Bile acid transport in cultured rat hepatocytes

Abstract

The mechanisms of bile acid uptake have been studied with primary monolayer cultures of rat hepatocytes. Hepatocytes were incubated with taurocholic acid (TC), glycocholic acid (GC), cholic acid (CA), glycochenodeoxycholic acid (GCDC), chenodeoxycholic acid (CDCA), deoxycholic acid (DOCA), lithocholic acid (LCA), or cholylglycylhistamine (CCH), a neutral bile acid derivative for 10 s to 60 min in medium containing sodium chloride, sodium chloride with 1 mM ouabain, or choline chloride. Cells were washed free of radioactive tracer, cell-associated radioactivity was quantitated, and bile acid uptake rates, kinetic parameters of uptake, and steady-state bile acid content were calculated. Two mechanisms for bile acid uptake were identified. Uptake of TC, GC, CA, and GCDC occurred predominantly via a sodium-dependent, ouabain-suppressible saturable mechanism, presumably sodium-coupled transport. Estimates of apparent Km and Vmax for these bile acids were TC, 33 micro M and 0.36 nmol . min-1 . mg prot-1; GC, 18 micro M and 0.22 nmol . min-1 . mg prot-1; CA, 13 micro M and 0.10 nmol . min-1 . mg prot; and GCDC, 6 micro M and 0.21 nmol . min-1 . mg prot, respectively. Uptake via this sodium-coupled mechanism exhibited considerable substrate selectivity. It was enhanced by increased ring hydroxylation and amino acid conjugation and decreased by further conjugation with a neutral histamine group (CGH). In contrast, uptake of CDCA, DOCA, LCA, and CGH occurred primarily via a nonsaturable sodium-independent mechanism, possibly simple diffusion. This mechanism accounted for only a small portion of uptake of TC, GC, CA, and GCDC at low bile acid concentrations. Nonsaturable bile acid uptake rates appeared to correlate with decane-buffer partition coefficients and to be related to bile acid structure.

Key discoveries in bile acid chemistry and biology and their clinical applications: history of the last eight decades

“…Lithocholic acid, CDCA, DCA, UDCA, and presumably 3,6-dihydroxy bile acids are highly membrane permeable ( 221,245 ), and in our judgment do not require a transporter for rapid uptake into the hepatocyte. Cholic acid, in contrast, passes across cell membranes slowly and requires a transporter for rapid cellular entry.…”

“…In a study published that same year from the Erlinger laboratory in Paris, fi rst pass extraction of taurocholate in the dog was found to be 40-80% and saturable, consistent with uptake being carrier mediated ( 244 ). Conjugated bile acid uptake was saturable, and using either hepatocytes ( 245 ) or the isolated perfused rat liver ( 245 ) was sodium-dependent ( 246 ). Thus it seemed highly likely that one or more sodium-coupled transporters were present in the sinusoidal membrane and mediated the effi cient uptake of bile acids.…”

Bile acid transporters

“…The uptake of conjugated (i.e., N-acyl amidated with taurine or glycine) bile acids, such as taurocholate, at the sinusoidal membrane is mediated predominantly (>75%) by secondary active Na + -dependent transport. In contrast to conjugated bile acids, Na + -dependent uptake accounts for less than half of the uptake of unconjugated bile acids (20)(21)(22). The Na + -dependent sinusoidal membrane uptake is mediated by the NTCP, whereas members of the organic anion transporting polypeptide (OATP) family are responsible for the Na + -independent transport.…”

Bile acids mobilise internal Ca2+ independently of external Ca2+ in rat hepatocytes

“…Since the characteristics of taurolithocholate uptake by liver cells have never been established, experiments were first directed towards investigating the kinetic properties of bile acid influx ( n = 6), a value not different from the non-conjugated lithocholate influx measured in cultured rat hepatocytes [21]. However, extrapolation of uptake to time zero revealed the presence of a bound fraction of taurolithocholate.…”