| Gastroparesis is one of the most common functional gastrointestinal diseases,and motilin receptor has become one of the most important potential therapeutic targets for gastroparesis due to its role in mediating gastric empties and phase Ⅲ of gastric migrating motor complex（MMC Ⅲ）.In fact,motilin is not only an important hormone for the start of MMC Ⅲ but also has the important function of regulating the digestive tract-brain-pancreatic axis.In addition,the results of the flow probes of ultrasound transit-time blood-flow meters have shown that motilin regulated the blood flow of the dog’s left gastric artery（LGA）simultaneously with the gastric MMC Ⅲ under physiological conditions,and this process depends on motilin receptor.The immunological and molecular biological experiments have shown direct evidence of motilin receptor expression on the endothelial cell membrane of the dog’s gastrointestinal arteries.However,the intracellular signal transduction pathway of endothelial motilin receptor-mediated gastric artery relaxation has not been systematically studied.This in vitro study is divided into two parts.Firstly,based on the relaxation effect of motilin on the main arteries of the gastrointestinal tract in dogs,we compared the relaxation rates of motilin on LGA,superior mesenteric artery（SMA）,and inferior mesenteric artery（IMA）,and functionally proved the roles of the endothelium,the motilin receptor and the muscarinic receptor in motilin-induced vasorelaxation.Secondly,the intracellular signal transduction pathways after the activation of endothelial motilin receptors were investigated.This study is expected to provide a more theoretical basis for the studies of physiological and pathological mechanisms of gastrointestinal functions.Part Ⅰ: Roles of endothelium-derived motilin receptor in motilin-induced relaxation of dog gastrointestinal arteriesObjective: Compare the relaxation rates induced by motilin in LGA,SMA,and IMA in dogs.Observe the roles of the endothelium,motilin receptor,and muscarinic receptor in motilin-induced relaxation.Methods: Motilin’s effects on the gastrointestinal arteries were evaluated in a multi-wire myograph system.The effects of endothelium-denuded,motilin receptor antagonist GM-109,and muscarinic receptor antagonist atropine on this process were observed.Results:（1）The concentration-response curve of motilin(10-9 M to 10-5 M)showed an "S" shape;The calculated half effective concentration（EC50）is 9.0 ± 0.8×10-8 M.The concentration of EC50 motilin(9 × 10-8 M)was used in the subsequent experiments.（2）The relaxation rates of LGA,SMA and IMA induced by motilin were 33.8% ± 1.3%,23.7% ± 3.4% and 15.3% ± 1.0%,respectively.There were statistically significant differences in the relaxation rate among the three arteries（p < 0.05）.（3）Denuded endothelium completely inhibited the relaxing effects induced by motilin(10-5 M)（p < 0.05）.（4）GM-109(10-10 to 10-5 M)inhibited LGA relaxation induced by motilin,and its concentration-response curve showed an inverted "S" shape.GM-109(10-6 M)significantly inhibited motilin-induced(10-8 M-10-6 M)vasorelaxation,with the inhibition rates ranging from 77.7% ± 2.8% to 89.3% ± 2.1%.A higher concentration of GM-109(10-5 M)inhibited the relaxing effects induced by motilin（p < 0.05）in LGA,SMA,and IMA.