ABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Cholagogues and Choleretics/therapeutic use , Cholestasis/metabolism , Cholestasis/prevention & control , Hepatocytes/metabolism , ATP-Binding Cassette Transporters/drug effects , Animals , Cholestasis/etiology , Humans , Models, Biological , Protein Transport/drug effects , Protein Transport/physiology , RatsABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bile Acids and Salts/metabolism , Cholagogues and Choleretics/antagonists & inhibitors , Cholestasis/chemically induced , Taurolithocholic Acid/adverse effects , ATP Binding Cassette Transporter, Subfamily B, Member 11 , ATP-Binding Cassette Transporters/antagonists & inhibitors , Actins/metabolism , Animals , Biological Transport , Cholagogues and Choleretics/pharmacokinetics , Cholestasis/metabolism , Male , Rats , Rats, Wistar , Taurolithocholic Acid/pharmacokineticsABSTRACT
BACKGROUND: Tauroursodeoxycholate (TUDC) provides partial protection against taurolithocholate (TLC) induced cholestasis, possibly by inducing a signalling cascade activating protein kinase C (PKC). The potential protective effects of beta muricholic acid (beta-MC), another 7-beta-hydroxylated bile salt, have not previously been studied in TLC cholestasis. AIMS: To study the effect of beta-MC on TLC induced cholestasis and also to investigate further the effects of agents affecting intracellular signalling, notably DBcAMP (a cell permeable cAMP analogue) and several protein kinase inhibitors. METHODS: Functional studies were carried out analysing the proportion of hepatocyte couplets able to accumulate the fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF) into their sealed canalicular vacuole (cVA of CLF assay). RESULTS: It was found that both beta-MC and DBcAMP were as effective as TUDC in protecting against TLC induced cholestasis. The PKC inhibitors staurosporin and H7 but not the specific protein kinase A (PKA) inhibitor KT5720 abolished the protective effects of TUDC and beta-MC. BAPTA/AM, a chelator of intracellular Ca(2+), significantly decreased the protective effect of both bile salts, and that of DBcAMP. PKC and PKA inhibitors had no effect on protection with DBcAMP. CONCLUSIONS: Beta-MC was as effective as TUDC in protecting against TLC cholestasis. Mobilisation of Ca(2+) and activation of PKC, but not of PKA, are involved in the anticholestatic effect of the two 7-beta-hydroxylated bile salts. The hepatoprotective effects of DBcAMP involved Ca(2+) mobilisation, but not PKC or PKA activation.
Subject(s)
Bucladesine/metabolism , Carbazoles , Cholagogues and Choleretics/therapeutic use , Cholestasis/prevention & control , Cholic Acids/therapeutic use , Egtazic Acid/analogs & derivatives , Signal Transduction , Taurochenodeoxycholic Acid/therapeutic use , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Analysis of Variance , Animals , Calcium/metabolism , Chelating Agents/pharmacology , Cholestasis/chemically induced , Cholestasis/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Egtazic Acid/pharmacology , Enzyme Activation , Enzyme Inhibitors/metabolism , Indoles/pharmacology , Liver/metabolism , Male , Protein Kinase C/antagonists & inhibitors , Pyrroles/pharmacology , Rats , Staurosporine/pharmacology , Taurolithocholic AcidABSTRACT
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.
