RESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácidos e Sais Biliares/metabolismo , Colagogos e Coleréticos/antagonistas & inibidores , Colestase/induzido quimicamente , Ácido Taurolitocólico/efeitos adversos , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Actinas/metabolismo , Animais , Transporte Biológico , Colagogos e Coleréticos/farmacocinética , Colestase/metabolismo , Masculino , Ratos , Ratos Wistar , Ácido Taurolitocólico/farmacocinéticaRESUMO
In this study we analyzed the effect resulting from a short-term (1 h) bile duct obstruction in bile acid-depleted or taurocholate-replenished rats on liver cytochrome P450 enzyme system activity. Rats were depleted of endogenous bile acids and then subjected to a biliary obstruction for 1 h. Some of these depleted-obstructed rats were replenished previously to the obstruction with exogenous taurocholic acid (TC) and the others were treated with the solvent alone. To study the isolated effect of the bile acid, other rats were also previously depleted and then replenished with TC but they were obstructed briefly (20 min). CYP3A2-linked activity was evaluated in vivo with the aminopyrine breath test and in vitro by the measurement of nifedipine oxidase microsomal activity. The results suggested that bile flow suppression per se might inhibit this CYP-linked activity and that bile acid retention is not involved at least as a sole determinant.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase Extra-Hepática/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Fígado/metabolismo , Aminopirina , Animais , Ácidos e Sais Biliares/análise , Ductos Biliares/cirurgia , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/metabolismo , Ligadura , Masculino , Oxigenases de Função Mista/análise , Ratos , Ratos Wistar , Esteroide Hidroxilases/metabolismo , Fatores de TempoRESUMO
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.
Assuntos
Colestase/induzido quimicamente , Colestase/prevenção & controle , Congêneres do Estradiol , Etinilestradiol , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Silimarina/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Fosfatase Alcalina/sangue , Animais , Bile/efeitos dos fármacos , Bile/fisiologia , Ácidos e Sais Biliares/antagonistas & inibidores , Ácidos e Sais Biliares/metabolismo , Membrana Celular/efeitos dos fármacos , Elasticidade , Congêneres do Estradiol/farmacologia , Etinilestradiol/farmacologia , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Ratos , Ratos WistarRESUMO
Adaptive hepatic changes were investigated in rats with mild stenosis of the common bile duct and in sham-operated controls. The studies were performed 24 h and 7-12 days postoperatively. A continuous intravenous infusion of taurocholic acid at stepwise-increasing rates was performed to explore the responses to bile acid effects. During the infusion, bile flow and the outputs of bile acids, phospholipids, cholesterol, alkaline phosphatase and gamma glutamyl transpeptidase were studied. At the end of the infusion, hepatic morphometric measurements were performed. In other experimental sets, biliary excretions of horseradish peroxidase, a marker of microtubule-dependent vesicular transport in the hepatocyte, and sulphobromophthalein, a well-known organic anion model, were studied. In other rats, bile acid pool size and composition were determined by depletion of bile. The results in rats with mild stenosis maintained for 24 h showed a greater susceptibility to the toxicity of taurocholic acid, as revealed by the abrupt decrement in bile flow at high rates of infusion, and increased outputs of phospholipids and canalicular enzymes. Conversely, rats with mild stenosis maintained for 7-12 days showed decreased bile acid maximum secretory rate and biliary outputs of phospholipids and canalicular enzymes, as well as hepatocyte hypertrophy. These findings may explain the limited hepatic and systemic repercussion of experimental mild stenosis of the common bile duct and help us to understand the early stages of constriction of the common bile duct in man.
Assuntos
Adaptação Fisiológica/fisiologia , Colestase Extra-Hepática/patologia , Doenças do Ducto Colédoco/patologia , Fígado/enzimologia , Fosfatase Alcalina/metabolismo , Animais , Ácidos e Sais Biliares/análise , Ácidos e Sais Biliares/metabolismo , Colestase Extra-Hepática/metabolismo , Doenças do Ducto Colédoco/metabolismo , Constrição Patológica , Peroxidase do Rábano Silvestre/farmacocinética , Concentração de Íons de Hidrogênio , Injeções Intravenosas , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Sulfobromoftaleína/farmacocinética , Ácido Taurocólico/farmacologia , gama-Glutamiltransferase/metabolismoRESUMO
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.
