Biomedical subjects

B Stieger

Publications and source records attributed to B Stieger.

At least 55 records · Page 3Linked to original sources

PubMed1999

Polyspecific substrate uptake by the hepatic organic anion transporter Oatp1 in stably transfected CHO cells.

The rat liver organic anion transporting polypeptide (Oatp1) has been extensively characterized mainly in the Xenopus laevis expression system as a polyspecific carrier transporting organic anions (bile salts), neutral compounds, and even organic cations. In this study, we extended this characterization using a mammalian expression system and confirm the basolateral hepatic expression of Oatp1 with a new antibody. Besides sulfobromophthalein [Michaelis-Menten constant (Km) of approximately 3 microM], taurocholate (Km of approximately 32 microM), and estradiol- 17beta-glucuronide (Km of approximately 4 microM), substrates previously shown to be transported by Oatp1 in transfected HeLa cells, we determined the kinetic parameters for cholate (Km of approximately 54 microM), glycocholate (Km of approximately 54 microM), estrone-3-sulfate (Km of approximately 11 microM), CRC-220 (Km of approximately 57 microM), ouabain (Km of approximately 3,000 microM), and ochratoxin A (Km of approximately 29 microM) in stably transfected Chinese hamster ovary (CHO) cells. In addition, three new substrates, taurochenodeoxycholate (Km of approximately 7 microM), tauroursodeoxycholate (Km of approximately 13 microM), and dehydroepiandrosterone sulfate (Km of approximately 5 microM), were also investigated. The results establish the polyspecific nature of Oatp1 in a mammalian expression system and definitely identify conjugated dihydroxy bile salts and steroid conjugates as high-affinity endogenous substrates of Oatp1.

Animals↗

PubMed1999

Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration.

The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichlorofluorescein were excreted into and concentrated in the bile canalicular lumen of day 5 sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca(2+)-free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca(2+)-free buffer. The cumulative uptake of [(3)H]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca(2+)-free buffer, due to accumulation of taurocholate in canalicular spaces. When [(3)H]taurocholate was preloaded in the day 5 sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca(2+)-free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from approximately 8% at day 0 to approximately 60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.

Animals↗

PubMed1999

Localization of the organic anion transporting polypeptide 2 (Oatp2) in capillary endothelium and choroid plexus epithelium of rat brain.

In this study we investigated the distribution of a recently cloned polyspecific organic anion transporting polypeptide (Oatp2) in rat brain by nonradioactive in situ hybridization histochemistry and immunofluorescence microscopy. The results demonstrate that Oatp2 is expressed in brain capillary and in plexus epithelial cells. At the blood-brain barrier (BBB), Oatp2 expression could be co-localized with the endothelial marker vWF (von Willebrand factor) but not with the astrocyte marker GFAP (glial fibrillary acidic protein). In choroid plexus epithelial cells, Oatp2 could be localized to the basolateral cell pole, whereas the first member of the Oatp gene family of membrane transporters to be cloned (Oatp1) co-localized with the alpha(1)-subunit of Na,K-ATPase at the apical plasma membrane domain. Because Oatp1 and Oatp2 have been previously shown to mediate transmembrane transport of a wide variety of amphipathic organic compounds, including many drugs and other xenobiotics, the histochemical localization of Oatp2 at the BBB and of Oatp1 and Oatp2 in the choroid plexus imply a role for these transporters in the active exchange of amphipathic solutes between the blood, brain, and cerebrospinal fluid compartments. (J Histochem Cytochem 47:1255-1263, 1999)

Animals↗

PubMed1999

Regulation of bile salt export pump mRNA levels by dexamethasone and osmolarity in cultured rat hepatocytes.

The major canalicular bile salt export pump (Bsep) of mammalian liver is downregulated by endotoxin. This study reports on the effects of dexamethasone and osmolarity on Bsep mRNA expression in cultured rat hepatocytes and its functional relevance in rat liver. Expression of Bsep mRNA in rat hepatocytes 24 and 48 h after isolation was dependent on the presence of dexamethasone (100 nM) in the culture medium. Bsep was functionally active at the pseudocanalicular membrane in cells cultured for 4 days in medium containing dexamethasone. Hypoosmolarity (205 mosmol/l) led to an induction of Bsep mRNA levels, whereas expression was decreased by hyperosmolarity (405 mosmol/l). Also the decay of Bsep mRNA following dexamethasone withdrawal was osmosensitive. In rat liver, dexamethasone counteracted the lipopolysaccharide (LPS)-induced down-regulation of Bsep mRNA levels after 12 hours and abolished the LPS-induced inhibition of taurocholate excretion. These results indicate that glucocorticoids are strong inducers of Bsep in liver. Furthermore, Bsep mRNA levels are osmosensitively regulated. The data suggest a longterm control of Bsep mRNA by osmolarity in addition to the short-term effects on canalicular bile acid excretion, which were reported recently.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Hepatic uptake of the magnetic resonance imaging contrast agent gadoxetate by the organic anion transporting polypeptide Oatp1.

