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Bile acid regulation of hepatic physiology. I. Hepatocyte transport of bile acids
Abstract
Bile acids are cholesterol derivatives that serve as detergents in bile and the small intestine. Approximately 95% of bile acids secreted by hepatocytes into bile are absorbed from the distal ileum into the portal venous system. Extraction from the portal circulation by the hepatocyte followed by reexcretion into the bile canaliculus completes the enterohepatic circulation of these compounds. Over the past few years, candidate bile acid transport proteins of the sinusoidal and canalicular plasma membranes of the hepatocyte have been identified. The physiology of hepatocyte bile acid transport and its relationship to these transport proteins is the subject of this Themes article.
... Under normal conditions bile acids are taken up by hepatocytes from portal circulation by high affinity sodium-dependent, sodium taurocholate cotransporting polypeptide (NTCP) and to a lesser extent by the sodium-independent organic anion-transporting polypeptides (OATPs) in the sinusoidal membrane. 1,2 Within the hepatocyte, returning bile acids are mixed with newly synthesized bile acids and are secreted into bile. Conjugated bile acids are secreted from the hepatocyte canalicular membrane into bile by the bile salt export pump (BSEP; ABCB11) which is an adenosine triphosphate-binding cassette transporter. ...
... A small amount is also secreted by multidrug resistance protein 2 (MRP2). 1,2 In cholestasis, bile flow from the liver to the intestine is impaired and as a consequence toxic bile acids and other metabolites are retained within the hepatocyte. In children, cholestasis with early onset accounts for a large proportion of the cases with severe liver disease with high mortality and morbidity. ...
Background
Primary human hepatocytes are a useful in vitro model system to examine hepatic biochemical pathways, liver disorders and/or pharmacotherapies. This system can also be used for transport studies to investigate uptake and excretion of bile acids. Proper modeling of hepatic function requires careful attention to media components, and culture substrates and conditions.
Objectives
To examine the effects of different culture media and conditions on bile acid transport in cultured human hepatocytes.
Methods and Results
Hepatocytes cultured in Williams' medium E showed an increase in both uptake and excretion of taurocholate compared to cells cultured in Dulbecco's Modified Eagle Medium (DMEM). Supplementation of DMEM with glutathione or ascorbic acid did not compensate for the lower transport. The difference can be explained by lower mRNA expression of the transporter proteins sodium taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP; ABCB11) when cultured in DMEM. Hepatocytes cultured in DMEM also display fewer and smaller bile canaliculi. Following extended time in culture supplementation of Williams' medium E with dexamethasone increased the expression of NTCP and BSEP.
Conclusion
Williams' medium E is superior to DMEM for transport studies in primary human hepatocytes. Supplementation with dexamethasone increase mRNA levels of NTCP and BSEP.
... uptake mechanism that is poorly understood. Hepatic cholesterol (delivered either via LDLR or SR-BI) can be oxygenated, converted into bile acids, and secreted into the intestine via canalicular transporters ( 10 ). While the majority of bile acids are reabsorbed in the intestines, a proportion is eliminated in the feces, thereby ridding the body of excess cholesterol ( 10 ) ( Fig. 1 ). ...
... Hepatic cholesterol (delivered either via LDLR or SR-BI) can be oxygenated, converted into bile acids, and secreted into the intestine via canalicular transporters ( 10 ). While the majority of bile acids are reabsorbed in the intestines, a proportion is eliminated in the feces, thereby ridding the body of excess cholesterol ( 10 ) ( Fig. 1 ). ...
High-density lipoproteins play a central role in systemic cholesterol homeostasis by stimulating the efflux of excess cellular cholesterol and transporting it to the liver for biliary excretion. HDL has long been touted as the good cholesterol because of the strong inverse correlation of plasma HDL cholesterol levels with coronary heart disease. However, the disappointing outcomes of recent clinical trials involving therapeutic elevations of HDL cholesterol have called this moniker into question and revealed our lack of understanding of this complex lipoprotein. At the same time, the discovery of microRNAs (miRNAs) that regulate HDL biogenesis and function have led to a surge in our understanding of the post-transcriptional mechanisms regulating plasma levels of HDL. Furthermore, HDL has recently been shown to selectively transport miRNAs and thereby facilitate cellular communication by shuttling these potent gene regulators to distal tissues. Finally, that miRNA cargo carried by HDL may be altered during disease states further broadened our perspective of how this lipoprotein can have complex effects on target cells and tissues. The unraveling of how these tiny RNAs govern HDL metabolism and contribute to its actions promises to reveal new therapeutic strategies to optimize cardiovascular health.
... Summarizing, structural elements positively correlated with the potency toward the HeLa cells are: The most potent compounds among 1e33 posses HBDs as the primary (8,19,24,28,29) or secondary amide (9, 20, 22, 25, 30e33) hydrogen, or as carboxylic group (7,21,26), all described by variables having the highest impact on model. ...
... Majority of 1e33 are cholic acid derivatives (Fig. S8 in Supplementary material). It is known that various Na þ -dependent transporters [17,18] are responsible for the uptake of the bile acids (Na þ /taurocholate cotransporting polypeptide (Ntcp1), organic anion transporting polypeptide (Oatp1), microsomal epoxide hydrolase (mEH)) [19,20], and they can carry some of the functionalized cholic acid derivatives into the cell [21]. ...
An alignment-free 3D QSAR study on antiproliferative activity of the thirty-three 1,2,4,5-tetraoxane derivatives toward two human dedifferentiated cell lines was reported. GRIND methodology, where descriptors are derived from GRID molecular interaction fields (MIF), were used. It was found that pharmacophoric pattern attributed to the most potent derivatives include amido NH of the primary or secondary amide, and the acetoxy fragments at positions 7 and 12 of steroid core which are, along with the tetraoxane ring, common for all studied compounds. Independently, simple multiple regression model obtained by using the whole-molecular properties, confirmed that the hydrophobicity and the H-bond donor properties are the main parameters influencing potency of compounds toward human cervix carcinoma (HeLa) and human malignant melanoma (FemX) cell lines. Corollary, similar structural motifs are found to be important for the potency toward both examined cell lines.
... However, substrate accumulation is dependent on transport into and out of cells as well affinities for intracellular binding partners. The endogenous situation is complicated by the presence of multiple uptake transporters, such as sodiumindependent organic anion transporting polypeptides (oatps) (33), by export transporters residing on the sinusoid and bile canaliculus and by the regulation of ntcp, which may be related to its intracellular distribution (2). In this report we utilized fluorescent bile acid derivatives to analyze organic anion transport by directly quantifying microscope images of hepatocytes and cultured cell lines captured with low-excitation intensity, low-light CCD cameras, and multi-channel semiautomated image acquisition. ...
... Neither ntcp-nor oatp1a1-expressing cells showed significant accumulation of CGamF by these methods, and HeLa cells did not take up any of the dyes in the absence of transporter because accumulation was not seen in the absence of Zn induction (Fig. 4A, uninduced data). Ntcp-mediated accumulation of CDCGamF was also dependent on the presence of extracellular sodium (Fig. 4B), as expected because of the sodium-dependent nature of ntcp-mediated but not oatp-mediated transport (11,33). ...
Sodium taurocholate-cotransporting polypeptide (ntcp) is considered to be a major determinant of bile acid uptake into hepatocytes. However, the regulation of ntcp and the degree that it participates in the accumulation of specific substrates are not well understood. We utilized fluorescent bile acid derivatives and direct quantitation of fluorescent microscopy images to examine the regulation of ntcp and its role in the cell-to-cell variability of fluorescent bile acid accumulation. Primary-cultured rat hepatocytes rapidly accumulated the fluorescent bile acids, chenodeoxycholylglycylamidofluorescein (CDCGamF), 7-β- nitrobenzoxadiazole 3-α hydroxy 5-β cholan-24-oic acid (NBD-CA), and cholyl-glycylamido-fluorescein (CGamF). However, in stably transfected HeLa cells, ntcp preferred CDCGamF, whereas the organic anion transporter, organic anion transporting polypeptide 1 (oatp1a1), preferred NBD-CA, and neither ntcp nor oatp1a1 showed strong accumulation of CGamF by these methods. Ntcp-mediated transport of CDCGamF was inhibited by taurocholate, cyclosporin, actin depolymerization, and an inhibitor of atypical PKC-ζ. The latter two agents altered the cellular distribution of ntcp as visualized in ntcp-green fluorescent protein-transfected cells. Although fluorescent bile acid accumulation was reproducible by the imaging assays, individual cells showed variable accumulation that was not attributable to changes in membrane permeability or cell viability. In HeLa cells, this was accounted for by variable levels of ntcp, whereas, in hepatocytes, ntcp expression was uniform, and low accumulation was seen in a large portion of cells despite the presence of ntcp. These studies indicate that single-cell imaging can provide insight into previously unrecognized details of anion transport in the complex environment of polarized hepatocytes.
... Additionally, the human organic aniontransporting polypeptides (OATP) OATP1B1 and OATP1B3 transport specific types of bile acids in the liver while excluding others. Specifically, CA, CDCA, and DCA are transported by these polypeptides, while the secondary bile acids LCA and UDCA are not (22). Furthermore, kinetic analyses revealed that conjugated bile acids are the preferred substrates as compared to unconjugated bile acids (23). ...
Bile acids are critical for the digestion and absorption of lipids and fat-soluble vitamins; however, evidence continues to emerge supporting additional roles for bile acids as signaling molecules. After they are synthesized from cholesterol in the liver, primary bile acids are modified into secondary bile acids by gut flora contributing to a diverse pool and making the composition of bile acids highly sensitive to alterations in gut microbiota. Disturbances in bile acid homeostasis have been observed in patients with Inflammatory Bowel Diseases (IBD). In fact, a decrease in secondary bile acids was shown to occur because of IBD-associated dysbiosis. Further, the increase in luminal bile acids due to malabsorption in Crohn’s ileitis and ileal resection has been implicated in the induction of diarrhea and the exacerbation of inflammation. A causal link between bile acid signaling and intestinal inflammation has been recently suggested. With respect to potential mechanisms related to bile acids and IBD, several studies have provided strong evidence for direct effects of bile acids on intestinal permeability in porcine and rodent models as well as in humans. Interestingly, different bile acids were shown to exert distinct effects on the inflammatory response and intestinal permeability that require careful consideration. Such findings revealed a potential effect for changes in the relative abundance of different bile acids on the induction of inflammation by bile acids and the development of IBD. This review summarizes current knowledge about the roles for bile acids as inflammatory mediators and modulators of intestinal permeability mainly in the context of inflammatory bowel diseases.
... Hepatocyte transport of bile acids and regulation of bile acid synthesis have been reviewed by Wolkoff and Cohen (2003) and Fuchs (2003). Detailed analyses of bile acid concentrations in healthy subjects and patients with liver disease have recently been published (Bathena et al., 2013(Bathena et al., , 2015bHaag et al., 2015). ...
Drug-drug interactions are a major concern not only during clinical practice, but also in drug development. Due to limitations of in vitro-in vivo predictions of transporter-mediated drug-drug interactions, multiple clinical Phase I drug-drug interaction studies may become necessary for a new molecular entity to assess potential drug interaction liabilities. This is a resource-intensive process and exposes study participants, who frequently are healthy volunteers without benefit from study treatment, to the potential risks of a new drug in development. Therefore, there is currently a major interest in new approaches for better prediction of transportermediated drug-drug interactions. In particular, researchers in the field attempt to identify endogenous compounds as biomarkers for transporter function, such as hexadecanedioate, tetradecanedioate, coproporphyrins I and III, or glycochenodeoxycholate sulfate for hepatic uptake via organic anion transporting polypeptide 1B or N1-methylnicotinamide for multidrug and toxin extrusion protein-mediated renal secretion. We summarize in this review the currently proposed biomarkers and potential limitations of the substances identified to date. Moreover, we suggest criteria based on current experiences, which may be used to assess the suitability of a biomarker for transporter function. Finally, further alternatives and supplemental approaches to classic drug-drug interaction studies are discussed. © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
... There are two routes for cholesterol excretion in vivo: One route can undergo reduction, cleavage, and hydroxylation for synthesis of deoxycholic, chenodeoxycholic, and lithocholic acids, followed by bile salt formation through conjugation with glycine or taurine for fecal excretion. However, the other route involves direct excretion of cholesterol in the neutral form [6,7]. T.Y.C. and Y.F.L. contributed equally to the present study. ...
OBJECTIVES
The objectives were to study the effect of several vegetable oils (camellia oil, soybean oil, palm oil) and blended oil composed of soybean oil and camellia oil on blood lipids reduction and antioxidative activity.
METHODS
Forty male hamsters were administered with AIN-93G diet for 1 week, followed by dividing into 5 groups, with the first group (control) being fed with low-fat diet containing 5% oil for six weeks and the remaining 4 groups each with high-fat diets containing 14% palm oil, 14% camellia oil, 14% soybean oil and 14% blended oil (8.4% soybean oil and 5.6% camellia oil) along with 0.2% cholesterol and 0.1% bile acid. After feeding period, the hamsters were sacrificed and analyzed.
RESULTS
Hamsters fed with high-fat diet raised serum triacylglycerol, total cholesterol and aspartate aminotransferase without affecting alanine aminotransferase. Compared to palm oil-containing diet, the other 3 high-fat diets reduced serum total cholesterol, low-density lipoprotein cholesterol and low-density lipoprotein-to-high-density lipoprotein cholesterol (LDL-C/HDL-C) ratio with an opposite trend for liver total cholesterol. However, compared to control, the serum HDL-C level was raised for all 4 high-fat diets. The higher the degree of oil unsaturation, the higher the serum thiobarbituric acid reactive substances and the lower the liver triacylglycerol level and activities of fatty acid synthase, glucose 6-phosphate dehydrogenase and malic enzymes. Also, both soybean oil and blended oil lowered the antioxidative activity of liver.
CONCLUSION
Camellia oil and blended oil were more efficient in elevating serum HDL-C and decreasing LDL-C/HDL-C ratio in hamsters than soybean oil.
... Primary bile acids such as chenodeoxycholic acid (CDCA) and cholic acid (CA), and secondary bile acids such as lithocholic acid (LCA) and deoxycholic acid (DOCA) are conjugated with the amino acids taurine and glycine and secreted as sodium or potassium salts into the biliary canaliculi via the ABC biliary transporter proteins [50] . Phosphatidylcholine, the predominant biliary phospholipid, is synthesized in hepatocytes and transported into the biliary canaliculi by the flippase multidrug resistant protein 3 (MDR3) [51] . ...