Subject(s)
Cholestasis/chemically induced , Cholestasis/prevention & control , Estradiol Congeners , Ethinyl Estradiol , Membrane Transport Proteins , Multidrug Resistance-Associated Proteins , Silymarin/pharmacology , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/metabolism , Alkaline Phosphatase/blood , Animals , Bile/drug effects , Bile/physiology , Bile Acids and Salts/antagonists & inhibitors , Bile Acids and Salts/metabolism , Cell Membrane/drug effects , Elasticity , Estradiol Congeners/pharmacology , Ethinyl Estradiol/pharmacology , Hepatocytes/drug effects , Liver/drug effects , Liver/metabolism , Male , Multidrug Resistance-Associated Protein 2 , Rats , Rats, WistarABSTRACT
BACKGROUND/AIMS: S-adenosyl-L-methionine (SAMe) and tauroursodeoxycholate (TUDC) exert an additive ameliorating effect on taurolithocholate (TLC)-induced cholestasis. The aims were to investigate the protective effect of SAMe on 17beta-estradiol-glucuronide (17betaEG) cholestasis and to find out whether SAMe and TUDC may exert an additive, ameliorating effect. METHODS: Hepatocyte couplet function was assessed by canalicular vacuolar accumulation (cVA) of cholyllysylfluorescein (CLF). Cells were co-treated with 17betaEG and SAMe, TUDC, or both (protection study), or treated with 17betaEG and then with SAMe, TUDC or both (reversion study) before CLF uptake. Couplets were also co-treated with SAMe and dehydroepiandrosterone (DHEA), a competitive substrate for the sulfotransferase involved in 17betaEG detoxification. The effects of 17betaEG, SAMe and TUDC were also examined on intracellular distribution of F-actin. RESULTS: Both SAMe and TUDC significantly protected against, and reversed, 17betaEG-induced cholestasis, but their effects were not additive. DHEA abolished the protective effect of SAMe. 17BetaEG did not affect the uptake of CLF into hepatocytes at the concentrations used, and also, it did not affect the intracellular distribution of F-actin. CONCLUSIONS: 17BetaEG does not affect the uptake of CLF into hepatocytes. SAMe and TUDC protect and reverse 17betaEG-induced cholestasis, but without an additive effect. Protection by SAMe may involve facilitating the sulfation of 17betaEG.
Subject(s)
Cholestasis/drug therapy , Cholestasis/prevention & control , S-Adenosylmethionine/pharmacology , Taurochenodeoxycholic Acid/pharmacology , Actins/metabolism , Animals , Biological Transport, Active/drug effects , Cholestasis/chemically induced , Cholestasis/metabolism , Cholic Acids/pharmacokinetics , Dehydroepiandrosterone/pharmacology , Estradiol/analogs & derivatives , Estradiol/toxicity , Fluoresceins/pharmacokinetics , Hepatocytes/drug effects , Hepatocytes/metabolism , In Vitro Techniques , Male , Rats , Rats, Wistar , S-Adenosylmethionine/administration & dosage , Taurochenodeoxycholic Acid/administration & dosageABSTRACT
Hormonal control of the restoration of hepatocanalicular polarity in short-term cultured hepatocyte couplets was analyzed. One hour following isolation, couplets were unable to accumulate the fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), and showed a nonpolarized distribution of F-actin and mrp2 over the cell body. A progressive, time-dependent restoration of couplet-polarized function and morphology was reached after 4 hours of culture. Both dibutyryl cyclic adenosine monophosphate (DBcAMP) and the Ca(2+)-elevating compound, thapsigargin, accelerated restoration of normal couplet morphology and function. The DBcAMP-mediated stimulus was inhibited by the Ca(2+) chelator, 1, 2-bis-(o-aminophenoxy)-ethene-N,N,N',N'-tetra-acetate tetra-(acetomethyl)ester (BAPTA/AM), but not by the protein kinase A (PKA) inhibitors, KT5720 or H89, suggesting that Ca(2+) elevation rather than PKA activation is involved. N-(6-aminohexyl-5-chloro-1-napththalenesulfonamide (W-7), a calmodulin inhibitor, and the protein kinase C (PKC) activator, phorbol dibutyrate, inhibited both the basal and the DBcAMP-stimulated recovery of functional polarity, whereas staurosporine and Gö 6976, 2 PKC inhibitors, accelerated the basal recovery of polarized function. Disruption of the microtubule cytoskeleton by colchicine induced only minor changes under basal, but not under DBcAMP-stimulated, conditions. The Golgi complex disruptor, brefeldin A, significantly delayed, and the microfilament-disrupting agent, cytochalasin D, fully blocked, both processes. However, DBcAMP stimulated trafficking of vesicles containing CLF to the pericanalicular region under the last condition. Our results indicate that restoration of couplet polarity following isolation occurs via a Ca(2+)-calmodulin-mediated mechanism, which depends on microfilament, but not on microtubule integrity. A second pathway is activated by DBcAMP activation via Ca(2+)-calmodulin formation, whose requirements with respect to cytoskeletal components are opposite. PKC has a negative regulatory role in both pathways.
