Evidence for an ATP-dependent bile acid transport protein other than the canalicular liver ecto-ATPase in rats.

BACKGROUND & AIMS: Canalicular secretion is rate limiting in overall blood-to-bile transport of bile acids. Studies using transfected cells have implicated the canalicular ecto-adenosine triphosphatase (ecto-ATPase) in adenosine triphosphate (ATP)-dependent bile acid transport. However, the structural features of this ecto-ATPase are not those anticipated for an in-to-out ATP-dependent transporter. The aim of this study was to explore the possible existence of an ATP-dependent bile acid transport mechanism distinct from ecto-ATPase. METHODS: Bile acid transport activity and ecto-ATPase expression were analyzed in primary rat hepatocytes, rat hepatoma HTC cells, and specially adapted HTC (HTC-R) cells using plasma membrane vesicles and Northern blot, slot blot, ribonuclease protection assay, and Western blot analyses. RESULTS: Plasma membranes isolated from HTC-R cells exhibited ATP-dependent taurocholate transport, which was many-fold greater than that in HTC cells. Hepatocytes showed the highest transport rates. Protein and RNA analyses showed very low expression of ecto-ATPase in HTC and HTC-R cells compared with hepatocytes. There was no difference between the two cell types at both the RNA and protein level. CONCLUSIONS: These findings show the presence in HTC-R cells and, apparently in hepatocytes, of one or more proteins other than the ecto-ATPase that mediate ATP-dependent transport of bile acids.

Thyroid hormone export regulates cellular hormone content and response.

Actions of thyroid hormones (THs) are determined by intracellular free hormone concentration. Here we report that enhanced TH extrusion via a saturable, cold-sensitive mechanism lowers intracellular TH and causes TH resistance in hepatoma cells. Since these cells overexpress multidrug resistance P-glycoproteins and TH extrusion and resistance are blunted by verapamil, P-glycoproteins may mediate this resistance. Verapamil-inhibitable TH efflux was also found in primary hepatocytes, cardiocytes, and fibroblasts. These findings demonstrate that TH extrusion can modulate TH availability and action in mammalian cells.

Enhanced secretion of glycocholic acid in a specially adapted cell line is associated with overexpression of apparently novel ATP-binding cassette proteins.

Secretion of anionic endo- and xenobiotics is essential for the survival of animal and plant cells; however, the underlying molecular mechanisms remain uncertain. To better understand one such model system--i.e., secretion of bile acids by the liver--we utilized a strategy analogous to that employed to identify the multidrug resistance (mdr) genes. We synthesized the methyl ester of glycocholic acid (GCE), which readily enters cells, where it is hydrolyzed to yield glycocholic acid, a naturally occurring bile acid. The rat hepatoma-derived HTC cell line gradually acquired resistance to GCE concentrations 20-fold higher than those which inhibited growth of naive cells, yet intracellular accumulation of radiolabel in resistant cells exposed to [14C]GCE averaged approximately 25% of that in nonresistant cells. As compared with nonresistant cells, resistant cells also exhibited (i) cross-resistance to colchicine, a known mdr substrate, but not to other noxious substances transported by hepatocytes; (ii) increased abundance on Northern blot of mRNA species up to 7-10 kb recognized by a probe for highly conserved nucleotide-binding domain (NBD) sequences of ATP-binding cassette (ABC) proteins; (iii) increased abundance, as measured by RNase protection assay, of mRNA fragments homologous to a NBD cRNA probe; and (iv) dramatic overexpression, as measured by Western blotting and immunofluorescence, of a group of 150- to 200-kDa plasma membrane proteins recognized by a monoclonal antibody against a region flanking the highly conserved NBD of mdr/P-glycoproteins. Finally, Xenopus laevis oocytes injected with mRNA from resistant cells and incubated with [14C]GCE secreted radiolabel more rapidly than did control oocytes. Enhanced secretion of glycocholic acid in this cell line is associated with overexpression of ABC/mdr-related proteins, some of which are apparently novel and are likely to include a bile acid transport protein.