... In turn, the propensity exists for the unsaturated fatty acids to be absorbed toward the proximal small intestine while the more difficult saturates are recovered distally (Vodovar et al., 1965;Honda et al., 2009). Although the emulsified lipid core progressively depletes, bile acids are largely retained at the surface to eventually be abraded and form micelles of their own distally and be absorbed in the ileum (Kvietys et al., 1981;Cabrel et al., 1987;Campanelli et al., 1987;Wolkoff and Cohen, 2002). Bile acids also can be lost into the large intestine when deconjugated and reduced to monohydroxy units as diffusible oxygen diminishes to favor terms aiding anaerobe activity (Tellier et al., 1987;Kim and Lee, 2005). ...
The small intestinal mucosa acts to recover nutrients from the lumen while providing a barrier against potential hazards. Its unstirred water layer (USWL) at the lumen interface involves membrane associated mucin linearly protruding from underlying microvilli that entangles secretory mucin released from local goblet cells. Both mucin sources are dominated by repetitive O-glycosylated areas dependant on threonine, serine, glycine, and proline. Secretory mucin differs from membrane attached mucin by further employing multiple cystines that interconnect these areas into a net-like molecular sieve. All of the glycosylated areas have ionizable acidic groups credited with reducing pH from that in the lumen to create a micro environment favoring enzymes finalizing digestion while optimizing nutrient terms for absorption. Erosion of the USWL and/or abuse of the membrane due to lumen threats require continuous repair. The aforementioned amino acids are necessary in substantial amounts while vitamin B6 collaborates with vitamin A as meaningful cofactors for mucin synthesis. Marginal inadequacies of these nutrients during inordinate demand are expected to impair mucin replacement. In turn, marginal increases in feed conversion likely occur while fostering the probability of necrotic enteritis together with gizzard erosions. Abuse of the absorptive membrane is of particular concern from fatty acid hydroperoxides because of their continual presence in feed and inability of the USWL to provide protection. These hydroperoxides threaten membrane integrity by their inclusion in micelles during digestive events with fat thereby permitting transit through the USWL. Once coalesced with membrane phospholipids, structural aberrations are visualized as interfering with nutrient recovery while enabling leakage of cell contents to potentiate wet excreta. Inclusion of dietary vitamin E along with vitamin A into micelles with fatty acid hydroperoxides provides relief by quenching further peroxidation. Assuring cystine, threonine, glycine, and serine that are directly available as such together with vitamins A, E, and B6 represents one approach toward optimizing maintenance of the intestinal mucosa.
... Current understanding of cholesterol biosynthesis and deposition indicates three main pathways: (1) regulation of HMG-CoA reductase activity and levels; (2) regulation of excess intracellular non-esterified cholesterol through the activity of acyl-CoA:cholesterol acyltransferase; and (3) regulation of plasma cholesterol levels via LDL receptor-mediated uptake and HDL-mediated reverse transport (7) . In addition, pathways involved in bile acid metabolism and cholesterol absorption can also influence the circulating levels of cholesterol as well as its deposition (11,12) . However, the majority of previous studies have been concentrated on regulation and reduction of disease-state cholesterol levels; the molecular events related to changes in circulating cholesterol level are less understood. ...
Hypercholesterolaemia is a risk factor for CVD, which is a leading cause of death in industrialised societies. The biosynthetic pathways for cholesterol metabolism are well understood; however, the regulation of circulating cholesterol by diet is still not fully elucidated. The present study aimed to gain more comprehensive understanding of the relationship between circulating cholesterol levels and molecular effects in target tissues using the hamster model. Male golden Syrian hamsters were fed with chow or diets containing 36 % energy from fat with or without 1 % cholesteyramine (CA) as a modulator of circulating cholesterol levels for 35 d. It was revealed that the expression of lanosterol 14α-demethylase (
CYP51
) instead of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase mRNA expression was responsive to circulating cholesterol in hamsters fed hypercholesterolaemic diets. The high-fat diet increased circulating cholesterol and down-regulated
CYP51
, but not HMG-CoA reductase. The CA diet decreased cholesterol and increased
CYP51
expression, but HMG-CoA reductase expression was not affected. The high-fat diet and CA diet altered the expression level of cholesterol, bile acids and lipid metabolism-associated genes (LDL receptor, cholesterol 7α-hydroxylase (
CYP7A1
), liver X receptor (
LXR
) α, and ATP-binding cassette subfamily G member 5/8 (
ABCG5/8
)) in the liver, which were significantly correlated with circulating cholesterol levels. Correlation analysis also showed that circulating cholesterol levels were regulated by LXR/retinoid X receptor and PPAR pathways in the liver. Using the hamster model, the present study provided additional molecular insights into the influence of circulating cholesterol on hepatic cholesterol metabolism pathways during hypercholesterolaemia.
... The two transporter mechanisms mediating the hepatic uptake of xenobiotics and endobiotics from the circulation consist of the sodium-dependent and sodium-independent transporter mechanisms (7,26). Bile salts circulate in plasma tightly bound to albumin and lipoproteins such as high-density lipoprotein (62). More than 80% of conjugated bile salts undergo single-pass extraction by the liver, predominantly via the sodium dependent transport via the sodium-taurocholate cotransporting polypeptide NTCP (SLC10A1) (53). ...
The orphan nuclear receptors Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) have been proposed to play an important role in the detoxification of xeno- and endobiotics by regulating the expression of detoxifying enzymes and transporters. We showed that the combined loss of PXR and CAR resulted in a significantly heightened sensitivity to bile acid toxicity in a sex-specific manner. The increased sensitivity in males was associated with genotype-specific suppression of bile acid transporters and loss of bile acid-mediated down regulation of small heterodimer partner, whereas the transporter suppression was modest or absent in the female DKO mice. The liver X receptors (LXRs), including the alpha and beta isoforms were identified as sterol sensors that regulate cholesterol and lipid homeostasis and macrophage functions. We found that activation of LXRĄ in transgenic mice or with LXR ligands confers a female-specific resistance to lithocholic acid (LCA)-induced hepatotoxicity and bile duct ligation (BDL)-induced cholestasis. In contrast, LXR alpha and beta double knockout mice (LXR DKO) exhibited heightened cholestatic sensitivity. The LCA and BDL resistance in transgenic mice was associated with an increased expression of bile acid detoxifying sulfotransferase 2A (SULT2A) and selected members of the bile acid transporters. We also showed that genetic or pharmacological activation of the orphan nuclear receptor liver X receptor (LXR) sensitized mice to cholesterol gallstone disease (CGD) induced by a high cholesterol lithogenic diet. LXR-promoted CGD was associated with increased expression of several canalicular transporters that efflux cholesterol and phospholipids, leading to higher biliary concentrations of cholesterol and phospholipids. The biliary bile salt concentration was reduced in these mice, resulting in increased cholesterol saturation index (CSI). Interestingly, the lithogenic effect of LXR was completely abolished in the low-density lipoprotein receptor (LDLR) null background or when the mice were treated with Ezetimibe, a cholesterol-lowering drug that blocks the intestinal dietary cholesterol absorption. We propose that LXRs have evolved to have dual function in maintaining cholesterol and bile acid homeostasis.
... Hepatic uptake, biliary excretion and intestinal reabsorption are mediated by specific transport proteins. More than 80% of conjugated bile salts, which circulate in plasma tightly bound to albumin and lipoproteins (Wolkoff and Cohen, 2003) undergo single-pass extraction by the liver. ...
... In regard to Ntcp, initial studies using Xenopus laevis oocytes showed a 95% reduction of sodium-dependent taurocholate uptake following inhibition of Ntcp expression by antisense nucleotides [40] suggesting that Ntcp is the main bile acid transport mediator, however these studies failed to establish if mEH was expressed and targeted to the plasma membrane of this system. In addition the oligonucleotide used was not specific for Ntcp [41]. Studies in the fch/ fch mouse, which is a model of erthropoietic protoporphyria, demonstrated that Ntcp and the sodium-independent bile acid transporter, OATP-1 were undetectable, while mEH levels remained unchanged (unpublished observation). ...
Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes. These compounds are involved in cholesterol homeostasis, lipid digestion, excretion of xenobiotics and the regulation of several nuclear receptors and signaling transduction pathways. Previous studies have demonstrated the critical role of GATA-4, a C/EBPα-NF/Y complex and an HNF-4α/CAR/RXR/PSF complex in the transcriptional regulation of the mEH gene (EPHX1). Studies also identified heterozygous mutations in human EPHX1 that resulted in a 95% decrease in mEH expression levels which was associated with a decrease in bile acid transport and severe hypercholanemia. In the present investigation we demonstrate that EPHX1 transcription is significantly inhibited by two heterozygous mutations observed in the Old Order Amish population that present numerous hypercholanemic subjects in the absence of liver damage suggesting a defect in bile acid transport into the hepatocyte. The identity of the regulatory proteins binding to these sites, established using biotinylated oligonucleotides in conjunction with mass spectrometry was shown to be poly(ADP-ribose)polymerase-1 (PARP-1) bound to the EPHX1 proximal promoter and a linker histone complex, H1.2/Aly, bound to a regulatory intron 1 site. These sites exhibited 71% homology and may represent potential nucleosome positioning domains. The high frequency of the H1.2 site polymorphism in the Amish population results in a potential genetic predisposition to hypercholanemia and in conjunction with our previous studies, further supports the critical role of mEH in mediating bile acid transport into hepatocytes.
... This would result in the expression of the primary basolateral bile acid uptake system (NTCP) being reduced in individuals with human cholestatic liver diseases. [21][22][23] Conversely, the results of this study demonstrated that significant up-regulation of NTCP mRNA occurred in the cases of early cholestasis following LDLT. This event would induce a significant increase of the hepatocellular damage. ...
Background : Intrahepatic cholestasis can occur early after living donor liver transplantation (LDLT). We investigated the changes in the expressions of the bile acid transporters and the liver histology in the patients who suffered with early cholestasis (EC) following LDLT. Methods : The histological differences between 15 graft livers with EC after LDLT and 5 graft livers with biliary stricture following LDLT were evaluated. The hepatic mRNA levels of the bile canaliculi transporters (BSEP, MRP2, MRP3, MDR1, MDR3, NTCP) in 40 (20 graft livers, 20 matched donor livers) liver biopsy tissues were analyzed by performing real-time reverse-transcription polymerase chain reaction (RT-PCR). Results : Microscopic examination revealed hepatocellular and/or bile canalicular cholestasis around acinar zone 3 in the livers of the patients with EC. In the livers with biliary stricture, the cholestasis was dominantly observed in the hepatocytic cytoplasm and in the bile ductules around the portal area rather than around acinar zone 3. The BSEP and MRP2 mRNA levels in the EC livers were significantly reduced by 44% and 23%, respectively (p=0.000), compared to the matched donor livers. The levels of MDR3 and NTCP mRNA in the EC livers increased by 738% (p=0.000) and 281% (p<0.01), respectively. The change of the expressions of the bile acid transporters in the patients with biliary stricture was less significant than that in the EC group. Conclusions : These results suggest that the altered expressions of the bile acid transporters may play a role in the pathogenesis of EC following LDLT.
... Bile acids are formed in liver from cholesterol, excreted into the bile, and mostly reabsorbed into the intestine. In enterohepatic circulation of bile acids, NTCP and BSEP have important roles in the uptake by the liver and in biliary excretion of bile acids, respectively (Wolkoff and Cohen, 2003), and single nucleotide polymorphisms in the BSEP gene have been responsible for progressive familial intrahepatic cholestasis type 2, which is characterized by cholestasis and jaundice (Strautnieks et al., 1998;Jansen et al., 1999). ...
It is useful to identify endogenous substrates for the evaluation of drug - drug interactions (DDIs) via transporters. In this study, we investigated the utility of bilirubins, substrates of organic anion transporting polypeptides (OATPs) and multidrug resistance-associated protein 2 (MRP2), and bile acids, substrates of sodium taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP), as biomarkers for the inhibition of transporters. In rats administered 20 and 80 mg/kg rifampicin, the plasma levels of bilirubin glucuronides were elevated, gradually decreased, and almost returned to the baseline level at 24 h after administration without an elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). This result indicates the transient inhibition of rOatps and/or rMrp2. Although the correlation between free plasma concentrations and IC50 values of rOatps depended on the substrates used in vitro studies, the inhibition of rOatps by rifampicin was confirmed in vivo study using valsartan as a substrate of rOatps. In rats administered 10 and 30 mg/kg cyclosporin A, the plasma levels of bile acids were elevated and persisted for up to 24 h after administration without an elevation of ALT and AST. This result indicates the continuous inhibition of rNtcp and/or rBsep, although there were differences between the free plasma or liver concentrations, and IC50 values of rNtcp or rBsep, respectively. This study suggests that the monitoring of bilirubins and bile acids in plasma is useful to evaluate the inhibitory potential for their corresponding transporters.
The American Society for Pharmacology and Experimental Therapeutics.
... Instead, microcystins accumulate in the liver via the bile acid transport system [7,14,17,18]. The bile acid transport system is comprised of proteins that actively transport peptides and biliary acids into hepatocytes [19]. The methyl-dehydroalanine (Mdha) and 3-amino-9-methyoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (ADDA) groups are integral to binding of microcystins to protein phosphatases in organisms ( Figure 1) [20]. ...
Microcystins are secondary metabolites produced by cyanobacteria that act as hepatotoxins in higher organisms. These toxins can be altered through abiotic processes, such as photodegradation and adsorption, as well as through biological processes via metabolism and bacterial degradation. Some species of bacteria can degrade microcystins, and many other organisms metabolize microcystins into a series of conjugated products. There are toxicokinetic models used to examine microcystin uptake and elimination, which can be difficult to compare due to differences in compartmentalization and speciation. Metabolites of microcystins are formed as a detoxification mechanism, and little is known about how quickly these metabolites are formed. In summary, microcystins can undergo abiotic and biotic processes that alter the toxicity and structure of the microcystin molecule. The environmental impact and toxicity of these alterations and the metabolism of microcystins remains uncertain, making it difficult to establish guidelines for human health. Here, we present the current state of knowledge regarding the alterations microcystins can undergo in the environment
... The most conclusive evidence that ntcp plays an important role in bile acid transport came from a study in which Xenopus laevis oocytes microinjected with rat liver cRNA in the presence of ntcp antisense RNA, had a 95% reduction in taurocholate uptake as compared to oocytes studied in the absence of the anti-sense construct (66,119). However, since this study was performed, more complete databases indicate that the antisense oligonucleotide that was used to inhibit ntcp expression was not specific for ntcp and could target other rat and Xenopus proteins in addition to ntcp (205). Of great interest is the recent observation that NTCP serves as the hepatocyte receptor for Hepatitis B virus (3,81,101,130,131,192,193,(214)(215)(216). ...