Subject(s)
Bile Canaliculi/metabolism , Carrier Proteins/metabolism , Cytoskeleton/physiology , Hepatocytes/metabolism , Signal Transduction/physiology , Actins/physiology , Animals , Anion Transport Proteins , Bile Canaliculi/physiology , Cell Membrane/metabolism , Cells, Cultured , Cellular Senescence/physiology , Cholic Acids/pharmacokinetics , Cytoplasm/metabolism , Fluoresceins/pharmacokinetics , Fluorescent Dyes/pharmacokinetics , Hormones/physiology , Male , Rats , Rats, Wistar , Vacuoles/physiologyABSTRACT
Hepatocyte couplets, by retaining functional and morphological polarity similar to that of hepatocytes in situ, are a valuable in vitro model to study mechanisms of bile secretion, cholestasis and hepatocellular injury. They have been useful in studies of the hormonal control of bile formation and are suitable for morphological studies. The availability of periportal- and perivenous-enriched couplet populations now allows a zonal perspective. Their contribution to our understanding of regulatory aspects of hepatobiliary dysfunction due to toxicological or cholestatic insult, as well as its reversion by using hepatoprotective agents, is still at an early stage. The next few years should see further exiting contributions to our understanding of hepatobiliary function and dysfunction.
Subject(s)
Cell Culture Techniques/methods , Liver/cytology , Liver/metabolism , Animals , Cholic Acids/pharmacology , Cytoskeleton/metabolism , Fluoresceins/pharmacology , Fluorescent Dyes/pharmacology , Rats , Tight Junctions/physiology , Vacuoles/metabolismABSTRACT
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.
Subject(s)
Bile Acids and Salts/metabolism , Biliary Tract/drug effects , Protective Agents/pharmacology , Silymarin/pharmacology , Analysis of Variance , Animals , Biliary Tract/metabolism , Male , Rats , Rats, WistarABSTRACT
We have recently shown that protein kinase C (PKC) activation induces similar morphological and functional alterations in couplets to that caused by increments of intracellular Ca(2+). Since certain PKC isoforms are activated by Ca(2+), we tested whether the PKC inhibitor H-7 can counteract the alterations induced by this ion in couplets. The Ca(2+) ionophore A23187, which can mobilize Ca(2+) from extracellular and intracellular sources, decreased, in a dose-dependent manner, the percentage of couplets accumulating the fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF) in their canalicular vacuoles, i.e., in the canalicular vacuolar accumulation test (cVA of CLF), a measure of the overall capability of the couplets to secrete and retain CLF. To a similar extent, A23187 also decreased the percentage of couplets retaining CLF once secreted, i.e., in the canalicular vacuole retention test (cVR of CLF), a measure of tight junctional integrity. ATP (50 microM), another Ca(2+)-elevating compound, altered canalicular function in a similar extent to A23187. All these functional changes were prevented by H-7 in a dose-dependent manner. Canalicular dysfunction was accompanied by bleb formation and extensive redistribution of F-actin from the pericanalicular area to the cell body, which was also fully prevented by H-7; the intracellular Ca(2+) chelator, 1, 2-bis(o-aminophenoxy)-ethene-N,N,N',N'-tetraacetate tetrakis-(acetomethylester), (BAPTA/AM) (20 microM) had virtually the same preventive effects as H-7. Both H-7 and BAPTA/AM not only prevented but also reversed the decrease in cVA of CLF and blebbing induced by A23187. Thus, H-7 can both prevent and reverse Ca(2+)-mediated hepatocellular injury.
Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Calcium/metabolism , Enzyme Inhibitors/pharmacology , Liver/drug effects , Protein Kinase Inhibitors , Actins/metabolism , Adenosine Triphosphate/pharmacology , Animals , Calcimycin/pharmacology , Cell Membrane/metabolism , Chelating Agents/pharmacology , Cholic Acids , Dose-Response Relationship, Drug , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Fluoresceins , Fluorescence , Fluorescent Dyes , In Vitro Techniques , Ionophores/pharmacology , Liver/enzymology , Liver/ultrastructure , Male , Rats , Rats, Wistar , Tight Junctions/drug effects , Vacuoles/metabolismABSTRACT
BACKGROUND/AIMS: Fluorescent bile acids have proved useful for characterizing bile salt transport mechanisms. The aim of this study was to further validate the use of lysyl-fluorescein conjugated bile acid analogues as surrogate bile acids. METHODS: We analyzed biliary excretion kinetics of cholyl lysyl fluorescein (CLF), lithocholyl lysyl fluorescein (LLF) and sulfo-lithocholyl lysyl fluorescein (sLLF), both in the isolated rat hepatocyte couplet model and in TR- rats with a selective canalicular transport defect of non-bile acid organic anions. RESULTS: CLF and LLF, which like their natural nonsulfated bile acid congeners are expected to be handled by the canalicular bile salt export pump, were transferred into the bile canaliculus much faster than sLLF, a putative substrate for the canalicular multispecific organic anion transporter in both the in vivo and the in vitro models employed. The contention that different transport systems are involved in sulfated and non-sulfated lysyl fluorescein conjugated bile acids biliary excretion was supported further by studies using TR- rats, in which the cumulative biliary excretion of sLLF was reduced to 6% as compared with that of normal Wistar rats, in good agreement with values for its naturally-occurring radiolabeled parent compound sulfoglycolithocholate. In contrast, CLF and LLF were reduced to 66% and 52%, similar values to these for their congeners, [14C] glycocholate and [14C] lithocholate. CONCLUSION: The close similarity in behavior of lysyl fluorescein conjugated bile acids to that of their naturally-occurring parent compounds in these different models gives support for both sulfated and nonsulfated lysyl fluorescein conjugated bile acids as substitute molecules for studies of bile acid transport.
Subject(s)
Bile Acids and Salts/metabolism , Bile Canaliculi/metabolism , Liver/metabolism , Sulfates/metabolism , Animals , Anion Transport Proteins , Carrier Proteins/metabolism , Cholic Acids , Contrast Media , Fluoresceins , Fluorescence , Liver/cytology , Male , Rats , Rats, Mutant Strains/metabolism , Rats, WistarABSTRACT
The monohydroxy bile acid, taurolithocholate (TLC), causes cholestasis in vivo and in isolated perfused livers. It is also cholestatic in vitro and, in this study using isolated rat hepatocyte couplets, causes a reduction of the accumulation of (fluorescent) bile acid in the canalicular vacuoles (cVA) of this polarized cell preparation. The hepatoprotective bile acid, tauroursodeoxycholate (TUDCA), partially protects against the action of TLC when added at the same time. It also partially reverses the cholestatic effect if added after the cells have been exposed to TLC. A second hepatoprotective compound, S-adenosyl-L-methionine (SAMe) also not only partially protects against the action of TLC when added at the same time, but it too is able to partially reverse the cholestatic effect. Neither hepatoprotective agent is fully effective alone, but their effects are additive. In combination, a full restoration of cVA is observed in moderate cholestasis, but not in severe cholestasis. We discuss briefly some possible mechanisms involved in the additive mode of action of both hepatoprotective compounds. In summary, we show for the first time that SAMe and TUDCA can exert an additive effect in the amelioration of TLC-induced cholestasis in isolated rat hepatocyte couplets. This finding may be of possible clinical relevance.
