Involvement of a cyclic adenosine monophosphate-dependent signal in the diet-induced canalicular trafficking of adenosine triphosphate-binding cassette transporter g5/g8.

UNLABELLED: The adenosine triphosphate-binding cassette (ABC) half-transporters Abcg5 and Abcg8 promote the secretion of neutral sterol into bile. Studies have demonstrated the diet-induced gene expression of these transporters, but the regulation of their trafficking when the nutritional status changes in the liver remains to be elucidated. Here, we generated a novel in vivo kinetic analysis that can monitor the intracellular trafficking of Abcg5/Abcg8 in living mouse liver by in vivo transfection of the genes of fluorescent protein-tagged transporters and investigated how hypernutrition affects the canalicular trafficking of these transporters. The kinetic analysis showed that lithogenic diet consumption accelerated the translocation of newly synthesized fluorescent-tagged transporters to intracellular pools in an endosomal compartment and enhanced the recruitment of these pooled gene products into the bile canalicular membrane in mouse liver. Because some ABC transporters are reported to be recruited from intracellular pools to the bile canaliculi by cyclic adenosine monophosphate (cAMP) signaling, we next evaluated the involvement of this machinery in a diet-induced event. Administration of a protein kinase A inhibitor, N-(2-{[3-(4-bromophenyl)-2-propenyl]amino}ethyl)-5-isoquinolinesulfonamide, decreased the canalicular expression of native Abcg5/Abcg8 in lithogenic diet-fed mice, and injection of a cAMP analog, dibutyryl cAMP, transiently increased their levels in standard diet-fed mice, indicating the involvement of cAMP signaling. Indeed, canalicular trafficking of the fluorescent-tagged Abcg5/Abcg8 was enhanced by dibutyryl cAMP administration. CONCLUSION: These observations suggest that diet-induced lipid loading into liver accelerates the trafficking of Abcg5/Abcg8 to the bile canalicular membrane through cAMP signaling machinery.

Hepatocyte apical bulkheads provide a mechanical means to oppose bile pressure.

Hepatocytes grow their apical surfaces anisotropically to generate a 3D network of bile canaliculi (BC). BC elongation is ensured by apical bulkheads, membrane extensions that traverse the lumen and connect juxtaposed hepatocytes. We hypothesize that apical bulkheads are mechanical elements that shape the BC lumen in liver development but also counteract elevated biliary pressure. Here, by resolving their structure using STED microscopy, we found that they are sealed by tight junction loops, connected by adherens junctions, and contain contractile actomyosin, characteristics of mechanical function. Apical bulkheads persist at high pressure upon microinjection of fluid into the BC lumen, and laser ablation demonstrated that they are under tension. A mechanical model based on ablation results revealed that apical bulkheads double the pressure BC can hold. Apical bulkhead frequency anticorrelates with BC connectivity during mouse liver development, consistent with predicted changes in biliary pressure. Our findings demonstrate that apical bulkheads are load-bearing mechanical elements that could protect the BC network against elevated pressure.

Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4(-/-) mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts. METHODS: To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts. RESULTS: Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1-CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice. CONCLUSIONS: ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1-CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice.

LKB1 is a 'master' protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins.

Phosphoinositide 3-kinase/protein kinase B signaling pathway is involved in estradiol 17ß-D-glucuronide-induced cholestasis: complementarity with classical protein kinase C.

UNLABELLED: Estradiol 17ß-D-glucuronide (E(2)17G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). We assessed whether phosphoinositide 3-kinase (PI3K) is involved in E(2)17G-induced cholestasis. E(2)17G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E(2)17G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2-Morpholin-4-yl-8-phenylchromen-4-one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E(2)17G was extensively prevented by WM; this effect was fully blocked by the microtubule-disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Gö6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E(2)17G-induced cholestasis. In isolated perfused rat liver, an intraportal injection of E(2)17G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [(3)H]taurocholate and glutathione until the end of the perfusion period. Unlike Gö6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule-dependent manner. CONCLUSION: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E(2)17G through sustained internalization of canalicular transporters endocytosed via cPKC.

The bile salt export pump: clinical and experimental aspects of genetic and acquired cholestatic liver disease.