Many of the compounds taken up by the liver are organic anions that circulate tightly bound to protein carriers such as albumin. The fenestrated sinusoidal endothelium of the liver permits these compounds to have access to hepatocytes. Studies to characterize hepatic uptake of organic anions through kinetic analyses, suggested that it was carrier-mediated. Attempts to identify specific transporters by biochemical approaches were largely unsuccessful and were replaced by studies that utilized expression cloning. These studies led to identification of the organic anion transport proteins (oatps), a family of 12 transmembrane domain glycoproteins that have broad and often overlapping substrate specificities. The oatps mediate Na(+)-independent organic anion uptake. Other studies identified a seven transmembrane domain glycoprotein, Na(+)/taurocholate transporting protein (ntcp) as mediating Na(+)-dependent uptake of bile acids as well as other organic anions. Although mutations or deficiencies of specific members of the oatp family have been associated with transport abnormalities, there have been no such reports for ntcp, and its physiologic role remains to be determined, although expression of ntcp in vitro recapitulates the characteristics of Na(+)-dependent bile acid transport that is seen in vivo. Both ntcp and oatps traffic between the cell surface and intracellular vesicular pools. These vesicles move through the cell on microtubules, using the microtubule based motors dynein and kinesins. Factors that regulate this motility are under study and may provide a unique mechanism that can alter the plasma membrane content of these transporters and consequently their accessibility to circulating ligands. © 2014 American Physiological Society. Compr Physiol 4: 1715-1735, 2014.
... This process is described in detail in several recent review articles (Trauner 2003). Bile salts circulate in plasma are tightly bound to albumin and lipoproteins such as high-density lipoprotein (Wolkoff and Cohen 2003). NTCP accounts for the bulk (about 90 %) of BA uptake and is the first cloned BA transporter . ...
Bile acids entering into enterohepatic circulating are primary acids synthesized from cholesterol in hepatocyte. They are secreted actively across canalicular membrane and carried in bile to gallbladder, where they are concentrated during digestion. About 95 % BAs are actively taken up from the lumen of terminal ileum efficiently, leaving only approximately 5 % (or approximately 0.5 g/d) in colon, and a fraction of bile acids are passively reabsorbed after a series of modifications in the human large intestine including deconjugation and oxidation of hydroxy groups. Bile salts hydrolysis and hydroxy group dehydrogenation reactions are performed by a broad spectrum of intestinal anaerobic bacteria. Next, hepatocyte reabsorbs bile acids from sinusoidal blood, which are carried to liver through portal vein via a series of transporters. Bile acids (BAs) transporters are critical for maintenance of the enterohepatic BAs circulation, where BAs exert their multiple physiological functions including stimulation of bile flow, intestinal absorption of lipophilic nutrients, solubilization, and excretion of cholesterol. Tight regulation of BA transporters via nuclear receptors (NRs) is necessary to maintain proper BA homeostasis. In conclusion, disturbances of enterohepatic circulation may account for pathogenesis of gallstones diseases, including BAs transporters and their regulatory NRs and the metabolism of intestinal bacterias, etc.
... Hepatobiliary transport processes are crucial for the secretion and elimination of toxic compounds (e.g., drugs, carcinogens, and endobiotics) (6,7). Numerous drugs act as both substrates and inhibitors of hepatic transporters such that unexpected and unwanted interactions are frequently observed (8,9), including toxic concentrations of bile acids or substrate drugs in the blood or liver (10). ...
Unlabelled:
Hepatic transport of (99m)Tc-mebrofenin through organic anion transport protein 1a and 1b (Oatp1a/1b) and multidrug resistance protein 2 (Mrp2) was investigated by small-animal SPECT. On the basis of the results, a noninvasive method to visualize and quantify disturbances in hepatic transport is proposed.
Methods:
Friend virus B wild-type mice (untreated, bile duct-ligated, vehicle- or rifampicin-treated) and strain-matched knockout mice unable to express the uptake transporters Oatp1a/1b (Slco1a/1b(-/-)/(-/-)) or the efflux transporter Mrp2 (Abcc2(-/-)) were intravenously injected with (99m)Tc-mebrofenin (n = 3 per group). After dynamic small-animal SPECT and short CT acquisitions, time-activity curves of the liver and of the gallbladder and intestines were obtained and correlated with direct blood samples.
Results:
Normal hepatobiliary clearance of (99m)Tc-mebrofenin was severely impaired in the bile duct-ligated animal, as evidenced by elevated hepatic tracer levels. In Slco1a/1b(-/-)/(-/-) mice, a lower area under the curve (AUC) for the liver (P = 0.014) was obtained and no activity was detected in the gallbladder and intestines. Renal rerouting was observed, along with an increase in the blood AUC (P = 0.01). Abcc2(-/-) mice had a higher liver AUC (P = 0.009), a delayed emergence time of (99m)Tc-mebrofenin in the gallbladder (P = 0.009), and a lower AUC for the gallbladder and intestines (P = 0.001). The blood curve was similar to that of wild-type mice. (99m)Tc-mebrofenin disposition was altered after rifampicin treatments. We observed a dose-dependent delayed time point at which tracer maximized in liver, an increased AUC for liver, and a lower AUC for gallbladder and intestines (P = 0.042, 0.034, and 0.001, respectively, highest dose). Emergence in the gallbladder occurred later (P = 0.009, highest dose), and blood AUC was higher (P = 0.006).
Conclusion:
The current study visualized and quantified hepatic uptake and biliary efflux of (99m)Tc-mebrofenin. Our results demonstrated the possibility of discriminating, on a quantitative level, between lack of functional activity of sinusoidal uptake versus that of biliary efflux transporters.
... Together with CYPs, these transporters mediate clearance of drugs from liver. Bsep, Mdr1a, and Mrp2 are efflux transporters which are present on the apical canalicular membrane [34,35]. Oatp1 and ntcp are located in the basolateral membrane and act as influx transporters. ...
... Moreover, condition in which the content of cMOAT on the canalicular domain of hepatocytes has been decreased, another transporter protein, Mrp 3 , localized on the basolateral domain of plasmatic membrane, increases the TCA efflux from hepatocytes. It is suggested that this process is a compensatory mechanism for deletion of bile acids from hepatocytes ( Wolkoff and Cohen 2003). ...
One of the synthetic analogues of antidiuretic hormone-desmopressin is used in patients with central diabetes insipidus and in those with coagulation disorders. However, its effects on bile secretion are not fully defined. We investigated the effect of desmopressin on bile formation and determined the role of V1a vasopressin receptors in the action of desmopressin on choleresis. Rats were injected intraportally with a bolus of desmopressin; the changes of bile flow, the content of free and conjugated cholates were compared with control animal group. Selective antagonist of V1a receptors was injected 10 minutes before desmopressin treatment and the findings were compared with the results after desmopressin injection alone. Desmopressin increased bile flow, secretion of total cholates like amino acids conjugated, while diminished free bile acids content. Secreted bile volume and conjugated bile acids content were reduced in V1a receptors antagonist+desmopressin-treated rats. In contrast, free bile acids content was more than the results in desmopressin-treated rats. Desmopressin at concentrations nearly equal to physiological concentrations of natural hormone in blood shows its choleretic effect. Antagonist of V1a vasopressin receptors modulates desmopressin action. This certifies the role of these receptors in the action of desmopressin on different processes of bile formation.
... The liverspecific transporters, which showed more than twofold higher mRNA expression in liver than in kidney, included Ntcp/Slc10a1 and Oatp1b2/Slco1b2, which have major roles in the uptake of bile acids from the systemic circulation into hepatocytes in an Na þ -dependent and independent manner, respectively. 19 Oatp1b2 also functions in the elimination pathway of xenobiotics, particularly amphipathic organic anions, with structural diversities. 20 The four transporters with more than twofold higher expression in the kidney than in the liver were amino-acid transporters, B 0 AT1/Slc6a19, XT3/Slc6a20, LAT2/Slc7a8, and b 0, þ AT/Slc7a9. ...
DNA methylation-dependent gene silencing is one of the most characterized mechanisms in epigenetic regulation of gene expression. This process is thought to influence the ability of hepatocyte nuclear factor 1 (HNF1) to transactivate organic anion transporter expression in the liver and kidney. To evaluate this further we profiled 282 mouse solute carrier transporters by examining regions near their transcription start sites for tissue-dependent differentially methylated regions (T-DMR) using restriction tag-mediated amplification to determine T-DMR disparity between the liver and kidney. Forty-two of these were associated with T-DMR tags hypomethylated in the kidney but hypermethylated in the liver. Computational analysis found a canonical HNF1-binding motif within 1 kbp of the promoter region of 13 carriers including the amino acid transporters Slc6a19, Slc6a20, Slc7a8 and Slc7a9; all expressed predominantly in the kidney. Bisulfite genomic sequencing found that CpG dinucleotides neighboring the T-DMR tags were hypomethylated in the kidney compared with the liver. The Hnf1alpha promoter region itself contained a T-DMR hypomethylated in the liver and kidney but hypermethylated in the cerebrum, consistent with the tissue distribution of Hnf1alpha. Taken together, our results show a central role of DNA methylation in the kidney-specific expression of amino acid transporters thus determining both the tissue distribution of their master regulator, Hnf1alpha, and its interaction with downstream genes.
... Following the rapid structural alteration, unconjugated bile acids are passively reabsorbed at the large intestine (Mekhjian et al., 1979). Together with those conjugated bile acids actively uptaken by ASBT at the distal ileum (Kramer et al., 2001; Wang et al., 2001), bile acids are bound to plasma albumin and returned back to the liver through portal venous circulation (Cowen et al., 1975; Wolkoff and Cohen, 2003). Bile acids are then efficiently absorbed by hepatocytes via the basolateral transporters. ...
... We used D-REAM to investigate the DNA methylation status in the 5Ј-flanking region of almost all mouse SLC and ABC transporter genes in the liver, kidney, and cerebrum and found that the liver-specific transporters, organic anion transporting polypeptide 1b2 (Oatp1b2)/Slco1b2, Na ϩ -taurocholate-cotransporting polypeptide (Ntcp)/Slc10a1, bile salt export pump (Bsep)/Abcb11, Abcg5, and Abcg8, possess T-DMRs hypomethylated in the liver. Oatp1b2 and Ntcp mediate the sodium-independent and -dependent uptake of amphipathic organic anions and bile acids, respectively (Wolkoff and Cohen, 2003;Evers and Chu, 2008). Bsep is an ABC transporter that plays a pivotal role in the canalicular efflux of bile acids (Trauner and Boyer, 2003). ...
Tissue-specific expression of transporters is tightly linked with their physiological functions through the regulation of the membrane transport of their substrates. We hypothesized that epigenetic regulation underlies the tissue-specific expression of mouse liver-specific transporters (Oatp1b2/Slco1b2, Ntcp/Slc10a1, Bsep/Abcb11, and Abcg5/g8). We examined their DNA methylation and histone modification profiles near the transcriptional start site (TSS) in the liver, kidney, and cerebrum. Genome-wide DNA methylation profiling with tissue-dependent differentially methylated region profiling with restriction tag-mediated amplification and subsequent bisulfite genomic sequencing demonstrated that the CpG dinucleotides around the TSS of Oatp1b2 (from -515 to +149 CpGs), Ntcp (from -481 to +495 CpGs), Bsep (from -339 to +282 CpGs), and Abcg5/g8 (from -161 to +5 CpGs for Abcg5, i.e., from -213 to -48 CpGs for Abcg8) were hypomethylated in the liver and hypermethylated in the kidney and cerebrum. The opposite pattern was observed for Pept2/Slc15a2 (from -638 to +4 CpGs), which was expressed in the kidney and cerebrum but not in the liver. These DNA methylation profiles are consistent with the tissue distribution of these transporters. A chromatin immunoprecipitation assay demonstrated that the histone H3 associated with Oatp1b2, Ntcp, Bsep, and Abcg5/g8 promoters was hyperacetylated in the liver but was acetylated very little in the kidney and cerebrum, whereas the upstream region of Pept2 was hyperacetylated only in the kidney and cerebrum. These results suggest the involvement of epigenetic systems in the tissue-specific expression of mouse transporters Oatp1b2, Ntcp, Bsep, Abcg5/g8, and Pept2.
... Transport of solutes from blood to bile is a vital liver function. It is through bile synthesis and transport that xenobiotics of environmental origin and endogenous metabolic by-products are either safely removed from the system, or, with systemic accumulation, result in morbidity and mortality [4,[68][69][70][71][72][73][74]. Inhibition/impairment of bile transport (cholestasis) commonly results in morbidity and mortality in mammals. ...
A novel transparent stock of medaka (Oryzias latipes; STII), recessive for all pigments found in chromatophores, permits transcutaneous imaging of internal organs and tissues in living individuals. Findings presented describe the development of methodologies for non invasive in vivo investigation in STII medaka, and the successful application of these methodologies to in vivo study of hepatobiliary structure, function, and xenobiotic response, in both 2 and 3 dimensions.
Using brightfield, and widefield and confocal fluorescence microscopy, coupled with the in vivo application of fluorescent probes, structural and functional features of the hepatobiliary system, and xenobiotic induced toxicity, were imaged at the cellular level, with high resolution (< 1 microm), in living individuals. The findings presented demonstrate; (1) phenotypic response to xenobiotic exposure can be investigated/imaged in vivo with high resolution (< 1 microm), (2) hepatobiliary transport of solutes from blood to bile can be qualitatively and quantitatively studied/imaged in vivo, (3) hepatobiliary architecture in this lower vertebrate liver can be studied in 3 dimensions, and (4) non invasive in vivo imaging/description of hepatobiliary development in this model can be investigated.
The non-invasive in vivo methodologies described are a unique means by which to investigate biological structure, function and xenobiotic response with high resolution in STII medaka. In vivo methodologies also provide the future opportunity to integrate molecular mechanisms (e.g., genomic, proteomic) of disease and toxicity with phenotypic changes at the cellular and system levels of biological organization. While our focus has been the hepatobiliary system, other organ systems are equally amenable to in vivo study, and we consider the potential for discovery, within the context of in vivo investigation in STII medaka, as significant.
