| 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 musles both maintain the closure condition of LES, whose regulatory mechanism is very sophisticated. The mechanism involves nervous system, humoral system and spontaneous myogenic factors. Gastrointestinal peptide hormone,as major component of humoral system, plays an essential part in the regulation of the LES. Cholecystokinin(CCK) is the gastrointestinal polypeptide hormone, which can mediate gallbladder contraction, pancreatic secretion and gastric emptying and. Nowadays, CCK becomes a tipical species of braingut peptide and can take an important part as a neurotransmitter and a neuromodulator, whose various kinds of physiological functions are realized by acting on its receptor. CCK receptor is divided into distinct subtypes of CCK receptor, CCKA receptor and CCKB receptor, according to its different affinity to CCK analogues, gastrin and specific antagonists. Some people think gastrin receptor and CCKB receptor are called CCKB/gastrin receptor. The aim of present study is to investigate the bonding force of 3H-CCK-8s to CCKA receptor and CCKB receptor and to decide a CCK receptor and a muscle strip playing predominant role in the regulation of human LES.Methods: Smooth muscles of the sling and clasp fibers which are not invaded by carcinoma were obtained from 7 patients who underwent subtotal esophagectomy for middle thoracic esophageal cancer in the Fourth Hospital, Hebei Medical University between December 2005 and May 2006. A 8-12mm long and 4-6mm wide muscle strip was carefully dissected from each specimens.1 The musle was homogenated by the tissue homogenizer with TBS buffer added in it, then the homogenate is 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 is quantitated and then frozen at -80℃for future use.3 Western blotting was used to demonstrated the existence of CCKA receptor and CCKB receptor in human LES.4 3H-CCK-8s as labeled ligand bound to receptor fully at 4℃vibration incubation in 200ul reaction system for 10 hours, and then saturation test was carried out. Then CR1409 and CR2945 were separately added to reaction system and to carry out competitive inhibition test. The binding ligand and free ligand were separated by sucking filtration with underpreeure using 49 type nitro fiberglass filter membrane. The filter membrane, at which ligand binding to receptor membrane was located, was put in the LS-6500 type liquid scintillation counter. The research result were analyzed by Graphpad Prism4 software and SPSS11.5 statistical analysis software.Result:1 The expression of CCKA rexeptor and CCKB receptor was shown in western blotting. The IOD of CCKA rexeptor and CCKB receptor was 22.650±0.642 and 13.195±0.423 in clasp fibres and there was statistical difference(t=12.299, p=0.000). But there is no statistical difference of the expression of CCKA rexeptor between in clasp fibres and in slling fibres(t=0.263, p=0.806) and it is the same to CCKB receptor.2 The saturation curve was got. The maximum binding weight of 3H-CCK-8s to CCK receptor (Bmaxs) in the sling fibres was 595.750±3.231 cpm. And the dissociation constant of CCK receptor (Kds) was 1.671±0.024mmol/L. But the maximum combining weight of 3H-CCK-8s to CCK receptor (Bmaxc), located in the clasp fibres of LES was 500.000±10.087cpm, and the dissociation constant of CCK receptor (Kdc) is 1.437±0.024mmol/L. We can find that there are significant differences between clasp fibres and sling fibes on the maximum combining weight (t=9.040, p=0.000) and dissociation constant (t=6.898, p=0.000) of 3H-CCK-8s to CCK receptor, which is of statistical significance. Meanwhile we saw that the CCK receptor of sling fibres bind more 3H-CCK-8s.3 The competitive inhibition curve was got in competitive inhibition test. The 50 % inhibiting concentration of CR1409 ( EC50SA)and CR2945(EC50SB) to combining weight between 3H-CCK-8s and CCK receptor is 2.798±0.104×10-9 mol/L and 8.147±0.551×10-9 mol/L in sling fibres, and the corresponding figures were 3.165±0.187×10-9 mol/L and 9.583±0.501×10-9 mol/L respectively in the clasp fibres. There were statistical differences in the inhibitory action between CR1409 and CR2945 on the binding of 3H-CCK-8s to CCK receptor in the sling fibres (t=9.53,p=0.001) or in the clasp fibres(t=11.99,p=0.000). But there were no statistical differences between clasp fibres and sling fibres on the 50%inhibitory concentration of CR1409 and CR2945(t=1.72,p=0.161).Conclusion:1 The binding affinity of 3H-CCK-8s to CCK receptor is specific and the CCK receptor in the sling fibres exceeds the clasp fibres in the affinity to 3H-CCK-8s.2 CCKA receptor antagonist CR1409 inhibits the binding of 3H-CCK-8s to CCK receptor more obviously than CCKB receptor antagonist CR2945.3 There are no differences between clasp fibres and sling fibres in the binding of antagonists and its receptors and no differences of CCKA receptor and CCKB receptor.4 CCKA receptor distributes more on the sling fibres than on the clasp fibres, which leads to the differences between clasp fibres and sling fibres on the maximum combining weight and dissociation constant of 3H-CCK-8s and CCK receptor. CCKA receptor probably plays an important role in mediating the contractile function and diastolic function of LES. |
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