Localization of the ammonium transporters, Rh B glycoprotein and Rh C glycoprotein, in the mouse liver.
OBJECTIVE
Hepatic ammonium metabolism is critical for maintenance of normal health. Three mammalian members of an ammonium transporter family have recently been identified: Rh A glycoprotein (RhAG), Rh B glycoprotein (RhBG), and Rh C glycoprotein (RhCG). This study examined which of these are expressed in the mouse liver and in which cells they are expressed.


METHODS
Normal Balb/c mice were used. Messenger RNA (mRNA) expression was detected using either conventional or real-time reverse-transcription polymerase chain reaction (RT-PCR). Protein expression was examined using immunoblot analysis and either immunohistochemical or immunofluorescent microscopy.


RESULTS
We confirmed hepatic RhBG mRNA expression using real-time RT-PCR. Immunoblot analysis identified expression of a approximately 45-kilodalton protein. Immunohistochemical and immunofluorescent microscopy identified basolateral RhBG immunoreactivity in 1-2 cell layers of hepatocytes surrounding central veins. No immunoreactivity was identified in periportal or midzonal hepatocytes. Perivenous hepatocyte-specific expression was confirmed by colocalization with glutamine synthetase. A second ammonium transporter, RhCG, was expressed but at substantially lower levels. Real-time RT-PCR quantified hepatic RhCG mRNA expression at approximately 0.4% of RhBG mRNA expression. Immunoblot analysis confirmed RhCG protein expression, and immunofluorescence microscopy identified RhCG expression in bile duct epithelia. In contrast to RhBG and RhCG, RhAG mRNA was not identified by RT-PCR.


CONCLUSIONS
RhBG and RhCG are expressed by the mouse liver. Basolateral RhBG is expressed by perivenous hepatocytes, where it may mediate ammonium uptake, and RhCG immunoreactivity is present in bile duct epithelial cells, where it may contribute to ammonium secretion into bile fluid.
North Florida/South Georgia Veterans Health System, and University of Florida College of Medicine, Gainesville, Florida 32610-0224, USA. weineid@ufl.edu
I David
ID
Weiner
R Tyler
RT
Miller
Jill W
JW
Verlander
D000818:Animals; D001798:Blood Proteins; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D027682:Cation Transport Proteins; D015870:Gene Expression / Q000502:physiology; D005974:Glutamate-Ammonia Ligase / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis
D001798:Blood Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; C486033:RhBG protein, mouse; C401210:Rhag protein, mouse; C485880:Rhcg protein, mouse; D005974:Glutamate-Ammonia Ligase