Subject(s)
Cholestasis/chemically induced , S-Adenosylmethionine/pharmacology , Taurochenodeoxycholic Acid/pharmacology , Taurolithocholic Acid/toxicity , Animals , Bile Acids and Salts/metabolism , Bile Canaliculi/ultrastructure , Cholestasis/prevention & control , Fluorescent Dyes , Male , Rats , Rats, Wistar , Vacuoles/drug effects , Vacuoles/metabolismABSTRACT
The effect of vasopressin (VP) on canalicular function and hepatocellular morphology, with particular regard to actin cytoskeletal organization and the concomitant plasma membrane bleb formation, was studied in isolated rat hepatocyte couplets. VP induced the concentration-dependent formation of multiple plasma membrane blebs as well as simultaneous impairment in both canalicular vacuolar accumulation (cVA) and retention (cVR) of the fluorescent bile acid, cholyl-lysyl-fluorescein (CLF), which evaluate couplet secretory function and tight-junction integrity, respectively. These effects were mimicked by the protein kinase C (PKC) activator, phorbol dibutyrate (PDB), but not by the protein kinase A (PKA) activator, dibutyryl-cAMP. VP-induced bleb formation and canalicular dysfunction were fully prevented by the protein kinase inhibitor, H-7, but not by the PKA inhibitor, KT5720, further suggesting a specific role of PKC. VP-induced alterations were also prevented by pretreatment with the Ca2+-buffering agent, BAPTA/AM, but not with the calmodulin-dependent protein kinase II antagonist, calmidazolium. Neither the Ca2+-activated neutral protease inhibitor, leupeptin, nor the antioxidants, alpha-tocopherol or deferoxamine, were able to prevent either VP-induced plasma membrane blebbing or canalicular dysfunction. The Ca2+-ionophore, A23187, mimicked the VP-induced alterations, but its harmful effects were completely prevented by H-7. Bleb formation induced by VP and PDB was accompanied by an extensive redistribution of filamentous actin from the pericanalicular area to the cell body, and this effect was fully prevented by H-7. These results suggest that VP-induced canalicular and cytoskeletal dysfunction is mediated by PKC and that classical (Ca2+-dependent) PKC appear to be involved because intracellular Ca2+ is required for VP to induce its harmful effects.
Subject(s)
Actins/metabolism , Bile Canaliculi/physiology , Carbazoles , Cytoskeleton/ultrastructure , Liver/physiology , Protein Kinase C/metabolism , Tight Junctions/physiology , Vasopressins/pharmacology , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , Antioxidants/pharmacology , Bile Canaliculi/drug effects , Bile Canaliculi/ultrastructure , Bucladesine/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cells, Cultured , Chelating Agents/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoskeleton/drug effects , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Indoles/pharmacology , Leupeptins/pharmacology , Liver/drug effects , Liver/ultrastructure , Male , Microscopy, Electron, Scanning , Phorbol 12,13-Dibutyrate/pharmacology , Pyrroles/pharmacology , Rats , Rats, Wistar , Tight Junctions/drug effects , Tight Junctions/ultrastructure , Vacuoles/drug effects , Vacuoles/physiology , Vacuoles/ultrastructureABSTRACT
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.
