Optimization of Canalicular ABC Transporter Function in HuH-7 Cells by Modification of Culture Conditions.

Human hepatoma cell lines are useful for evaluation of drug-induced hepatotoxicity, hepatic drug disposition, and drug-drug interactions. However, their applicability is compromised by aberrant expression of hepatobiliary transporters. This study was designed to evaluate whether extracellular matrix (Matrigel) overlay and dexamethasone (DEX) treatment would support cellular maturation of long-term HuH-7 hepatoma cell cultures and improve the expression, localization, and activity of canalicular ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and bile salt export pump (BSEP/ABCB11). Matrigel overlay promoted the maturation of HuH-7 cells toward cuboidal, hepatocyte-like cells displaying bile canaliculi-like structures visualized by staining for filamentous actin (F-actin), colocalization of MRP2 with F-actin, and by accumulation of the MRP2 substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) within the tubular canaliculi. The cellular phenotype was rather homogenous in the Matrigel-overlaid cultures, whereas the standard HuH-7 cultures contained both hepatocyte-like cells and flat epithelium-like cells. Only Matrigel-overlaid HuH-7 cells expressed MDR1 at the canaliculi and excreted the MDR1 probe substrate digoxin into biliary compartments. DEX treatment resulted in more elongated and branched canaliculi and restored canalicular expression and function of BSEP. These findings suggest that hepatocyte polarity, elongated canalicular structures, and proper localization and function of canalicular ABC transporters can be recovered, at least in part, in human hepatoma HuH-7 cells by applying the modified culture conditions. SIGNIFICANCE STATEMENT: We report the first demonstration that proper localization and function of canalicular ABC transporters can be recovered in human hepatoma HuH-7 cells by modification of cell culture conditions. Matrigel overlay and dexamethasone supplementation increased the proportion of hepatocyte-like cells, strongly augmented the canalicular structures between the cells, and restored the localization and function of key canalicular ABC transporters. These results will facilitate the development of reproducible, economical, and easily achievable liver cell models for drug development.

Claudin-3 regulates bile canalicular paracellular barrier and cholesterol gallstone core formation in mice.

BACKGROUND & AIMS: Most cholesterol gallstones have a core consisting of inorganic and/or organic calcium salts, although the mechanisms of core formation are poorly understood. We examined whether the paracellular permeability of ions at hepatic tight junctions is involved in the core formation of cholesterol gallstones, with particular interest in the role of phosphate ion, a common food additive and preservative. METHODS: We focused on claudin-3 (Cldn3), a paracellular barrier-forming tight junction protein whose expression in mouse liver decreases with age. Since Cldn3-knockout mice exhibited gallstone diseases, we used them to assess the causal relationship between paracellular phosphate ion permeability and the core formation of cholesterol gallstones. RESULTS: In the liver of Cldn3-knockout mice, the paracellular phosphate ion permeability through hepatic tight junctions was significantly increased, resulting in calcium phosphate core formation. Cholesterol overdose caused cholesterol gallstone disease in these mice. CONCLUSION: We revealed that in the hepatobiliary system, Cldn3 functions as a paracellular barrier for phosphate ions, to help maintain biliary ion homeostasis. We provide in vivo evidence that elevated phosphate ion concentrations play a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease under cholesterol overdose. LAY SUMMARY: Herein, we reveal a new mechanism for cholesterol gallstone formation, in which increased paracellular phosphate ion permeability across hepatobiliary epithelia causes calcium phosphate core formation and cholesterol gallstones. Thus, altered phosphate ion metabolism under cholesterol overdose plays a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease.

Quantification of Drug-Induced Inhibition of Canalicular Cholyl-l-Lysyl-Fluorescein Excretion From Hepatocytes by High Content Cell Imaging.

