RESUMO
AIMS: The aim of this study was to investigate the transport of two kinds of bile acids by organic anion transporting polypeptide 1B3 (OATP1B3) using first-trimester trophoblasts. The mechanisms of damage to fetuses with intrahepatic cholestasis of pregnancy were investigated, providing new potential strategies for targeted therapies aimed at reducing fetal risk. MATERIAL AND METHODS: The expression of OATP1B3 was knocked down by lentiviral vector-mediated RNA interference, and silencing efficiency was assessed using real-time polymerase chain reaction and Western blotting. The cytotoxicity of two bile acids (glycocholic acid [GCA] and glycochenodeoxycholic acid [GCDCA]) was assessed using the MTT method. Transport of bile acids was assessed by establishing an in vitro trophoblast monolayer model using polyester Transwell-clear inserts, and the concentration of bile acids in the upper compartment was assessed using high-pressure liquid chromatography. RESULTS: GCA and GCDCA (10 and 20 µM) were not cytotoxic to the SWAN cell line (P > 0.05). RNAi treatment decreased the mRNA and protein expressions of OATP1B3 by 94.42% and 49.51%, respectively (P < 0.05). The bile acid transport curves were similar in the control and negative RNAi groups, whereas those in the RNAi group differed significantly from those in the control and negative RNAi groups. The concentration of GCA and GCDCA in the upper compartment was significantly lower in the RNAi group than in the control and negative RNAi groups. CONCLUSIONS: OATP1B3 expression in trophoblasts was confirmed indirectly by its ability to transport the bile acids GCA and GCDCA.
Assuntos
Ácido Glicoquenodesoxicólico/farmacocinética , Ácido Glicocólico/farmacocinética , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Trofoblastos/metabolismo , Transporte Biológico , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colestase Intra-Hepática/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Ácido Glicoquenodesoxicólico/farmacologia , Ácido Glicocólico/farmacologia , Humanos , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Gravidez , Complicações na Gravidez/metabolismo , Primeiro Trimestre da Gravidez , RNA Mensageiro/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
Cooperative ligand binding to human ileal bile acid binding protein (I-BABP) was studied using the stopped-flow fluorescence technique. The kinetic data obtained for wild-type protein are in agreement with a four-step mechanism where after a fast conformational change on the millisecond time scale, the ligands bind in a sequential manner, followed by another, slow conformational change on the time scale of seconds. This last step is more pronounced in the case of glycocholate (GCA), the bile salt that binds with high positive cooperativity and is absent in mutant I-BABP proteins that lack positive cooperativity in their bile salt binding. These results suggest that positive cooperativity in human I-BABP is related to a slow conformational change of the protein, which occurs after the second binding step. Analogous to that in the intestinal fatty acid binding protein (I-FABP), we hypothesize that ligand binding in I-BABP is linked to a disorder-order transition between an open and a closed form of the protein.
Assuntos
Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/farmacocinética , Hidroxiesteroide Desidrogenases/química , Hidroxiesteroide Desidrogenases/farmacocinética , Íleo/química , Íleo/metabolismo , Sítios de Ligação/genética , Ácido Glicoquenodesoxicólico/química , Ácido Glicoquenodesoxicólico/farmacocinética , Ácido Glicocólico/química , Ácido Glicocólico/farmacocinética , Humanos , Hidroxiesteroide Desidrogenases/genética , Ligantes , Modelos Biológicos , Modelos Estatísticos , Ligação Proteica/genética , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
The isolated perfused rat liver was used to examine the hepatic extraction, biliary secretion and effect on bile flow of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid. The naturally occurring taurine and glycine conjugates of these bile acids were used for comparisons. The 2-fluoro-beta-alanine conjugates were extracted by the liver to a similar extent as the taurine and glycine conjugates. The biliary secretion rate and increase in bile flow were similar for all the cholic acid conjugates. On the other hand, the maximal biliary secretion rate of the 2-fluoro-beta-alanine conjugate of chenodeoxycholate was similar to that of the glycochenodeoxycholate, but 47% lower than that of taurochenodeoxycholate. In addition, the 2-fluoro-beta-alanine conjugate of chenodeoxycholate produced a decrease in bile flow that was comparable to that observed with the glycochenodeoxycholate (54% vs. 74%), but which was greater than that produced by the taurochenodeoxycholate (12%). In summary, these data demonstrate that the biological properties of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid are not markedly different from those of the naturally occurring taurine and glycine conjugates. These data also suggest that the amino acid moiety can influence the biliary secretion and cholestatic properties of chenodeoxycholic acid conjugates.
