The Role And Mechanism Of Calcium-sensitive Receptors In Regulating Feeding And Gastrointestinal Motility

Objective Obesity and some gastrointestinal disorders are important factors that threaten human health and become the research hotspot in medical field.Calcium-sensing receptor（Ca SR）is a typical G-protein-coupled receptor for sensing nutrients.In addition to being activated by calcium ions(Ca²⁺),it is also activated by polyamines and amino acids,which are involved in the regulation of feeding and gastrointestinal motility.In the central region,Ca SR is richly expressed in the area postrema（AP）,which is closely associated with feeding,gastric emptying,and intestinal motility.Therefore,in this paper,we explored the effects and potential mechanisms of Ca SR in the AP on feeding and gastrointestinal motility.Method1.Male C57BL/6 mice were used to observe the effects of Ca SR agonist R568 injected into the AP on food intake,gastric emptying rate and intestinal transport time.2.After microinjection of R568 into the AP,the contents of glutamic acid,γ-aminobutyric acid（GABA）in the AP,oxytocin（OT）in the paraventricular nucleus（PVN）,Ach in the dorsal nucleus of vagus nerve（DMV）and neurotransmitters in gastrointestinal tissue were detected by enzymoly-linked immunosorbent assay（ELISA）.3.Immunofluorescence（IF）was used to observe the co-staining of OT and c-fos in the PVN,GABA-,VGlu T1 positive neurons and Ca SR in the AP,GABA-A receptor/ionic glutamate receptor（NMDAR）and OT in the PVN,Ch AT with c-fos and Ch AT with NMDAR in the DMV.4.After PVN was damaged,R568 was injected into the AP to observe the changes of food intake in 0-24h.PVN was damaged,and the effect on R568 inhibiting feeding was observed.5.Inhibition of GABA neurons by chemogenetic experiment was used to observe the effect on R568-induced feeding behavior and gastrointestinal motility changes.6.GABA-A receptor blocker and glutamate receptor blockers（Ionic/metabolic type）were injected into the AP to observe their effects on food intake and gastrointestinal motivity changes induced by R568.7.Using anterograde tracking technique combined with IF,the anterograde virus-carrying Ca MKlla promoter was injected into the AP and the fiber projection of AP to DMV Ch AT neurons was observed under fluorescence microscope.ResultPart one Effect of Ca SR on feeding in the AP and its mechanism1.The results showed that compared with DMSO group,the food intake was significantly reduced within 0-2h after microinjection of R568 in the AP（P<0.05）.2.ELISA results showed that compared with DMSO group,the contents of glutamic acid and GABA in the AP were significantly increased in R568 group,and the content of OT in the PVN also increased significantly（P<0.05）.3.IF results showed that microinjection of R568 into the AP increased the expression of co-staining of OT and c-fos positive neurons（P<0.05）.4.After PVN was damaged,the anorexia effect induced by R568 was inhibited（P<0.05）.5.IF results showed that there was co-staining of Ca SR and VGlu T1 neurons in the AP.However,Ca SR and GABA neurons did not co-stain.6.The results of chemogenetic method showed that the anorexia effect of R568 was weakened after inhibition of glutamate neurons in the AP（P<0.05）.7.The anorexia effect of R568 was weakened after microinjection of GABA-A receptor blockers and glutamate receptor inhibitors（ionic）into the PVN（P<0.05）.8.IF results showed there are co-staining of GABA-A receptors,ionic glutamate receptors（NMDAR）and OT in the PVN.Part two Effect and mechanism of Ca SR on gastrointestinal motility in the AP9.After microinjection of R568 into the AP,the gastric emptying rate was significantly decreased（P<0.05）,and intestinal transmission time was significantly prolonged（P<0.05）.10.ELISA results showed that the content of Ach in the DMV increased significantly after microinjection of R568 in the AP（P<0.05）.11.IF result showed that the number of Ch AT and c-fos positive neurons and the co-staining of Ch AT and c-fos positive neurons increased significantly after microinjection of R568 into the AP（P<0.05）.12.Immunofluorescence results showed that after injection of FG in the DMV,there was co-staining of FG,VGlu T1 and Ca SR in the AP,and anterograde tracing results showed that the virus carrying Ca MKlla promoter was injected into the AP,and there was co-staining of anterograde fibers and Ch AT-positive neuron in the DMV.13.The results of chemogenetics showed the inhibitory effect of R568 on gastrointestinal motion was weakened by inhibition of glutamate neurons in the AP（P<0.05）.14.The inhibitory effect of R568 on gastrointestinal motion was weakened by microinjection of ionic glutamate receptor blocker into the DMV（P<0.05）;IF results showed significant co-staining of Ch AT and NMDAR in the DMV.15.ELISA results showed that after microinjection of R568 into the AP,Ach content in stomach was significantly decreased and n NOS content was increased（P<0.05）;In the proximal small intestine,Ach content was significantly increased（P<0.05）and in the distal small intestine,n NOS content was significantly decreased（P<0.05）;The content of dopamine（DA）in colon was significantly increased（P<0.05）.Conclusion1.Activation of Ca SR in the AP can inhibit feeding;Glutaminergic neurons in the AP mediated the anorexia of R568.GABA-A receptors and NMDAR in the PVN are involved in the action of R568.R568 also promotes OT synthesis in the PVN,which is mediated by NMDAR.2.Microinjection of R568 into the AP inhibited gastric emptying and prolonged intestinal transport time,this effect is related to glutaminergic neuron activation;Activation of DMV Ch AT neurons and increased Ach synthesis,which is mediated by NMDAR.Ch AT neurons further stimulate the synthesis of neurotransmitters in the gastrointestinal tract through vagus efferent fibers and inhibit gastrointestinal motility.