Subject(s)
Bile Acids and Salts/metabolism , Colchicine/toxicity , Gout Suppressants/toxicity , Liver/drug effects , Alkaline Phosphatase/blood , Animals , Bile Acids and Salts/physiology , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Isomerism , L-Lactate Dehydrogenase/blood , Liver/pathology , Lumicolchicines/toxicity , Male , Rats , Rats, Wistar , Taurochenodeoxycholic Acid/metabolism , Taurocholic Acid/analogs & derivatives , Taurocholic Acid/metabolismABSTRACT
A simple, fast method to evaluate acute changes of tight junctional permeability in isolated hepatocyte couplets is proposed. The method consists of the recording of the number of canalicular vacuoles able to retain the previously accumulated fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF), as visualized by inverted fluorescent microscopy, following acute exposure to the compounds under study. The method was validated by (i) making a systematic documentation of the effect on CLF retention of a variety of hormonal modulators (vasopressin and phorbol esters), as well as several cholestatic (taurolithocholic acid, cyclosporin A, and estradiol 17 beta-glucuronide) and hepatotoxic agents (menadione, A23187, and t-butyl hydroperoxide), all known to affect biliary permeability in intact liver, and (ii) carrying out a comparative analysis of the results obtained with those recorded using rapid canalicular access of horseradish peroxidase (HRP) as an alternative procedure. The compounds tested all decreased canalicular vacuolar retention of CLF in a dose-dependent manner. Vasopressin- and phorbol ester-induced decline in CLF retention were prevented by pretreatment with the protein kinase C inhibitors H-7 and staurosporine, thus confirming a role for this enzyme in canalicular permeability regulation. A significant direct correlation (r = 0.934, p < 0.001) was obtained when the decrease in canalicular retention of CLF was compared with the increment in the canalicular access of HRP. Image analysis revealed that cellular fluorescence was not increased following exposure to these compounds, suggesting a paracellular rather than transcellular route for CLF egress. These results all support canalicular vacuolar retention of CLF as a suitable method to readily evaluate acute changes in tight junctional permeability in isolated hepatocyte couplets induced by physiological modulators or hepatotoxic agents.
Subject(s)
Liver/drug effects , Protein Kinases/metabolism , Tight Junctions/drug effects , Toxicity Tests/methods , Animals , Bile Canaliculi/drug effects , Bile Canaliculi/metabolism , Cell Membrane Permeability , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Cholic Acids , Dose-Response Relationship, Drug , Fluoresceins , Horseradish Peroxidase , Liver/metabolism , Male , Microscopy, Fluorescence , Rats , Rats, Wistar , Taurolithocholic Acid/pharmacology , Tight Junctions/metabolism , Vasopressins/pharmacology , Vitamin K/pharmacologyABSTRACT
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.
Subject(s)
Biliary Tract/drug effects , Cell Membrane Permeability/drug effects , Cholestasis/chemically induced , Coloring Agents/toxicity , Animals , Bile/chemistry , Bile/physiology , Biliary Tract/metabolism , Bromcresol Green/administration & dosage , Bromcresol Green/analysis , Bromcresol Green/toxicity , Cholestasis/metabolism , Coloring Agents/administration & dosage , Injections, Intravenous , Intercellular Junctions/metabolism , Liver Function Tests , Male , Phenolsulfonphthalein/administration & dosage , Phenolsulfonphthalein/analysis , Phenolsulfonphthalein/toxicity , Rats , Rats, Wistar , Rose Bengal/administration & dosage , Rose Bengal/analysis , Rose Bengal/toxicity , Sucrose/metabolism , Sulfobromophthalein/administration & dosage , Sulfobromophthalein/analysis , Sulfobromophthalein/toxicity , Time FactorsABSTRACT
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.
