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 B max from 1116 to 853 fmol mg protein and increased the K D 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 p K i of 6.91 ± 0.03, which was similar to that of AF-DX 116 (11[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11-dihydro-6 H-pyrido[2,3-b] [1,4]benzodiazepine-6-one); (p K i = 6.90 ± 0.04), whereas hexahydrodifenidol (HDD) and its p-fluoro-derivative (p-FHHSiD) showed higher affinities than pirenzepine, having p K i values of 7.45 ± 0.05 and 7.17 ± 0.06, respectively. The antagonist 4-diphenylacetoxy- N-methylpiperidine methobromide (4-DAMP) showed a p K i of 8.25 ± 0.03, which did not differ significantly from the affinity shown by methoctramine (p K i = 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 M 2 subtype muscarinic receptor. NEM treatment altered the affinities of the mAchR towards specific antagonists, such as methoctramine ( K i increased 3 times), and the results indicated that the alkylated mAchR behaves as a chemically modified M 2 subtype. This suggests the presence of thiol groups controlling the antagonist binding activity of this muscarinic receptor subtype.