RESUMEN
Bile formation is an osmotic process driven by the vectorial transport of actively transferred biliary components across the basolateral (sinusoidal) and apical (canalicular) hepatocyte membranes, the latter being the rate-limiting step of the overall blood-to-bile transfer. The ATP-binding cassette (ABC) superfamily of membrane transporters comprises novel ATP-dependent carriers that mediate canalicular transfer of several endogenous and exogenous substrates, and therefore play a key role in bile formation. Gene expression, as well as the balance between vesicular targeting and internalization of these transporters to/from the canalicular membrane are highly regulated processes. This balance is affected in several models of hepatocellular cholestasis, and these alterations may either initiate or perpetuate the cholestatic manifestations. This review describes the regulation of the normal activity of hepatocellular ABC transporters, focusing on the involvement of transcription factors and signaling pathways in the regulation of carrier synthesis, dynamic localization and phosphorylation status. Its alteration in different experimental models of cholestasis, such as those induced by estrogens, lipopolysaccharide (endotoxin), monohydroxylated bile salts and oxidative stress, is also reviewed. Finally, several experimental therapeutic approaches based upon the administration of compounds known/thought to induce carrier synthesis (e.g., protein synthesis inducers), to counteract etiological factors responsible for the cholestatic disease (e.g., corticoids in lipopolysaccharide-induced cholestasis) or to stimulate exocytic insertion of canalicular transporters (e.g., cAMP, silymarin or tauroursodeoxycholate) are described with respect to their ability to prevent cholestatic alterations; the role of signaling molecules as putative downstream mediators of their effects are also discussed.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Colagogos y Coleréticos/uso terapéutico , Colestasis/metabolismo , Colestasis/prevención & control , Hepatocitos/metabolismo , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Animales , Colestasis/etiología , Humanos , Modelos Biológicos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , RatasRESUMEN
BACKGROUND: Taurolithocholate induced cholestasis is a well established model of drug induced cholestasis with potential clinical relevance. This compound impairs bile salt secretion by an as yet unclear mechanism. AIMS: To evaluate which step/s of the hepatocellular bile salt transport are impaired by taurolithocholate, focusing on changes in localisation of the canalicular bile salt transporter, Bsep, as a potential pathomechanism. METHODS: The steps in bile salt hepatic transport were evaluated in rats in vivo by performing pharmacokinetic analysis of (14)C taurocholate plasma disappearance. Bsep transport activity was determined by assessing secretion of (14)C taurocholate and cholyl-lysylfluorescein in vivo and in isolated rat hepatocyte couplets (IRHC), respectively. Localisation of Bsep and F-actin were assessed both in vivo and in IRHC by specific fluorescent staining. RESULTS: In vivo pharmacokinetic studies revealed that taurolithocholate (3 micro mol/100 g body weight) diminished by 58% canalicular excretion and increased by 96% plasma reflux of (14)C taurocholate. Analysis of confocal images showed that taurolithocholate induced internalisation of Bsep into a cytosolic vesicular compartment, without affecting F-actin cytoskeletal organisation. These effects were reproduced in IRHC exposed to taurolithocholate (2.5 micro M). Preadministration of dibutyryl-cAMP, which counteracts taurolithocholate induced impairment in bile salt secretory function in IRHC, restored Bsep localisation in this model. Furthermore, when preadministered in vivo, dibutyryl-cAMP accelerated recovery of both bile flow and bile salt output, and improved by 106% the cumulative output of (14)C taurocholate. CONCLUSIONS: Taurolithocholate impairs bile salt secretion at the canalicular level. Bsep internalisation may be a causal factor which can be prevented by dibutyryl-cAMP.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Ácidos y Sales Biliares/metabolismo , Colagogos y Coleréticos/antagonistas & inhibidores , Colestasis/inducido químicamente , Ácido Taurolitocólico/efectos adversos , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Actinas/metabolismo , Animales , Transporte Biológico , Colagogos y Coleréticos/farmacocinética , Colestasis/metabolismo , Masculino , Ratas , Ratas Wistar , Ácido Taurolitocólico/farmacocinéticaRESUMEN
