Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in gastrointestinal system.
OBJECTIVE
To clone and characterize the porcine aquaporins (AQPs) in the gastrointestinal system.


METHODS
A PCR-based cloning strategy and RACE were used to clone full-length AQP coding sequence from reversely transcribed pig liver cDNA. Stopped-flow light scattering and a YFP-based fluorescence method were used to measure the osmotic water permeability of erythrocytes and the stably transfected CHO cells. RT-PCR, Northern blot, and immunohistochemistry were used to determine the gastrointestinal expression and localization of cloned AQPs. Protein expression in transfected cells and red blood cells was analyzed by Western blot.


RESULTS
An 813 bp cDNA encoding a 271 amino acid porcine aquaporin (designated pAQP1) was cloned from liver mRNA (pAQP1 has a 93% identity with human AQP1 and contains two NPA motifs conserved in AQP family, one consensus sequence for N-linked glycosylation, and one mercury-sensitive site at cysteine 191). RT-PCR analysis revealed extensive expression of pAQP1 mRNA in porcine digestive glands and gut. Northern blot showed a single 3.0 kb transcript in selected digestive organs. pAQP1 protein was localized at central lacteals of the small intestine, microvessles of salivary glands, as well as epithelium of intrahepatic bile ducts by immunoperoxydase. High osmotic water permeability that is inhibitable by HgCl2 was detected in porcine erythrocytes and CHO cells stably transfected with pAQP1 cDNA. Immunoblot analysis of porcine erythrocytes and pAQP-transfected CHO cells revealed an unglycosylated 28 ku band and larger glycosylated proteins.


CONCLUSIONS
pAQP1 is the first porcine aquaporin that can be molecularly identified so far. The broad distribution of pAQP1 in epithelium and endothelium of porcine digestive organs may suggest an important role of channel-mediated water transport in fluid secretion/absorption as well as in digestive function and pathophysiology of the gastrointestinal system.
Jin
Shun-Ying
SY
Membrane Channel Research Laboratory, Northeast Normal University, 5268 Renmin Street,Changchun 130024, Jilin Province, China.
Liu
Yan-Li
YL
Xu
Li-Na
LN
Jiang
Yong
Y
Wang
Ying
Y
Yang
Bao-Xue
BX
Yang
Hong
H
Ma
Tong-Hui
TH
D000595:Amino Acid Sequence; D000818:Animals; D051398:Aquaporin 1 / Q000032:analysis* / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D016466:CHO Cells; D002463:Cell Membrane Permeability; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000032:analysis / Q000235:genetics; D004912:Erythrocytes / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D041981:Gastrointestinal Tract / Q000737:chemistry* / Q000166:cytology; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D008099:Liver / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014162:Transfection
D012333:RNA, Messenger; D051398:Aquaporin 1; D004247:DNA