Calcium-sensitive Receptor Agonist R568 Modulates Gastric Motility And Feeding And Potential Mechanism Invloved In Mice

Objective:In recent years,obesity,diabetes,gastrointestinal motility disorders have become a common problem for people.Exploring the regulatory mechanisms of food intake and gastrointestinal function will help us to find targets for more effective treatment of metabolic diseases.Previous studies have shown that calcium-sensitive receptor(Ca SR),expressed in endocrine cells of the gastrointestinal tract and enteric nervous system(ENS),plays an important role in fluid and electrolyte transport in the gastrointestinal tract,and its role has been paid increasing attention in the regulation of gastrointestinal movement.We aimed to investigate the effect of Ca SR agonist R568 on gastric motility and feeding and its potential mechanism.Methods:Effect of intragastric administration of R568 on gastric emptying and potential mechanism1.To observe the effects of calcium sensitive receptor agonist R568 by oral gavage on gastric emptying of mice and the effect of calcium sensitive receptor blocker NPS-2143on the effect of R568 by phenol red excretion experiment.2.Fluorescence immunohistochemistry was used to observe the co-expression of Ca SR and nitric oxide synthase(n NOS)/choline acetyltransferase(Ch AT)positive neurons in the gastric myenteric plexus and the effect of R568 by oral gavage on the expression of Ch AT neurons and n NOS neurons in the myenteric plexus of the stomach.3.To observe the effects of R568 by oral gavage on the contents of acetylcholine(Ach),nitric oxide(NO)released in the gastric tissues with mucous membrane scrapped by enzym-linked immunosorbent assay(ELISA).4.To observe the role of vagal afferent in the regulation of the effect of R568 on gastric emptying by capsaicin subcutaneous injected.5.Fluorescence immunohistochemistry was used to observe the effects of R568 by oral gavage on the expression of c-fos neurons in the the nucleus of the solitary tract(NTS).6.To observe the effects of R568 by oral gavage on the release of gastrin in mice by ELISA.Effect of R568 on gastric smooth muscle strips and potential mechanism7.The effect of R568 with different doses on the contraction of smooth muscle strips of gastric corpus and antrum was observed in vitro.The effect of NPS-2143,M receptor blocker(atropine),nitric oxide synthase inhibitor(L-NAME),Na~+channel blocker(TTX)on the action of R568 were studied to explore the potential mechanism of R568.8.Fluorescence immunohistochemistry was use to observe the expression of Ca SR in the myenteric plexus in gastric corpus and gastric antrum.9.Fluorescence immunohistochemistry was used to observe the co-expression of Ca SR and c-kit in the myenteric plexus in gastric corpus and gastric antrum.10.The effects of R568 on the discharge frequency and amplitude of Cajal cells(ICC-MY)were observed by intracellular recording.Effect of intragastric administration of R568 on food intake and potential mechanism11.To observe the effect of R568 on the food intake of mice by gavage alone or pre-administration of NPS-2143 or cholecystokinin receptor antagonist(L-364,718).12.To observe the effects of R568 by gavage on the contents of ghrelin,cholecystokinin(CCK),peptide YY(PYY)and glucagon-like peptide-1(GLP-1)in serum and stomach/small intestines of mice by ELISA.13.Fluorescence immunohistochemistry was used to observe the effects of R568 on the expression of c-fos neurons in the paraventricular nucleus(PVN),the lateral hypothalamus(LHA)and the arcuate nucleus(ARC)of mice.Results:Effect of intragastric administration of R568 on gastric emptying and potential mechanism1.The effect of R568 by gavage on gastric emptying showed that the gastric emptying rate of mice was decreased in a dose-dependent manner by intragastric administration of1.5μg/g,3.0μg/g,6.0μg/g or 12μg/g R568(P<0.05～0.01).Compared with 3.0μg/g R568 group,pre-administration of 20μg/g NPS-2143 completely blocked the inhibitory effect of R568 on gastric emptying(P<0.05).In this study,3.0μg/g R568(ED50)was selected for subsequent experiments.2.Effect of R568 by gavage on the expression of neurons in ENS which participate in regulating the gastric motility:Fluorescence immunohistochemical staining showed that there was the expression of Ca SR immunoreactive neurons in the myenteric plexus in mice and some Ca SR immunoreactive neurons were co-expressed with n NOS or Ch AT immunopositive neurons.Compared with the control group,the expression of Ch AT immunoreactive neurons in gastric antrum was significantly decreased(P<0.01),and the expression number of n NOS immunoreactive neurons was significantly increased(P<0.05)with R568 intragastric administrated.3.Effects of R568 by