Investigation Of Affinity Of Muscarinic Receptors To Its Ligands In Human Lower Esophageal Sphincter

Objective: Lower esophageal sphincter (LES) is the incrassate muscle bundle located at the esophagogastric junction, which is composed of sling fibers at the greater curvature and clasp fibers at the lesser curvature. The two muscles both maintain the closure condition of the LES, and form the high pressure zone of LES. In normal conditions LES works as a single-way valve: the food could be emptied from the esophagus to the stomach passing the"valve", but the the contents of stomach could not be allowed to flow into the esophagus. In some special conditions, however, the LES relaxes and allowing the content of the stomach flows back to the esophagus, such as belching and vomiting. The regulation of the LES is a very complex process involving neurohumoral and myogenic factors. Acetylcholine is the most important neurotransmitter, which play its regulatory role through the muscarinic receptor (M receptor) distributed in the smooth muscle cells. M receptor widely exists in smooth muscles including the gastrointestinal tract, and plays a major role in maintaining smooth muscle contraction. Up to know, the family of M receptor have been identified four subtypes including M1, M2, M3, and M4 with pharmacological method, and five subtypes including m1,m2, m3,m4 and m5 with molecular biological method. The main subsets of M receptor existing in the membrane of smooth muscle fiber are M2 and M3. M2 consists of 75% to 80% of the number of all subsets of M receptor, but M3 receptor is the major functional subtype. This study investigated the distribution of M2 and M3 in sling and clasp fibers and affinity between the receptors and their ligand by radio ligand binding assay (RLBA). The regulatory function of the two receptor subsets to human LES was also analyzed.Methods: Smooth muscles of the sling and clasp fibers which were not invaded by carcinoma were obtained from 8 patients who underwent subtotal esophagectomy for middle thoracic esophageal cancer in the Fourth Hospital, Hebei Medical University from May 2007 to December 2007. An 8-12mm long and 2-4mm wide muscle strip was carefully dissected from each specimen.1. The muscle was homogenated by the tissue homogenizer with TBS buffer and then the homogenate was centrifuged at 1000×g for 10 minutes.2. The supernatant was discarded and the receptor membrane was prepared using Membrane Protein Extraction. The receptor membrane was quantitated and then frozen at -80℃for future use.3. Labeled ligand, 3H-QNB and antagonist atropine was put in reaction system with the cubage of 250μl, so that 3H-QNB and M receptor fully combined at 37℃for 40 minutes. Then saturation test was carried out.Methoctramine and 4-DAMP as antagonist of M2 and M3 respectively, the competitive inhibition test was carried out by repeating the above course with above reaction system.4. The competitive inhibition test using M2 antagonist, methoctramine and M3 antagonist, 4-DAMP was carried out by repeating the above reaction system.5. The combined and dissociated ligand were seperated by nitro fiberglass filter membrane which was sucked with a negative pressure. The filter membrane containing receptors binding with ligand was counted in liquid scintillation counter.The obtained data were converted to concentration- effectiveness curve and competitive inhibition plot by Graphpad software and Prism4.03 software. The equilibrium dissociation constant (Kd), maximal bonding (Bmax) and IC50were calculated Statistical analysis was performed with SPSS13.0 software package, and the difference was cansidered significance if P value was less than 0.05.Result:1. Saturation test: The saturation curves of M receptor in sling fibers and clasp fibres were obtained respectively. The Bmaxs of 3H-QNB and M receptor in the sling fibres was 568.2±5.28cpm and the Kds was 1.049±0.054mmol/L. The corresponding number in clasp fibers was 557.9±10.41cpm and 1.033±0.045mmol/L, respectively. There were no significant differences between the 2 muscle fibers in Bmax (t=0.782,P=0.391)and Kd (t=0.395,P=0.540)respectively.2. Competitive inhibition test: The competitive inhibition curves of the antagonists of M2 and M3 were obtained, respectively. The IC50 of metoctrmine in the sling fibres was 5.422±0.112×10-6 mol/L compared to 1.658±0.071×10-6 mol/L of 4-DAMP ( t=6.26 , P=0.025 ) .The IC50 of metoctrmine in the clasp fibres was 1.993±0.065×10-6 mol/L compared to 5.680±0.105×10-6 mol/L (t=7.21,P =0.037). But the difference of inhibition function of Methoctramine in sling fibers and clasp fibers has no statistical significanc(et=0.079,P=0.783), and 4-DAMP had a similar effects (t=0.024, P=0.878)Conclusion:1. Combination between 3H-QNB and M receptor in human LES is specific,and there are no significant differences in the affinity to 3H-QNB of M receptors between the two muscles.2. The inhibition effects of 4-DAMP, the antagonist of M3 on the combinatin of 3H-QNB and M3 in the LES is stronger than the effects of Methoctramine, the antagonist of M2, on the combination of 3H-QNB and M2. This suggest that M3 plays more important pharmacological role in regulation of human LES by stronger affinity to its ligand .3. Both Methoctramine and 4-DAMP have no singnificant differences in inhibition effects between sling fibers and clasp fibers.