Q000378:metabolism*
chemical_list: D003959:Diamines; D010277:Parasympathomimetics; D011976:Receptors, Muscarinic; D011813:Quinuclidinyl Benzilate; C054938:methoctramine; D005033:Ethylmaleimide