Ursodeoxycholic acid (UDCA) is a natural substance physiologically produced in the liver. Initially used to dissolve gallstones, it is now successfully used in treating primary biliary cirrhosis and as adjuvant therapy for various hepatobiliary cholestatic diseases. However, the mechanisms underlying its beneficial effects are not entirely clear. Evidence suggests three mechanisms of action for UDCA that could benefit humans with cholestatic liver disease (CLD): protection of cholangiocytes against hydrophobic bile acid (BA) cytotoxicity, stimulation of hepatobiliary excretion, and protection of hepatocytes against BA-induced apoptosis. These mechanisms may act individually or together to potentiate them. At the molecular level, it has been observed that UDCA can generate modifications in the transcription and translation of proteins essential in the transport of BA, correcting the deficit in BA secretion in CLD, in addition to activating signaling pathways to translocate these transporters to the sites where they should fulfill their function. Inhibition of BA-induced hepatocyte apoptosis may play a role in CLD, characterized by BA retention in the hepatocyte. Thus, different mechanisms of action contribute to the improvement after UDCA administration in CLD. On the other hand, the effects of UDCA on tissues that possess receptors that may interact with BAs in pathological contexts, such as skeletal muscle, are still unclear. The objective of this work seeks to describe the main molecular mechanisms by which UDCA acts in the human body, emphasizing the interaction in other tissues than the liver.
Ambiguity associated with cholesterol portrays its significance and concern. Cholesterol is a steroid biomolecule of animal cells with several important functions in living system, such as steroid hormone production, structural architecture of cell membranes, production of vitamin D, sources of bile salts and bile acids. Its sources include endogenous (de novo production by body’s tissues) and exogenous (dietary), contributing to cholesterol pool in the body. Cholesterol homeostasis is essentially regulated by the body. This review focuses on the following; basis of cholesterol, biological functions of cholesterol, structural description of cholesterol, Biosynthesis of cholesterol, cholesterol and its derivatives e.g bile acids, bile salts, steroid hormone, absorption and utilization of dietary cholesterol and current advancement in cholesterol management against risk factors. Hypercholesterolaemia is known to be an important precondition to the etiology of cardiovascular disorders which include atherosclerosis, stroke and coronary heart diseases. Interventions for the management and prevention of hypercholesterolaemia currently advocated include pharmaceuticals (drugs) and non pharmaceuticals, but more concern on non pharmacological therapeutic interventions such as the use of Medicinal Plants and herbs, Nutraceuticals, Diet and Exercises (lifestyle) and Functional and Mediterranean Foods. It is thus glaring that the disease linked implication of cholesterol can be prevented and managed using the appropriate interventions. Peer Review History: Received 4 November 2020; Revised 12 Decembe; Accepted 2 January, Available online 15 January 2021 UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency. Received file: Comments of reviewer(s): Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 8.0/10 Reviewer(s) detail: Dr. Rawaa Souhil Al-Kayali, Aleppo University, Syria, rawah67@hotmail.com Dr. U. S. Mahadeva Rao, Universiti Sultan Zainal Abidin, Terengganu Malaysia, raousm@gmail.com Similar Articles: USE OF DIOSGENIN, YAMOGENIN, TIGOGENIN AND NEOTIGOGENIN FOR TREATMENT OF HYPERLIPIDEMIA BY INHIBITING CHOLESTEROL ABSORPTION IN GIT
this work is therefore aimed at determining the level of lipid profile in automobile mechanics.
The liver is the body’s largest internal organ. It plays a vital role in many metabolic processes. The liver has a unique vascular supply with most of its blood coming from the portal venous circulation. The distribution of the portal vein and hepatic artery (which supplies the liver), hepatic vein (which drains the liver), and bile ducts (transport out of the liver) form a unique pattern. This architectural pattern is important to keep in mind as it impacts various metabolic processes of the liver, disease occurrence, and surgical options for intervention (if required). The liver performs complex functions of synthesizing and metabolizing carbohydrates, protein, and lipids. In addition, the liver plays a significant role in modification of proteins and drugs to their biologically active form (which can be used by the body). In addition to modification, the liver is involved in detoxification and filtration of drugs out of the body. Due to the myriad processes the liver is involved in, there are no specific tests or tools that can be used to comprehensively evaluate its function.
Microcystins (MCYs) are cyanobacterial heptapeptides known for their high toxicity in eukaryotic cells and for their potential human health hazards. They are potent and specific inhibitors of type 1 and 2A, serine-threonine protein phosphatases (PP1 and PP2A) and as such, interfere with key cellular and metabolic events. Moreover, they induce oxidative stress involving reactive oxygen species (ROS) generation. Their cytoskeletal effects involve both mitotic and differentiated eukaryotic cells. The main objective of the present review is to summarize the most important cytoskeletal effects of MCY on human, animal and plant cells known to date and to give an insight into the cellular and molecular background of these alterations. Disruptions of microtubule (MTs), microfilament (MF) and intermediate filament (IF) organization have all been described, having consequences on cell shape, tissue integrity and functionality and mitotic division. Most of these subcellular changes are closely related to PP1 and PP2A inhibition and involve misfunctioning of cytoskeleton associated proteins. However, several cytoskeletal alterations are likely to be related to the induction of oxidative stress. MCY induced changes in MT, MF and IF assembly may have severe human health consequences. The main target of cyanotoxin in human/ animal cells is liver and cytoskeletal disruption alters structure and functioning of hepatocytes. However, many other cell types undergo alterations similar to those observed in hepatocytes. Both PP1/PP2A inhibition and ROS generation are involved and the activation of mitogen activated protein kinases (MAPKs) seem to play a crucial role in the molecular events leading to cytoskeletal disruption.
The role of microRNAs (miRs) in cellular processes such as differentiation, apoptosis, and proliferation as well as their alteration in diseases such as cancer are well known. However, there is little knowledge about the role of miRs in lipid metabolism and associated disorders. Recently, it has been shown that these molecules play an important role in lipid homeostasis by regulating post-transcriptional level expression of genes involved in this metabolism. It has also been observed that lipoproteins, mainly high-density lipoproteins (HDL), are capable of transporting miRs, enabling cellular communication between distant tissues, establishing a mutual regulation. Understanding the regulatory mechanisms involved in these processes opens up new possibilities for the development of therapeutic approaches for metabolic and cardiovascular disorders.
The liver is a complex organ with multiple functions and no single test can be used to assess its function. Such complexity is exemplified by the absence of any long-term means of artificial support short of organ transplantation, in contrast to the options available when loss of other vital organs occur. A variety of investigations are available to evaluate liver function but several have become obsolete due to their cost, complexity and substitution by modern imaging techniques and are not discussed here. This chapter focuses on the principal aspects of liver function and reviews common liver biochemical tests. From this chapter, readers should gain an excellent knowledge of the cardinal functions of the liver, be comfortable with the interpretation of abnormal liver biochemistries and also be cognizant of the limitations of these investigations. A thorough history and physical examination is always the first step in the evaluation of abnormalities in liver function and should guide physicians towards a prompt and cost-effective evaluation and diagnosis.
Lipid droplets (LDs) are phylogenetically conserved cytoplasmic organelles that store neutral lipids within a phospholipid monolayer. LDs compartmentalize lipids and may help to prevent cellular damage caused by their excess or bioactive forms. FIT2 is a ubiquitously expressed transmembrane endoplasmic retiuculum (ER) membrane protein that has previously been implicated in LD formation in mammalian cells and tissue. Recent data indicate that FIT2 plays an essential role in fat storage in an in vivo constitutive adipose FIT2 knockout mouse model, but the physiological effects of postnatal whole body FIT2 depletion have never been studied. Here we show that tamoxifen-induced FIT2 deletion using a whole body ROSA26CreERT2-driven FIT2 knockout (iF2KO) mouse model leads to lethal intestinal pathology including villus blunting and death of intestinal crypts, and loss of lipid absorption. iF2KO mice lose weight and die within two weeks after the first tamoxifen dose. At the cellular level, LDs failed to form in iF2KO enterocytes after acute oil challenge, and instead accumulated within the ER. Intestinal bile acid (BA) transporters were transcriptionally dysregulated in iF2KO mice, leading to the buildup of BAs within enterocytes. These data support the conclusion that FIT2 plays an essential role in regulating intestinal health and survival postnatally.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Bile is an essential fluid that delivers bile acids, lipids, and metabolites to the intestine. It is formed by a complex mechanism that requires hepatocyte secretion of bile acids and other solutes, such as glutathione and bicarbonate, and bile ductular secretion of bicarbonate. Approximately 95% of bile acids is recovered from the intestine and then reused (enterohepatic circulation). Although there is a small amount of passive absorption of bile acids in the proximal small intestine, the major site of absorption is the distal ileum where there is an active bile acid transport mechanism. Much of what we know about hepatocyte transport mechanisms that are essential for the enterohepatic cycling of bile acids has been learned from patients with specific inheritable defects in this process. Discovery of the transporters mediating enterohepatic cycling has opened new windows into diagnosis and treatment of hepatobiliary disorders.
Epidemiological studies have shown that low plasma levels of High Density Lipoprotein Cholesterol (HDL-C) are associated with an increased risk for myocardial infarction. These studies suggested that by increasing HDL-C levels one could reduce cardiovascular risk. However, emerging evidence from studies in animals and humans indicate that high levels of HDL-C are not sufficient to confer atheroprotection but that the functionality of the HDL particles is equally important. The picture is complicated further by the finding that HDL functionality is compromised in patients with chronic inflammatory diseases such as Coronary Artery Disease (CAD), diabetes and rheumatoid arthritis. Despite these obstacles, HDL raising is still a promising strategy for the reduction of CAD risk. Low HDL-C can be caused by inactivating mutations in apoA-I, ATP Binding Cassette Transporter A1 (ABCA1) or Lecithin-Cholesterol Acyl Transferase (LCAT) which affect HDL biogenesis and maturation whereas high HDL can be caused by mutations in Cholesteryl Ester Transfer Protein (CETP) or Scavenger receptor Class B Type I (SR-BI). Recent studies suggest that heterogeneity in HDL levels in the population is polygenic in origin. One approach to raise plasma HDL-C is to increase the rate of HDL biosynthesis by capitalizing on the mechanisms that control the transcription of genes that play key roles in HDL biogenesis. We review some of the genetic and non-genetic factors that affect plasma HDL levels and functions and discuss the mechanisms that regulate HDL metabolism at the level of gene transcription in the liver focusing on apoA-I, ABCA1 and apoM.
This chapter discusses cellular mechanisms involved in hepatic solute transport, bile formation and cholestasis. Bile formation involves vectorial transport of solutes from blood to bile, and depends on the coordinated activities of various solute transporters located at the basolateral and apical membranes of hepatocytes and cholangiocytes. Cholestasis is the result of compromised vectorial transport of solutes destined for bile. Cholestasis can be divided into two major categories—extrahepatic and intrahepatic cholestasis. Extrahepatic cholestasis results from overt extrahepatic blockage or from duct obstruction visible at the microscopic level. After bile duct obstruction, constituents of bile are secreted for a period of time. The back pressure of obstruction may disrupt canalicular tight junctions leading to regurgitation of biliary contents into the bloodstream. While physical obstruction to bile flow underlies extrahepatic cholestasis, the mechanism of intrahepatic cholestasis entails alterations in cellular mechanisms involved in bile formation. Intrahepatic cholestasis is produced by a number of agents, like estrogen, anabolic steroids, certain bile acids, or endotoxins. Hepatic solute transport is, in turn, dependent on cellular mechanisms responsible for the synthesis and delivery of transporters to the appropriate membrane domains, maintenance of both ion gradients, and adequate energy supply.
The expression and activities of hepatobiliary transporter genes is a critical component of liver function. Both sinusoidal
and canalicular membrane transporters are responsible for the coordinated transport of a wide variety of organic anions, drugs,
toxins, endobiotics and bile acids that ultimately are metabolized by hepatocytes and secreted into bile. Over the past decade,
a tremendous amount of new information has been uncovered regarding the molecular identification of hepatobiliary transporter
genes responsible for the delivery of the principal solutes in bile, and therefore the mechanisms underlying the generation
of bile. Along with the cloning and identification of critical hepatobiliary transporter genes has come the capability of
exploring, and possibly modifying, the molecular mechanisms driving alterations in transponer gene expression in health and
disease. Regulation at the level of mRNA transcription initiation is becoming increasingly recognized as the predominant means
governing transporter gene expression. An expanding role for Hepatocyte nuclear factor la and several members of the nuclear
receptor superfamily of ligand-regulatable transcription factors permits coordination of expression of multiple transporter
genes. In particular, the recently-identified nuclear receptor for bile acids, FXR, serves as both sensor and effector to
help maintain intracellular bile acid homeostasis. These findings have significant importance in our current understanding,
and as a means of directing future therapy, of liver diseases where cholestasis is a prominent feature.
Bile salt export pump (BSEP/ABCB11), a member of the family of ATP-binding cassette transporters, is localized on the canalicular membrane of hepatocytes and mediates the efficient biliary excretion of bile acid.The secretion of bile acid into bile by BSEP provides the primary osmotic driving force for bile flow generation. Intrahepatic cholestasis resulting from dysfunction of BSEP can be caused by a mutation in the gene encoding this protein orby acquired factors,such as the side effects of xenobiotics and drugs. In some pathophysiological states,inhibition of BSEP function isassociated with its reduced expression on the canalicular membrane caused by impaired trafficking and sorting of BSEP.This fact has generated interest in better understanding the trafficking and sorting mechanism of BSEP.This review describes the molecular characteristics and physiological roles of BSEP, the trafficking and sorting machinery of BSEP, and themechanisms responsible for disturbance ofBSEP, which causes intrahepatic cholestasis.
The liver parenchyma functions as both an exocrine gland producing excretory products to be secreted into the biliary duct
system, and an endocrine gland, synthesising products to be directly delivered to the blood. Delicate canaliculi (BC) bordered
by the apical liver cell surfaces build up the initial parts of the bile transport system visible in the central part of both
panels A and B.
