Basolateral Plasma Membrane Vesicles Research Articles

Modulation of Na+/Mg2+ exchanger stoichiometry ratio by Cl− ions in basolateral rat liver plasma membrane vesicles

The Na+/Mg²+ exchanger represents the main Mg²+ extrusion mechanism operating in mammalian cells including hepatocytes. We have previously reported that this exchanger, located in the basolateral domain of the hepatocyte, promotes the extrusion of intravesicular trapped Mg²+ for extravesicular Na+ with ratio 1. This electrogenic exchange is supported by the accumulation of tetraphenyl-phosphonium within the vesicles at the time when Mg²+ efflux occurs. In this present study, the role of extra- and intra-vesicular Cl⁻ on the Na+/Mg²+ exchange ratio was investigated. The results reported here suggest that Cl⁻ ions are not required for the Na+ to Mg²+ exchange to occur, but the stoichiometry ratio of the exchanger switches from electrogenic (1Na(in)+ :1Mg(out)²+) in the presence of intravesicular Cl⁻ to electroneutral (2Na(in)+ :1 Mg(out)²+) in their absence. In basolateral liver plasma membrane vesicles loaded with MgCl₂ labeled with ³⁶Cl⁻, a small but significant Cl⁻ efflux (~30 nmol Cl⁻/mg protein/1 min) is observed following addition of NaCl or Na-isethionate to the extravesicular medium. Both Cl⁻ and Mg²+ effluxes are inhibited by imipramine but not by amiloride, DIDS, niflumic acid, bumetanide, or furosemide. In vesicles loaded with Mg-gluconate and stimulated by Na-isethionate, an electroneutral Mg²+ extrusion is observed. Taken together, these results suggest that the Na+/Mg²+ exchanger can operate irrespective of the absence or the presence of Cl⁻ in the extracellular or intracellular environment. Changes in trans-cellular Cl⁻ content, however, can affect the modus operandi of the Na+/Mg²+ exchanger, and consequently impact "cellular" Na+ and Mg²+ homeostasis as well as the hepatocyte membrane potential.

Substrate specificity of rat Na+/taurocholate cotransporting polypeptide

Human Na+/taurocholate cotransporting polypeptide (NTCP) has recently been proposed to be a multispecific transporter that mediates uptake of xenobiotics including some statins in addition to its prototypical substrate (bile acids). In rat basolateral plasma membrane vesicles Na+‐dependent taurocholate uptake was competitively inhibited by several glitazones, suggesting that these drugs could be substrates of rat Ntcp. Therefore we used CHO cells that stably express rat Ntcp to screen for drugs and other xenobiotics that inhibit sodium dependent taurocholate uptake and therefore might be substrates of Ntcp. We tested selected inhibitors as substrates using LC‐MS/MS analysis. We confirmed that troglitazone and rosiglitazone are strong inhibitors of rat Ntcp (IC50 = 13 μM for troglitazone, 8 μM for rosiglitazone). Furthermore, we determined that several additional drugs including losartan, ritonavir, saquinavir, fluvastatin, ketoconazole, diclofenac and carbenoxolone inhibited Ntcp mediated taurocholate transport in a concentration dependent pattern. The so far tested troglitazone, rosiglitazone, ritonavir, saquinavir and pravastatin however are not substrates for rat Ntcp although pravastatin is transported by human NTCP. In conclusion, rat Ntcp is inhibited by numerous drugs but its substrate specificity seems to be more restricted than the substrate specificity of human NTCP.

Effect of thiazolidinediones on bile acid transport in rat liver

The thiazolidinedione derivatives, troglitazone, rosiglitazone, and pioglitazone, are novel insulin-sensitizing drugs that are useful in the treatment of type 2 diabetes. However, hepatotoxicity associated with troglitazone led to its withdrawal from the market in March 2000. In view of case reports of hepatotoxicity from rosiglitazone and pioglitazone, it is unclear whether thiazolidinediones as a class are associated with hepatotoxicity. Although the mechanism of troglitazone-associated hepatotoxicity has not been elucidated, troglitazone and its major metabolite, troglitazone sulfate, competitively inhibit adenosine triphosphate (ATP)-dependent taurocholate transport in isolated rat canalicular liver plasma membrane vesicles mediated by the canalicular bile salt export pump (Bsep). These results suggest that cholestasis may be a factor in troglitazone-associated hepatotoxicity. To determine whether this effect is 1) limited to canalicular bile acid transport and 2) is specific to troglitazone, the effect of troglitazone, rosiglitazone, and ciglitazone on bile acid transport was examined in rat basolateral (blLPM) and canalicular (cLPM) liver plasma membrane vesicles. In cLPM vesicles, troglitazone, rosiglitazone, and ciglitazone (100 μM) all significantly inhibited ATP-dependent taurocholate transport. In blLPM vesicles, these three thiazolidinediones also significantly inhibited Na +-dependent taurocholate transport. Inhibition of bile acid transport was concentration dependent and competitive in both cLPM and blLPM vesicles. In conclusion, these findings are consistent with a class effect by thiazolidinediones on hepatic bile acid transport. If hepatotoxicity is associated with this effect, then hepatotoxicity is not limited to troglitazone. Alternatively, if hepatotoxicity is limited to troglitazone, other mechanisms are responsible for its reported hepatotoxicity.