The primary transporter responsible for bile salt secretion is the bile salt export pump (BSEP, ABCB11), a member of the ATP-binding cassette (ABC) superfamily, which is located at the bile canalicular apical domain of hepatocytes. In humans, BSEP deficiency results in several different genetic forms of cholestasis, which include progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), as well as other acquired forms of cholestasis such as drug-induced cholestasis (DIC) and intrahepatic cholestasis of pregnancy (ICP). Because bile salts play a pivotal role in a wide range of physiologic and pathophysiologic processes, regulation of BSEP expression has been a subject of intense research. The authors briefly describe the molecular characteristics of BSEP and then summarize what is known about its role in the pathogenesis of genetic and acquired cholestatic disorders, emphasizing experimental observations from animal models and cell culture in vitro systems.

Motility of bile canaliculi in the living animal: implications for bile flow.

Modern fluorescence microscopic techniques were used to image the bile canalicular system in the intact rat liver, in vivo. By combining the use of sodium fluorescein secretion into bile, with digitally enhanced fluorescence microscopy and time-lapse video, it was possible to capture and record the canalicular motility events that accompany the secretion of bile in life. Active bile canalicular contractions were found predominantly in zone 1 (periportal) hepatocytes of the liver. The contractile movements were repetitive, forceful, and appeared unidirectional moving bile in a direction towards the portal bile ducts. Contractions were not seen in the network of canaliculi on the surface of the liver. Cytochalasin B administration resulted in reduced canalicular motility, progressive dilation of zone 1 canaliculi, and impairment of bile flow. Canalicular dilations invariably involved the branch points of the canalicular network. The findings add substantively to previous in vitro studies using couplets, and suggest that canalicular contractions contribute physiologically to bile flow in the liver.

Pro-inflammatory cytokines enhance dilatation of bile canaliculi caused by cholestatic antibiotics.

Many drugs can induce liver injury, characterized by hepatocellular, cholestatic or mixed hepatocellular-cholestatic lesions. While an inflammatory stress is known to aggravate hepatocellular injury caused by some drugs much less evidence exists for cholestatic features. In this study, the influence of pro-inflammatory cytokines (IL-6, IL-1ß and TNF-α), either individually or combined, on cytotoxic and cholestatic properties of antibiotics was evaluated using differentiated HepaRG cells. Six antibiotics of various chemical structures and known to cause cholestasis and/or hepatocellular injury in clinic were investigated. Caspase-3 activity was increased with all these tested hepatotoxic drugs and except with erythromycin, was further augmented in presence of cytokines mainly when these were co-added as a mixture. TNF-α and IL-1ß aggravated cytotoxicity of TVX more than IL-6. Bile canaliculi (BC) dilatation induced by cholestatic drugs was increased by co-treatment with IL-6 and IL-1ß but not with TNF-α. Reduced accumulation of carboxy-dichlorofluorescein, a substrate of the multi-drug resistance-associated protein 2, in antibiotic-induced dilatated BC, was further extended in presence of individual or mixed cytokines. In conclusion, our data demonstrate that pro-inflammatory cytokines either individually or in mixture, can modulate cholestatic and/or cytotoxic responses to antibiotics and that the extent of these effects is dependent on the cytokine and the cholestatic antibiotic.

Hepatic secretion of phospholipid vesicles in the mouse critically depends on mdr2 or MDR3 P-glycoprotein expression. Visualization by electron microscopy.

Hepatocellular secretion of bile salts into the biliary space induces phospholipid and cholesterol secretion, but the mechanism for integrated lipid secretion is poorly understood. Knockout mice unable to make the canalicular membrane mdr2 P-glycoprotein exhibit normal rates of bile salt secretion, yet are virtually incapable of secreting biliary phospholipid and cholesterol. As the mdr2 P-glycoprotein is thought to mediate transmembrane movement of phospholipid molecules, this mouse model was used to examine the mechanism for biliary phospholipid secretion. In wild-type mdr2 (+/+) mice, ultrarapid cryofixation of livers in situ revealed abundant unilamellar lipid vesicles within bile canalicular lumina. Although 74% of vesicles were adherent to the external aspect of the canalicular plasma membrane, bilayer exocytosis was not observed. Vesicle numbers in mdr2 (+/-) and (-/-) mice were 55 and 12% of wild-type levels, respectively. In a strain of mdr2 (-/-) mice which had been "rescued" by heterozygous genomic insertion of the MDR3 gene, the human homologue of the murine mdr2 gene, vesicle numbers returned to 95% of wild-type levels. Our findings indicate that biliary phospholipid is secreted as vesicles by a process largely dependent on the action of the murine mdr2 P-glycoprotein or human MDR3 P-glycoprotein. We conclude that mdr2-mediated phospholipid translocation from the internal to external hemileaflet of the canalicular membrane permits exovesiculation of the external hemileaflet, a vesiculation process promoted by the detergent environment of the bile canalicular lumen.