Assuntos
Sistema Biliar/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colestase/induzido quimicamente , Corantes/toxicidade , Animais , Bile/química , Bile/fisiologia , Sistema Biliar/metabolismo , Verde de Bromocresol/administração & dosagem , Verde de Bromocresol/análise , Verde de Bromocresol/toxicidade , Colestase/metabolismo , Corantes/administração & dosagem , Injeções Intravenosas , Junções Intercelulares/metabolismo , Testes de Função Hepática , Masculino , Fenolsulfonaftaleína/administração & dosagem , Fenolsulfonaftaleína/análise , Fenolsulfonaftaleína/toxicidade , Ratos , Ratos Wistar , Rosa Bengala/administração & dosagem , Rosa Bengala/análise , Rosa Bengala/toxicidade , Sacarose/metabolismo , Sulfobromoftaleína/administração & dosagem , Sulfobromoftaleína/análise , Sulfobromoftaleína/toxicidade , Fatores de TempoRESUMO
Several techniques for developing incomplete obstruction of the common bile duct have been described but none of them properly represents a compression or constriction of the bile duct. In this study, a mild stenosis of the common bile duct was achieved in the rat by means of a double ligature including a cannula that could be easily slipped out of the ligatures. Sham-operated rats were used as controls. The studies, performed 7-10 days postoperatively, indicated that in ligated rats a duct constriction was produced, made evident by an increase of the biliary pressure, an upstream dilatation of the bile duct, an increase of the liver volume constituted by portal tracts, and ductular proliferation. Serum parameters were practically similar in ligated and control rats, except for a slight increase in serum bilirubin. Following intravenous injection of sodium taurocholate there were rapid increases of bile flow and bile salt output in both groups, but choleresis induced by sodium taurocholate was higher in ligated rats than in controls. The clearances of [14C]erythritol and [14C]sucrose suggested that ductular water contributing to bile flow and changes in biliary permeability were not involved in ligated rats. The limited repercussion of humoral effects and hepatic behaviour seen in ligated rats despite the morphological alterations induced make the mild stenosis of the bile duct a good model for the study of early stages of compression or constriction of the biliary tract.
Assuntos
Doenças do Ducto Colédoco/etiologia , Animais , Bile/fisiologia , Ácidos e Sais Biliares/metabolismo , Bilirrubina/sangue , Colagogos e Coleréticos/farmacologia , Doenças do Ducto Colédoco/patologia , Doenças do Ducto Colédoco/fisiopatologia , Constrição Patológica , Modelos Animais de Doenças , Ligadura , Masculino , Pressão , Ratos , Ratos Wistar , Sulfobromoftaleína/farmacologia , Ácido Taurocólico/farmacologia , Fatores de TempoRESUMO
Microsomal membranes from rat small intestine exhibit a higher cholesterol/phospholipid ratio and a lower phosphatidyl-choline/sphingomyelin ratio than those of the liver, which could negatively influence membrane-bound enzymes like bilirubin UDP-glucuronosyltransferase. To study the effect of in vitro modifications in the lipid composition of intestinal microsomes on bilirubin glucuronidating activity, two strategies were employed. On one hand, microsomal lipids were modified in order to mimic those of the liver tissue; on the other hand, cholesterol content of microsomal membranes was increased or decreased with respect to the normal value. Lipid changes were carried out by both an enzyme-mediated and a detergent-mediated procedure. Irrespective of the methodology employed, when a depletion in the cholesterol content was produced, enzyme activity increased about 40%, and when lipid composition approached that of the liver tissue, which not only decreased cholesterol but also modified phospholipid classes, enzyme activity increased about 80%. Both lipid modifications produced a 'fluidification' of microsomal membranes measured by fluorescence anisotropy of 1,6-diphenylhexatriene, being the effect of the approach to the liver higher than that of the decrease of cholesterol. In turn, the enrichment in cholesterol of microsomal membranes led to a decrease of enzyme activity of about 20% and to a 'rigidization' of the membranes. The present findings suggest that in rat intestine, bilirubin glucuronidation is strongly influenced by microsomal lipids. In particular, there seems to be an inverse association between enzyme activity and the cholesterol content of membrane.