We describe the use of a commercially available high content cell imaging algorithm (Cellomics Arrayscan Spot Detector) to quantify biliary excretion of the fluorescent probe substrate cholyl-l-lysyl-fluorescein (CLF) from rat hepatocytes cultured in collagen/matrigel sandwich configuration and to explore inhibition of this process by a variety of test compounds. The method provided robust, reproducible data. Twenty-nine pharmaceuticals inhibited biliary CLF efflux from hepatocytes and a broad range of potencies of inhibition were observed (IC50 values ranged between <1 and 794 µM). Thirteen drugs that inhibited CLF efflux also inhibited hepatocellular uptake of the probe substrate [(3)H]-taurocholate. Although no clear correlation between the potencies of inhibition of the 2 processes was evident, these data highlight the need to consider possible uptake transporter inhibition when interpreting hepatocyte CLF inhibition data. It has been reported that CLF is transported by MRP2. The CLF efflux inhibition data correlated closely with published data on inhibition by the drugs of the bile salt export pump (Bsep), which suggests that the tested drugs inhibit both Bsep and Mrp2. Calculation of the ratios between the maximum human plasma concentrations of the drugs and their CLF efflux inhibition IC50 values raised the possibility that for many, but not all, of them the in vitro effects may be functionally significant in vivo and that Mrp2 inhibition might be a drug-induced liver injury (DILI) risk factor. These data indicate that imaging hepatocyte CLF inhibition is a promising new method for quantification of biliary efflux inhibition by drugs, which could aid assessment of compound-related DILI risk.

Prevalência da baixa densidade mineral óssea em mulheres na pós-menopausa tratadas de câncer de mama / Prevalence of low bone mineral density in postmenopausal breast cancer survivors

OBJETIVO: Avaliar a prevalência da baixa densidade mineral óssea (DMO) em mulheres na pós-menopausa tratadas de câncer de mama. MÉTODOS: Estudo de corte transversal que incluiu 115 mulheres tratadas de câncer de mama atendidas em Hospital Universitário do Sudeste do Brasil. Foram incluídas mulheres com amenorreia há 12 meses ou mais e 45 anos ou mais de idade, tratadas de câncer de mama e livres de doença há pelo menos 5 anos. A DMO foi mensurada pelos raios-X de dupla energia em coluna lombar (L1 a L4) e colo de fêmur. Considerou-se baixa DMO quando valores de T-score de coluna total e/ou colo de fêmur <-1,0 Score de Delphi (DP) (osteopenia e osteoporose). Por meio de entrevista, foram avaliados fatores de risco para baixa DMO. Na análise estatística, empregaram-se os testes do χ2 ou Exato de Fisher. RESULTADOS: A média de idade das pacientes foi 61,6±10,1 anos e o tempo de menopausa, 14,2±5,6 anos, com tempo médio de seguimento de 10,1±3,9 anos. Considerando coluna e colo de fêmur, 60% das mulheres tratadas de câncer de mama apresentavam baixa DMO. Avaliando os fatores de risco para baixa DMO, foi encontrada diferença significativa na distribuição percentual quanto à idade (maior porcentagem de mulheres com mais de 50 anos e baixa DMO), história pessoal de fratura prévia (11,6% com baixa DMO e nenhuma com DMO normal) e índice de massa corpórea. Maior frequência de obesidade foi observada entre mulheres com DMO normal (63%) quando comparadas àquelas com baixa DMO (26,1%; p<0,05). CONCLUSÃO: Mulheres na pós-menopausa tratadas de câncer de mama apresentaram elevada prevalência de baixa DMO (osteopenia e/ou osteoporose). .

