Identification of a single sinusoidal bile salt uptake system in skate liver

Abstract

To identify the sinusoidal bile acid uptake system(s) of skate liver, photoaffinity labeling and kinetic transport studies were performed in isolated plasma membranes as well as intact hepatocytes. In both preparations photoaffinity labeling with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan-24-oyl)-2-aminoethanesulfonate revealed the presence of a predominant bile salt binding polypeptide with an apparent molecular weight of 54,000. The labeling of this polypeptide was inhibited by taurocholate and cholate in a concentration-dependent manner and was virtually abolished by 1 mM of the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Kinetic studies of hepatic uptake with taurocholate, cholate, and the photoreactive bile salt derivative indicated the involvement of a single transport system, and all three substrates mutually competed with the uptake of each other. Finally, irreversible inhibition of the bile salt uptake system by photoaffinity labeling of hepatocytes with high concentrations (250 microM) of photolabile derivative reduced the Vmax but not the Km of taurocholate uptake. These findings strongly indicate that a single polypeptide with an apparent molecular weight of 54,000 is involved in sinusoidal bile salt uptake into skate hepatocytes. These findings contrast with similar studies in rat liver that implicate both a 54,000- and 48,000-K polypeptide in bile salt uptake and are consistent with a single Na+-independent transport mechanism for hepatic bile salt uptake in this primitive vertebrate.

Phylogenic and ontogenic expression of hepatocellular bile acid transport.

“…Under low stringency conditions, no homologous mRNA can be found in any of the livers from nonmammalian species. The absence of mRNA for this transporter in nonmammalian liver is in keeping with previously published functional studies in isolated hepatocytes from marine elasmobranchs that demonstrate uptake of taurocholate into skate liver only by sodium-independent transport mechanisms (11,24). The absence of a functional Na+-dependent bile acid transporter in this lower vertebrate species was further confirmed by expressing this transport system in oocytes following injection of skate liver mRNA.…”

“…A 55-kDa organic anion-binding protein has also been identified in rat liver cell sinusoidal plasma membrane subfractions using photoactivated [35S]BSP (28). When liver plasma membranes from lower vertebrates (small skate) are photolabeled with 7,7-azotaurocholate, only a 54-to 55-kDa protein is labeled and Scatchard analysis reveals only one binding site (11). These studies are in contrast to the studies in the rat where two proteins are labeled and two binding sites are detected by Scatchard plots (11).…”

“…When liver plasma membranes from lower vertebrates (small skate) are photolabeled with 7,7-azotaurocholate, only a 54-to 55-kDa protein is labeled and Scatchard analysis reveals only one binding site (11). These studies are in contrast to the studies in the rat where two proteins are labeled and two binding sites are detected by Scatchard plots (11). Thus it seems likely that there are two transporters for the hepatic uptake of organic anions in mammalian species but that transport of these ligands in lower vertebrates occurs only by the multispecific sodiumindependent transporter(s) that is facilitated by chloride ions.…”

“…Bile acids (specifically, taurocholate) are transported from portal blood into hepatocytes preferentially by the sodium-dependent system (5-7), whereas bilirubin and a prototype substrate, sulfobromophthalein (BSP), utilize sodium-independent mechanisms (9). Phylogenic studies of taurocholate transport suggest that the sodiumdependent transporter is absent in hepatocytes from lower vertebrates (cartilaginous fishes) (10,11), whereas studies in liver plasma membrane vesicles from the developing fetus of the rat indicate that expression of this transporter is first detected at 20 days of gestation in this mammalian species (12). Sodium-dependent transporters for bile acid uptake in liver have been identified functionally in a number of mammalian species, including man (13).…”

An evolutionarily ancient Oatp: insights into conserved functional domains of these proteins

“…Because the predicted size of skate Oatp is 76 kDa, a finding also demonstrated by the in vitro translation studies, the larger size on Western blotting suggests that skate Oatp is glycosylated and possibly also phosphorylated when fully expressed in skate liver. A similar phenomenon was observed for human OATP8, which has a predicted size of 77 kDa but appeared as a 120-kDa band in a Western blot analysis (22) Uptake studies in skate Oatp cRNA-injected Xenopus oocytes, using a panel of substrates, demonstrate that this skate Oatp is a multispecific transporter whose substrate profile overlaps most closely with human OATP-C (SLC21A6) and rat Oatp4 (Slc21a10) (36), the two liver-specific members of this family (25) (6,13), this skate Oatp may also be a major determinant of hepatic bile salt uptake in elasmobranchs.…”