Assuntos
Glucuronosiltransferase/metabolismo , Mucosa Intestinal/metabolismo , Lipídeos/análise , Microssomos/metabolismo , Animais , Ativação Enzimática , Intestinos/ultraestrutura , Masculino , Microssomos Hepáticos/metabolismo , Ratos , Ratos WistarRESUMO
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.
Assuntos
Corantes/farmacologia , Ducto Colédoco/efeitos dos fármacos , Fígado/efeitos dos fármacos , Sulfobromoftaleína/farmacologia , Junções Íntimas/efeitos dos fármacos , Animais , Bile/metabolismo , Ducto Colédoco/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Infusões Intravenosas , Fígado/metabolismo , Masculino , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Wistar , Sacarose/metabolismoRESUMO
The effect of oral administration of the bile acid ursodeoxycholic acid on rat hepatic and intestinal microsomal UDP-glucuronosyltransferase was studied. The bile acid was administered during 8 days at a daily dose of 500 mg/kg body weight. Enzyme activity was assessed in native and activated microsomes, using bilirubin and p-nitrophenol as substrates. Activation was achieved either by including UDP-N-acetylglucosamine in the incubation mixture or by preincubating native microsomes with an optimal concentration of Lubrol Px. Irrespective of activation status of the microsomes, ursodeoxycholic acid treatment increased enzyme activities toward both substrates in intestine, but not in liver. The analysis of the degree of activation by Lubrol Px revealed that, at least for bilirubin, ursodeoxycholic acid decreased the latency of the intestinal enzyme. The analysis of the lipid composition of microsomes showed several changes in response to ursodeoxycholic acid in intestine but not in liver. Thus, a decrease in cholesterol/phospholipid ratio and an increase in the unsaturation index of total-lipid fatty acids, which correlated well with a membrane "fluidification," were observed. These modifications appear to be related to the lower latency of bilirubin UDP-glucuronosyltransferase in intestine from treated rats and could be responsible, at least in part, for the improvement of enzyme activity in this group. Whatever the mechanism involved, the increment of intestinal UDP-glucuronosyltransferase activities toward both substrates may be relevant as a complement to the hepatic enzymes in those liver diseases in which ursodeoxycholic acid is used as a therapeutic agent.
Assuntos
Glucuronosiltransferase/metabolismo , Intestinos/enzimologia , Fígado/enzimologia , Ácido Ursodesoxicólico/farmacologia , Animais , Bile/efeitos dos fármacos , Bilirrubina/metabolismo , Colagogos e Coleréticos/farmacologia , Ácidos Graxos/metabolismo , Imunoensaio de Fluorescência por Polarização , Técnicas In Vitro , Intestinos/efeitos dos fármacos , Metabolismo dos Lipídeos , Fígado/efeitos dos fármacos , Masculino , Fluidez de Membrana/efeitos dos fármacos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Nitrofenóis/metabolismo , Ratos , Ratos WistarRESUMO
The hepatic transport of organic anions was evaluated in taurolithocholate-induced cholestasis in rats. Taurolithocholate (3 mumol per 100 g body wt., i.v.) diminished bile flow by 61%, whereas biliary excretion of bile salts was normalized after 80 min. Tm studies of sulfobromophthalein revealed reduced biliary excretion (-58%) and increased hepatic content of the dye (+75%). Conjugation pattern in bile showed that free sulfobromophthalein was increased by 57%, suggesting that hepatic conjugation was also impaired. This finding, however, could not fully explain the reduced sulfobromophthalein excretion since Tm of its non-metabolizable analog phenol-3,6-dibromophthalein was also decreased (-41%). Compartmental analysis of plasma decay of both dyes revealed that, whereas hepatic uptake was unaltered, canalicular excretion was reduced and reflux from the liver into plasma was increased by the cholestatic agent. Studies on transport of phenol-3,6-dibromophthalein by isolated hepatocytes showed that while uptake was unaffected, the treatment reduced (-36%) the release from hepatocytes preloaded with the dye. Neither glutathione S-transferase activity nor binding of sulfobromophthalein to cytosolic proteins was altered when evaluated in vitro, suggesting that reduced conjugation and enhanced sinusoidal reflux were not due to an irreversible effect of taurolithocholate on this enzyme. In conclusion, taurolithocholate impairs the hepatic transport of organic anions by impairing canalicular excretion and intrahepatic conjugation, as well as by increasing transfer from the liver into the plasma.