Osmoregulation of bile formation.

Bile secretion by liver parenchymal cells is the result of vectorial transcellular transport of solutes and involves the coordinated action of transport proteins at the basolateral (sinusoidal) and apical (canalicular) membranes of the hepatocyte. A complex network of signals controls uptake and efflux transporters on a long- and a short-term timescale, including regulation at the level of gene transcription, protein translation and maturation, covalent modification, and dynamic localization of transporter proteins, as well as substrate availability. Evidence has shown that the hepatocellular hydration state exerts powerful control on the transcellular transport of solutes, such as conjugated bile acids and glucuronide and glutathione conjugates. This is of physiological significance because liver cell hydration is a dynamic parameter, which changes within minutes under the influence of hormones, nutrients, and oxidative stress. Thus, osmoregulation of bile formation is of physiological and pathophysiological interest.

A Predictive 3D Multi-Scale Model of Biliary Fluid Dynamics in the Liver Lobule.

Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow in vivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury.

Rho-kinase/myosin light chain kinase pathway plays a key role in the impairment of bile canaliculi dynamics induced by cholestatic drugs.

Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury; however, the mechanisms underlying such injuries are poorly understood. In this study of human HepaRG and primary hepatocytes, we found that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid (BA) efflux and require alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. Short exposure to 6 cholestatic compounds revealed that BC constriction and dilation were associated with disruptions in the ROCK/MLCK/myosin pathway. At the studied concentrations, cyclosporine A and chlorpromazine induced early ROCK activity, resulting in permanent MLC2 phosphorylation and BC constriction. However, fasudil reduced ROCK activity and caused rapid, substantial and permanent MLC2 dephosphorylation, leading to BC dilation. The remaining compounds (1-naphthyl isothiocyanate, deoxycholic acid and bosentan) caused BC dilation without modulating ROCK activity, although they were associated with a steady decrease in MLC2 phosphorylation via MLCK. These changes were associated with a common loss of BC contractions and failure of BA clearance. These results provide the first demonstration that cholestatic drugs alter BC dynamics by targeting the ROCK/MLCK pathway; in addition, they highlight new insights into the mechanisms underlying bile flow failure and can be used to identify new predictive biomarkers of drug-induced cholestasis.

Intracellular sites involved in the biogenesis of bile canaliculi in hepatic cells.

Studies in hepatoma cells and hepatocytes have revealed that the biogenesis of bile canalicular membrane involves microvilli-lined vesicles (MLV), which are formed in well differentiated cells. The vesicles grow as a function of time and are presumably vectorially transported to cell surface contact sites of attached cells. We demonstrate that a fluorescent head group-labeled lipid analog, N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), after its exogenous insertion into the plasma membrane of HepG2 cells at 4 degrees C, accumulates in these microvilli-lined vesicles at 37 degrees C. This shows that the MLV are a target for plasma membrane-derived lipids. Furthermore, also the Golgi apparatus is involved in the formation of the vesicles. After initial accumulation of the fluorescent sphingolipid precursor, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (C6-NBD)-ceramide in the Golgi apparatus at 37 degrees C, prolonged incubation at 37 degrees C results in the appearance of NBD fluorescence in the microvilli-lined vesicles. The transport route for the Golgi-derived material to the developing bile canalicular vesicle is not an indirect pathway, i.e. involving transcytosis via the basolateral plasma membrane. This could be demonstrated by including bovine serum albumin (BSA) in the incubation media, a lipid scavenger that will remove any C6-NBD-lipids exposed at the basolateral membrane. At these conditions, lipid trafficking between the Golgi complex and MLV still occurred. We further demonstrate that the targeting from the Golgi apparatus to the bile canaliculus is also operational in isolated human hepatocytes. The latter results suggests that the Golgi complex is involved in both the formation of bile canaliculi and in bile secretion in fully differentiated cells.

Bile canalicular contraction is coincident with reorganization of pericanalicular filaments and co-localization of actin and myosin-II.