The purpose of this study was to determine the 12-h fasting preprandial and 2-h postprandial serum bile acid concentration (SBAC) reference intervals for healthy, adult rhesus macaques (Macaca mulatta). We hypothesized that the mean 2-h postprandial SBAC would be significantly higher than the mean preprandial SBAC. We included 40 (24 male, 16 female) macaques after confirming that their health records, physical examinations, CBC, serum chemistry panels, and urinalyses were all within normal limits. In addition, hepatitis A titers were determined, an ultrasound examination of the liver was performed, and two 16-gauge ultrasound guided percutaneous liver biopsies were collected and submitted for histopathology. Macaques were confirmed healthy according to hepatitis A screens and sonographic and histologic evaluation of hepatic tissue. Within 2 wk of the screening procedures, preprandial and postprandial SBACs were measured. Preprandial SBAC (mean ± 1 SD) was 11.1 ± 1.9 μmol/L and postprandial SBAC was 19.7 ± 8.0 μmol/L, which was significantly higher than the preprandial value. Sex and hepatitis titers did not significantly influence preprandial and postprandial SBAC. The current study indicates that the SBAC reference values for rhesus macaques are higher than those reported for humans and companion animals.
IntroductionHepatocyte Uptake of Bile AcidsHepatocyte Uptake of Non-Bile Acid Organic AnionsStudies of OATP1A1 as a Prototypical Member of the OATP FamilySummaryAcknowledgmentsReferences
Small aquarium fishes are increasingly used as animal models, and one of these, the Japanese Medaka (Oryzias latipes), is frequently utilized for toxicity testing. While these vertebrates have many similarities with their terrestrial counterparts, there are differences that must be considered if these organisms are to be used to their highest potential. Commonly, testing may employ either the developing embryo or adults; both are easy to use and work with. To illustrate the utility and breadth of toxicity testing possible using medaka fish, we present protocols for assessing neurotoxicity in developing embryos, evaluating toxicant effects on sexual phenotype after treatment with endocrine-disrupting chemicals by sexual genotyping, and measuring hepatotoxicity in adult fish after treatment with a model hepatotoxicant. The methods run the gamut from immunohistology through PCR to basic histological techniques.
Although inflammation is known to impose changes in the expression and activity of drug transporters, little is known about the impact of inflammatory stimuli on these transporters during pregnancy. Our objective was to study the effect of viral-induced inflammation on key maternal hepatic and placental drug transporters and their endogenous substrates. Acute inflammation was induced in pregnant Sprague-Dawley rats (gestation day 17-18, n = 5-6/group) by single intraperitoneal doses of polyinosinic/polycytidylic acid [poly(I:C)] (2.5 or 5.0 mg/kg) with saline as a control. Tissues were harvested 24 h later. Expression of transporters was measured via real-time polymerase chain reaction and Western blotting. Maternal plasma levels of cytokines, bile acids, and bilirubin and fetal levels of bile acids were examined. Plasma concentrations of interferon-γ, tumor necrosis factor-α, and interleukin-6 were significantly induced in poly(I:C)-treated rats, compared with controls (p < 0.001). Significant down-regulation of placental Abcb1a/b, Abcc1, Abcc3, Abcg2, Slco1a4, and Slco4a1 mRNA and of hepatic Abcc2, Abcg2, Slco1a4, Slc10a1, and Cyp3a2 mRNA was observed in poly(I:C)-treated rats. Hepatic Abcb1b and Abcc3 mRNA levels were significantly induced. Hepatic protein levels of P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance protein were significantly down-regulated relative to those for controls (p < 0.05). Total bile acids in maternal plasma were significantly increased at the higher dose of poly(I:C). In summary, the poly(I:C) model of viral infection imposes significant changes in the expression of key drug transporters in placental and hepatic tissues of pregnant rats. Because many clinically important endogenous and exogenous compounds are substrates of these transporters, inflammation-mediated changes in transporter expression could affect their maternal disposition and fetal exposure.
Microsomal epoxide hydrolase (mEH) is a conserved enzyme that is known to hydrolyze many drugs and carcinogens, and a few endogenous steroids and bile acids. mEH-null mice were produced and found to be fertile and have no phenotypic abnormalities thus indicating that mEH is not critical for reproduction and physiological homeostasis. mEH has also been implicated in participating in the metabolic activation of polycyclic aromatic hydrocarbon carcinogens. Embryonic fibroblast derived from the mEH-null mice were unable to produce the proximate carcinogenic metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens. They were also resistant to DMBA-mediated toxicity. Using the two-stage initiation-promotion skin cancer bioassay, the mEH-null mice were found to be highly resistant to DMBA-induced carcinogenesis. In a complete carcinogenesis bioassay, the mEH mice were totally resistant to tumorigenesis. These data establish in an intact animal model that mEH is a key genetic determinant in DMBA carcinogenesis through its role in production of the ultimate carcinogenic metabolite of DMBA, the 3,4-diol-1,2-epoxide.
Canalicular secretion of bile salts is a vital function of the vertebrate liver, yet the molecular identity of the involved ATP-dependent carrier protein has not been elucidated. We cloned the full-length cDNA of the sister of P-glycoprotein (spgp; Mr approximately 160,000) of rat liver and demonstrated that it functions as an ATP-dependent bile salt transporter in cRNA injected Xenopus laevis oocytes and in vesicles isolated from transfected Sf9 cells. The latter demonstrated a 5-fold stimulation of ATP-dependent taurocholate transport as compared with controls. This spgp-mediated taurocholate transport was stimulated solely by ATP, was inhibited by vanadate, and exhibited saturability with increasing concentrations of taurocholate (Km approximately 5 microM). Furthermore, spgp-mediated transport rates of various bile salts followed the same order of magnitude as ATP-dependent transport in canalicular rat liver plasma membrane vesicles, i.e. taurochenodeoxycholate > tauroursodeoxycholate = taurocholate > glycocholate = cholate. Tissue distribution assessed by Northern blotting revealed predominant, if not exclusive, expression of spgp in the liver, where it was further localized to the canalicular microvilli and to subcanalicular vesicles of the hepatocytes by in situ immunofluorescence and immunogold labeling studies. These results indicate that the sister of P-glycoprotein is the major canalicular bile salt export pump of mammalian liver.
Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5'-O-(thiotriphosphate) ([35S]ATP gamma S) allows the identification of ATP-binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATP gamma S and with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]-cholan-24-oyl-2'- aminoethanesulfonate followed by immunoprecipitation with a monoclonal antibody (Be 9.2) revealed the identity of the ATP-binding and the bile salt-binding canalicular membrane glycoprotein with the apparent Mr of 110,000 (gp110). The isoelectric point of this glycoprotein was 3.7. Transport of bile salt was studied in vesicles enriched in canalicular and sinusoidal liver membranes. Incubation of canalicular membrane vesicles with [3H] taurocholate in the presence of ATP resulted in an uptake of the bile salt into the vesicles which was sensitive to vanadate. ATP-dependent taurocholate transport was also observed in membrane vesicles from mutant rats deficient in the ATP-dependent transport of cysteinyl leukotrienes and related amphiphilic anions. Substrates of the P-glycoprotein (gp170), such as verapamil and doxorubicin, did not interfere with the ATP-dependent transport of taurocholate. Reconstitution of purified gp110 into liposomes resulted in an ATP-dependent uptake of [3H]taurocholate. These results demonstrate that gp110 functions as carrier in the ATP-dependent transport of bile salts from the hepatocyte into bile. This export carrier is distinct from hitherto characterized ATP-dependent transport systems.
Phalloidin, a bicyclic heptapeptide, and antamanide, a monocyclic decapeptide from the poisonous mushroom Amanita phalloides, interact with bile-salt-binding polypeptides of the hepatocyte membrane, as demonstrated by photoaffinity labeling using the photolabile bile salt derivative 7,7,-azo-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid, either unconjugated or taurine conjugated. With the photolabile derivatives of phalloidin, N-delta-(4-[(1-azi-2,2,2-trifluoroethyl) benzoyl]-beta-alanyl)-delta-aminophalloin, (N epsilon-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]lys6)-anta manide, the same membrane polypeptides with apparent MrS of 54,000 and 48,000 were labeled as with the photolabile derivatives of unconjugated and conjugated bile salts. The presence of bile salts decreased markedly the extent of labeling of these phalloidin- and antamanide-binding polypeptides. These results indicate that hepatic uptake systems for bile salts, phallotoxins, and the cycloamanide antamanide are identical, thus explaining the organotropism of phallotoxins.
In an effort to characterize the hepatocyte bile acid transport system, a photoreactive derivative of taurocholate, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid (7-ADTC) has been synthesized and its transport properties compared to those of the natural substrate. Both the bile acid and its synthetic analog were shown to be transported against an electrochemical gradient as well as a chemical gradient. Transport as a function of concentration and the presence of sodium indicated that both substrates were taken up by a sodium-dependent and a sodium-independent route. Taurocholate had Km values of 26 and 57 microM and Vmax values of 0.77 and 0.15 nmol/mg of protein/min, respectively. In comparison, 7-ADTC had very similar kinetic properties with Km values of 25 and 31 microM and Vmax values of 1.14 and 0.27 nmol/mg of protein/min. Each compound was shown to inhibit competitively the transport of the other, suggesting that these substrates utilized a common membrane carrier. The transport properties of the photoreactive anion transport inhibitor, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) were also characterized in the hepatocyte system. Transport occurred via a sodium-dependent and a sodium-independent route with Km values of 210 and 555 microM and Vmax values of 0.57 and 1.62 nmol/mg of protein/min. As in the case of 7-ADTC, NAP-taurine and taurocholate were also shown to be mutual competitive inhibitors. In the absence of light, 7-ADTC was a reversible inhibitor of taurocholate uptake. Upon irradiation, irreversible photoinactivation of the taurocholate uptake system was observed. These results indicate that 7-ADTC and NAP-taurine can be utilized as photoaffinity probes for the identification of the bile acid carrier protein(s) in hepatocyte plasma membranes.
The interaction of human serum albumin with twelve bile acids (ba) has been studied by equilibrium dialysis technique using 3H- and 14C-labeled bile acids. The physiological bile acids studied were: cholic, chenodeoxycholic, deoxycholic, lithocholic, ursodeoxycholic, and 7-ketolithocholic acids, all in the free and conjugated (with glycine and taurine) forms. For each bile acid studied, the interaction was characterized by two classes of binding sites, the first consisting of 2--4 sites and the second of 8--30. K1 values (liter/mol) for the different bile acids were: cholic acid, 0.3 x 10(4); chenodeoxycholic acid, 5.5 x 10(4); deoxycholic acid, 4.0 x 10(4); ursodeoxycholic acid, 3.8 x 10(4); 7-ketolithocholic acid, 1.9 x 10(4); lithocholic acid, 20 x 10(4). The affinity constant of a bile acid for albumin decreases with an increase in the number of hydroxy groups and also with the replacement of 7-hydroxy by 7-keto groups. The affinity constant is similar for glycine and taurine conjugated bile acids, but is slightly higher for unconjugated than conjugated forms.
Recently two different bile-acid carriers for the hepatocellular sodium-dependent uptake of taurocholate have been described. The first transport system was isolated and characterized by functional expression cloning in Xenopus laevis oocytes. The corresponding cDNA clone, named Ntcp for Na+/taurocholate co-transporting polypeptide, codes for a protein of 362 amino acids and shows no similarity to previously known sequences. The transport function of this carrier system is well documented by expression in Xenopus laevis oocytes and by transient and stably transfected cell lines. In addition, several lines of evidence implied that the well-known xenobiotic-metabolizing enzyme microsomal epoxide hydrolase (mEH, EC 3.3.2.3) is also able to mediate sinusoidal uptake of taurocholate. Furthermore, it was claimed that the same enzyme also mediates the uptake of the conjugated bile acid into the smooth endoplasmic reticulum (ER). No direct proof of the transport function of mEH by its heterologous expression has yet been published. In the present work we used a stable transfected cell line that expressed high levels of heterologous mEH for uptake studies of various bile acids and the loop diuretic bumetanide. The uptake of the conjugated bile acid taurocholate, of the non-conjugated bile acid cholate and of the organic anion bumetanide was measured in the transfected as well as in the non-transfected parental cell line. These organic anions represent the main substrates of the known transport systems for organic anions in the rat liver. The results show that the microsomal epoxide hydrolase is unable to transport taurocholate, cholate or bumetanide. Furthermore, Western-blot analysis revealed the expression of mEH in hepatoma tumor cell lines, which show no transport activity for these organic anions. These results show that it is unlikely that mEH can mediate the transport of these substrates.
Previous studies have suggested that the enzyme microsomal epoxide hydrolase (mEH) is able to mediate sodium-dependent transport of bile acids such as taurocholate into hepatocytes (von Dippe, P., Amoui, M., Alves, C., and Levy, D.(1993) Am. J. Physiol. 264, G528-G534). In order to characterize directly the putative transport properties of the enzyme, a pCB6 vector containing the cDNA for this protein (pCB6-mEH) was transfected into Madin-Darby canine kidney (MDCK) cells, and stable transformants were isolated that could express mEH at levels comparable with the levels expressed in hepatocytes. Sodium-dependent transport of taurocholate was shown to be dependent on the expression of mEH and to be inhibited by the bile acid transport inhibitor 4,4'-diisothiocyanostilbene-2,2'disulfonic acid (DIDS), as well as by other bile acids. Kinetic analysis of this system indicated a Km of 26.3 microM and a Vmax of 117 pmol/mg protein/min. The Km value is essentially the same as that observed in intact hepatocytes. The transfected MDCK cells also exhibited sodium-dependent transport of cholate at levels 150% of taurocholate in contrast to hepatocytes where cholate transport is only 30% of taurocholate levels, suggesting that total hepatocyte bile acid transport is a function of multiple transport systems with different substrate specificities, where mEH preferentially transports cholate. This hypothesis is further supported by the observation that a monoclonal antibody that partially protects (26%) taurocholate transport from inhibition by DIDS in hepatocytes provides almost complete protection (88%) from DIDS inhibition of hepatocyte cholate transport, suggesting that taurocholate is also taken up by an alternative system not recognized by this antibody. Additional support for this concept is provided by the observation that the taurocholate transport system is almost completely protected (92%) from DIDS inhibition by this antibody in MDCK cells that express mEH as the only bile acid transporter. These results demonstrate that mEH is expressed on the surface of hepatocytes as well as on transfected MDCK cells and is able to mediate sodium-dependent transport of taurocholate and cholate.
Recent studies indicate that wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase, interferes with bile acid secretion in rat liver; taurocholate induces recruitment of ATP-dependent transporters to the bile canalicular membrane, and PI 3-kinase products are important in intracellular trafficking.