Assuntos
Ânions/farmacocinética , Colestase/induzido quimicamente , Colestase/metabolismo , Fígado/metabolismo , Ácido Taurolitocólico , Animais , Bile/metabolismo , Transporte Biológico , Citosol/metabolismo , Fígado/citologia , Masculino , Proteínas/metabolismo , Ratos , Ratos Wistar , Sulfobromoftaleína/análogos & derivados , Sulfobromoftaleína/farmacocinéticaRESUMO
Ursodeoxycholic acid and its endogenous metabolite tauroursodeoxycholic acid inhibited in vitro the microsomal bilirubin UDP-glucuronosyltransferase from rat liver. The magnitude of the inhibition correlated well with the loss of integrity of microsomal vesicles, suggesting that bile salts needed to reach the lumen to exert their inhibitory effects. The endogenous bile acids cholic acid, chenodeoxycholic acid and deoxycholic acid also exhibited inhibitory effects on bilirubin glucuronidation in digitonin-disrupted microsomes. Ursodeoxycholic acid inhibitory capacity was similar to that of chenodeoxycholic acid and deoxycholic acid but greater than that of cholic acid, the major endogenous bile salt. Kinetic studies, performed in detergent-activated preparations, showed that the inhibitions produced by ursodeoxycholic and tauroursodeoxycholic acids were competitive toward both bilirubin and UDP-glucuronic acid. The estimated Ki(app) for both substrates did not differ statistically between ursodeoxycholic and tauroursodeoxycholic acids. Both bile salts were weak inhibitors toward bilirubin but rather strong inhibitors toward UDP-glucuronic acid.
Assuntos
Glucuronosiltransferase/antagonistas & inibidores , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Ácido Ursodesoxicólico/farmacologia , Animais , Ácidos e Sais Biliares/farmacologia , Digitonina/farmacologia , Ativação Enzimática , Glucuronatos/metabolismo , Glucuronosiltransferase/metabolismo , Cinética , Masculino , Membranas/efeitos dos fármacos , Membranas/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Ratos , Ratos Wistar , Ácido Tauroquenodesoxicólico/farmacologiaRESUMO
Taurolithocholate (TLC), a natural bile salt, induces selective impairment on canalicular membrane of the hepatocyte, which seems to be a major determinant of its cholestatic effect in experimental animals. In order to extend existing studies about the effects of TLC on bile secretion, we examined in TLC-treated rats the biliary excretion of compounds that are transported to canalicular membrane via vesicles, such as lipids and proteins. The single intravenous injection of TLC (3 mumol/100 g body wt.) inhibited transiently the biliary bile salt excretion, while the biliary excretion of lipids (i.e., cholesterol and phospholipids) and proteins remained inhibited even though the biliary excretion and composition of bile salts were normalized. Under such a condition, TLC also inhibited the transcellular vesicular pathway to the exogenous protein horseradish peroxidase entry into bile, without altering the paracellular biliary access of the protein. The hepatic uptake of horseradish peroxidase was unaffected by TLC-treatment. The results indicate that TLC can inhibit the biliary excretion of compounds that reach the canaliculus via a vesicular pathway, such as lipids and proteins, by a mechanism not related to a defective bile salt excretion. Possible explanations for these findings are discussed.