We examined the relationships between actin-myosin interaction and bile canalicular contraction using a new experimental model: cytoskeleton-enriched canalicular membranes (CCM). In CCM, the bile canaliculus compartment is isolated complete with membrane-attached pericanalicular actin filaments and the surrounding intermediate filament sheath. Immunofluorescence and immunoelectron microscopy showed that actin and myosin-II were distributed over pericanalicular microfilaments that insert into adherens (belt) junctions; intermediate filaments predominantly inserted into desmosomes. The addition of "contraction solution" (1 microM Ca2+, 1 mM ATP) resulted in closure of CCM lumens, which was interpreted as canalicular contraction. Contraction was also associated with shortening and/or twisting of canaliculi. Rearrangement of actin filaments and myosin-II with co-localization of actin and myosin was observed. Evidence is also provided for attachment of actin-myosin-II aggregates to intermediate filaments coincident with contraction, suggesting a key scaffold function for intermediate filaments of the canaliculus. Attention is drawn to the overall similarity of structure-function dynamics in hepatic apical membranes to those described in intestinal brush border membrane preparations. The results are consistent with dynamic actin-myosin interaction with co-localization of actin and myosin-II in filament clumps coincident with canalicular contraction.

Relationship between age-related increases in rat liver lipid peroxidation and bile canalicular plasma membrane fluidity.

Aging is associated with increased cellular levels of lipid peroxides and reactive oxygen species. This increase in the radical species may affect membrane fluidity and consequently membrane functions. We attempted to determine whether age-related increases in lipid peroxide and reactive oxygen species levels affect the annular fluidity of the bile canalicular plasma membrane in young (2-3 weeks old), adult (20 weeks old) and old (100 weeks old) rats. Hepatic levels of lipid peroxides measured with thiobarbituric acid reactive substances (TBARS), reactive oxygen species and reduced glutathione were significantly higher in the old rats than in the young and adult rats. Arachidonic acid (AA) levels increased and docosahexaenoic acid (DHA) levels decreased in the isolated canalicular plasma membrane of the old rats, and the ratio of DHA to AA thus decreased significantly with rat aging. Hepatic TBARS levels correlated negatively with molar ratios of DHA to AA. The annular fluidity of the bile canalicular plasma membrane decreased significantly in the old rats compared with that in the young and adult rats. These results suggest that the age-related decrease in DHA content of the bile canalicular plasma membrane and the molar ratio of DHA to AA may be associated with age-related deterioration of membrane annular fluidity.

A simple method for the correction of biliary excretion curves distorted by the biliary dead space.

Biliary solute concentrations measured at the tip of the cannula suffer a delay with respect to bile flow due to the transit time through the biliary tree volume. This study proposes a simple method, which is valid under variable bile flow conditions, to correct the distortion introduced by the biliary tree volume on the kinetic curves of the biliary excretion rate. The biliary transit time (tt) was calculated as the time needed to excrete a bile volume equal to the biliary tree volume by means of the interpolation of biliary cumulative volume versus time curves. Such tt permits one to estimate the canalicular concentration at time t, interpolating the biliary concentration curves at time t-tt. The product between the estimated canalicular concentration and the bile flow allows the calculation of the corrected biliary excretion rate. This method was evaluated by a comparison between biliary excretion rate curves of [14C]taurocholate [( 14C]TC) injected as a bolus under basal and sodium dehydrocholate (DHC)-induced choleresis conditions. Since the canalicular excretion rate of [14C]TC is considered independent of bile flow, the significant differences observed in its excretion kinetics under both conditions were attributed to distortion due to the biliary tree volume. After the correction, both curves showed a significant overlapping. This result indicates that the method improves the time-course representation of canalicular events in biliary excretion kinetic studies.

Differential response of the permeability of the rat liver canalicular membrane to sucrose and mannitol following in vivo acute single and multiple exposures to microwave radiation (2.45 GHz) and radiant-energy thermal stress.

Both acute and chronic exposures to microwave radiation altered the function of the rat canalicular membrane. A single acute exposure to microwave radiation [80 mW/cm2, 2.45 GHz, continuous wave, 30 min exposure (SAR approximately equal to 72 W/kg)] or a matched radiant-energy thermal load, both designed to raise core body temperature approximately 3 degrees C, decreased the permeability of the canalicular membrane of male Sprague-Dawley rats to sucrose. The change in canalicular membrane permeability was demonstrated by a significant increase in the percentage of [3H]sucrose recovered in bile following its administration by a segmented retrograde intrabiliary injection. Similar acute exposures to microwave and radiant-energy thermal sources produced no significant alterations in canalicular membrane permeability to [14C]mannitol. In both acute exposure protocols, a rapidly reversible increase in bile flow rate was observed. Four exposures (30 min/day x 4 days) to either microwave radiation (80 mW/cm2) or a matched radiant-energy thermal load resulted in a significant depression in bile flow rate at normothermic temperatures. Animals receiving multiple exposures to microwave radiation had significant decreases in canalicular membrane permeability to both [3H]sucrose and [14C]mannitol, while similar exposure to radiant-energy thermal load alone altered canalicular membrane permeability to [3H]sucrose. An examination of the hepatic clearance of sucrose and mannitol following acute microwave exposure demonstrated no significant differences. Thus acute single exposure to microwave and radiant-energy thermal loads produced similar alterations in canalicular membrane permeability. Conversely, multiple exposures produced nonreversible changes in bile flow rate and canalicular membrane permeability, with microwave exposure producing greater alterations in the function of the canalicular membrane than an equivalent radiant-energy thermal load.

