Molecular Mechanisms Of Calcium Signaling Mediated Intestinal Epithelial Ion Transport Through TRPV4-constituted SOCE And Its Role In Irritable Bowel Syndrome With Diarrhea

Part?A universal role of serasol TRPV4-constituted SOCE mechanism in secretagogues-stimulated intestinal epithelial anion secretionBackground:As little is currently known about the role of Ca²⁺signaling in small intestinal epithelial anion secretion,we aimed to study its regulatory role in secretagogue-stimulated small intestinal anion secretion and the underlying molecular mechanisms.Methods and results:The intestinal anion secretion from native mouse duodenal epithelia was examined with Ussing chambers to monitor PGE₂-,5-HT-,and CCh-induced short-circuit currents(I_sc).PGE₂(10?M)and5-HT(10?M)induced mouse duodenal I_sc,which was markedly attenuated by serosal Ca²⁺-free solution and selective blockers of the store-operated Ca²⁺channels on serosal side of the duodenum.PGE₂-and 5-HT-induced duodenal I_sc was also inhibited by the ER Ca²⁺chelator TPEN.However,dantrolene,a selective blocker of ryanodine receptors,inhibited PGE₂-induced duodenal I_sc,while Li Cl,an inhibitor of IP₃ production,inhibited 5-HT-induced I_sc.Moreover,the duodenal I_sc response to the serosal applications of both PGE₂ and 5-HT was significantly attenuated in TRPV4 knockout mice.Finally,mucosal application of carbachol(100?M)also induced duodenal I_sc via selective activation of muscarinic receptors,which was significantly inhibited in serosal Ca²⁺-free solution but neither in mucosal Ca²⁺-free solution nor by nifedipine.Conclusion:Therefore,the serosal TRPV4-constituted SOCE mechanism is likely universal for the most common and important secretagogues-induced and Ca²⁺-dependent intestinal anion secretion.These findings will enhance our knowledge about GI epithelial physiology and the associated GI disease,such as diarrhea and constipation.Part ? 5-HT via TRPV4-constituted SOCE regulates mouse distal colonic epithelial ion secretionBackground: The first part studied the role of 5-HT mediating anion secretion in the mouse duodenum.Simultaneously,the study of 5-HT in the mouse colon also focused on c AMP and c GMP.The 5-HT mediated ion secretion mechanism through Ca2+ signals in the distal colon has not been fully elucidated.We aimed to study the Ca2+ regulation of 5-HT in the distal colon anion secretion and its underlying molecular mechanism.Methods and results: We used the Ussing Chamber to examine the intestinal anion secretion of natural mouse colonic epithelium to monitor 5-HT-induced short-circuit current(Isc);real-time calcium imaging was used to determine the IEC cell calcium signal response to 5-HT;q PCR was performed to determine the expression of 5-HT receptors in the distal colon of mice.It is proved that 1)5-HT activates 5-HT4 R on the mouse colon's serosal side to stimulate Ca2+-dependent ion secretion.The basis of ion secretion is Cl-and HCO3-,mainly Cl-;2)serosal side 5-HT in the distal colon mainly stimulates anion secretion through the serosal SOCE mechanism initiated by IP3R/ER Ca2+ release;3)CARC channel may be the SOCE mechanism in the Ca2+-dependent anion secretion process;4)TRPV4 channel may represent the serosal side SOCE/CARC channel mediates the molecular composition of calcium-dependent anion secretion;5)5-HT-induced Ca2+ secretes Cl-mainly through activation of CaCC in the distal colon of mice.Conclusion:our results indicate that the calcium ion signal is still the key to induce anion secretion in the distal colon.The SOCE mechanism of TRPV4 on the serosal side may play an important role.The secretion of epithelial anions mediated by calcium ions in the distal colon is mainly Cl-, and is mainly secreted by CaCC,which provides new insights into the molecular mechanism of anion secretion.Part ? Study on the role of 5-HT-mediated ion secretion in diarrhea of irritable bowel syndromeBackground: The previous study have clarified the role of 5-HT in mediating anion secretion in the duodenum and distal colon of mice under physiological conditions,but under pathological conditions,the mouse with irritable bowel syndrome(IBS),the mechanism of ion secretion in IBS has not yet been fully elucidated.We aimed to study the role of 5-HT in IBS colonic anion secretion and its underlying molecular mechanism.Methods and results: We chose the water avoidance stress(WAS)model and the DSS-induced irritable bowel syndrome(IBS)model to build animal models and used the Ussing Chamber to detect the transepithelial resistance(TEER)and 5-HT-induced of model mice Short-circuit current(Isc);FITC-Dextran was used to determine the permeability of the colonic mucosa of model mice;q PCR was performed to determine the expression of 5-HT receptors in the distal colon of model mice.Proved that 1)WAS mice have significantly increased defecation volume and no obvious pathological changes in the intestines.The model can represent IBS symptoms.2)Both TEER and FITC-Dextran in the 3-day acute phase of WAS mice indicate increased permeability.3)The ion secretion induced by 5-HT on the distal serosal side of the colon of WAS mice is less than that of sham mice.4)The expression of 5-HT receptors in the distal colon of WAS mice decreased.Conclusion:our results show that the WAS mouse model can represent irritable bowel syndrome diarrhea-type,which stool volume increase without obvious pathological changes.The intestinal permeability of WAS mice was significantly increased.The decrease of 5HT stimulated ion secretion in WAS is related to the decrease of 5-HT receptor expression in the colon of WAS mice.This provides us with new insights into the pathogenesis of irritable bowel syndrome.