Gadoxetate is a new hepatobiliary magnetic resonance imaging contrast agent. It is specifically taken up by hepatocytes, and its uptake can be inhibited by the coadministration of bromosulfophthalein, suggesting an involvement of one or several of the cloned organic anion transporting polypeptides Oatp1, Oatp2, and/or OATP. In this study, we demonstrated saturable uptake of gadoxetate by Oatp1 cRNA-injected Xenopus laevis oocytes (Km approximately 3.3 mM). In contrast, gadoxetate was not taken up by Oatp2 or OATP cRNA-injected oocytes. Oatp1-mediated gadoxetate uptake (100 microM) could be inhibited by 10 microM bromosulfophthalein (45%), 200 microM taurocholate (92%), 100 microM rifamycin SV (97%), and 100 microM rifampicin (51%). These results show that gadoxetate is a low-affinity substrate of Oatp1. Oatp1-mediated gadoxetate transport demonstrated a similar apparent Km value and cis-inhibition pattern as previously determined in rats in vivo, indicating that Oatp1 is significantly involved in gadoxetate uptake into rat liver.

Animals↗

PubMed1999

Carrier-mediated hepatic uptake of peptidic endothelin antagonists in rats.

The endothelin antagonist BQ-123, an anionic cyclopentapeptide, is taken up by rat hepatocytes through active transport systems. Here, we have examined the hepatocellular uptake mechanism for several BQ-123 derivatives with anionic charges using isolated rat hepatocytes. BQ-485, a linear peptide, BQ-518, a cyclic peptide, and compound A, a cyclic peptide with a cationic moiety, were taken up by hepatocytes in a concentration-dependent manner. The uptake of BQ-485 was most efficient, whereas compound A showed comparable uptake with BQ-123. The uptake of these peptides was Na(+)- and energy-dependent, suggesting that active transport mechanisms are involved in their uptake into hepatocytes. BQ-485, BQ-518, and compound A can almost completely inhibit both the Na(+)-dependent and -independent uptake of [(3)H]BQ-123, with inhibition constants (K(i)) that are comparable to the Michaelis-Menten constants (K(m)) for their Na(+)-dependent and -independent uptake, respectively. Inhibition by BQ-485 was competitive, and the uptake of BQ-485 can be inhibited by BQ-123, with K(i) values that are comparable with the K(m) values for BQ-123 uptake. The uptake of BQ-123 by COS-7 cells transfected with either Na(+)-dependent taurocholate-cotransporting polypeptide (Ntcp) or Na(+)-independent basolateral organic anion-transporting polypeptide (oatp1) was minimal. Thus, these three peptides share the transporters that also recognize BQ-123 but appear to differ from Ntcp and oatp1.

Anesthetics, Inhalation↗

PubMed1998

The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver.

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Characterization of a sodium-dependent transport system for butyrobetaine into rat liver plasma membrane vesicles.

Butyrobetaine transport into the liver was studied using isolated rat hepatocyte plasma membrane vesicles. In the presence of a sodium chloride gradient, an overshoot could be observed, indicating active sodium-dependent transport. A similar overshoot was recorded in the presence of lithium, but not of potassium, cesium, or choline chloride. Investigation of several sodium salts revealed that an overshoot could only be observed in the presence of chloride, but not of nitrate, thiocyanate, sulfate, or gluconate. An osmolarity plot in the presence of sodium chloride revealed a slope different from zero and a positive intercept, indicating active transport and nonspecific binding, respectively. In agreement with the osmolarity plot, the kinetic characterization of butyrobetaine transport revealed a binding and a saturable component. The saturable component could be described by Michaelis-Menten kinetics, with a Km of 4.88 +/- 0.70 mmol/L and a Vmax of 4.16 +/- 0.73 picomoles per milligram of protein per second. Butyrobetaine transport could be inhibited significantly (30%) by 250 micromol/L propionylcarnitine, but not by D- or L-carnitine, other acylcarnitines (acetylcarnitine, isovalerylcarnitine, palmitoylcarnitine), trimethyllysine, or quinine. Butyrobetaine transport activity was also expressed in Xenopus laevis oocytes by injecting mRNA isolated from rat liver or kidney. After 5 days of cultivation, the endogenous butyrobetaine transport activity was increased by 82% in oocytes injected with liver mRNA and by 99% in oocytes injected with kidney mRNA. The studies show that butyrobetaine is transported actively across the basolateral plasma membrane of hepatocytes and that this transport is driven by sodium and chloride gradients. This transport is quite specific for butyrobetaine and is not rate-limiting for carnitine biosynthesis.