We investigated the role of PI 3-kinase in bile acid secretion by studying the in vivo effect of taurocholate, colchicine, and wortmannin on bile acid secretion, kinase activity, and protein levels in canalicular membrane vesicle (CMV) and sinusoidal membrane vesicle (SMV) fractions from rat liver. Treatment of rats or perfusion of isolated liver with taurocholate significantly increased PI 3-kinase activity in both membrane fractions. Taurocholate increased protein content of ATP-dependent transporters, which were detected only in CMVs, whereas increased levels of p85 and a cell adhesion molecule, cCAM 105, were observed in both fractions.
Colchicine prevented taurocholate-induced changes in all proteins studied, as well as the increase in PI 3-kinase activity in CMVs, but it resulted in further accumulation of PI 3-kinase activity, p85, and cCAM 105 in SMVs. These results indicate that taurocholate-mediated changes involve a microtubular system.
Wortmannin blocked taurocholate-induced bile acid secretion. The effect was more profound when wortmannin was administered prior to treatment with taurocholate. When wortmannin was given after taurocholate, the protein levels of each ATP-dependent transporter were maintained in CMVs, whereas the levels of p85 and cCAM decreased in both membrane fractions. Perfusion of liver with wortmannin before taurocholate administration blocked accumulation of all proteins studied in CMVs and SMVs.
These results indicate that PI 3-kinase is required for intracellular trafficking of itself, as well as of ATP-dependent canalicular transporters.
Microsomal epoxide hydrolase (mEH) is a bifunctional membrane protein that plays a central role in the metabolism of xenobiotics and in the hepatocyte uptake of bile acids. Numerous studies have established that this protein is expressed both in the endoplasmic reticulum and at the sinusoidal plasma membrane. Preliminary evidence has suggested that mEH is expressed in the endoplasmic reticulum (ER) membrane with two distinct topological orientations. To further characterize the membrane topology and targeting of this protein, an N-glycosylation site was engineered into mEH to serve as a topological probe for the elucidation of the cellular location of mEH domains. The cDNAs for mEH and this mEH derivative (mEHg) were then expressed in vitro and in COS-7 cells. Analysis of total expressed protein in these systems indicated that mEHg was largely unglycosylated, suggesting that expression in the ER was primarily of a type I orientation (Ccyt/Nexo). However, analysis, by biotin/avidin labeling procedures, of mEHg expressed at the surface of transfected COS-7 cells, showed it to be fully glycosylated, indicating that the topological form targeted to this site originally had a type II orientation (Cexo/Ncyt) in the ER. The surface expression of mEH was also confirmed by confocal fluorescence scanning microscopy. The sensitivity of mEH topology to the charge at the N-terminal domain was demonstrated by altering the net charge over a range of 0 to +3. The introduction of one positive charge led to a significant inversion in mEH topology based on glycosylation site analysis. A truncated form of mEH lacking the N-terminal hydrophobic transmembrane domain was also detected on the extracellular surface of transfected COS-7 cells, demonstrating the existence of at least one additional transmembrane segment. These results suggest that mEH may be integrated into the membrane with multiple transmembrane domains and is inserted into the ER membrane with two topological orientations, one of which is targeted to the plasma membrane where it mediates bile acid transport.
Progressive familial intrahepatic cholestasis (PFIC), an inherited liver disease of childhood, is characterized by cholestasis and either normal or increased serum gamma-glutamyltransferase activity. Patients with normal gamma-glutamyltransferase activity have mutations of the FIC1 locus on chromosome 18q21 or mutations of the BSEP gene on chromosome 2q24. Also, patients with bile acid synthesis defects have low gamma-glutamyltransferase activity. We investigated expression of the bile salt export pump (BSEP) in liver samples from patients with a PFIC phenotype and correlated this with BSEP gene mutations.
BSEP and multidrug resistance protein 2 (MRP2) expressions were studied by immunohistochemistry in liver specimens of 28 patients and BSEP gene mutation analysis in 19 patients. Bile salt kinetics were studied in 1 patient.
Sixteen of 28 liver samples showed no canalicular BSEP staining. Staining for MRP2 showed a normal canalicular pattern in all but 1 of these samples. Ten of 19 patients showed BSEP gene mutations; BSEP protein expression was lacking in all 10 patients. No mutations were found in 9 of 19 patients, and in all except 1, BSEP protein expression was normal. Bile salt concentration in bile of BSEP-negative/MRP2-positive PFIC patients was 0.2 +/- 0.2 mmol/L (n = 9; <1% of normal) and in BSEP-positive PFIC patients 18.1 +/- 9.9 mmol/L (n = 3; 40% of normal). The kinetic study confirmed the dramatic decrease of bile salt secretion in BSEP-negative patients.
The findings show a close correlation between BSEP gene mutations and canalicular BSEP expression. Biliary secretion of bile salts is greatly reduced in BSEP-negative patients.
Recent studies implicate a role in hepatocyte organic anion transport of a plasma membrane protein that has been termed oatp1 (organic anion transport protein 1). Little is known regarding mechanisms by which its transport activity is modulated in vivo. In previous studies (Campbell, C. G., Spray, D. C., and Wolkoff, A. W. (1993) J. Biol. Chem. 268, 15399-15404), we demonstrated that hepatocyte uptake of sulfobromophthalein was down-regulated by extracellular ATP. We have now found that extracellular ATP reduces the V(max) for transport of sulfobromophthalein by rat hepatocytes; K(m) remains unaltered. Reduced transport also results from incubation of hepatocytes with the phosphatase inhibitors okadaic acid and calyculin A. Immunoprecipitation of biotinylated cell surface proteins indicates that oatp1 remains on the cell surface after exposure of cells to ATP or phosphatase inhibitor, suggesting that loss of transport activity is not caused by transporter internalization. Exposure of (32)P-loaded hepatocytes to extracellular ATP results in serine phosphorylation of oatp1 with the appearance of a single major tryptic phosphopeptide; oatp1 from control cells is not phosphorylated. This phosphopeptide comigrates with one of four phosphopeptides resulting from incubation of cells with okadaic acid. These studies indicate that the phosphorylation state of oatp1 must be an important consideration when assessing alterations of its functional expression in pathobiological states.
We have previously shown that cloned rat multidrug resistance-associated protein 3 (Mrp3) has the ability to transport organic anions such as 17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG) and has a different substrate specificity from MRP1 and MRP2 in that glutathione conjugates are poor substrates for Mrp3 (Hirohashi, T., Suzuki, H., and Sugiyama, Y. (1999) J. Biol. Chem. 274, 15181-15185). In the present study, the involvement of Mrp3 in the transport of endogenous bile salts was investigated using membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 cDNA. The ATP-dependent uptake of [(3)H]taurocholate (TC), [(14)C]glycocholate (GC), [(3)H]taurochenodeoxycholate-3-sulfate (TCDC-S), and [(3)H]taurolithocholate-3-sulfate (TLC-S) was markedly stimulated by Mrp3 transfection in LLC-PK1 cells. The extent of Mrp3-mediated transport of bile salts was in the order, TLC-S > TCDC-S > TC > GC. The K(m) and V(max) values for the uptake of TC and TLC-S were K(m) = 15.9 +/- 4.9 microM and V(max) = 50.1 +/- 9.3 pmol/min/mg of protein and K(m) = 3.06 +/- 0.57 microM and V(max) = 161.9 +/- 21.7 pmol/min/mg of protein, respectively. At 55 nM [(3)H]E(2)17betaG and 1.2 microM [(3)H]TC, the apparent K(m) values for ATP were 1.36 and 0.66 mM, respectively. TC, GC, and TCDC-S inhibited the transport of [(3)H]E(2)17betaG and [(3)H]TC to the same extent with an apparent IC(50) of approximately 10 microM. TLC-S inhibited the uptake of [(3)H]E(2)17betaG and [(3)H]TC most potently (IC(50) of approximately 1 microM) among the bile salts examined, whereas cholate weakly inhibited the uptake (IC(50) approximately 75 microM). Although TC and GC are transported by bile salt export pump/sister of P-glycoprotein, but not by MRP2, and TCDC-S and TLC-S are transported by MRP2, but not by bile salt export pump/sister of P-glycoprotein, it was found that Mrp3 accepts all these bile salts as substrates. This information, together with the finding that MRP3 is extensively expressed on the basolateral membrane of human cholangiocytes, suggests that MRP3/Mrp3 plays a significant role in the cholehepatic circulation of bile salts.
Mutations in the sister of P-glycoprotein (Spgp) or bile salt export pump (BSEP) are associated with Progressive Familial Intrahepatic Cholestasis (PFIC2). Spgp is predominantly expressed in the canalicular membranes of liver. Consistent with in vitro evidence demonstrating the involvement of Spgp in bile salt transport, PFIC2 patients secrete less than 1% of biliary bile salts compared with normal infants. The disease rapidly progresses to hepatic failure requiring liver transplantation before adolescence. In this study, we show that the knockout of spgp gene in mice results in intrahepatic cholestasis, but with significantly less severity than PFIC2 in humans. Some unexpected characteristics are observed. Notably, although the secretion of cholic acid in mutant mice is greatly reduced (6% of wild-type), total bile salt output in mutant mice is about 30% of wild-type. Also, secretion of an unexpectedly large amount of tetra-hydroxylated bile acids (not detected in wild-type) is observed. These results suggest that hydroxylation and an alternative canalicular transport mechanism for bile acids compensate for the absence of Spgp function and protect the mutant mice from severe cholestatic damage. In addition, the spgp(-/-) mice display a significant increase in the secretion of cholesterol and phospholipids into the bile. This latter observation in spgp(-/-) mice suggests that intrahepatic, rather than intracanalicular, bile salts are the major driving force for the biliary lipid secretion. The spgp(-/-) mice thus provide a unique model for gaining new insights into therapeutic intervention for intrahepatic cholestasis and understanding mechanisms associated with lipid homeostasis.
cAMP-mediated stimulation of hepatic bile acid uptake is associated with dephosphorylation and translocation of Na+-taurocholate (TC) cotransporting peptide (NTCP) to the plasma membrane. Although translocation of NTCP may be facilitated by dephosphorylation, the mechanism of dephosphorylation is unknown. The ability of cAMP to translocate and dephosphorylate NTCP is, in part, dependent on cAMP-mediated increases in cytosolic Ca2+ concentration ([Ca2+]), indicating that a Ca2+/calmodulin-dependent protein phosphatase (PP2B) may be involved. Thus we studied the role of PP2B using the inhibitor cypermethrin (CM). Freshly isolated hepatocytes were pretreated with 1-5 nM CM for 30 min followed by 15 min incubation with 10 microM 8-(4-chlorophenylthio)cAMP. CM (5 nM) and FK-506 (5 microM) inhibited cAMP-stimulated TC uptake by 80 and 75%, respectively, without affecting basal TC uptake. CM also reversed cAMP-mediated NTCP dephosphorylation and translocation to 80 and 15% of the basal level, respectively. cAMP stimulated PP2B activity by 60%, and this effect was completely inhibited by 5 nM CM. PP2B dephosphorylated NTCP immunoprecipitated from control but not from cAMP-treated hepatocytes. The effect of CM was not due to any changes in cAMP-mediated increases in cytosolic [Ca2+] or decreases in mitogen-activated protein kinase (extracellular regulated kinases 1 and 2) activity. Taken together, these results suggest that cAMP dephosphorylates NTCP by activating PP2B in hepatocytes, and PP2B-mediated dephosphorylation of NTCP may be involved in cAMP-mediated NTCP translocation to the plasma membrane.
Mutations in the sister of P-glycoprotein (Spgp) or bile salt export pump (BSEP) are associated with Progressive Familial Intrahepatic Cholestasis (PFIC2). Spgp is predominantly expressed in the canalicular membranes of liver. Consistent with in vitro evidence demonstrating the involvement of Spgp in bile salt transport, PFIC2 patients secrete less than 1% of biliary bile salts compared with normal infants. The disease rapidly progresses to hepatic failure requiring liver transplantation before adolescence. In this study, we show that the knockout of spgp gene in mice results in intrahepatic cholestasis, but with significantly less severity than PFIC2 in humans. Some unexpected characteristics are observed. Notably, although the secretion of cholic acid in mutant mice is greatly reduced (6% of wild-type), total bile salt output in mutant mice is about 30% of wild-type. Also, secretion of an unexpectedly large amount of tetra-hydroxylated bile acids (not detected in wild-type) is observed. These results suggest that hydroxylation and an alternative canalicular transport mechanism for bile acids compensate for the absence of Spgp function and protect the mutant mice from severe cholestatic damage. In addition, the spgp−/− mice display a significant increase in the secretion of cholesterol and phospholipids into the bile. This latter observation in spgp−/− mice suggests that intrahepatic, rather than intracanalicular, bile salts are the major driving force for the biliary lipid secretion. The spgp−/− mice thus provide a unique model for gaining new insights into therapeutic intervention for intrahepatic cholestasis and understanding mechanisms associated with lipid homeostasis.
Purpose. It has been shown that plasma concentration and urinary excretion of bile acids is elevated under the cholestatic/hyperbilirubinemic conditions. Previously, it was demonstrated that the plasma concentration of bile acids was elevated in the multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The purpose of the present study was to compare the sinusoidal efflux clearance of taurocholate (TC) between Mrp2-deficient Eisai hyperbilirubinemic rats (EHBR) and normal rats.
Method. Hepatic disposition of the [3H]TC was examined in the perfused liver. Apparent efflux clearance (PSnet, eff) of [3H]TC from hepatocytes to outflow across the sinusoidal membrane was defined as the amount of [3H]TC excreted into the outflow from the liver divided by hepatic AUC of [3H]TC. Additionally, influx clearance (PSinf) was also determined by multiple indicator dilution method because PSnet, eff is also affected by PSinf.
Results. PSnet, eff was significantly higher in EHBR than that in Sprague-Dawley (SD) rats (16.6 1.7 vs. 6.1 1.3 L/min/g liver, P inf was comparable between SD rats and EHBR. Kinetic analysis suggested that the intrinsic clearance for the efflux of [3H]TC across the sinusoidal membrane in EHBR was higher than that in SD rats (10.4 1.0 v.s. 23.3 1.7 L/min/g liver, P
Conclusions. Enhanced sinusoidal efflux of TC in EHBR may be related to the altered disposition of bile acids in the mutant rats. Because Mrp3 transports TC and its expression is induced on the basolateral membrane of Mrp2-deficient rats, the enhanced sinusoidal efflux of TC in EHBR may be accounted for, at least partially, by the increased expression of Mrp3.