The effect of silymarin (SIL) on 17alpha-ethynylestradiol (EE)-induced cholestasis was evaluated in rats. EE (5 mg/kg, subcutaneously, daily, for 5 days) decreased both the bile-salt-dependent and the bile-salt-independent fractions of the bile flow. The decrease in the former was associated to a reduction in the bile salt pool size (-58%), and this effect was completely prevented by SIL. This compound also counteracted the inhibitory effect induced by EE on HCO(3)(-) but not glutathione output, 2 major determinants of the bile-salt-independent bile flow. EE decreased the secretory rate maximum (SRM) of tauroursodeoxycholate, (-71%) and bromosulfophthalein (BSP; -60%), as well as the expression of the BSP canalicular carrier, mrp2; SIL failed to increase mrp2 expression, and had only a marginal beneficial effect on both tauroursodeoxycholate and BSP SRM values. However, the two-compartment model-based kinetic constant for BSP canalicular transfer was significantly improved by SIL (+262%). SIL decreased rather than increased CYP3A4, the cytochrome P450 isoenzyme involved in the oxidative metabolism of EE, and had no inhibitory effect on the UDP-glucuronosyltrasferase isoforms involved in the formation of its 17beta-glucuronidated, more cholestatic metabolite. Pretreatment of isolated rat hepatocyte couplets with silibinin, the major, active component of SIL, counteracted the estradiol 17beta-glucuronide-induced decrease in the percentage of couplets secreting apically the fluorescent bile acid analogue, cholyl-lysyl-fluorescein. These results show that SIL protects against EE-induced cholestasis by normalizing mainly the decrease in the bile salt pool size and HCO(3)(-) output, and probably by counteracting the cholestatic effect of its cholestatic, glucuronidated metabolite.
Asunto(s)
Colestasis/inducido químicamente , Colestasis/prevención & control , Congéneres del Estradiol , Etinilestradiol , Proteínas de Transporte de Membrana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Silimarina/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Fosfatasa Alcalina/sangre , Animales , Bilis/efectos de los fármacos , Bilis/fisiología , Ácidos y Sales Biliares/antagonistas & inhibidores , Ácidos y Sales Biliares/metabolismo , Membrana Celular/efectos de los fármacos , Elasticidad , Congéneres del Estradiol/farmacología , Etinilestradiol/farmacología , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Ratas , Ratas WistarRESUMEN
The effect of the hepatoprotector silymarin on bile secretion, with particular regard to bile salt secretion, was studied in Wistar rats. Silymarin (25, 50, 100, and 150 mg/kg/day, i.p., for 5 days) induced a dose-dependent increase in bile flow and bile salt secretion, the maximal effect being reached at a dose of 100 mg/kg/day (+17 and +49%, for bile flow and bile salt output, respectively; P < 0.05). Assessment of bile salt composition in bile revealed that stimulation of the bile salt secretion was accounted for mainly by an increase in the biliary secretion of beta-muricholate and, to a lesser extent, of alpha-muricholate, chenodeoxycholate, ursodeoxycholate, and deoxycholate. The maximum secretory rate (T(m)) of bile salts, as assessed by infusing the non-hepatotoxic bile salt tauroursodeoxycholate i.v. at stepwise-increasing rates, was not influenced by silymarin. The flavonolignan also increased the endogenous bile salt pool size (+53%, P < 0.05) and biliary bile acid excretion after bile acid pool depletion (+54%, P < 0.05), a measure of de novo bile salt synthesis. These results suggest that silymarin increases the biliary excretion and the endogenous pool of bile salts by stimulating the synthesis, among others, of hepatoprotective bile salts, such as beta-muricholate and ursodeoxycholate.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Sistema Biliar/efectos de los fármacos , Sustancias Protectoras/farmacología , Silimarina/farmacología , Análisis de Varianza , Animales , Sistema Biliar/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
Colchicine, a microtubule-disrupting agent, induces hepatotoxicity in experimental animals at the doses commonly employed to explore vesicular transport in the liver. The effect of manipulations of the bile salt pool on colchicine-induced hepatotoxicity was studied in rats to determine the role of bile salts in this phenomenon. Leakage of enzyme markers of liver-cell damage into plasma and bile induced by colchicine pre-treatment displayed a sigmoidal log dose-effect curve, the half-maximal effect being reached at 0.12 micromol per 100 g body wt. Lumicolchicine, instead, showed no harmful effect. Maximal increment of biliary LDH discharge induced by colchicine was reduced from 950 +/- 124% to 216 +/- 29% by bile diversion leading to a marked reduction in bile salt output, and this