Animals↗

PubMed1998

Sodium taurocholate cotransporting polypeptide is a serine, threonine phosphoprotein and is dephosphorylated by cyclic adenosine monophosphate.

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.

Amino Acids↗

PubMed1998

Bile acid and xenobiotic transporters in liver.

Identification of transporters involved in bile formation in liver is rapidly progressing. It is now clear that these transporters are also important in drug disposition in the body. Significant recent advances include the cloning of an ATP-dependent bile acid transporter, related to the p-glycoprotein family, in the canalicular plasma membrane of hepatocytes. In addition, liver transporter genes responsible for hereditary forms of cholestatic liver disease have been identified and found to belong to the superfamily of ATP-binding cassette proteins.

Animals↗

PubMed1998

Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration.

PURPOSE: This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions. METHODS: Hepatocytes isolated from male Wistar rats (230-280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein. Na+/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5. RESULTS: By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was approximately 13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na+-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 microM) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent Km values were comparable for day-0 and day-5 hepatocytes (3241 microM). In contrast, the Vmax values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were approximately 5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film. CONCLUSIONS: A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.

Animals↗

PubMed1998

Taurocholate induces preferential release of phosphatidylcholine from rat liver canalicular vesicles.

AIMS/BACKGROUND: Biliary phospholipid secretion involves predominant segregation of canalicular phosphatidylcholine into bile. We tested the hypothesis that micellar concentrations of the major physiologic bile salt taurocholate can preferentially solubilize phosphatidylcholine from the canalicular rat liver plasma membrane. METHODS: Subcellular fractions from rat liver and kidney were isolated with standardized procedures, incubated in vitro with taurocholate or 3-[(3-cholamidopropyl)dimethylammonio]-propane-1-sulphonate (CHAPS) and released phospholipids determined after centrifugation. RESULTS: After incubation of canalicular (cLPM) and basolateral (blLPM) rat liver plasma membrane vesicles with 6 and 8 mM taurocholate, the proportion of phosphatidylcholine released was about two-fold higher as compared with its relative contribution to the overall lipid composition of the membranes. Quantitatively, this taurocholate-induced preferential phosphatidylcholine release was about four-fold higher in cLPM (117 nmol) as compared with blLPM (28 nmol). Comparison of membranes from different organs showed that increased sphingomyelin content reduced taurocholate-induced phosphatidylcholine release. Furthermore, phosphatidylcholine release from cLPM did not fit an inverse exponential relationship between membrane sphingomyelin content and phosphatidylcholine release from different starting material, indicating that cLPM is especially prone to taurocholate-induced phosphatidylcholine release. In contrast, in rat liver microsomes and kidney brush border membranes, taurocholate released phospholipids in proportion of their membrane contents, indicating an unspecific membrane solubilizing effect only. Similarly, CHAPS had an unselective lipid solubilizing effects in cLPM and blLPM. CONCLUSION: These results support the concept that the very last step of canalicular phospholipid secretion is mediated in vivo by bile salt-induced vesiculation of phosphatidylcholine-enriched microdomains from the outer leaflet of cLPM.

Animals↗

PubMed1998

Substrate specificity of the rat liver Na(+)-bile salt cotransporter in Xenopus laevis oocytes and in CHO cells.