Binding and transport characteristics for uptake of taurocholic acid by isolated rat liver cells were studied.
An adsorption of taurocholate to the cell surface is terminated in less than 15 s. A K s of 0.55 mM and a total binding capacity of 3.8 nmol/mg cell protein is determined.
The rate of uptake of taurocholate follows Michaelis‐Menten kinetics with K m = 19 μM and V = 1.7 nmol/mg protein min.
There is a broad pH optimum for uptake between pH 6.5–8.0.
The activation energy amounts to 29 kcal/mol. At high taurocholate concentration an unusual upward bend is observed in the Arrhenius plot.
Taurocholate uptake is competitively inhibited by taurochenodeoxycholate ( K i = 9 μM). It is noncompetitively inhibited by bromosulfophthalein ( K i = 3 μM).
At physiological taurocholate concentrations a 200‐fold intracellular accumulation of taurocholate is observed.
Uptake is inhibited by about 75% by either antimycin A, carbonylcyanide m ‐chlorophenyl‐hydrazone, ouabain.
Replacement of extracellular Na ⁺ by either K ⁺ or sucrose results in a 75% decrease of uptake.
It is concluded that taurocholate uptake is a carrier‐mediated process, and suggested that the energy for intracellular accumulation is made available by cotransport of Na ⁺ .
The uptake of 14C-labeled cholic, taurocholic, and chenodeoxycholic acid by the perfused rat liver was studied to characterize the mechanism responsible for hepatic uptake of bile acids. A rapid-injection multiple indicator-dilution technique and the three-compartment model of Goresky were employed. The kinetics of hepatic uptake of the three bile acids could be described by the Michaelis-Menten equation. The maximal uptake velocities (Vmax) were 24.9 +/- 2.2 (mean +/- SD), 20.8 +/- 1.2, 1.2, and 11.4 +/- 0.9 nmol/s-g liver for cholic, taurocholic, and chenodeoxycholic acid, respectively. The corresponding apparent half-saturation constants (Km) were 526 +/- 125, 258 +/- 43, and 236 +/- 48 nmol/g liver. Competitive inhibition could be demonstrated between cholate and taurocholate as well as between cholate and chenodeoxycholate. Substitution of 94% of the Na+ in the perfusion medium decreased the Vmax and the apparent Km of taurocholate uptake by 68 and 55%, respectively. These findings are consistent with the hypothesis that bile acids are taken up into the hepatocyte by Na+-dependent carrier-mediated transport.
The secretion of bile by the liver is primarily determined by the ability of the hepatocyte to transport bile acids into the bile canaliculus. A carrier-mediated process for the transport of taurocholate, the major bile acid in humans and rats, was previously demonstrated in canalicular membrane vesicles from rat liver. This process is driven by an outside-positive membrane potential that is, however, insufficient to explain the large bile acid concentration gradient between the hepatocyte and bile. In this study, we describe an ATP-dependent transport system for taurocholate in inside-out canalicular membrane vesicles from rat liver. The transport system is saturable, temperature-dependent, osmotically sensitive, specifically requires ATP, and does not function in sinusoidal membrane vesicles and right side-out canalicular membrane vesicles. Transport was inhibited by other bile acids but not by substrates for the previously demonstrated ATP-dependent canalicular transport systems for organic cations or nonbile acid organic anions. Defects in ATP-dependent canalicular transport of bile acids may contribute to reduced bile secretion (cholestasis) in various developmental, inheritable, and acquired disorders.
To clarify the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on the transcytotic vesicle pathway, we measured the biliary excretion of bile acid, phospholipid, and horseradish peroxidase (HRP) in the isolated perfused rat liver (IPRL) with or without infusion of N6,2'-O-dibutyryl-cAMP (DBcAMP). A linear relationship between bile flow and bile acid excretion was observed in both control and DBcAMP-infused livers. DBcAMP increased the y-axis intercept from 1.10 +/- 0.16 to 1.48 +/- 0.19 microliters.min-1.g liver-1 (P less than 0.01) and the slope from 6.5 +/- 1.99 to 10.77 +/- 1.71 microliters/mumol bile acid (P less than 0.01). DBcAMP also increased the biliary excretion of bile acid and phospholipid during a 1.0 mumol/min infusion of taurocholate. When HRP was pulse loaded for 1 min, HRP appeared in bile in early (4-6 min) and late (20-25 min) peaks. DBcAMP markedly increased the late peak of HRP from 0.33 +/- 0.08 to 1.15 +/- 0.32 ng.min-1.g liver-1 (P less than 0.01), a phenomenon blocked by colchicine. An electron-microscopic morphometric analysis indicated that DBcAMP increased both the density and %area of HRP-containing vesicles in the pericanalicular area, compared with controls, 18 min after a 1-min pulse of HRP. DBcAMP had no effect on the uptake rate of HRP in 4-h primary hepatocyte cultures but stimulated biliary excretion of HRP when preloaded in the IPRL. These findings indicate that cAMP regulates excretory function in part by stimulating the microtubule-dependent transcytotic vesicle transport system.
The effect of taurocholate on transcytotic vesicular pathways labeled with horseradish peroxidase was assessed in isolated perfused rat liver preparations. Forty-five minutes after a horseradish peroxidase load in a recirculating system, continuous infusion of taurocholate but not taurodehydrocholate significantly increased horseradish peroxidase excretion in bile by 50% compared with controls. When horseradish peroxidase (25 mg) was pulse loaded for 1 minute in control perfusions, it appeared in bile in early (4-6 minutes) and late (20-25 minutes) peaks, the latter accounting for 90% of total horseradish peroxidase output. Taurocholate infusion significantly increased horseradish peroxidase output in both early and late peaks, whereas only a small increase in the early peak was observed with taurodehydrocholate. Colchicine pretreatment increased the early peak in bile but abolished the second peak. Electron micrographs from control livers revealed the accumulation of horseradish peroxidase-containing vesicles in pericanalicular regions at early (2 minutes) as well as late (18 minutes) periods. When a morphometric analysis of electron micrographs was performed from pericanalicular regions 2 minutes after a 1-minute pulse of horseradish peroxidase (500 mg), taurocholate but not taurodehydrocholate increased both the density and percent area of horseradish peroxidase-containing vesicles compared with controls. In contrast, colchicine pretreatment had no effect on the density of the early-appearing vesicles, although their individual sizes were reduced. Taurocholate but not taurodehydrocholate also increased the percent of tubular structures in the pericanalicular region. These findings indicate that taurocholate stimulates both early and late transcytotic vesicle pathways and therefore probably microtubule-independent vesicle pathway is present in hepatocytes that must be distinguished from paracellular routes.
The apparent target size of the sodium-dependent taurocholate transporter in basolateral rat liver plasma membrane vesicles, showing overshooting taurocholate uptake in the presence of sodium was estimated by radiation inactivation. Radiation at -105 to -120 degrees C and 2.5 Mrad/min causes a dose-dependent monoexponential reduction of the overshoot of taurocholate uptake in the presence of sodium. In contrast, taurocholate transport in the absence of sodium and taurocholate permeation at 4 degrees C remained totally unaffected by the radiation dose, indicating that the passive permeability of the membrane towards taurocholate remained unaffected. Radiation inactivation by high-energy electrons provides information about the size of the functional unit of the transporter in situ. The target size determined represents the size of the radiation-sensitive mass which is compact enough for significant energy transfer to occur within all parts of the transport system. The minimal function molecular mass was determined to be 170 kDa for the sodium-dependent taurocholate transporter. To prove the validity of radiation inactivation data four internal standard enzymes were tested under identical conditions.
Recent studies have suggested that the canalicular bile salt transport system of rat liver corresponds to a 100-kDa membrane glycoprotein. In the present study we attempted to functionally reconstitute the 100-kDa protein into artificial proteoliposomes. Canalicular membrane proteins were solubilized with octyl glucoside in the presence of asolectin phospholipids. The extracts were treated with preimmune serum or the 100-kDa protein selectively immunoprecipitated with a polyclonal antiserum. Proteins remaining in the supernatant were then incorporated into proteoliposomes by gel-filtration chromatography. Canalicular proteoliposomes containing the 100-kDa protein exhibited transstimulatable taurocholate uptake that could be inhibited by 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS). In contrast, no DIDS-sensitive transstimulatable taurocholate uptake was found in 100-kDa protein-free canalicular proteoliposomes. However, when the immunoprecipitated 100-kDa protein was dissociated from the antibodies and exclusively incorporated into liposomes, reconstitution of DIDS-sensitive transstimulatable and electrogenic taurocholate anion transport was again positive. Although incorporation of solubilized basolateral membrane proteins into liposomes also resulted in a prompt reconstitution of Na+ gradient-driven taurocholate uptake, the anti-100-kDa antibodies had no effects on the reconstituted transport activity of basolateral proteins. Thus, the findings establish that the previously characterized canalicular-specific 100-kDa protein is directly involved in the transcanalicular secretion of bile salts.
Bile acid (BA) hydrophobicity, evaluated by the octanol-water partition coefficient, decreases along the series deoxycholic acid-chenodeoxy cholic acid-hyodeoxycholic acid-ursodeoxycholic acid-cholic acid (CA)-ursocholic acid (UCA). In vitro experiments carried out using dialysis techniques (to determine the maximum BA binding) and ultrafiltration of plasma pre-incubated with 0.1 mM BA (to assess the distribution of BA between the different lipoprotein fractions) showed that the maximum binding of BA to plasma and lipoproteins follows the same order of hydrophobicity. The fraction not bound to proteins, greater with the hydrophilic BA (UCA and CA), is distributed in the lipoprotein fractions and in particular in high density lipoproteins.
The mechanisms of bile acid uptake have been studied with primary monolayer cultures of rat hepatocytes. Hepatocytes were incubated with taurocholic acid (TC), glycocholic acid (GC), cholic acid (CA), glycochenodeoxycholic acid (GCDC), chenodeoxycholic acid (CDCA), deoxycholic acid (DOCA), lithocholic acid (LCA), or cholylglycylhistamine (CCH), a neutral bile acid derivative for 10 s to 60 min in medium containing sodium chloride, sodium chloride with 1 mM ouabain, or choline chloride. Cells were washed free of radioactive tracer, cell-associated radioactivity was quantitated, and bile acid uptake rates, kinetic parameters of uptake, and steady-state bile acid content were calculated. Two mechanisms for bile acid uptake were identified. Uptake of TC, GC, CA, and GCDC occurred predominantly via a sodium-dependent, ouabain-suppressible saturable mechanism, presumably sodium-coupled transport. Estimates of apparent Km and Vmax for these bile acids were TC, 33 micro M and 0.36 nmol . min-1 . mg prot-1; GC, 18 micro M and 0.22 nmol . min-1 . mg prot-1; CA, 13 micro M and 0.10 nmol . min-1 . mg prot; and GCDC, 6 micro M and 0.21 nmol . min-1 . mg prot, respectively. Uptake via this sodium-coupled mechanism exhibited considerable substrate selectivity. It was enhanced by increased ring hydroxylation and amino acid conjugation and decreased by further conjugation with a neutral histamine group (CGH). In contrast, uptake of CDCA, DOCA, LCA, and CGH occurred primarily via a nonsaturable sodium-independent mechanism, possibly simple diffusion. This mechanism accounted for only a small portion of uptake of TC, GC, CA, and GCDC at low bile acid concentrations. Nonsaturable bile acid uptake rates appeared to correlate with decane-buffer partition coefficients and to be related to bile acid structure.
Uptake of the bile acid taurocholate by hepatocytes is coupled to Na+ influx. The stoichiometry of uptake, however, is uncertain, as is the influence of the transmembrane electrical potential difference (PD) on this process. In this study, we examined the relationship between taurocholate extraction and PD (measured using intracellular microelectrodes) in perfused liver, and we measured taurocholate-induced transport current in cultured hepatocytes using patch-clamp recording techniques. In the perfused liver under basal conditions, PD averaged -28.4 +/- 0.6 (SE) mV, and extraction of 1, 50, and 300 microM taurocholate was 0.95 +/- 0.02, 0.98 +/- 0.01, and 0.41 +/- 0.03, respectively. When the Na+ chemical gradient was decreased by replacing perfusate Na+ with choline, the membrane depolarized to -17.2 +/- 1.1 mV, and taurocholate extraction markedly decreased at all taurocholate concentrations (P < 0.01). When perfusate Na+ concentration was held constant at 137 mM, membrane depolarization induced by substitution of gluconate for perfusate Cl- (-17.9 +/- 0.6 mV) or Cl- for nitrate (-10.3 +/- 2.1 mV) significantly decreased extraction of 300 microM taurocholate. Abrupt exposure to taurocholate produced a concentration-dependent membrane depolarization in the presence of Na+, but not in its absence (P < 0.001). In cultured hepatocytes, exposure to 100 microM taurocholate produced an inward current of -0.056 +/- 0.016 pA/pF at a holding potential of -40 mV. This current was Na+ dependent, and it increased twofold as holding potential was changed from -20 to -50 mV.(ABSTRACT TRUNCATED AT 250 WORDS)
A Na(+)-dependent bile acid (Na+/taurocholate co-transporting polypeptide; Ntcp) and a Na(+)-independent bromosulphophthalein (BSP)/bile acid uptake system (organic-anion-transporting polypeptide; oatp) have been cloned from rat liver by using functional expression cloning in Xenopus laevis oocytes. To evaluate the extent to which these cloned transporters could account for overall hepatic bile acid and BSP uptake, we used antisense oligonucleotides to inhibit the expression of Ntcp and oatp in Xenopus laevis oocytes injected with total rat liver mRNA. An Ntcp-specific antisense oligonucleotide co-injected with total rat liver mRNA blocked the expression of Na(+)-dependent taurocholate uptake by approx. 95%. In contrast, an oatp-specific antisense oligonucleotide when co-injected with total rat liver mRNA had no effect on the expression of Na(+)-dependent taurocholate uptake, but it blocked Na(+)-independent uptake of taurocholate by approx. 80% and of BSP by 50%. Assuming similar expression of hepatocellular bile acid and organic anion transporters in Xenopus laevis oocytes, these results indicate that Ntcp and oatp respectively represent the major, if not the only, Na(+)-dependent and Na(+)-independent taurocholate uptake systems in rat liver. By contrast, the cloned oatp accounts for only half of BSP transport, suggesting that there must be additional, non-bile acid transporting organic anion uptake systems in rat liver.
