Characteristics And Mechanisms Of CaSR Sensing Protein And Aromatic Amino Acids In Stomach Of Pigs

As major energy sources,dietary protein is the essential material of life and the main bearer of life activities.Protein can be broken down into peptide or amino acids in the gastrointestinal tract.Peptide or amino acidscan not only be absorbedas nutriments,but also the important signaling molecules to activate a variety of sensors and transporters in the gut.The activitation of sensorsand transporters can regulate the proliferation of intestinal epithelial cells,the secretion of gastric juice and gastrointestinal hormone,the digestion and absorption of nutrients and the gastrointestinal physiological function.Calcium sensing receptor(CaSR)is a member of the family C of G protein-coupled receptors,which is widely expressed throughout the gastrointestinal tract,especially in the stomach fundus corpus,and G cell,D cell of the antrum.In previous studies,aromatic amino acids,for examples,phenylalanine(Phe)and tryptophan(Trp)can activate CaSR in the stomach,regulate H+-K+-ATPase activity and the secretion of the gut hormone,leading to the gastric acid secretion.However,the effects of amino acids and proteins on the expression and function of gastric CaSR are mainly focused on humans and rodents in the current studies.Livestock,especially the related studies in porcine stomach have not been reported yet.At present,whether CaSR presentsin porcine gastric tissue,whether it can mediate amino acids on the regulation of functional enzyme activity and the secretion of gastrointestinal hormones in the stomach need to be verified.Therefore,this study combined two parts(in vivo and vitro)to explore a series of relative issues.1 Effects of different dietary protein content on CaSR expression in gastrointestinal of pig,gastrointestinal hormone secretion and the parameters of gastric functionThe objectives of this experiment were to investigate the effect of different dietary protein content on the expression of calcium-sensing receptor(CaSR)in gastrointestinal tract,gastrointestinal hormone secretion and the activity of pepsin and H+-K+-ATPase,and to explore the relationship between CaSR expression of small intestine and gastrointestinal hormone secretion,CaSR expression of stomach and the activity of pepsin and H+-K+-ATPase,respectively.Eighteen Duroc×Landrace × Yorkshire weaned pigs(35 days of age,initial body weight 9.57 ± 0.64kg)were randomly assigned into one of three dietary crude protein(CP)levels:normal protein group(NP),low protein group(MP),and very low protein group(LP).Pigs in each group were fed corn-soybean meal basal diet.Diets designated as NP group contained 20%,18%and 16%CP to meet nutrient requirements for weaned pigs,growing pigs and finishing pigs according to the National Research Council(NRC 2012).Pigs in MP group were fed diets containing 17%,15%and 13%CP,while pigs in LP group were fed diets containing 14%,12%and 10%CP.Diets for both MP and LP group were supplemented with L-lysine,L-methionine,L-threonine,and L-tryptophan to meet the requirements for weaned,growing and finishing pigs.After 125 experimental days,all pigs were slaughtered.Precaval vein blood,gastrointestinal tissue,mucosa and content were collected for analysis of gastrointestinal hormones,the activity of pepsin and H+-K+-ATPase,and CaSR expression.With the reduction of dietary protein,a significant increase of CaSR expression in stomach was revealed(P<0.05),while a significant decrease of CaSR expression in jejunum and ileum was observed(P<0.05).MP feeding decreased the concentration of GIP and PYY in serum(P<0.05)whereas the concentration of CCK was increased by MP feeding(P<0.05).The activity of pepsin and H+-K+-ATPase were decreased by LP diets(P<0.05).Correlation analysis showed that the expression of CaSR in intestine were positively correlated to the concentration of GIP and PYY,and the expression of CaSR in stomach were positively correlated tothe activity of pepsin and H+-K+-ATPase.The findings indicate that reduction of dietary protein decreased CaSR expression,which affected the gut hormone secretion and activity of pepsin and H+-K+-ATPase in stomach.2 Phe-induced the secretion of gut hormone and the activation of H+-K+-ATPase mediated by CaSR in pigstomachIn the present study,we examined whether the calcium-sensing receptor(CaSR)mediated Phenylalanine(Phe)-induced the gut hormone secretion and H+-K+-ATPase activity in stomach antrum or fundus corpus of swine using superfusion system in vitro.The stomach of swine was obtained from the local slaughter house and sliced into small pieces,subsequently perfused with Krebs-Henseleit buffer(KHB)as basal solution for 120 minutes.Gastrin and Somatostatin(SS)secretion and H+-K+-ATPase activityin were measured in stomach antrum or fundus corpus under various Phe concentrations(OmM,50mM,80mM),CaSR agonist(cinacalcet)or antagonist(NPS 2143),with or without intracellular Ca2+ and inositol 1,4,5-triphosphate receptor(IP3R)antagonist.Results showed that 80mM phenylalanine(Phe)and cinacalcet significantly promoted the secretion of SS,gastrin and the activation of H+-K+-ATPase(P<0.05),increased the CaSR gene expression(P<0.05).NPS2143 inhibited phenylalanine-induced the secretion of SS,gastrin,the activation of H+-K+-ATPase(P<0.05),and CaSR gene(P<0.05).Ca2+ is the natural ligand of CaSR,which promoted the phenylalanine-induced the gut hormones secretion and H-K-ATPase activity(P<0.05),increased the expression of CaSR gene(P<0.05).Furthermore,2-aminoethyl diphenyl-borinate(2-APB),as IP3 specific inhibitor,decreased gastrin and SS release stimulated by Phe in swine stomach antrum(P<0.05).In conclusion,our data demonstrates that the gastrin,SS secretion and H-K-ATPase activity could be induced by Phe in stomach of swine in vitro,and the gut hormones secretion was mediated by CaSR and its downstream signaling molecular IP3.3 Trp-induced secretion of gut hormone and the activation of H+-K+-ATPase mediated by CaSR in stomach of pigsThe current study aimed to clarify the effect of Trp on the H+-K+-ATPase activity,the gut hormone secretion and the roles of CaSR and its downstream signal pathway in the gut hormone secretion by the perfusion of pig stomach in vitro.Different concentrations(0,20 mM and 50 mM)of Trp,CaSR agonist and antagonist,inositol 1,4,5-triphosphate receptor(IP3R)antagonist and[Ca2+],at a final concentration in the perfusate were applied to investigate the effect of Trp on gut hormone secretion and H+-K+-ATPase activity.Results revealed that 20 mM,50mM Trp and the CaSR agonist cinacalcet promoted the secretion of SS,gastrin and the activation of H-K-ATPase(P<0.05),elevated the mRNA level of CaSR in the presence of 1.8 mM extracellular Ca2+([Ca2+]e)(P<0.05).However,NPS2143 attenuated the SS,gastrin release(P<0.05)and H-K+-ATPase activity,reduced the mRNA level of CaSR(P<0.05).And the 2-APB,aninhibitor of IP3R,inhibits Trp-stimulated gastrin,SS release in swine stomach antrum(P<0.05).Taken together,the SS,gastrin secretion and H+-K+-ATPase activity could be induced by Trp in the stomach of swine in vitro,and the secretion of gut hormones were mediated by CaSR and its downstream signaling molecular IP3R.