Bile acid inhibition of taurocholate uptake by rat hepatocytes: role of OH groups

Abstract

To define further the structural specificity of the taurocholate uptake site, we studied the ability of a variety of taurine-conjugated bile acids with differing hydroxyl substituents on the sterol moiety to inhibit [14C]taurocholate uptake. Rat hepatocytes isolated by collagenase perfusion were incubated in a tris(hydroxymethyl)aminomethane-phosphate buffer containing [14C]taurocholate (2.5-100 microM) in the presence or absence of inhibitor bile acid. Stronger inhibitors were studied at a fixed concentration of 5 microM, weaker ones at 25 microM. Initial uptake velocity was measured by sedimenting an aliquot of cells through silicone oil into 3 N KOH every 15 s for 1 min. Uptake velocity (nmol X mg protein-1 X min-1) could then be related to taurocholate concentration and a Vmax and Km could be determined by applying a nonlinear least squares fit to the data obtained with or without inhibitor. The kinetic parameters allowed the determination of the type of inhibition and of inhibition constants (Ki) of the various test bile acids. The data indicate that bile acids containing a 6- or 7-OH group exhibit competitive inhibition, whereas bile acids with no 6- or 7-OH group exhibit noncompetitive inhibition. Of the compounds exhibiting competitive inhibition, Ki varied with the number of hydroxyl groups on the sterol moiety. We conclude that the presence or absence of a 6- or 7-OH group dictates the mechanism of inhibition; the number of hydroxyl substituents determines the potency of competitive inhibition.

Taurohyocholate, taurocholate, and tauroursodeoxycholate but not tauroursocholate and taurodehydrocholate counteract effects of taurolithocholate in rat liver

“…At least at the concentrations used and in this albumin-free system no relevant uptake inhibition for TLC by any of the bile salts tested was observed. This might be different in other systems when using similar concentrations [9].…”

“…(i) The uptake of TLC could be reduced since TLC and TC have the same uptake site [9]. (ii) TC might form micelles and thus transport TLC in micellar form through the membrane [8].…”

Transport of organic anions in the liver. An update on bile acid, fatty acid, monocarboxylate, anionic amino acid, cholephilic organic anion, and anionic drug transport

“…Despite the lower extraction ratio, affinity constant (Km), and Vmax for uptake, taurine-conjugated mono-and dihydroxylated bile acids (7c¢-OH; 7~-OH; 3~,7~-OH; 3oL7b-OH; 3o~,12~-OH; 3o~,6o~-OH) are stronger inhibitots of the uptake of the trihydroxy bile acid TCA than all taurine-conjugated trihydroxy bile acids tested (3~,7cql2o~-OH; 3~,713,12o~-OH; 3cq6o~,7~-OH) (Bellentani et al 1987). It appears that the fewer the hydroxyl groups, the stronger is the inhibition.…”

“…Studies on structure-activity relationships of bile acid uptake were performed with isolated (Iga and Klaassen 1982b;Bartholomew and Billing 1983;Hardison et al 1984;Bellentani et al 1987) and cultured rat hepatocytes (Van Dyke et al 1982b). The conclusions were: (a) conjugation by taurine, glycine and probably sulfate markedly increase hepatic clearance and uptake Vmax; (b) the relative rate of hepatic clearance (in the rat) is in the order of TCA_>GCA_>CA_>DCA_>CDCA; (c) side chain length at C17 significantly modulates affinity; (d) all taurine-conjugated bile acids (mono, di-, and trihydroxylated) are competitive inhibitors of TCA uptake with the exception of those lacking a hydroxyl group in the C7 position (taurolithocholic acid (TLCA), and TDCA); and (e) unconjugated and conjugated trihydroxy bile acids are mutual competitive inhibitors.…”

Characterization of the transport of a synthetic bile salt, iodinated cholyl-glycyl-tyrosine, in isolated cultured rat hepatocytes

“…TCA is considered a good probe for transport through the sodium-dependent bile salt transporter (21, whereas indocyanine green is believed to be an equally good probe for sodium-independent organic anion transport (5). However, research based on classical kinetic methodologies yields data that are difficult to interpret because some overlap may exist among different compounds used as markers of transport (20,23). The data are so complicated that it has been suggested there are actually families of carriers, each with varying affinities for different ligands (9).…”