The bile canalicular membrane contains four specific ATP-dependent transport processes that are involved in secretion of bile acids, non-bile acid organic anions (mrp1), phospholipids (mdr2), and organic cations (mdr3). The aim of this study was to determine how the canalicular presence of these transport proteins is regulated. Canalicular membrane vesicles (CMV) were prepared from livers of rats treated with taurocholate (TC) and/or dibutyryl-adenosine 3',5'-cycle monophosphate (DBcAMP) with and without pretreatment with colchicine. Transport studies were performed with radiolabeled substrates. Changes in the relative amounts of transport proteins were determined by Western blots. Compared with controls, the specific activity of each of the transport processes was enhanced 1.5- and 3-fold with TC and DBcAMP treatments, respectively. Western blots revealed the same increases with mdr2 and mdr3. Pretreatment of rats with colchicine prevented these responses fully with TC treatment, whereas only partial prevention was obtained with DBcAMP treatment. Besides the ATP-dependent transporters, the relative specific activities of the canalicular membrane ectoenzyme markers, leucine aminopeptidase and gamma-glutamyltranspeptidase, were also affected the same way. These results suggest that TC and DBcAMP increase the specific activity of the canalicular ATP-dependent transport proteins and some canalicular membrane ectoenzymes by stimulating an increase in the relative amounts of these proteins in the membrane.
The human Dubin-Johnson syndrome (DJS) is a rare autosomal recessive liver disorder characterized by chronic conjugated hyperbilirubinemia. Patients have impaired hepatobiliary transport of non-bile salt organic anions. A highly similar phenotype has been described for a mutant Wistar rat strain, the transport-deficient (TR-) rat, which is defective in the canalicular multispecific organic anion transporter (cmoat). This protein mediates adenosine triphosphate-dependent transport of a broad range of endogenous and xenobiotic compounds across the (apical) canalicular membrane of the hepatocyte. The complementary DNA (cDNA) encoding rat cmoat has recently been cloned, and the mutation underlying the defect in TR- rats has been identified. In the present study, we have isolated the human homologue of rat cmoat, human cMOAT, and analyzed the corresponding cDNA from fibroblasts of a DJS patient for mutations. Our results show that a mutation in this gene is the cause of DJS.
Na+/taurocholate (Na+/TC) cotransport in hepatocytes is mediated primarily by Na+/TC cotransporting polypeptide (Ntcp), and cyclic adenosine monophosphate (cAMP) stimulates Na+/TC cotransport by inducing translocation of Ntcp to the plasma membrane. The aim of the present study was to determine if Ntcp is a phosphoprotein and if cAMP alters Ntcp phosphorylation. Freshly prepared hepatocytes from rat livers were incubated with carrier-free 32PO4 for 2 hours, followed by incubation with 10 micromol/L 8-chlorophenylthio adenosin 3':5'-cyclic monophosphate (CPT-cAMP) for 15 minutes. Subcellular fractions isolated from 32P-labeled hepatocytes were subjected to immunoprecipitation using Ntcp antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography to determine if Ntcp is phosphorylated. Ntcp immunoprecipitated from plasma membranes isolated from nonlabeled hepatocytes was subjected to immunoblot analysis using anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine antibody to determine whether Ntcp is a serine, threonine, or tyrosine phosphoprotein. Hepatocytes were loaded with bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid (MAPTA), a Ca2+ buffering agent, and the effect of CPT-cAMP on TC uptake, cytosolic [Ca2+], and ntcp phosphorylation and translocation was determined. In addition, the effect of cAMP on protein phosphatases 1 and 2A (PP1/2A) was determined in homogenates and plasma membranes obtained from CPT-cAMP-treated hepatocytes. Phosphorylation study showed that phosphorylated Ntcp is detectable in plasma membranes, and cAMP treatment resulted in dephosphorylation of Ntcp. Immunoblot analysis with phosphoamino antibodies revealed that Ntcp is a serine/threonine, and not a tyrosine, phosphoprotein, and cAMP inhibited both serine and threonine phosphorylation. In MAPTA-loaded hepatocytes, CPT-cAMP failed to stimulate TC uptake, failed to increase cytosolic [Ca2+], and failed to induce translocation and dephosphorylation of Ntcp. cAMP did not alter the activity of PP1/2A in either homogenates or in plasma membranes. Taken together, these results suggest that Ntcp is a serine/threonine phosphoprotein and is dephosphorylated by cAMP treatment. Activation of PP1/2A is not involved in cAMP-mediated dephosphorylation of Ntcp. Both translocation and dephosphorylation of Ntcp may be involved in the regulation of hepatic Na+/TC cotransport.
Liver regeneration in response to various forms of injury or surgical resection is a complex process resulting in restoration of the original liver mass and maintenance of liver-specific functions such as bile formation. However, liver regeneration is frequently associated with cholestasis, whose molecular pathogenesis remains unknown.
To study the molecular mechanisms leading to cholestasis, expression of all major hepatic organic anion transporters contributing to bile formation was determined for up to 2 weeks in rats after 70% partial hepatectomy.
Inversely related to serum bile acid levels, basolateral transporters including the sodium-taurocholate cotransporter (Ntcp) and the organic anion transporting polypeptides Oatp1 and Oatp2 were markedly down-regulated at both protein and steady-state mRNA levels by 50%-60% of controls (P < 0.05) during early replicative stages of regeneration (12 hours to 2 days) with a slightly delayed time course for Oatp2. Expression of all basolateral transporters returned to control values between 4 and 4 days after partial hepatectomy. In contrast, protein and mRNA expression of both the canalicular ATP-dependent bile salt export pump (Bsep) and the multiorganic anion transporter Mrp2 remained unchanged or were slightly increased during liver regeneration, but also returned to control values 7-14 days after partial hepatectomy.
The data suggest a differential regulation of basolateral and canalicular organic anion transporters in the regenerating liver. Unaltered expression of Bsep and Mrp2 provides a potential molecular mechanism for regenerating liver cells to maintain or even increase bile secretion expressed per weight of remaining liver. However, down-regulation of basolateral organic anion transporters might protect replicating liver cells by diminishing uptake of potentially hepatotoxic bile salts, because the remaining liver initially cannot cope with the original bile acid pool size.
Biliary excretion of certain bile acids is mediated by multidrug resistance associated protein 2 (Mrp2) and the bile salt export pump (Bsep). In the present study, the transport properties of several bile acids were characterized in canalicular membrane vesicles (CMVs) isolated from Sprague--Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose Mrp2 function is hereditarily defective and in membrane vesicles isolated from Sf9 cells infected with recombinant baculovirus containing cDNAs encoding Mrp2 and Bsep. ATP-dependent uptake of [(3)H]taurochenodeoxycholate sulfate (TCDC-S) (K(m)=8.8 microM) and [(3)H]taurolithocholate sulfate (TLC-S) (K(m)=1.5 microM) was observed in CMVs from SD rats, but not from EHBR. In addition, ATP-dependent uptake of [(3)H]TLC-S (K(m)=3.9 microM) and [(3)H]taurocholate (TC) (K(m)=7.5 microM) was also observed in Mrp2- and Bsep-expressing Sf9 membrane vesicles, respectively. TCDC-S and TLC-S inhibited the ATP-dependent TC uptake into CMVs from SD rats with IC(50) values of 4.6 microM and 1.2 microM, respectively. In contrast, the corresponding values for Sf9 cells expressing Bsep were 59 and 62 microM, respectively, which were similar to those determined in CMVs from EHBR (68 and 33 microM, respectively). By co-expressing Mrp2 with Bsep in Sf9 cells, IC(50) values for membrane vesicles from these cells shifted to values comparable with those in CMVs from SD rats (4.6 and 1.2 microM). Moreover, in membrane vesicles where both Mrp2 and Bsep are co-expressed, preincubation with the sulfated bile acids potentiated their inhibitory effect on Bsep-mediated TC transport. These results can be accounted for by assuming that the sulfated bile acids trans-inhibit the Bsep-mediated transport of TC.
Maturity-onset diabetes of the young type 3 (MODY3) is caused by haploinsufficiency of hepatocyte nuclear factor-1alpha (encoded by TCF1). Tcf1-/- mice have type 2 diabetes, dwarfism, renal Fanconi syndrome, hepatic dysfunction and hypercholestrolemia. Here we explore the molecular basis for the hypercholesterolemia using oligonucleotide microchip expression analysis. We demonstrate that Tcf1-/- mice have a defect in bile acid transport, increased bile acid and liver cholesterol synthesis, and impaired HDL metabolism. Tcf1-/- liver has decreased expression of the basolateral membrane bile acid transporters Slc10a1, Slc21a3 and Slc21a5, leading to impaired portal bile acid uptake and elevated plasma bile acid concentrations. In intestine and kidneys, Tcf1-/- mice lack expression of the ileal bile acid transporter (Slc10a2), resulting in increased fecal and urinary bile acid excretion. The Tcf1 protein (also known as HNF-1alpha) also regulates transcription of the gene (Nr1h4) encoding the farnesoid X receptor-1 (Fxr-1), thereby leading to reduced expression of small heterodimer partner-1 (Shp-1) and repression of Cyp7a1, the rate-limiting enzyme in the classic bile acid biosynthesis pathway. In addition, hepatocyte bile acid storage protein is absent from Tcf1-/- mice. Increased plasma cholesterol of Tcf1-/- mice resides predominantly in large, buoyant, high-density lipoprotein (HDL) particles. This is most likely due to reduced activity of the HDL-catabolic enzyme hepatic lipase (Lipc) and increased expression of HDL-cholesterol esterifying enzyme lecithin:cholesterol acyl transferase (Lcat). Our studies demonstrate that Tcf1, in addition to being an important regulator of insulin secretion, is an essential transcriptional regulator of bile acid and HDL-cholesterol metabolism.
Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.
It has been shown that plasma concentration and urinary excretion of bile acids is elevated under the cholestatic/ hyperbilirubinemic conditions. Previously, it was demonstrated that the plasma concentration of bile acids was elevated in the multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The purpose of the present study was to compare the sinusoidal efflux clearance of taurocholate (TC) between Mrp2-deficient Eisai hyperbilirubinemic rats (EHBR) and normal rats.
Hepatic disposition of the [3H]TC was examined in the perfused liver. Apparent efflux clearance (PSnet, eff) of [3H]TC from hepatocytes to outflow across the sinusoidal membrane was defined as the amount of [3H]TC excreted into the outflow from the liver divided by hepatic AUC of [3H]TC. Additionally, influx clearance (PSinf) was also determined by multiple indicator dilution method because PSnet, eff is also affected by PSinf.
PSnet, eff was significantly higher in EHBR than that in Sprague-Dawley (SD) rats (16.6 +/- 1.7 vs. 6.1 +/- 1.3 microL/min/g liver, P < 0.01). In contrast, PSinf was comparable between SD rats and EHBR. Kinetic analysis suggested that the intrinsic clearance for the efflux of [3H]TC across the sinusoidal membrane in EHBR was higher than that in SD rats (10.4 +/- 1.0 v.s. 23.3 +/- 1.7 microL/min/g liver, P < 0.01).
Enhanced sinusoidal efflux of TC in EHBR may be related to the altered disposition of bile acids in the mutant rats. Because Mrp3 transports TC and its expression is induced on the basolateral membrane of Mrp2-deficient rats, the enhanced sinusoidal efflux of TC in EHBR may be accounted for, at least partially, by the increased expression of Mrp3.
Bile salts are the major organic solutes in bile and undergo extensive enterohepatic circulation. Hepatocellular bile salt uptake is mediated predominantly by the Na(+)-taurocholate cotransport proteins Ntcp (rodents) and NTCP (humans) and by the Na(+)-independent organic anion-transporting polypeptides Oatp1, Oatp2, and Oatp4 (rodents) and OATP-C (humans). After diffusion (bound by intracellular bile salt-binding proteins) to the canalicular membrane, monoanionic bile salts are secreted into bile canaliculi by the bile salt export pump Bsep (rodents) or BSEP (humans). Both belong to the ATP-binding cassette (ABC) transporter superfamily. Dianionic conjugated bile salts are secreted into bile by the multidrug-resistance-associated proteins Mrp2/MRP2. In bile ductules, a minor portion of protonated bile acids and monomeric bile salts are reabsorbed by non-ionic diffusion and the apical sodium-dependent bile salt transporter Asbt/ASBT, transported back into the periductular capillary plexus by Mrp3/MRP3 [and/or a truncated form of Asbt (tAsbt)], and subjected to cholehepatic shunting. The major portion of biliary bile salts is aggregated into mixed micelles and transported into the intestine, where they are reabsorbed by apical Oatp3, the apical sodium-dependent bile salt transporter (ASBT), cytosolic intestinal bile acid-binding protein (IBABP), and basolateral Mrp3/MRP3 and tAsbt. Transcriptional and posttranscriptional regulation of these enterohepatic bile salt transporters is closely related to the regulation of lipid and cholesterol homeostasis. Furthermore, defective expression and function of bile salt transporters have been recognized as important causes for various cholestatic liver diseases.
ATP-binding cassette (ABC) transporters located in the hepatocyte canalicular membrane of mammalian liver are critical players in bile formation and detoxification. Although ABC transporters have been well characterized functionally, only recently have several canalicular ABC transporters been cloned and their molecular nature revealed. Subsequently, development of specific antibodies has permitted a detailed investigation of ABC transporter intrahepatic distribution under varying physiological conditions. It is now apparent that there is a complex array of ABC transporters in hepatocytes. ABC transporter molecules reside in intrahepatic compartments and are delivered to the canalicular domain following increased physiological demand to secrete bile. Insufficient amounts of ABC transporters in the bile canalicular membrane result in cholestasis (i.e., bile secretory failure). Therefore, elucidation of the intrahepatic pathways and regulation of ABC transporters may help to understand the cause of cholestasis at a molecular level and provide clues for novel therapies.
- Plm Jansen
- M Muller
- E Sturm
- Genes
Jansen PLM, Muller M, and Sturm E. Genes and cholestasis.
Hepatology 34: 1067–1074, 2001.
- Plm Jansen
- M Muller
- E Sturm
- Genes
- Cholestasis
Jansen PLM, Muller M, and Sturm E. Genes and cholestasis.
Hepatology 34: 1067–1074, 2001.
- Plm Jansen
- M Muller
- E Sturm
Jansen PLM, Muller M, and Sturm E. Genes and cholestasis.
Hepatology 34: 1067-1074, 2001.