Assuntos
Bile/metabolismo , Metabolismo dos Lipídeos , Proteínas/metabolismo , Ácido Taurolitocólico/farmacologia , Animais , Bile/efeitos dos fármacos , Peroxidase do Rábano Silvestre/metabolismo , Masculino , Ratos , Ratos EndogâmicosRESUMO
Polyethylene glycol molecular weight 900 (PEG-900) has been used as a marker of vectorial water transport into bile canaliculus. However, the mechanisms by which this compound is excreted have not been clarified. To gain more information on this process, we studied the biliary excretion of [3H]PEG-900 in rats during choleresis induced by canalicular choleretics. In addition, the effects of the microtubule inhibitors colchicine and vinblastine, and of the acidotropic agent chloroquine, on PEG-900 excretion were studied to determine whether a vesicular pathway is involved. Continuous i.v. infusion of either dehydrocholate (DHC, a non-micelle forming bile salt choleretic) or 4-methylumbelliferone (4-MU, a non-bile salt canalicular choleretic) at stepwise-increasing rates [0.7, 1.0 and 1.2 mumol.min-1.(100 g body wt)-1] induced a gradual increment in bile flow, whereas a transient increment of [3H]PEG-900 excretion was observed only during DHC-induced choleresis. Furthermore, studies in which two consecutive i.v. injections of DHC (10 mumol/100 g body wt) were administered showed that [3H]PEG-900 excretion induced by a second administration of DHC was 54% lower than that induced by the first one, despite a similar excretion in bile flow. Finally, colchicine (0.5 mumol/100 g body wt), vinblastine (0.5 mumol/100 g body wt) and chloroquine (50 mg/kg body wt) pretreatments inhibited the DHC-induced increment in biliary [3H]PEG-900 output, while DHC-induced choleresis was almost unaffected. Conversely, excretion of [14C]sucrose, when coadministered with [3H]PEG-900, was not impaired by the treatments. These results suggest that, unlike sucrose, PEG-900 excretion is not associated with canalicular water movements. Instead, it may be related to a vesicular transport process followed by a bile acid-stimulated discharge of secretory vesicles into bile through the lysosomal compartment.
Assuntos
Ácidos e Sais Biliares/metabolismo , Bile/metabolismo , Fígado/metabolismo , Polietilenoglicóis/farmacocinética , Animais , Canalículos Biliares/metabolismo , Transporte Biológico , Cloroquina/farmacologia , Colchicina/farmacologia , Ácido Desidrocólico/antagonistas & inibidores , Ácido Desidrocólico/farmacologia , Masculino , Peso Molecular , Polietilenoglicóis/metabolismo , Ratos , Ratos Endogâmicos , Sacarose/metabolismo , Umbeliferonas/farmacologia , Vimblastina/farmacologiaRESUMO
Conjugation has been considered the rate-limiting step for bilirubin hepatic transport, and bypass of this metabolic step could explain why photobilirubins can be rapidly cleared by the liver. In this paper we assessed whether photoirradiation may enhance the bilirubin overall hepatic transport in the isolated perfused Wistar rat liver, a model possessing intact transport and conjugating systems. Bilirubin was administered as a bolus so as to reach a perfusate concentration of approximately 10 microM (bilirubin/albumin molar ratio 1:17). Perfusate light exposure (0.56.10(15) quanta s-1 cm-2) yielded 7-10% of configurational photoisomers, which were further identified as (4Z,15E/4E,15Z)-bilirubin IX alpha. Under such conditions, the perfusate removal rate was increased by 39% over that from dark conditions. Likewise, biliary excretion, estimated as total bilirubin recovery at 60 min, was also increased (+48%). This later improvement was mainly produced at the expense of unconjugated bilirubin, which most likely derived from its configurational photoisomers that, once excreted into bile, readily re-isomerized to the parent compound. In addition, this increment was partially due to a delayed improvement of monoglucuronide pigment excretion. The calculated hepatic pigment content was significantly higher under light conditions. A direct assessment of hepatic content of different bilirubin moieties at 20 min after bilirubin administration confirmed that such an increment was fully accounted for by unconjugated pigment. Our finding that hepatic pigment content rose (despite a higher biliary excretion) when the bilirubin was irradiated suggests a higher net uptake of photoisomers than native pigment. This observation, and the finding that bilirubin photoisomers were usually excreted without undergoing conjugation even if the metabolic system is active, contribute to explain the greater appearance of unconjugated bilirubin in Wistar rat bile under light exposure.