gavage on the release of Ach/NO in mice:ELISA results showed that compared with the control group,the contents of Ach and NO in the gastric corpus were not significantly different with that in the R568 treated group(P>0.05);However,the content of Ach was significantly decreased(P<0.01),and the content of NO was significantly increased(P<0.05)in gastric antrum in the R568 treated group.4.Role of afferent vagus nerve in the effect of R568 on gastric emptying:The results showed that capsaicin which could impair the function of afferent vagus nerve blocked the inhibitory effect of R568 on gastric emptying partially(P<0.05).5.Effect of R568 on the expression of c-fos in the NTS of mice:Fluorescence immunohistochemical staining showed that compared with the control group,R568 could significantly increase the expression of c-fos protein in the NTS(P<0.05).6.Effect of R568 on gastrin expression in mice:ELISA results showed that compared with the control group,the expression of gastrin was significantly increased after R568was administrated(P<0.05).Effect of R568 on gastric smooth muscle strips and potential mechanism7.The study of contractions of smooth muscle strips in vitro showed that R568 had no significant effect on the contractile activity of gastric corpus muscle strips(P>0.05).R568inhibited the contraction of gastric antrum muscle strips in a dose-dependent manner(P<0.05～0.01).When the gastric antrum muscle strips were incubated with NPS-2143 in advance,the inhibitory effect of R568 on the gastric antrum muscle strips could be completely blocked(P<0.01).When the gastric antrum muscle strips were incubated with L-NAME/atropine or L-NAME and atropine in advance,the inhibitory effect of R568 on the gastric antrum muscle strips could be partially blocked(P<0.05～0.01).The inhibitory effect of R568 on the contraction of gastric antrum muscle strips was also weakened by TTX(P<0.01).8.The expression of Ca SR in the ENS of stomach:Fluorescence immunohistochemical staining results showed that the expression of Ca SR neurons in the myenteric plexus of gastric antrum was significantly higher compared with that of gastric corpus(P<0.01).9.The effect of R568 on gastric ICC excitability:Fluorescence immunohistochemical staining results showed that c-kit immunopositive cells and Ca SR immunopositive cells were co-expressed in the gastric myentric plexus of mice.Compared with the control group,the discharge frequency and amplitude of gastric ICC were significantly reduced with R568(P<0.01).Effect of intragastric administration of R568 on food intake and potential mechanism10.Feeding study showed that compared with the control group,the food intake of mice in R568 group was significantly decreased for 0-1 h and 1-2 h(P<0.01).Compared with R568 group,the 0-1 h/1-2 h food intake of R568+L-364,718 group was significantly increased(P<0.05);NPS-2143+R568 group significantly increased the 0-1 h/1-2 h food intake(P<0.05).11.The effects of R568 by gavage on the contents of Ghrelin,cholecystokinin(CCK),PYY and glucagon-like peptid-1(GLP-1)in mice:Compared with the control group,ghrelin in the serum and gastric tissues were significantly decreased(P<0.05,P<0.01);the contents of CCK in serum and proximal small intestine were significantly increased(P<0.05,P<0.01)and there were no significant changes of PYY and GLP-1 in serum and distal small intestine in R568 treated group(P>0.05).12.Effect of R568 by gavage on the expression of c-fos immunopositive neurons in the hypothalamus of mice:Compared with the control group,fluorescence immunohistochemical staining showed that the expression of c-fos immunopositive neurons in the PVN,LHA and ARC of mice was significantly increased in R568 treated group(P<0.01).Conclusion:Activation of gastrointestinal Ca SR inhibited gastric emptying and contraction of gastric smooth muscle strips in mice.The decrease of gastric emptying is closely related to the inhibition of cholinergic nervous system and the activation of nitrergic nervous system in ENS;This effect is also mediated by vagal afferent nerve.The inhibition of the smooth muscle strips of gastric antrum is not only related to the inhibition of the cholinergic nervous system and the activation of the nitrergic nervous system of ENS,but also related to the inhibition of the excitability of Cajal cells in the myenteric plexus.The activation of Ca SR inhibited the feeding of mice in a short time.This effect is related to the decrease of ghrelin secretion,the increase of CCK secretion and the regulation of excitability of neurons in hypothalamic PVN,LHA and ARC by R568.Our researches expand the theory of energy metabolism balance and provide experimental basis for clinical treatment of energy metabolism disorders related diseases.