It has been proposed that the hepatocellular Na(+)-dependent bile salt uptake system exhibits a broad substrate specificity in intact hepatocytes. In contrast, recent expression studies in mammalian cell lines have suggested that the cloned rat liver Na(+)-taurocholate cotransporting polypeptide (Ntcp) may transport only taurocholate. To characterize its substrate specificity Ntcp was stably transfected into Chinese hamster ovary (CHO) cells. These cells exhibited saturable Na(+)-dependent uptake of [3H]taurocholate [Michaelis constant (K(m)) of approximately 34 microM] that was strongly inhibited by all major bile salts, estrone 3-sulfate, bumetanide, and cyclosporin A. Ntcp cRNA-injected Xenopus laevis oocytes and the transfected CHO cells exhibited saturable Na(+)-dependent uptake of [3H]taurochenodeoxycholate (Km of approximately 5 microM), [3H]tauroursodeoxycholate (Km of approximately 14 microM), and [14C]glycocholate (Km of approximately 27 microM). After induction of gene expression by sodium butyrate, Na(+)-dependent transport of [3H]estrone 3-sulfate (Km of approximately 27 microM) could also be detected in the transfected CHO cells. However, there was no detectable Na(+)-dependent uptake of [3H]bumetanide or [3H]cyclosporin A. These results show that the cloned Ntcp can mediate Na(+)-dependent uptake of all physiological bile salts as well as of the steroid conjugate estrone 3-sulfate. Hence, Ntcp is a multispecific transporter with preference for bile salts and other anionic steroidal compounds.

Animals↗

PubMed1998

Expression, distribution, and activity of Na+,K+-ATPase in normal and cholestatic rat liver.

Hepatocellular Na+,K+-ATPase is an important driving force for bile secretion and has been localized to the basolateral plasma membrane domain. Cholestasis or impaired bile flow is known to modulate the expression, domain specificity, and activity of various transport systems involved in bile secretion. This study examined Na+, K+-ATPase after ethinylestradiol (EE) treatment and after bile duct ligation (BDL), two rat models of cholestasis. It applied quantitative immunoblotting, biochemical and cytochemical determination of enzyme activity, and immunocytochemistry to the same livers. The data showed a good correlation among the results of the different methods. Neither EE nor BDL induced alterations in the subcellular distribution of Na+,K+-ATPase, which was found in the basolateral but not in the canalicular (apical) plasma membrane domain. Protein expression and enzyme activity showed a small (approximately 10%) decrease after EE treatment and a similar increase after BDL. These modest changes could not be detected by immunofluorescence, immuno EM, or cytochemistry. The data, therefore, demonstrate that Na+,K+-ATPase is only slightly affected by EE and BDL. They suggest that other components of the bile secretory apparatus that take effect downstream of the primary basolateral driving force may play a more prominent role in the pathogenesis of cholestasis.

Animals↗

PubMed1998

Transport of temocaprilat into rat hepatocytes: role of organic anion transporting polypeptide.

The mechanism for hepatic uptake of temocaprilat, an angiotensin-converting enzyme inhibitor that is predominantly excreted into bile was studied using isolated rat hepatocytes and COS-7 cells expressing the organic anion transporting polypeptide (oatp1). The uptake of temocaprilat into isolated rat hepatocytes exhibited saturation with a Km of 20.9 microM and a Vmax of 0.21 nmol/min/mg protein. This uptake was temperature sensitive and was significantly reduced by metabolic inhibitors, a sulfhydryl-modifying reagent and an anion-exchange inhibitor, although the replacement of Na+ with Li+ in the medium did not affect the uptake. [3H]Temocaprilat uptake was inhibited by estradiol-17beta-D-glucuronide and dibromosulphophthalein, typical substrates for the Na+-independent organic anion transporter, in a concentration-dependent manner, whereas excess estradiol-17beta-D-glucuronide did not completely inhibit the uptake. Temocaprilat uptake into COS-7 cells transfected with oatp1 cDNA revealed a concentration-dependency with a Km of 46.7 microM, a value comparable with that obtained in isolated hepatocytes. The contribution of oatp1 to carrier-mediated hepatic uptake of temocaprilat was less than 50% by correcting the uptake clearance with that of estradiol-17beta-D-glucuronide. A good linear correlation was observed for the inhibitory effect of angiotensin-converting enzyme inhibitors (benazeprilat, cilazaprilat, delaprilat and enalaprilat) between isolated hepatocytes and oatp1-expressing cells. These data suggest that oatp1, along with another transporter(s), mediates the uptake of angiotensin-converting enzyme inhibitors into rat hepatocytes.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1997

Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain.

