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

Abstract

Sodium taurocholate-cotransporting polypeptide (ntcp) is considered to be a major determinant of bile acid uptake into hepatocytes. However, the regulation of ntcp and the degree that it participates in the accumulation of specific substrates are not well understood. We utilized fluorescent bile acid derivatives and direct quantitation of fluorescent microscopy images to examine the regulation of ntcp and its role in the cell-to-cell variability of fluorescent bile acid accumulation. Primary-cultured rat hepatocytes rapidly accumulated the fluorescent bile acids, chenodeoxycholylglycylamidofluorescein (CDCGamF), 7-β- nitrobenzoxadiazole 3-α hydroxy 5-β cholan-24-oic acid (NBD-CA), and cholyl-glycylamido-fluorescein (CGamF). However, in stably transfected HeLa cells, ntcp preferred CDCGamF, whereas the organic anion transporter, organic anion transporting polypeptide 1 (oatp1a1), preferred NBD-CA, and neither ntcp nor oatp1a1 showed strong accumulation of CGamF by these methods. Ntcp-mediated transport of CDCGamF was inhibited by taurocholate, cyclosporin, actin depolymerization, and an inhibitor of atypical PKC-ζ. The latter two agents altered the cellular distribution of ntcp as visualized in ntcp-green fluorescent protein-transfected cells. Although fluorescent bile acid accumulation was reproducible by the imaging assays, individual cells showed variable accumulation that was not attributable to changes in membrane permeability or cell viability. In HeLa cells, this was accounted for by variable levels of ntcp, whereas, in hepatocytes, ntcp expression was uniform, and low accumulation was seen in a large portion of cells despite the presence of ntcp. These studies indicate that single-cell imaging can provide insight into previously unrecognized details of anion transport in the complex environment of polarized hepatocytes.

Transcription Factors with Conserved Binding Sites Near ATOH1 on the POU4F3 Gene Enhance the Induction of Cochlear Hair Cells

“…To evaluate the level of pou4f3 /GFP expression in each cell, the average brightness of individual HCs was measured on Image J (U. S. National Institutes of Health, Bethesda, Maryland USA; [16,19]. For each sample, 10 pou4f3 /GFP cells in the GER were measured for the brightness of GFP expression The average GFP brightness of the cells were standardized against the average GFP brightness of native HCs in the same images, to correct for difference in image exposure.…”

Dipeptidyl peptidase IV inhibitor lowers PPARγ agonist-induced body weight gain by affecting food intake, fat mass, and beige/brown fat but not fluid retention

“…Paraffin embedded fat tissue was cut into 5-m sections and stained with hematoxylin and eosin. Pictures were taken on an Olympus IX81 microscope, and adipocyte cross-sectional area was determined using Image J 1.45s (http://rsb.info.nih.gov/ij), as described previously (11 (27). Fluorescence signal was quantified by scoring the percent of pixels per image that exceeded an intensity threshold.…”

Sodium-dependent bile salt transporters of the SLC10A transporter family: more than solute transporters

“…This exemplifies that the modifications of NTCP substrates have to be carefully considered and support the concept that the side chain of bile salts is a critical determinant for their recognition by NTCP as a transported substrate (9). The following fluorescent bile salts have been shown to be substrates of rat or human NTCP: cholylglycylfluorescein and chenodeoxycholyllysyl-NBD (22), NBD-cholyltaurine (122), cholylglycylamidofluorescein and chenodeoxycholylglycylamidofluorescein (109,114). …”