Subject(s)
Coloring Agents/pharmacology , Common Bile Duct/drug effects , Liver/drug effects , Sulfobromophthalein/pharmacology , Tight Junctions/drug effects , Animals , Bile/metabolism , Common Bile Duct/metabolism , Horseradish Peroxidase/metabolism , Infusions, Intravenous , Liver/metabolism , Male , Permeability/drug effects , Rats , Rats, Wistar , Sucrose/metabolismABSTRACT
The effects of ursodeoxycholate and its taurine conjugate on biliary Tm of bilirubin were evaluated in rats. Ursodeoxycholate was administered at four different doses (4, 8, 12 or 16 mumol per 100 g body wt i.v., followed by an i.v. infusion of 0.3, 0.6, 0.9 or 1.2 mumol/min per 100 g body wt, respectively), whereas tauroursodeoxycholate was administered only at the maximal dose. A dose-dependent diminution of bilirubin Tm was observed during ursodeoxycholate administration, which ranged from no effect at the lowest dose to a virtual excretory blockage at the highest dose. This was associated with an increase in bilirubin concentrations in both plasma and liver as well as in the fractional amount of conjugated pigment in both sites, suggesting an impairment of bilirubin transfer at the canalicular level. Incomplete taurine conjugation of ursodeoxycholate well correlated with these effects. Unlike ursodeoxycholate, tauroursodeoxycholate had no inhibitory effect on bilirubin Tm, although a slight inhibition of bilirubin uptake and bilirubin conjugation became apparent. Taken together, these results suggest that ursodeoxycholate interferes with the hepatobiliary transport of bilirubin by impairing its transfer at the canalicular level and that incomplete taurine conjugation appears to be a key factor determining this effect.
Subject(s)
Bilirubin/metabolism , Liver/metabolism , Taurochenodeoxycholic Acid/pharmacology , Ursodeoxycholic Acid/pharmacology , Animals , Bile/drug effects , Bile/metabolism , Bile Acids and Salts/metabolism , Bilirubin/blood , Bilirubin/pharmacology , Biological Transport/drug effects , Dose-Response Relationship, Drug , Liver/drug effects , Male , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Glucuronosyltransferase/metabolism , Intestines/enzymology , Liver/enzymology , Ursodeoxycholic Acid/pharmacology , Animals , Bile/drug effects , Bilirubin/metabolism , Cholagogues and Choleretics/pharmacology , Fatty Acids/metabolism , Fluorescence Polarization Immunoassay , In Vitro Techniques , Intestines/drug effects , Lipid Metabolism , Liver/drug effects , Male , Membrane Fluidity/drug effects , Microsomes/drug effects , Microsomes/enzymology , Nitrophenols/metabolism , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Anions/pharmacokinetics , Cholestasis/chemically induced , Cholestasis/metabolism , Liver/metabolism , Taurolithocholic Acid , Animals , Bile/metabolism , Biological Transport , Cytosol/metabolism , Liver/cytology , Male , Proteins/metabolism , Rats , Rats, Wistar , Sulfobromophthalein/analogs & derivatives , Sulfobromophthalein/pharmacokineticsABSTRACT
The mechanisms involved in bile salt-induced choleresis are poorly known. To give an insight in this physiological process, bile salt-associated electrolyte secretion was studied following relief of a short-term (2h) biliary obstruction in the rat, an experimental model that shows an important diminution of bile salt choleretic efficiency. For this purpose, biliary excretion of total bile salts and electrolytes (sodium, chloride and bicarbonate) were studied in such a model during taurocholate infusion at increasing rates. The results showed that bile flow, bile salt output and electrolyte secretion stimulated by taurocholate administration were decreased in the rats that were subjected to biliary obstruction. Besides, the choleretic efficiency of the excreted bile salts, as estimated by the slope of the regression line of bile flow vs. bile salt output, was diminished by 46% (p < 0.005). Multiple regression analysis of bile flow vs. bile salt and electrolyte outputs allowed to detect a selective diminution of the fraction of bile flow related to bile salt-associated electrolyte secretion ("secretory fraction" of the choleretic efficiency of bile salts) (3.2 +/- 0.3 vs. 2.5 +/- 0.2 L/mol, p < 0.05) whereas the "osmotic fraction" of the choleretic efficiency of bile salts was not modified by the treatment (5.0 +/- 0.4 vs. 5.1 +/- 0.3 L/mol, p > 0.05). Since both chloride and bicarbonate biliary concentrations in the volume of bile stimulated by taurocholate were reduced by 53% and 52% respectively, a role of these anions in the generation of bile salt-induced choleresis was suggested. Possible mechanisms involved in such a process and in its early impairment during cholestasis are discussed.