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

Abstract

The uptake of tri-hydroxy conjugated bile salts by hepatocytes is principally by a sodium-dependent carrier. We examined the uptake kinetics of the high-specific-activity, hydroxylated, conjugated bile salt 125I-labeled cholyl-glycyl-tyrosine, to determine whether this synthetic bile salt was transported by the sodium-dependent bile salt system. 125I-labeled cholyl-glycyl-tyrosine was synthesized, and its transport kinetics were studied in freshly cultured rat hepatocytes. Uptake into hepatocytes was time and temperature dependent and was decreased by the inhibitors diisothiocyanodisulfonic acid stilbene, probenecid and carbonyl cyanide chlorophenyl hydrazone, demonstrating carrier mediation and energy dependence. At concentrations of iodinated cholyl-glycyl-tyrosine less than 10 mumol/L, uptake was 27% +/- 5% sodium dependent, whereas at concentrations from 10 mumol/L to 40 mumol/L uptake was 52% +/- 4% sodium dependent. The apparent affinity for uptake of 125I-labeled cholyl-glycyl-tyrosine was 8 +/- 2 mumol/L, and the maximal velocity was 50 +/- 20 pmol/micrograms DNA/min. Both taurocholate and indocyanine green inhibited uptake of 125I-labeled cholyl-glycyl-tyrosine. Indocyanine green inhibited the uptake of 125I-labeled cholyl-glycyl-tyrosine (Ki = 10 microns) more effectively than taurocholate (Ki = 20 microns). We conclude that 125I-labeled cholyl-glycyl-tyrosine is not a specific probe for either sodium-dependent bile salt or sodium-independent organic anion carriers, but appears to use both systems in a concentration-dependent manner in cultured rat hepatocytes.

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

“…2.1.4). In cultured hepatocytes uptake of 125I-labeled CGTyr was also by sodium-independent transport, which was even the major portion of total uptake on low substrate concentrations (Deutsch et al 1992). Uptake was inhibited to 50% by the uncoupler carbonylcyano chlorophenylhydrazone (CCCP).…”

“…Further inhibitors were probenecid and ICG in this cell model. The Km of total CGTyr uptake in he~atocyte cultures was only 8 _+ 2 gM, and the Vmax was 50 + 20 pmol mg -1 min- (Deutsch et al 1992). The low Vmax value is not surprising since in cultured hepatocytes bile acid uptake declines markedly.…”

“…Transport studies with 125I-labeled cholylglycyltyrosine (CGTyr) have been performed in intact rats (Mills et al 1986), intact rats and isolated rat hepatocytes (Suchy et al 1983), and isolated cultured rat hepatocytes (Deutsch et al 1992). The biliary elimination data indicate that this bile acid is handled very similarly to other conjugated bile acids.…”

“…However, the data from this study, particularly the proportion of sodium-dependent transport compared with the proportion of sodium-independent transport suggested that the iodinated CGTyr also has affinity to organic anion carriers which are not identical with the TCA transporter. Based on the relative abilities of CGTyr and iodinated CGTyr to inhibit uptake of 125I-labeled CGTyr the authors concluded that the addition of an iodine in the CGTyr molecule is responsible for shifting uptake toward the sodium-independent organic anion pathway (Deutsch et al 1992).…”