（5）Atropine,a muscarinic receptor antagonist,did not inhibit the relaxing effects induced by motilin（p < 0.05）.Conclusion: The relaxation rates of motilin in the gastrointestinal arteries were LGA > SMA > IMA;the process was dependent on the endothelium-derived motilin receptor but not the muscarinic receptor.Part Ⅱ: Roles of the signal transduction pathway of endothelium-derived motilin receptor in motilin-induced relaxation of dog LGAObjective: Reveal the G protein signal pathways coupled with endothelial motilin receptor;investigate the role of extracellular Ca2+.Investigate the roles of nitric oxide（NO）,prostacyclin（PGI2）,myoendothelial gap junction（MEGJ）,and K+ channel in motilin-induced smooth muscle cells relaxation of dog LGA;observe the effects of motilin on NO and cGMP levels in LGA tissues.Methods: The effects of the inhibitors of G protein signal pathways,different concentrations of Ca2+,the inhibitors of NOS-NO-s GC signal pathways,the cyclooxygenase inhibitor indomethacin,the blockers of the MEGJ and K+ channels on the relaxation of LGA induced by motilin in dogs were detected in a multi-wire myograph system.LGA tissues were divided into groups and incubated with motilin or motilin with blockers,respectively.The NO detection kit using the nitrate reduction method was used to detect the level of NO in LGA tissues.The cGMP level was detected by the cGMP ELISA kit.Results:（1）The G protein antagonist NEM(3 × 10-5 M)reduced the relaxation rate of LGA from 49.5% ± 2.4% to 23.4% ± 1.7%（p < 0.05）.The PLC inhibitor U73122 reduced the relaxation rate from 44.4% ± 5.2% to 4.9% ± 0.8%（p < 0.05）.The IP3 receptor antagonist and store-operated Ca2+ channel（SOCC）blocker 2-APB(3 × 10-4 M)reduced the relaxation rate from 38.5% ± 5.2% to 1.6% ± 0.2%（p < 0.05）.（2）The PKC inhibitor chelerythrine(10-6 M)increased the relaxation rate from 34.9% ± 2.9% to 39.7% ± 3.7% with significance（p < 0.05）.（3）The PKA inhibitor H89(5 × 10-6 M)increased the relaxation rate from 34.9% ± 2.9% to 39.7% ± 3.7% without significance（p > 0.05）.（4）With Krebs solution containing [Ca2+] of 2.5 × 10-3 M,1.25 × 10-3 M,0.625 × 10-3 M,and 0 M incubation,as the [Ca2+] decreased,motilin-induced relaxation rates also gradually reduced（p < 0.05）.（5）The NOS inhibitor L-NAME decreased the vasorelaxation from 35.5% ± 4.1% to 3.2% ± 0.6%（p < 0.05）.Similarly,the s GC inhibitor ODQ reduced relaxation rate the from 36.1% ± 3.8% to 3.4% ± 0.5%（p < 0.05）.（6）The cyclooxygenase inhibitor indomethacin(10-5 M)reduced relaxation rate from 39.5% ± 3.0% to 32.7% ± 3.4%（p < 0.05）.（7）The MEGJ inhibitor 18α-GA(7.5 × 10-5 M)decreased vasorelaxation from 35.2% ± 3.4% to 26.4% ± 2.9%（p < 0.05）.（8）A high [K+] solution containing 30 mM KCl decreased vasorelaxation from 42.4% ± 4.4% to 5.5% ± 1.4%（p < 0.05）.The non-specific KCa channel blocker TEA(10-2 M)reduced LGA relaxation from 36.5% ± 2.7% to 18.8% ± 1.9%（p < 0.05）.The KATP channel blocker glibenclamide(10-6 M)increased the relaxation rate from 41.3% ± 2.7% to 55.0% ± 4.3%（p < 0.05）.（9）The NO and the cGMP baseline levels were 2.5 ± 0.4 μmol g-1 protein and 1.9 ± 0.02 pmol mg-1 protein,respectively.Motilin(9 × 10-8 M)increased the levels of NO and cGMP by 1.3 and 1.5 times,respectively（p < 0.05）.GM-109(10-5 M)and L-NAME(10-4 M)significantly attenuated the synthesis of NO and cGMP（p < 0.05）.The effect of ODQ(10-5 M)on cGMP levels was similar to that of L-NAME（p < 0.05）.Conclusion:（1）Motilin activates the motilin receptor-G protein-PLC-IP3 signal transduction pathway,but not the G protein-PLC-DG-PKC and G protein-AC-PKA signal pathway.（2）The extracellular Ca2+ was involved in motilin-induced relaxation.（3）The NOS-NO-s GC pathway played an important role in the motilin-induced relaxation of LGA smooth muscle cells.The PGI2 and the EDHF were also involved in the process. |
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