Effects Of Natural Flavonoids On CFTR And CaCCs Chloride Channel Activities

Appropriate stool water content and intestinal motility are essential for normal defecation.Malfunction of intestinal fluid secretion and/or motility will lead to such diseases as constipation and secretary diarrhea.It is well established that cystic fibrosis transmembrane conductance regulator(CFTR)and calcium-activated chloride channels(Ca CCs)in the luminal membrane of intestinal epithelium are the major chloride channels for fluid secretion,and TMEM16 A type Ca CC in gastrointestinal(GI)smooth muscle plays important roles in modulation of GI motility.CFTR is a c AMP-activated Cl– channel expressed in epithelial cells in the airways,gastrointestinal tract,sweat gland,and reproductive organs.CFTR play critical roles in transepithelial salt and fluid transport.Numerous studies implicated that Ca CCs involved in many important physiological functions,the molecular identity of the first Ca CC was unveiled as TMEM16A(also called ANO1)in 2008 by three independent groups,ANO2 was subsequently confirmed.Roles of CFTR and Ca CCs as molecule targets in the therapy of constipation and secretory diarrhea have been highlighted.Previously we found many CFTR and Ca CCs modulators with different chemical identities including several flavonoids.In the present paper,we systematically investigated several flavionoids compounds on their CFTR and Ca CCs chloride channel modulation activities.The results showed that:(1)Cell-based fluorescence assay showed that chrysin,5,7,3',4'-Tetramethoxyflavone(TMF)and 5,7,3',4',5'-Pentamethoxyflavone(PMF)dose-dependently potentiated wild-type CFTR chloride channel activities,with EC50 values of30.63±0.26 ?M,9.06 ±0.15 ?M and 11.35±0.18 ?M,respectively.Further studies indicated activation of CFTR chloride channel by this compouns with characteristics of rapid,reversible and forskolin-dependent manners.Our results also showed that the activation of CFTR by these compounds could be totally reversed by the known CFTR inhibitors CFTRinh-172.(2)Short-circuit current studies showed that chrysin,TMF and PMF could potentate CFTR-mediated transepithelial current in mouse colonic mucosa,which confirmed the cell-based fluorescence results.(3)Cell-based fluorescence assay also revealed rapid and reversible inhibition of ATP-activated TMEM16 A channel activities by chrysin and nobiletin.IC50 values of the inhibition were 156.9±0.29 ?M(chrysin)and 91.78±0.17 ?M(nobiletin),respectively.(4)Both chrysin and nobiletin could dose-dependently inhibite TMEM16A-mediated curerents stimulated by TMEM16 A selective activator Eact as shown inthe Ussing chamber studies.(5)Short-circuit current measurement also showed that both chrysin and nobiletin could inhibited a molecule identity unknown Ca CC on the apical plasma membrane of enterocytes.In conclusion,the present study found several CFTR and Ca CCs modulators,the results added new molecule targets to the flavoinoid compounds.These compounds may have potential use in elucidating the roles of chloride channels in intestinal fluid secretion and motility under baseline conditions and in constipation models,and provides lead compounds for constipation and secretory diarrhea.