Heterogeneous accumulation of fluorescent bile acids in primary rathepatocytes does not correlate with their homogenous expression of ntcp

Sodium taurocholatecotransportingpolypeptide (ntcp) is considered to be a major determinantof bile acid uptake into hepatocytes. However, the regulationof ntcp and the degree that it participates in the accumulation ofspecific substrates are not well understood. We utilized fluorescentbile acid derivatives and direct quantitation of fluorescent microscopyimages to examine the regulation of ntcp and its role in the cell-to-cellvariability of fluorescent bile acid accumulation. Primary-cultured rathepatocytes rapidly accumulated the fluorescent bile acids, chenodeoxycholylglycylamidofluorescein(CDCGamF), 7-β- nitrobenzoxadiazole3-α hydroxy 5-β cholan-24-oic acid (NBD-CA), and cholylglycylamido-fluorescein (CGamF). However, in stably transfectedHeLa cells, ntcp preferred CDCGamF, whereas the organic aniontransporter, organic anion transporting polypeptide 1 (oatp1a1), preferredNBD-CA, and neither ntcp nor oatp1a1 showed strong accumulationof CGamF by these methods. Ntcp-mediated transport ofCDCGamF was inhibited by taurocholate, cyclosporin, actin depolymerization,and an inhibitor of atypical PKC-ζ. The latter two agentsaltered the cellular distribution of ntcp as visualized in ntcp-greenfluorescent protein-transfected cells. Although fluorescent bile acidaccumulation was reproducible by the imaging assays, individual cellsshowed variable accumulation that was not attributable to changes inmembrane permeability or cell viability. In HeLa cells, this wasaccounted for by variable levels of ntcp, whereas, in hepatocytes, ntcpexpression was uniform, and low accumulation was seen in a largeportion of cells despite the presence of ntcp. These studies indicatethat single-cell imaging can provide insight into previously unrecognizeddetails of anion transport in the complex environment ofpolarized hepatocytes.