PURPOSE: To evaluate the prevalence of low bone mineral density (BMD) in postmenopausal breast cancer survivors. METHODS: In this cross-sectional study, 115 breast cancer survivors, seeking healthcare at a University Hospital in Brazil, were evaluated. Eligibility criteria included women with amenorrhea ≥12 months and age ≥45 years, treated for breast cancer and metastasis-free for at least five years. BMD was measured by DEXA at the lumbar spine (L1-L4) and femoral neck. Low BMD was considered when total-spine and/or femoral-neck T-score values were <-1.0 Delphi Score (DP) (osteopenia and osteoporosis). The risk factors for low BMD were assessed by interview. Data were analyzed statistically by the χ2 test and Fisher's exact test. RESULTS: The mean age of breast cancer survivors was 61.6±10.1 years and time since menopause was 14.2±5.6 years, with a mean follow-up of 10.1±3.9 years. Considering spine and femoral neck, 60% of breast cancer survivors had low BMD. By evaluating the risk factors for low BMD, a significant difference was found in the percent distribution for age (higher % of women >50 years with low BMD), personal history of previous fracture (11.6% with low BMD versus 0% with normal BMD) and BMI. A higher frequency of obesity was observed among women with normal BMD (63%) compared to those with low BMD (26.1%) (p<0.05). CONCLUSION: Postmenopausal breast cancer survivors had a high prevalence of osteopenia and osteoporosis. .

Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport.

Fresh hepatocytes cultured in a sandwich configuration allow for the development of intact bile canaliculi and the ability to measure hepatic uptake and biliary clearance. A disadvantage of this model is its dependence upon hepatocytes from fresh tissue. Therefore, the ability to use cryopreserved human hepatocytes in this model would be a great advantage. Multiple variables were tested, and the recommended conditions for culturing cryopreserved human hepatocytes in a sandwich configuration in 24-well plates are as follows: BioCoat plates, a cell density of 0.35 x 10(6) cells/well in 500 microl, an overlay of Matrigel and InVitroGRO media. These conditions resulted in good hepatocyte morphology and the formation of distinct bile canaliculi. The function of multiple uptake and efflux transporters was tested in multiple lots of cryopreserved and fresh human hepatocytes. For taurocholate [Na+ taurocholate cotransporting polypeptide/organic anion transporting polypeptide (OATP) uptake/bile salt export pump efflux], the average apparent uptake, apparent intrinsic biliary clearance, and biliary excretion index among five cryopreserved hepatocyte lots was high, ranging from 11 to 17 pmol/min/mg protein, 5.8 to 10 microl/min/mg protein, and 41 to 63%, respectively. The corresponding values for digoxin (OATP-8 uptake/multidrug resistance protein 1 efflux) were 0.69 to 1.5 pmol/min/mg protein, 0.60 to 1.5 microl/min/mg protein, and 37 to 63%. Both substrates exhibited similar results when fresh human hepatocytes were used. In addition, substrates of breast cancer resistance protein and multidrug resistance-associated protein 2 were also tested in this model, and all cryopreserved lots showed functional transport of these substrates. The use of cryopreserved human hepatocytes in 24-well sandwich culture to form intact bile canaliculi and to exhibit functional uptake and efflux transport has been successfully demonstrated.

Use of canalicular membrane vesicles (CMVs) from rats, dogs, monkeys and humans to assess drug transport across the canalicular membrane.

INTRODUCTION: A novel application of a Ultrafree filter cartridge/centrifugation method was evaluated to determine uptake in canalicular membrane vesicles (CMVs) from SD rats, beagle dogs, cynomolgus monkeys (common safety species in the pharmaceutical industry) and humans to assess biliary transport. METHODS: CMVs prepared from fresh livers of rats, dogs, monkeys and humans (four donors) were characterized for enrichment, basolateral and Golgi contamination and orientation. The presence of MRP2 and p-glycoprotein (P-gp) were confirmed by Western blots. Uptake of [3H]-leukotriene C4 (LTC4) and [3H]-estradiol-17beta-d-glucuronide (E2-Gluc) was determined at a low substrate concentration and/or by kinetic measurements (K(m) and V(max)). Correlation of in vitro data with in vivo findings was achieved by determining the biliary clearance of E2-Gluc in rats after a single i.v. dose and with literature in vivo data for LTC4. RESULTS: CMVs were highly enriched and minimally contaminated based on marker enzyme activities. Uptake clearance among different species varied by approximately ten-fold (rat > dog = human > monkey) for LTC4 and less than two-fold for E2-Gluc. The lower uptake of LTC4 by human than rat CMVs may be attributed to a higher Km value for human than rat CMVs. Uptake of LTC4 or E2-Gluc by human CMVs showed little inter-subject variability (2-5-fold). Differences in in vitro uptake clearance (10-fold) between LTC4 and E2-Gluc in rat CMVs seemed to correlate with differences in their biliary clearance (4-fold) in rats, consistent with LTC4 and E2-Gluc being a high and a low clearance substrate, respectively. DISCUSSION: A novel application of a Ultrafree filter cartridge/centrifugation method was developed to determine uptake in CMVs from different preclinical animal safety species and humans, and may represent a useful approach to study the mechanism of biliary excretion during drug discovery and development.

