Expression Of Motilin Receptor On The Gastrotintestinal Artery And Enteric Nervous System In Dogs

BackgroundMotilin is one of the brain-gut peptides located in specific endocrine cells of the jejunum and duodenum. J.C. Brown first isolated it from pig duodenal mucosa. Motilin played a important role through combining with motilin receptors(MTLR). Miller P etc. revealed that motilin receptors from nerves or smooth muscles express different binding characteristics and pharmacological features, suggesting the existence of 2 subtypes of motilin receptors located on nerves (N) and muscles (M). But it is seldom reported that motilin receptor is located on the endothelium of artery. Our previous study showed the left gastric arterial (LGA) blood flow was stable during a quiescent phase (phase I) in conscious dogs. But the left gastric arterial (LGA) blood flow is increased as high as 240% of the resting levels, lasting 30 minutes. After 30 minutes into the stationary phase, an intravenous infusion of motilin in dog vein can induce increased blood flow in the left gastric artery, which resemble the spontaneously occurring periodic changes associated with phase Ⅲ-like contractions of the stomach and duodenum. When stopping the infusion of motilin, the elevation of LGA blood flow quickly returned to the quiescent levels. An intravenous infusion of GM-109 completely abolished the motilin induced LGA blood-flow changes and contractions of the stomach and duodenum. The spontaneous LGA blood-flow changes and contractions of the stomach and duodenum appeared promptly after the cessation of GM-109 infusion. But superior mesenteric artery(SMA) and inferior mesenteric arteries(IMA) have not similar changes. So we consider there may exist a new subtype for the motilin receptor i.e. endothelium of artery (E) through the research on dog motilin receptor expression in gastrointestinal arterial, which provided the structure basis for the further study of molecular mechanism of motilin’s action on regulating dog gastric blood flow during interdigestive period.The main physiological function of motilin is released periodically during the interdigestive, through the motilin receptor, to regulate the migrating motor complex (MMC) of gastrointestinal tract. MMC is the innate and basic form of motion for both Human and Mammals, which is of great physiological significance in the whole life activities. So studying the distribution, struction and function of the motilin receptor has potentially valuable for the treatment of gastrointestinal disorders.Objective1.To investigate whether motilin receptor was expressed on gastric artery and compare their expression on intestinal arteries in dogs in vitro.2. To study the distribution of the motilin receptor in gastrointestinal tract of dogs and compare the expression strength.Methods1. We used immunohistochemistry(S-P)and immunofluorescence double staining to detect the distribution of MTLR on LGA, SMA and IMA of dogs.2. We detected the expression of MTLR mRNA on LGA, SMA and IMA of dogs by RT-PCR.3. We detected the expression of MTLR protein on LGA, SMA and IMA of dogs by Western blot.4. We used immunohistochemistry(S-P) and RT-PCR to detect the distribution of MTLR in gastrointestinal tract of dogs and to compare the expression strength.Results1. Immunohistochemistry of the dog arteries revealed that the motilin receptor was localized on endothelium, LGA was strongly immunoreactive for motilin receptor compared to SMA and IMA, i.e. LGA endothelial cells appeared brown or tan particles (P<0.01)2. Immunofluorescence data demonstrated that motilin receptor was located on the membrane of the endothelium. In addition, the fluorescent intensity of LGA was significantly higher than SMA and IMA (P<0.01)3. We detected that motilin receptor mRNA was expressed on LGA, SMA and IMA. Nevertheless, motilin receptor mRNA on LGA is the most highly expressed with significant decrease on SMA, followed by IMA (P<0.01), consistent with immunohistochemical and immunofluorescence results.4. We detected that motilin receptor protein was expresssed on LGA, SMA and IMA. Nevertheless, motilin receptor protein on LGA is the most highly expressed with significant decrease on SMA, followed by IMA (P<0.01). This is consistent with the results of RT-PCR.5. Immunohistochemistry of the dogs’gastrointestinal tract revealed that motilin receptor was expressed in muscle fibers and my enteric plexus of dogs’gasric antrum, duodenum, jejunum, ileum, proximal colon, middle colon. Terminal colon was negative. Staining strength from deep to light was duodenum> ileum> jejunum> proximal colon> gasric antrum> middle colon> terminal colon (P<0.05)6. We detected that motilin receptor mRNA was expressed in gastrointestinal tract of the dogs except the terminal colon. Expression intensity was duodenum> ileum> jejunum> proximal colon> gasric antrum> middle colon> terminal colon (P<0.05)Conclusion1.Motilin receptor located on vascular endothelial cell membrane.2.Motilin receptor was preferentially expressed on the endothelium of LGA compared to SMA and IMA among the gastrointestinal arteries.3.Motilin receptor was expressed in muscle fibers and myenteric plexus throughout the gastrointestinal tract of dogs except the terminal colon.4.The expression intensity of the motilin receptor in gastrointestinal tract was duodenum> ileum> jejunum> proximal colon> gasric antrum> middle colon> terminal colon.