parameter was further decreased to 100 +/- 13% and 157 +/- 39% by subsequent repletion of the bile salt pool with the hydrophilic bile salts taurodehydrocholate and tauroursodeoxycholate, respectively. Conversely, infusion of taurocholate into non-bile salt depleted, colchicine-treated rats led to cholestasis and massive discharge of enzymes into both blood and bile. Our data show conclusively that colchicine-induced hepatotoxicity depends on the magnitude and composition of the bile salt flux traversing the liver. They also support the view that functional integrity of vesicular mechanisms presumably involved in membrane repair are indispensable to protect the hepatocytes from the damaging effect of bile salts during normal bile formation.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Colchicina/toxicidad , Supresores de la Gota/toxicidad , Hígado/efectos de los fármacos , Fosfatasa Alcalina/sangre , Animales , Ácidos y Sales Biliares/fisiología , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Isomerismo , L-Lactato Deshidrogenasa/sangre , Hígado/patología , Lumicolchicinas/toxicidad , Masculino , Ratas , Ratas Wistar , Ácido Tauroquenodesoxicólico/metabolismo , Ácido Taurocólico/análogos & derivados , Ácido Taurocólico/metabolismoRESUMEN
Changes in hepatic paracellular permeability were investigated during the development of cholephilic dye-induced cholestasis in rats. For this purpose, four dyes with different cholestatic potency (phenol red, sulfobromophthalein, bromcresol green and rose bengal) were infused at a high, potentially damaging dose (280 nmol/min per 100 g body wt., i.v.), and changes in paracellular permeability were continuously monitored by measuring the access into bile of the permeability probe -14C-sucrose. The cholestatic potency of the different dyes was: rose bengal > bromcresol green > sulfobromophthalein > phenol red. All dyes increased [14C]sucrose bile-to-plasma ratio, producing a displacement towards curves of higher permeability. The capability of the dyes to increase biliary permeability followed the same order as their respective cholestatic potencies. The possible implications of the present results for cholephilic dye-induced cholestasis are discussed.
Asunto(s)
Sistema Biliar/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Colestasis/inducido químicamente , Colorantes/toxicidad , Animales , Bilis/química , Bilis/fisiología , Sistema Biliar/metabolismo , Verde de Bromocresol/administración & dosificación , Verde de Bromocresol/análisis , Verde de Bromocresol/toxicidad , Colestasis/metabolismo , Colorantes/administración & dosificación , Inyecciones Intravenosas , Uniones Intercelulares/metabolismo , Pruebas de Función Hepática , Masculino , Fenolsulfonftaleína/administración & dosificación , Fenolsulfonftaleína/análisis , Fenolsulfonftaleína/toxicidad , Ratas , Ratas Wistar , Rosa Bengala/administración & dosificación , Rosa Bengala/análisis , Rosa Bengala/toxicidad , Sacarosa/metabolismo , Sulfobromoftaleína/administración & dosificación , Sulfobromoftaleína/análisis , Sulfobromoftaleína/toxicidad , Factores de TiempoRESUMEN
Changes in biliary permeability during cholephilic dye-induced choleresis, as assessed by measuring the movement into bile of two permeability probes, [14C]sucrose and horseradish peroxidase, were analyzed following an i.v. infusion (60 nmol/min per 100 g body wt) of the model cholephilic organic anion sulfobromophthalein in rats. Dye infusion led to a progressive increase of the [14C]sucrose bile-to-plasma ratio, which reached a maximum value after 100 min of dye infusion (+97%). Paracellular entry of horseradish peroxidase, as evaluated by the early peak of its biliary appearance curve, was also selectively increased (+69%), without changes in the later (transcytotic) access of the protein. Additional dose-response studies of biliary permeability to [14C]sucrose, using sulfobromophthalein and rose bengal, showed that this effect was dose-dependent and rapidly reversed by interruption of dye administration. The influence of hydrophobic/hydrophilic balance on this effect was also studied by infusing four dyes covering a broad range of hydrophobicity (phenol red, bromocresol green, sulfobromophthalein, and rose bengal), so as to attain a similar value of dye hepatic content at the end of the experiment (approximately 150 nmol/g liver wt). Under these conditions, a strong positive correlation was found between the increase in biliary permeability to [14C]sucrose and dye hydrophobicity. These results suggest that cholephilic dyes increase tight junctional permeability in a reversible and dose-dependent manner, and that this effect depends on the hydrophobic/hydrophilic balance of the dye.