Biliary secretory function in rats chronically intoxicated with aluminum.

The effects of a chronic aluminum (Al) exposure on biliary secretory function, with special emphasis on hepatic handling of non-bile salt organic anions, was investigated. Male Wistar rats received, intraperitoneally, either 27 mg/kg body weight of Al, as Al hydroxide [Al (+) rats], or the vehicle saline [Al (-) rats] three times a week for 3 months. Serum and hepatic Al levels were increased by the treatment (approximately 9- and 4-fold, respectively). This was associated with enhanced malondialdehyde formation (+110%) and a reduction in GSH content (-17%) and in the activity of the antioxidant enzymes catalase (-84%) and GSH peroxidase (-46%). Bile flow (-23%) and the biliary output of bile salts (-39%), cholesterol (-43%), and proteins (-38%) also decreased. Compartmental analysis of the plasma decay of the model organic anion bromosulphophthalein revealed that sinusoidal uptake and canalicular excretion of the dye were significantly decreased in Al (+) rats (-53 and -43%, respectively). Expression of multidrug resistance-associated protein 2 (Mrp2), the main, multispecific transporter involved in the canalicular excretion of organic anions, was also decreased (-40%), which was associated with a significant decrease in the cumulative biliary excretion of the Mrp2 substrate, dinitrophenyl-S-glutathione (-50%). These results show that chronic Al exposure leads to oxidative stress, cholestasis, and impairment of the hepatic handling of organic anions by decreasing both sinusoidal uptake and canalicular excretion. The alteration of the latter process seems to be causally related to impairment of Mrp2 expression. We have addressed some possible mechanisms involved in these deleterious effects.

Functional expression of the canalicular bile salt export pump of human liver.

BACKGROUND & AIMS: Hepatic bile salt secretion is an essential function of vertebrate liver. Rat and mouse bile salt export pump (Bsep) are adenosine triphosphate (ATP)-dependent bile salt transporters. Mutations in human BSEP were identified as the cause of progressive familial intrahepatic cholestasis type 2. BSEP protein is highly identical with its rat and mouse orthologs and has not yet been functionally characterized; the effect of BSEP mutations on its function has also not been studied. Therefore, the aim of this study was to functionally characterize human BSEP. METHODS: Complementary DNA for BSEP was isolated from human liver and expressed with the baculovirus system in Sf9 cells. ATP-dependent bile salt transport assays were performed with Sf9 cell vesicles expressing BSEP and a rapid filtration assay. RESULTS: Cloning of human BSEP required the inactivation of a bacterial cryptic promoter motif within its coding region. BSEP expressed in Sf9 cells transports different bile salts in an ATP-dependent manner with Michaelis constant values as follows: taurocholate, 7.9 +/- 2.1 micromol/L; glycocholate, 11.1 +/- 3.3 micromol/L; taurochenodeoxycholate, 4.8 +/- 1.7 micromol/L; tauroursodeoxycholate, 11.9 +/- 1.8 micromol/L. The rank order of the intrinsic clearance of bile salts was taurochenodeoxycholate > taurocholate > tauroursodeoxycholate > glycocholate. CONCLUSIONS: This study characterizes human BSEP as an ATP-dependent bile salt export pump with transport properties similar to its rat and mouse orthologs. Expression of BSEP in Sf9 cells will enable functional characterization of the consequences of mutations in the human BSEP gene.

