Quantitation of the Contractile Response Mediated by Two Receptors: M2 and M3 Muscarinic Receptor-Mediated Contractions of Human Gastroesophageal Smooth Muscle

Abstract

Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M 2 and M 3 muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M 2 receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M 3 density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtypeselective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M 2 and M 3 receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M 2 and M 3 receptors to contraction is presented. Using this new method of analysis, it was found that the M 2 muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M 3 receptor predominantly mediates contraction.Located at the junction of the tubular esophagus and the saccular stomach, the gastroesophageal junction (GEJ) is the area of transition from positive pressure in the abdominal cavity to the respiratory oscillations of negative and positive pressure in the thoracic cavity. It has the dual function of ensuring passage of a swallowed bolus and preventing gastroesophageal reflux. The existence of an anatomical sphincter at the GEJ has been disputed for more than half a century.The existence of a sphincter at the GEJ was proposed in an observational and anatomical study of cadavers (Lerche, 1950). With the use of manometry in 1956, this same area was described as a high-pressure zone (HPZ) rather than an anatomical sphincter (Code et al., 1956). Since then, much has been written about the HPZ in the lower esophagus. It is mainly composed of pressures from the extrinsic crural diaphragm and the intrinsic muscles of the stomach and the lower esophagus (McCray et al., 2000).In 1979, the arrangement of the smooth muscles around the GEJ was first described as consisting of "clasp" fibers at the lesser curvature and "sling" fibers at the greater curvature of the stomach, suggesting that these muscle fibers might produce the HPZ at the GEJ (Liebermann-Meffert et al., 1979). This theory laid the foundation for further studies on the physiology, pathology, and pharmacology of the GEJ. Over the span of the next three decades, the formation and regulation of the HPZ were closely s...

Pharmacologic Specificity of Nicotinic Receptor-Mediated Relaxation of Muscarinic Receptor Precontracted Human Gastric Clasp and Sling Muscle Fibers within the Gastroesophageal Junction

“…We previously reported that the bethanechol-induced maximal contraction is greater in the sling muscle fibers than in all other fibers studied in these subjects (Braverman et al, 2009). This result is in general agreement with previous studies showing that human gastric sling muscle fibers contract significantly greater to acetylcholine than human clasp muscle fibers (Tian et al, 2004).…”

Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells

“…Effect of the nonselective M 3 -R antagonist 4-DAMP on M 3 -R activation by BeCh in the IAS SMC. 4-DAMP caused significant and concentration-dependent rightward shift in the BeCh CRC in the IAS SMC.…”

“…1). To determine whether BeCh-induced shortening of the IAS SMC is via M 3 -R activation, we examined the effect of the M 3 -R antagonist darifenacin (3,23). Darifenacin (1 ϫ 10 Ϫ9 , 3 ϫ 10 Ϫ9 , and 1 ϫ 10 Ϫ8 M) caused concentration-dependent rightward parallel shift in the BeCh CRCs.…”

Comparison of human and porcine gastric clasp and sling fiber contraction by M2and M3muscarinic receptors

“…The results of the receptor density determinations are shown in Table 1 compared with our previous results in human tissue (4). As in the human tissue, the total and M 3 receptor density in the pig sling fibers were statistically significantly greater than the clasp fibers.…”