Assuntos
Bilirrubina/metabolismo , Fígado/metabolismo , Animais , Bilirrubina/efeitos da radiação , Bilirrubina/urina , Transporte Biológico , Cinética , Luz , Masculino , Taxa de Depuração Metabólica , Perfusão , Ratos , Ratos EndogâmicosAssuntos
Bilirrubina/metabolismo , Glucuronosiltransferase/biossíntese , Microssomos Hepáticos/efeitos dos fármacos , Fenobarbital/farmacologia , Espironolactona/farmacologia , Animais , Digitonina/farmacologia , Indução Enzimática , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Córtex Renal/efeitos dos fármacos , Córtex Renal/enzimologia , Masculino , Microssomos Hepáticos/enzimologia , Ratos , Ratos EndogâmicosRESUMO
The main factors involved in the impairment of formation of the bile salt-independent bile flow (BSIF) in streptozotocin (SZ)-treated rats were examined. Twenty-four hours after SZ injection (50 mg/kg body wt, i.v.) bile flow, bile salt output and biliary excretion of the major inorganic electrolytes (sodium, chloride and bicarbonate) were significantly diminished. The relationship between bile flow and bile salt output obtained during the administration of sodium taurocholate at stepwise-increasing rates indicated that bile salt-independent bile flow (y-intercept) was diminished by 37% in SZ-treated rats. The relationship between electrolyte output and bile salt output showed that the fractions of sodium, chloride and bicarbonate excreted independently of bile salt (y-intercept) decreased to 59%, 47% and 67% of the control values respectively, while the amount of electrolyte secreted per unit of bile salt secreted was unaffected in SZ-treated rats. The hepatic activity of Na+,K(+)-adenosine triphosphatase (Na+,K(+)-ATPase) was decreased by 59% (P less than 0.05) in SZ-treated rats. Nicotinamide administered prior to SZ prevented the hyperglycemia indicative of SZ-induced diabetes, but had no effect on the decrease in Na+,K(+)-ATPase activity caused by the drug. These results suggest that SZ itself, and not its diabetogenic effect, decreases the BSIF by a mechanism that involves impairment of the biliary electrolyte excretion, which could be the result of the inhibition of the hepatic Na+,K(+)-ATPase activity.