Effects of docosahexaenoic acid on annular lipid fluidity of the rat bile canalicular plasma membrane.

The role of docosahexaenoic acid (DHA) in the fluidity of the annular lipid regions and their associated membrane-bound proteins is still not as well understood as that in the global (bulk) lipid regions. We therefore studied the effects of dietary DHA on the relationship between annular and global lipid fluidity and membrane-bound enzymes such as 5'-nucleotidase and Mg(2)+-ATPase in the rat bile canalicular membrane. Dietary DHA caused significant increases in 5'-nucleotidase and Mg(2)+-ATPase activity and in global and annular lipid fluidity, a higher increase in fluidity in the annular lipids than the global lipids, and a decrease in the cholesterol-to-phospholipid molar ratio in the canalicular membrane. Plasma total cholesterol and LDL cholesterol decreased, and fecal cholesterol increased in the DHA-fed rats. No changes were observed in oxidative markers, but glutathione peroxidase increased in the liver with DHA feeding. Annular lipid fluidity, but not global lipid fluidity, correlated remarkably well with DHA, synchronously with the activities of 5'-nucleotidase and Mg(2)+-ATPase. The data indicate that the DHA-induced increase in annular lipid fluidity is responsible for the increases observed in the enzyme activity. We therefore concluded that the increased activity of membrane-bound enzymes and transporters induced by DHA and the concomitant increase in annular lipid fluidity comprise one of the mechanisms involved in DHA-induced clearance of plasma cholesterol.

Intravenous fish oil emulsion attenuates total parenteral nutrition-induced cholestasis in newborn piglets.

Total parenteral nutrition (TPN) causes intrahepatic cholestasis and membrane phospholipid changes. Fatty acid (FA) composition of bile and hepatocyte phospholipid is influenced by dietary FA composition. We hypothesized that altering FA composition of i.v. lipid emulsions modifies 1) severity of TPN-induced cholestasis; 2) hepatocyte membrane composition and function; 3) bile flow and composition. Newborn piglets received either sow's milk, TPN with i.v. soybean oil or TPN with i.v. fish oil (FO). After 3 wk, basal and stimulated bile flow were measured after bolus injections of 20, 50, and 100 micromol/kg of taurocholate (TCA). Bile was analyzed for bile acids, cholesterol, phospholipids, and phospholipid-FA. Sinusoidal and canalicular membrane PL-FA, fluidity, and Na+/K+-ATPase were measured. Although the soybean oil-fed animals developed cholestasis, the FO and milk group had similar liver and serum bilirubin. Basal and stimulated bile flow rates were impaired in the soybean oil but not in the FO group. Hepatocyte membrane FA composition reflected dietary FA. Changes in sinusoidal and canalicular membrane fluidity and sinusoidal Na+/K+-ATPase activity did not explain the effect of FO on TPN-induced cholestasis. Intravenous FO reduces TPN-induced cholestasis by unknown mechanisms.

Reconstitution of bile acid transport in a heterologous cell by cotransfection of transporters for bile acid uptake and efflux.

The rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP) is a 110-kDa transmembrane phosphoglycoprotein that is thought to have bile acid efflux, ecto-ATPase, and cell adhesion properties. Its extracellular amino-terminal domain is highly homologous to carcinoembryonic antigen (CEA), a glycophosphatidyl inositol-anchored membrane protein with cell adhesion properties and a marker for adenocarcinoma. In the current study, we examined the possibility of more clearly defining the role of CBATP in bile acid efflux by cotransfecting a heterologous cell, the COS cell, with cDNAs for a bile acid importer, the ileal bile acid transporter (IBAT), as well as for CBATP. The results show that when IBAT mediates uptake of [3H]taurocholate to a level 20-fold higher than that achieved previously by nonspecific pinocytosis, CBATP mediates time-, temperature- and concentration-dependent efflux. Efflux of [3H]taurocholate mediated by CBATP in the cotransfected COS cells is saturable and has curvilinear kinetic characteristics (Vmax = 400 pmol/mg protein/min, Km = 70 microM). It is inhibited by 4,4'-diisothiocyanostilbene-2,2-disulfonic acid and dependent on ATP but not dependent on membrane potential. Although CEA could not mediate bile acid efflux in COS cells cotransfected with IBAT and CEA, efflux of [3H]taurocholate was detected in COS cells cotransfected with IBAT and a chimeric molecule having the carboxyl-terminal tail and membrane spanning domain of CBATP and the amino-terminal extracellular tail of CEA. Taken together, these data provide further evidence that CBATP confers bile acid efflux properties on heterologous cells and that its cytoplasmic tail and membrane spanning segment are integral to this property. The data also establish a model system for more clearly defining the molecular determinants of bile acid transport mediated by this molecule.

