Synthesis and applicability of a photolabile 7,7-azi analogue of 3-sulfated taurine-conjugated bile salts.

For the identification of proteins involved in hepatobiliary transport, the photolabile derivative 7,7-ASLCT ((7,7-azi-3 alpha-sulfato-5 beta-cholan-24-oyl)-2'-aminoethanesulfonate) was synthesized. 7,7-ASLCT is taken up into liver and excreted into bile completely unmetabolized at a rate between the excretion rate of SLCT ((3 alpha-sulfato-5 beta-cholan-24-oyl)-2'- aminoethanesulfonate) and SCCT ((7 alpha-hydroxy-3 alpha-sulfato-5 beta- cholan-24-oyl)-2'-aminoethanesulfonate). The dependency of flux rate of uptake into freshly isolated hepatocytes on the concentration of 7,7-ASLCT in presence of Na+ (143 mM) and with Na+ depletion (1 mM) is best described by the assumption of two simple transport systems, the kinetic parameters of which are similar to those of SLCT. As studied in the presence of Na+, 7,7-ASLCT and SLCT exhibit a clearly competitive cross-inhibition of uptake with inhibition constants that are similar to the corresponding half-saturation constants. Photoaffinity labeling of isolated hepatocytes using 7,7-ASLCT (400 microM) resulted in the irreversible inhibition of the uptake of 7,7-ASLCT and SLCT to similar extents, confirming the kinetic data that 7,7-ASLCT is a true competing substrate for the uptake of SLCT. Because in intact rat liver 7,7-ASLCT and SLCT mutually inhibit their biliary excretion, the photolabile derivative shares with SLCT the same pathways in uptake and in excretion. Thus, 7,7-ASLCT should be appropriate for the study of hepatobiliary transport of sulfated and taurine-conjugated bile salts by photoaffinity labeling.