Assuntos
Bile/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas , Eletrólitos/metabolismo , Estreptozocina/farmacologia , Animais , Bile/metabolismo , Ácidos e Sais Biliares/fisiologia , Fígado/enzimologia , Hepatopatias/metabolismo , Hepatopatias/fisiopatologia , Masculino , Ratos , Ratos Endogâmicos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidoresRESUMO
Choleresis induced by dehydrocholate (DHC) stimulates the discharge into bile of lysosomes, which are implicated in the biliary excretion of proteins. Contrary to taurocholate-induced choleresis, DHC choleresis is not affected by microtubule (mt) inhibition. Therefore, the role of mt's in the biliary protein excretion during bile salt choleresis was analyzed in this study. Normal rats and rats treated with the mt poisons colchicine or vinblastine or with the acidotropic agent chloroquine (Cq) were used. The analysis of the protein component in bile was made on SDS-polyacrylamide gel, and the individual polypeptides were quantitated by densitometry. The excretion of bile polypeptides were compared with that of lysosomal acid phosphatase. Bile flow and bile salt output did not show changes on account of treatments. The biliary excretion of acid phosphatase was stimulated by DHC, and it was not affected by mt inhibitors but was markedly diminished by Cq. DHC choleresis produced different effects on the bile polypeptides. The biliary excretion of polypeptide of high molecular mass (84-140 kDa) was stimulated by DHC. Cq treatment increased their basal biliary excretions, whereas DHC-induced secretion was qualitatively and quantitatively similar to that of controls. The 69-kDa polypeptide (albumin) also increased during DHC-induced choleresis, but it showed a different excretory pattern. Cq treatment inhibited such an increase but no correlation with the excretory pattern of the lysosomal marker was found. The biliary excretion of polypeptides of low molecular mass (down to 14 kDa) suffered a transitory decrease and then a subsequent increase over basal values during the DHC choleresis.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bile/metabolismo , Ácido Desidrocólico/farmacologia , Fígado/metabolismo , Proteínas/metabolismo , Fosfatase Ácida/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Colchicina/farmacologia , Eletroforese em Gel de Poliacrilamida/métodos , Fígado/efeitos dos fármacos , Masculino , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Ratos , Ratos Endogâmicos , Dodecilsulfato de Sódio , Vimblastina/farmacologiaAssuntos
Glucuronosiltransferase/biossíntese , Microssomos Hepáticos/enzimologia , Fenobarbital/farmacologia , Espironolactona/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Sinergismo Farmacológico , Indução Enzimática/efeitos dos fármacos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos EndogâmicosRESUMO
The biliary protein excretion during sodium taurocholate induced choleresis was studied in normal rats and in rats treated with the lysosomotropic agent, chloroquine. The analysis of the protein component in bile was made on SDS-polyacrilamide gel, and the individual polypeptides were quantitated by densitometry. The excretion of bile polypeptides was compared with that of lysosomal acid phosphatase. The biliary excretion of polypeptides of molecular mass lower than and equal to 54 kDa was markedly stimulated by taurocholate-induced choleresis. Chloroquine treatment of rats diminished the biliary excretion of such polypeptides and also inhibited their excretion induced by taurocholate. The behaviour of these polypeptides was well correlated to that of the lysosomal marker. The biliary excretion of polypeptide bands of a higher molecular mass (up to 140 kDa) did not show major changes during taurocholate-induced choleresis in any of the groups. The results indicate that biliary excretion of proteins in the rat may be either stimulated by taurocholate or may be independent of the bile salt. The former requires the functional integrity of chloroquine-sensitive hepatocyte compartments, which may involve the lysosomes.
Assuntos
Bile/metabolismo , Cloroquina/farmacologia , Proteínas/metabolismo , Ácido Taurocólico/farmacologia , Fosfatase Ácida/metabolismo , Animais , Bile/efeitos dos fármacos , Ácidos e Sais Biliares/metabolismo , Eletroforese em Gel de Poliacrilamida , Fígado/enzimologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
The effect of streptozotocin (SZ) administration on sodium [14C]taurocholate (TC) transmural transfer was studied in the everted rat ileum. The excretion of fecal bile acids was also studied in living rats injected with that compound. The viability of the preparation used for the in vitro experiments was evaluated by light microscopy and by the rate of glucose uptake by tissue from the mucosal fluid. The results obtained showed that TC transfer to the serosal fluid was impaired after 24 h of SZ injection, as well as the active transport observed in control preparations. The amount of TC accumulated in the intestinal tissue was also diminished. In addition, total ATPase activity of tissue was decreased, and intracellular electrolyte concentration was altered. Therefore, a slower saturation of binding sites could be responsible for the effects of SZ on TC tissue accumulation, and a decreased ATPase activity for the impairment of the TC concentrative transport system. The results observed in vitro were supported by data in vivo because fecal bile acid excretion was significantly diminished in SZ-treated rats.