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?1?M?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.