The neurotoxin 1-methyl-4-phenylpyridinium is a substrate for the canalicular organic cation/H+ exchanger.

Hepatic organic cation transport consists, in part, of carrier-mediated sinusoidal uptake stimulated by an inside-negative membrane potential and canalicular excretion driven by electroneutral organic cation/H+ exchange. Intracellular organic cation transport involves sequestration into acidified organelles, also mediated by organic cation/H+ exchange. A sinusoidal organic cation transporter has been cloned; however, canalicular organic cation transport has not been characterized at the molecular level. On the assumption that hepatic organic cation/H+ exchange resembles monoamine transport in synaptic vesicles, we examined, using canalicular rat liver plasma membrane vesicles, the transport of 1-methyl-4-phenylpyridinium (MPP+), a neurotoxin taken up by a synaptic vesicular monoamine transporter that has been cloned. Under voltage-clamped conditions, an outwardly directed H+ gradient stimulated [3H]MPP+ uptake, compared with uptake under pH-equilibrated conditions, consistent with electroneutral MPP+/H+ exchange. Substrates for canalicular organic cation/H+ exchange cis-inhibited pH-dependent MPP+ uptake. Equilibrium exchange of [14C]tetraethylammonium was inhibited by MPP+ in a concentration-dependent manner, consistent with a direct interaction of MPP+ with the organic cation carrier. Carrier-mediated MPP+ uptake exhibited saturability, with kinetic parameters similarto those described for canalicular tetraethylammonium+/H+ exchange. Canalicular [3H]MPP+ uptake was ATP-independent and, thus, distinct from P-glycoprotein-mediated efflux. The finding that MPP+ is a substrate for canalicular organic cation/H+ exchange is applicable to studies, using degenerate oligonucleotides complementary to sequences conserved in neurotransmitter transporters, aimed at cloning this transporter.

Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes.

We have previously shown that the multi-drug resistance protein (MRP) mediates the ATP-dependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. In the present study we demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse transcription-PCR of human and normal rat liver mRNA resulted in two expected cDNA fragments of MRP. Four different antibodies against MRP reacted on immunoblots with the glycoprotein of about 190 kD from human canalicular as well as basolateral hepatocyte membrane preparations. A polyclonal antibody directed against the carboxy-terminal sequence of MRP detected the rat homolog of MRP in liver. Double immunofluorescence microscopy and confocal laser scanning microscopy showed the presence of human MRP and rat Mrp in the canalicular as well as in the lateral membrane domains of hepatocytes. The transport function of the mrp gene-encoded conjugate export pump was assayed in plasma membrane vesicles with leukotriene C4 as a high-affinity glutathione S-conjugate substrate. The deficient ATP-dependent conjugate transport in canalicular membranes from TR- mutant rat hepatocytes was associated with a lack of amplification of one of the mrp cDNA fragments and with a selective loss of Mrp on immunoblots of canalicular membranes. Double immunofluorescence microscopy of livers from transport-deficient TR- mutant rats localized Mrp only to the lateral but not to the canalicular membrane. Our results indicate that the absence of Mrp or an isoform of Mrp from the canalicular membrane is the basis for the hereditary defect of the hepatobiliary excretion of anionic conjugates by the transport-deficient hepatocyte.