A novel multispecific organic anion transporting polypeptide (oatp2) has been isolated from rat brain. The cloned cDNA contains 3,640 bp. The coding region extends over 1,983 nucleotides, thus encoding a polypeptide of 661 amino acids. Oatp2 is homologous to other members of the oatp gene family of membrane transporters with 12 predicted transmembrane domains, five potential glycosylation, and six potential protein kinase C phosphorylation sites. In functional expression studies in Xenopus laevis oocytes, oatp2 mediated uptake of the bile acids taurocholate (Km approximately 35 microM) and cholate (Km approximately 46 microM), the estrogen conjugates 17beta-estradiol-glucuronide (Km approximately 3 microM) and estrone-3-sulfate (Km approximately 11 microM), and the cardiac gylcosides ouabain (Km approximately 470 microM) and digoxin (Km approximately 0.24 microM). Although most of the tested compounds are common substrates of several oatp-related transporters, high-affinity uptake of digoxin is a unique feature of the newly cloned oatp2. On the basis of Northern blot analysis under high-stringency conditions, oatp2 is highly expressed in brain, liver, and kidney but not in heart, spleen, lung, skeletal muscle, and testes. These results provide further support for the overall significance of oatps as a new family of multispecific organic anion transporters. They indicate that oatp2 may play an especially important role in the brain accumulation and toxicity of digoxin and in the hepatobiliary and renal excretion of cardiac glycosides from the body.

Amino Acid Sequence↗

PubMed1997

Functional expression of the rat liver canalicular isoform of the multidrug resistance-associated protein.

The rat hepatocanalicular isoform (called mrp2) of the human multidrug resistance-associated protein (MRP) has been cloned and transiently expressed in COS-7 cells and in Xenopus laevis oocytes. In both systems mrp2 expression induced a markedly increased efflux of intracellularly formed [14C]2,4-dinitrophenyl-S-glutathione. Injection of mrp2 cRNA into oocytes also stimulated efflux of [3H(N)]leukotriene C4. Furthermore, mrp2 mRNA was markedly decreased in the liver of the transport mutant TR rat, which has a congenital defect in the biliary excretion of glutathione-S conjugates and of other divalent organic anions. The study provides a direct demonstration of mrp2-mediated transport function and supports the concept that mrp2 represents the canalicular multispecific organic anion transporter (cMOAT) of mammalian liver.

ATP-Binding Cassette Transporters↗

PubMed1997

Differential interaction of bile acids from patients with inborn errors of bile acid synthesis with hepatocellular bile acid transporters.

People with genetic or acquired defects in the biosynthesis of bile acids may suffer from cholestasis. Patients with a deficiency of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase from 3 beta, 7 alpha-dihydroxy- and 3 beta, 7 alpha, 12 alpha-trihydroxy-5-cholenoic acids, the sulfated and partially glycine-conjugated forms of which are found in their urine and bile. 3-Oxo-delta 4 bile acids are detected in the urine of patients with a deficiency of 5 beta-reductase. It has been postulated that these unusual bile acids might act as cholestatic agents in these patients. The aim of the present study was to test this hypothesis in an in vitro system, since the abnormal bile acids would be metabolized in in vivo experiments. Basolateral (sinusoidal) and canalicular plasma membrane vesicles were isolated from rat liver. A rapid filtration method was used to determine transport of cholyltaurine in the presence of model bile acids into the isolated vesicles. It was found that 3 beta, 7 alpha-dihydroxy-5-cholenoic acid and 7 alpha-hydroxy-3-oxo-4-cholenoic acid both inhibited the apical, ATP-dependent transport system for cholyltaurine in a competitive manner with K(m) values of 15 microM and 16 microM, respectively. Radioactively labeled 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine and 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine were not transported by the same transport system. The same types of experiments were performed with basolateral plasma membrane vesicles. It was found that, in contrast to the canalicular ATP-dependent bile acid transport system, only 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine was a competitive inhibitor of the sodium-dependent transport system for cholyltaurine with a K(m) of 16 microM. Studies with radioactively labeled 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine and 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine revealed that 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine was transported in a sodium-dependent manner into basolateral rat liver plasma membrane vesicles, whereas 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine was not transported in a sodium-dependent way. These results support the hypothesis that the unusual bile acids found in patients with defects in bile acid biosynthesis might act as cholestatic agents by inhibiting the canalicular ATP-dependent transport system for bile acids which constitutes the rate-limiting step in the overall process of bile acid transport across hepatocytes. Furthermore, the experiments demonstrated that, despite similar substrate specificities, the basolateral sodium-dependent and the apical ATP-dependent transport system for cholyltaurine might have different recognition sites for bile acids.

Animals↗