Pharmacological and molecular characterization of muscarinic receptor subtypes in human esophageal smooth muscle

Esophageal peristalsis is dependent on activation of muscarinic receptors, but little is known about the roles of specific receptor subtypes in the hu man esophagus. We examined muscarinic receptor expression and function in h uman esophageal smooth muscle obtained from patients undergoing resection f or cancer. [H-3] Quinuclidinyl benzylate (QNB)-specific binding was similar in longitudinal muscle (B-max = 106 +/- 22 fmol/mg of protein, K-d = 68 +/ - 9 pM) and circular muscle (B-max = 81 +/- 16 fmol/mg of protein, K-d = 79 +/- 15 pM). Subtype-selective antagonists inhibited [H-3] QNB similarly in muscle from both layers. Further analysis of antagonist inhibition of [H-3 ] QNB binding showed a major site (60-70%) with antagonist affinity profile consistent with the M2 subtype and a second site that could not be classif ied. Reverse transcription-polymerase chain reaction and immunoblotting dem onstrated the presence of all five known muscarinic receptor subtypes, and immunocytochemistry on acutely isolated smooth muscle cells confirmed the e xpression of each subtype on the muscle cells. Subtype-selective antagonist s had similar inhibitory effects on carbachol-evoked contractions in longit udinal muscle and circular muscle strips with pA(2) values of 9.5 +/- 0.1 a nd 9.6 +/- 0.2 for 4-diphenylacetoxy-N-methylpiperidine methiodide, 7.1 +/- 0.1 and 7.0 +/- 0.2 for pirenzepine, and 6.2 +/- 0.2 and 6.4 +/- 0.2 for m ethoctramine, respectively. We conclude that human esophageal smooth muscle expresses muscarinic receptor subtypes M1 through M5. The antagonist sensi tivity profile for muscle contraction is consistent with activation of the M3 subtype.