Bile canaliculi are defective in hepatic involvement of organ failure and recovery of liver function is due to their secondary regeneration.

OBJECTIVE: To investigate the morphological changes in the liver in patients with organ failure and hyperbilirubinemia and to correlate them to the outcome. DESIGN: A case series prospective study. SETTING: Intensive care units of two general hospitals. PATIENTS: Twelve patients in organ failure with predominant hepatic involvement, aged 16 to 69 years (mean 56 years). INTERVENTIONS: Liver biopsy was performed on all patients 3-15 days after organ failure. A second biopsy was also performed on all four surviving patients, as well as on 3 patients just before death at a mean time of 16 days (6-32) and 31 days (14-55), respectively, after the first biopsy. The samples were studied by electron microscopy and findings were assessed according to Rappaport's designation. MEASUREMENTS AND MAIN RESULTS: In the first biopsy it was shown that in zone III there was complete degeneration of bile canaliculi and hepatocytes in contrast to zone I. The grade of histological severity for zone III is positively correlated to the bilirubin concentration (p = 0.001). In the specimens from the second biopsy, it was shown that numerous, newly formed secondary bile canaliculi per 20 consecutive hepatocytes had developed in zone III in the surviving patients, whereas there was a complete absence of such canaliculi in the patients who died (mean +/- SD: 9.6 +/- 3.2 vs 0). CONCLUSIONS: It appears that the destruction of primary bile canaliculi is a striking anatomical defect in patients with organ failure and impaired bilirubin excretion. The restoration of liver function coincides with adequate formation of new secondary bile canaliculi in zone III, giving credence to the hypothesis that this formation is an important structural change responsible for the improvement in liver function.

Tumor necrosis factor-alpha and interleukin-6 reduce bile canalicular contractions of rat hepatocytes.

BACKGROUND: Surgeons sometimes encounter hyperbilirubinemia without mechanical obstruction of the biliary tree postoperatively. Many of these patients have bacterial infections and endotoxemia. Kupffer's cells stimulated by endotoxin secrete inflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. We hypothesized that TNF-alpha and IL-6 might be involved in the pathogenesis of hyperbilirubinemia. METHODS: Effects of TNF-alpha and IL-6 on the contractions of bile canaliculi (BC) of rat hepatocyte couplets were examined and time-lapse images using phase-contrast microscopy were taken. Bile was collected from rats treated with or without the cytokines. The livers, perfused with lanthanum after the injection of cytokines, were examined ultrastructurally using electron microscopy. RESULTS: The number of BC contractions decreased in the couplets treated with both cytokines. The rapid movement of a droplet from BC was observed at the intercellular space of the hepatocyte couplet treated with TNF-alpha. Systolic blood pressure and hepatic tissue blood flow of rats injected with TNF-alpha were not changed, whereas the hepatic tissue blood flow of rats treated with IL-6 decreased (Dunnett test, P <.05). Bile secretion was reduced in both groups of rats (Dunnett test, P <.05). In rats treated with TNF-alpha the total serum bile acid concentration increased and lanthanum temporarily accumulated in BC. CONCLUSIONS: These results suggest that TNF-alpha and IL-6 may reduce BC contractions and thereby decrease bile flow.

Role of bile acid measurement in pregnancy.

The diagnosis of obstetric cholestasis (OC) has serious implications for maternal, and especially fetal, health. Total serum bile acid concentration is an important investigation in any woman with itching in the absence of a rash during pregnancy. Results should be available within 1-2 working days. Pruritus plus raised total bile acids in the third trimester suggests a diagnosis of OC. Other biochemical abnormalities, usually a raised alanine aminotransferase, accompany or follow the finding of raised bile acids. Bile acids are sensitive, but not specific, markers for OC. The diagnosis is one of exclusion. The clinician should remain alert to other causes of liver dysfunction, either specific to pregnancy or not. Effective treatment is available that improves maternal biochemical indices and well-being. It is not clear whether such treatment reduces the risks to the fetus. More active management of OC pregnancies has reduced the associated fetal and perinatal mortality.