RESUMEN
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.
Asunto(s)
Marcadores de Afinidad/metabolismo , Compuestos Azo/farmacocinética , Hígado/metabolismo , Ácido Taurocólico/metabolismo , Ácido Taurolitocólico/análogos & derivados , Animales , Compuestos Azo/síntesis química , Unión Competitiva , Transporte Biológico , Hígado/citología , Masculino , Fotólisis , Ratas , Ratas Wistar , Ácido Taurolitocólico/síntesis química , Ácido Taurolitocólico/metabolismo , Ácido Taurolitocólico/farmacocinéticaRESUMEN
To identify the cytosolic proteins of rat hepatocytes involved in transcellular transport of sulfated and taurine-conjugated bile salts in comparison with only taurine-conjugated bile salts, photoaffinity labeling studies were performed with [3H]-7,7-ASLCT (7,7-azi-3 alpha-sulfatolithocholyl[2'-3H(N)]taurine), [3H]-7,7-ACT ((7,7-azi-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)- 2'-[2'-3H(N)]aminoethanesulfonate), and [3H]-7,7-ALCT (7,7-azilithocholyl[2'-3H(N)]taurine) using isolated hepatocytes and intact liver tissue. Photoaffinity labeling of isolated hepatocytes with [3H]-7,7-ASLCT on the one hand and with [3H]-7,7-ACT and [3H]-7,7-ALCT on the other resulted in a different labeling pattern of cytosolic polypeptides, without a relevant incorporation of radioactivity into subunits of glutathione transferases. This suggests that glutathione transferases play no role in the transport of dianionic or of monoanionic bile salts. With [3H]-7,7-ACT and [3H]-7,7-ALCT a moderate incorporation of radioactivity was found in polypeptides with apparent M(r)s of 33,000, 38,000, and 54,000, whereas with [3H]-7,7-ASLCT, a polypeptide with an apparent M(r) of 14,000, identified as H-FABP, was markedly and almost exclusively labeled. Photoaffinity labeling of specimens of intact liver tissue resulted in a labeling pattern of cytosolic polypeptides comparable to that obtained from photolabeled isolated hepatocytes. All results suggest that transcellular transport of dianionic sulfated as well as taurine-conjugated bile salts and of monoanionic taurine-conjugated bile salts follows different pathways. In intracellular transport of taurine-conjugated bile salts, several cytosolic polypeptides may have a function, whereas, in transport of taurine-conjugated 3 alpha-sulfato bile salts, only H-FABP appears to be involved.