The Function And Mechanism Of Voltage-gated Chloride Channel ClC-2 In The Regulation Of Duodenal Mucosal Ion Transport And Bicarbonate Secretion

Backgrouds:The mammalian duodenal epithelium has both absorptive and secretory functions.Its ability to secrete,particularly mucus and HCO₃^-,is critical to defend the vulnerable duodenal epithelium against the aggressive gastric factors of acid and pepsin.It has been widely accepted that cystic fibrosis transmembrane conductance regulator（CFTR）and Cl^-/HCO₃^-anion exchangers（AE）are primary apical HCO₃^-transporters involved in duodenal bicarbonate secretion（DBS）.Besides CFTR and AE,intestinal epithelial cells may also functionally express several other Cl^-channels capable of mediating intestinal anion secretion.Among these Cl^-channels are ClC-2 channels,which have been demonstrated to express in the apical or/and basolateral membrane of intestinal epithelia.ClC-2 channels are a member of the ClC superfamily of voltage-gated anion channels.It was previously reported that ClC-2channels would mediate colonic Cl^-secretion.So far,the function of ClC-2 channels in small intestinal epithelial anion secretion,particularly DBS has rarely investigated.Therefore,in the present study we examined whether ClC-2 channels play roles in the regulation of small intestinal epithelial anion secretion,whether ClC-2 or CFTR is the target of lubiprostone-induced DBS;and if so,what the underlying mechanisms are.Methods:Firstly,the immunofluorescence and quantitative real time RT-PCR assay were applied to detect the protein and mRNA expressions of ClC-2 in duodenal mucosae of mice.Then duodenal mucosae from mice were stripped of seromuscular layers and mounted in Ussing chambers.Both duodenal short-circuit current（Isc）and HCO₃^-secretion in vitro were simultaneously recorded.Duodenal mucosal bicarbonate secretion（DMBS）in vivo was measured by a CO₂-sensitive electrode and cytoplasmic free Ca²⁺([Ca²⁺]cyt)was measured by digital Ca²⁺imaging in duodenal epithelial cell lines SCBN.The transepithelial electrical resistance（TEER）of mouse duodenal mucosa was monitored by the short-circuit current（SSC）technique.Results:1.ClC-2 was preferentially located basolaterally in duodenal epithelium of mice.2.Lubiprostone（10-1000 nM）,a selective ClC-2 activator,concentration-dependently increased both duodenal Isc and DMBS only when applied basolaterally,but essentially no response when applied apically.3.Removal of extracellular Cl^-abolished lubiprostone-induced duodenal Isc,but did not alter HCO₃^-secretion even in the presence of DIDS,a Cl^-/HCO₃^-exchanger inhibitor（p>0.05,n=6）.4.Addition of glybenclamide,a CFTR channel blocker,abolished lubiprostone-evoked HCO₃^-secretion（p<0.01,n=6）.Moreover,lubiprostone-induced HCO₃^-secretion was impaired in CFTR^-/-mice compared to their wild type littermates（p<0.01,n=5）.5.Perfusion of duodenal lumen with lubiprostone（1μM）did not alter basal DMBS in vivo,but lubiprostone（0.1mg/kg,i.p）was able to induce DMBS,which was also significantly inhibited by Cd²⁺,a ClC-2 channel blocker（p<0.01,n=6）.6.[Ca²⁺]cyt level,Ca²⁺-activated K⁺channel and cAMP-mediated duodenal Isc and HCO₃^-secretion were unchanged by lubiprostone.Conclusions:Lubiprostone may selectively open basolateral ClC-2 channels in duodenal epithelium to supply HCO₃^-for apical transport through CFTR channels.Therefore,we have provided the first evidence for the functional expression of ClC-2 channels in duodenal epithelium and their novel role in the regulation of DBS.