Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in gastrointestinal system

AIM:To clone and characterize the porcine aquaporins(AQPs)in the gastrointestinal system.METHODS:A PCR-based cloning strategy and RACEwere used to clone full-length AQP coding sequencefrom reversely transcribed pig liver cDNA.Stopped-flowlight scattering and a YFP-based fluorescence methodwere used to measure the osmotic water permeabilityof erythrocytes and the stably transfected CHO cells.RT-PCR,Northern blot,and immunohistochemistrywere used to determine the gastrointestinal expressionand localization of cloned AQPs.Protein expression intransfected cells and red blood cells was analyzed byWestern blot.RESULTS:An 813 bp cDNA encoding a 271 aminoacid porcine aquaporin(designated pAQP1)was clonedfrom liver mRNA(pAQP1 has a 93% identity withhuman AQP1 and contains two NPA motifs conservedin AQP family,one consensus sequence for N-linkedglycosylation,and one mercury-sensitive site at cysteine191).RT-PCR analysis revealed extensive expressionof pAQP1 mRNA in porcine digestive glands and gut.Northern blot showed a single 3.0 kb transcript inselected digestive organs,pAQP1 protein was localizedat central lacteals of the small intestine,microvesslesof salivary glands,as well as epithelium of intrahepaticbile ducts by immunoperoxydase.High osmotic water permeability that is inhibitable by HgCl2was detected inporcine erythrocytes and CHO cells stably transfectedwith pAQP1 cDNA.Immunoblot analysis of porcineerythrocytes and pAQP-transfected CHO cells revealedan unglycosylated 28 ku band and larger glycosylatedproteins.CONCLUSION:pAQP1 is the first porcine aquaporinthat can be molecularly identified so far.The broaddistribution of pAQP1 in epithelium and endotheliumof porcine digestive organs may suggest an importantrole of channel-mediated water transport in fluidsecretion/absorption as well as in digestive function andpathophysiology of the gastrointestinal system.