Functional expression cloning of the canalicular sulfate transport system of rat hepatocytes.

We have cloned a single cDNA encoding the canalicular sulfate transporter of rat liver using Xenopus laevis oocytes as a functional expression system. The cloned cDNA sulfate anion transporter-1 (sat-1) expresses saturable Na(+)-independent sulfate uptake (Km approximately 0.14 mM) that can be inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS, IC50 = 28 microM) and oxalate, but not by succinate or cholate. These properties are very similar to sulfate uptake expressed in oocytes injected with total rat liver mRNA and to the bicarbonate/sulfate exchange system previously characterized in canalicular rat liver plasma membrane vesicles. The cloned sat-1 cDNA has a total length of 3726 base pairs (bp) with an open reading frame encompassing 2109 bp, a 5'-untranslated region of 367 bp, and a 3'-untranslated region of 1250 bp. The coding region predicts a protein of 703 amino acids with a calculated molecular mass of 75.4 kDa. Computer-based hydrophobicity analysis suggests the presence of 12 putative transmembrane spanning domains. Furthermore, three potential glycosylation sites are detected (Asn-158, Asn-163, Asn-587). Northern blot analysis indicates that similar sulfate anion transporters are also present in the kidney, muscle, and brain of rat and in the liver of the mouse. Using antisense oligonucleotides the mRNA-species of the sat-1 analogue in rat kidney has been characterized by hybrid depletion experiments (Markovich, D., Bissig, M., Sorribas, V., Hagenbuch, B., Meier, P. J., and Murer, H. (1994) J. Biol. Chem. 269, 3022-3026).

Hepatobiliary excretion of organic anions in double-mutant rats with a combination of defective canalicular transport and uridine 5'-diphosphate-glucuronyltransferase deficiency.

Mutant rats with a selective defect for the hepatobiliary excretion of organic anions (GT+TR- rats) are valuable models to study hepatic transport processes. However, retained conjugates in the livers of these rats may secondarily affect hepatic uptake, metabolism, and excretion of other compounds and this may confound the interpretation of test results. We have developed double mutants (GT-TR-) rats with both a conjugation and an excretion defect by cross-breeding uridine 5'-diphosphate-glucuronyl-transferase-deficient GT-TR+ Gunn rats with transport-deficient GT+TR- rats. Phenotypically, GT-TR- rats and Gunn rats are alike in that both have unconjugated hyperbilirubinemia. Intravenous administration of tetrabromosulphthalein, bilirubin diglucuronide, and bilirubin monoglucuronide revealed a significant difference in that the clearance of these compounds was reduced to 10%, 10%, and 20%, respectively, in GT-TR- rats when compared with Gunn rats. The hepatic elimination of tetrabromosulphthalein in GT-TR- rats and in GT+TR- rats is impaired to the same extent. Thus, both have a similar hepatic excretion defect. However, bile flow and bile acid excretion in GT+TR- rats are more depressed than in GT-TR- rats: bile flow, 88 +/- 3 vs. 36 +/- 1 microliters/min.kg and bile acid excretion, 3.4 +/- 0.2 vs. 1.5 +/- 0.1 mumol/min.kg in GT-TR- and GT+TR- rats, respectively. This suggests that accumulated glucuronides in the liver inhibit bile flow and bile acid excretion. To test whether conjugated bilirubin and the photoisomers of unconjugated bilirubin are excreted via the same transport pathways, the effect of phototherapy was studied in GT-TR- rats and in Gunn rats. Photoexposure caused a 120% increase in biliary excretion of bilirubin isomers in Gunn rats and only 40% in GT-TR- rats. This shows that the biliary excretion of bilirubin photoisomers is indeed affected by the hepatic excretion defect of GT-TR- rats and suggests that hepatic excretion of bilirubin photoisomers proceeds via the same route as other organic anions such as conjugated bilirubin and tetrabromosulphthalein.