BACKGROUND/AIMS: An Na+/taurocholate cotransporting polypeptide (Ntcp) has recently been cloned from rat liver. The aim of this study was to directly characterize the native Ntcp on the protein level and study its in situ distribution in rat liver. METHODS: A rabbit antiserum was raised against a fusion protein containing the maltose-binding protein and the C terminus of Ntcp. Native Ntcp was localized in situ by immunofluorescent techniques. Expression of Ntcp was directly correlated with taurocholate uptake measurements in stably transfected Chinese hamster ovary cells. RESULTS: Native Ntcp showed an apparent molecular weight of 51,000. After deglycosylation of isolated basolateral rat liver plasma membranes, the apparent molecular weight of Ntcp decreased to 33,500. In intact rat liver, Ntcp was selectively localized at the basolateral surface domain of hepatocytes. In short-term cultured hepatocytes, a positive surface immunoreaction was only obtained in detergent-permeabilized cell cultures. In stably transfected Chinese hamster ovary cells, the surface expression of immunopositive Ntcp was associated with Na(+)-dependent taurocholate uptake activity. CONCLUSIONS: Native Ntcp represents a glycoprotein of the basolateral hepatocyte plasma membrane with its C-terminal end facing the intracellular compartment. Furthermore, surface expression of Ntcp is a prerequisite for Na(+)-dependent taurocholate uptake to occur, thus providing further proof for its bile acid transport function in rat liver.
BACKGROUND/AIMS: An Na+/taurocholate cotransporting polypeptide (Ntcp) has recently been cloned from rat liver. The aim of this study was to directly characterize the native Ntcp on the protein level and study its in situ distribution in rat liver. METHODS: A rabbit antiserum was raised against a fusion protein containing the maltose-binding protein and the C terminus of Ntcp. Native Ntcp was localized in situ by immunofluorescent techniques. Expression of Ntcp was directly correlated with taurocholate uptake measurements in stably transfected Chinese hamster ovary cells. RESULTS: Native Ntcp showed an apparent molecular weight of 51,000. After deglycosylation of isolated basolateral rat liver plasma membranes, the apparent molecular weight of Ntcp decreased to 33,500. In intact rat liver, Ntcp was selectively localized at the basolateral surface domain of hepatocytes. In short-term cultured hepatocytes, a positive surface immunoreaction was only obtained in detergent-permeabilized cell cultures. In stably transfected Chinese hamster ovary cells, the surface expression of immunopositive Ntcp was associated with Na(+)-dependent taurocholate uptake activity. CONCLUSIONS: Native Ntcp represents a glycoprotein of the basolateral hepatocyte plasma membrane with its C-terminal end facing the intracellular compartment. Furthermore, surface expression of Ntcp is a prerequisite for Na(+)-dependent taurocholate uptake to occur, thus providing further proof for its bile acid transport function in rat liver.
Authors: Dongke Yu; Han Zhang; Daniel A Lionarons; James L Boyer; Shi-Ying Cai Journal: Am J Physiol Regul Integr Comp Physiol Date: 2017-01-11 Impact factor: 3.619