The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice

The main player in biliary cholesterol secretion is the heterodimeric transporter complex, ABCG5/ABCG8, the function of which is necessary for the majority of sterols secreted into bile. It is not clear whether the primary step in this process is flopping of cholesterol from the inner to the outer leaflet of the canalicular membrane, with desorption by mixed micelles, or decreasing of the activation energy required for cholesterol desorption from the outer membrane leaflet. In this study, we investigated these mechanisms by infusing Abcg8(+/+), Abcg8(+/-), and Abcg8(-/-) mice with hydrophilic and hydrophobic bile salts. In Abcg8(-/-) mice, this failed to substantially stimulate biliary cholesterol secretion. Infusion of the hydrophobic bile salt taurodeoxycholate also resulted in cholestasis, which was induced in Abcg8(-/-) mice at a much lower infusion rate compared with Abc8(-/-) and Abcg8(+/-) mice, suggesting a reduced cholesterol content in the outer leaflet of the canalicular membrane. Indeed, isolation of canalicular membranes revealed a reduction of 45% in cholesterol content under these conditions in Abcg8(-/-) mice. Our data support the model that ABCG5/ABCG8 primarily play a role in flopping cholesterol (and sterols) from the inner leaflet to the outer leaflet of the canalicular membrane.

Involvement of ERK1/2 and p38 in Mg2+ accumulation in liver cells

Activation of PKC signaling induces Mg(2+) accumulation in liver cells. To test the hypothesis that PKC induces Mg(2+) accumulation via MAPKs activation, hepatocytes were incubated in the presence of PD98059 and SB202190 as specific inhibitors of ERK1/2 and p38, respectively, and stimulated for Mg(2+) accumulation by addition of PMA or OAG. Accumulation of Mg(2+) within the cells was measured by atomic absorbance spectrophotometry in the acid extract of cell pellet. The presence of either inhibitor completely abolished Mg(2+) accumulation irrespective of the dose of agonist utilized while having no discernible effect on beta -adrenoceptor mediated Mg(2+) extrusion. A partial inhibition on alpha (1)-adrenoceptor mediated Mg(2+) extrusion was observed only in cells treated with PD98059. To confirm the inhibitory effect of PD98509 and SB202190, total and basolateral liver plasma membrane vesicles were purified in the presence of either MAPK inhibitor during the isolation procedure. Consistent with the data obtained in intact cells, liver plasma membrane vesicles purified in the presence of PD98509 or SB202190 lost the ability to accumulate Mg(2+)in exchange for intra-vesicular entrapped Na(+) while retaining the ability to extrude entrapped Mg(2+) in exchange for extra-vesicular Na(+). These data indicate that ERK1/2 and p38 are involved in mediating Mg(2+) accumulation in liver cells following activation of PKC signaling. The absence of a detectable effect of either inhibitor on beta -adrenoceptor induced, Na(+)-dependent Mg(2+) extrusion in intact cells and in purified plasma membrane vesicles further support the hypothesis that Mg(2+) extrusion and accumulation occur through distinct and differently regulated transport mechanisms.

Plasma membrane domain localization and transcytosis of the glucagon-induced hepatic system A carrier.

We have utilized canalicular (cLPM) and basolateral (blLPM) liver plasma membrane vesicles to investigate the domain localization of several Na(+)-dependent amino acid transporters. Neutral amino acid transport by systems N and ASC was measurable in both domains but was greater in blLPM vesicles. Sodium-dependent glutamate uptake (system X-) was preferentially localized to cLPM. The absolute activity and domain distribution of these three carriers remained unchanged after treatment of rats with a combination of glucagon and dexamethasone. A low level of basal system A activity was present in both the blLPM and cLPM. Glucagon-induced system A activity was first observed in blLPM vesicles approximately 60 min posthormone treatment and, in cLPM vesicles, approximately 30 min later. In situ perfusion of glucagon-treated rat liver with the membrane-impermeant protein modification reagent glutathione maleimide specifically inactivated blLPM system A activity and abolished the delayed arrival of hormone-induced activity to the cLPM. Transient perfusion of the liver with glutathione maleimide followed by a recovery period in vivo showed that the reagent did not irreversibly inactivate the transcytotic process and also provided an independent demonstration of the time delay between arrival of the carrier activity at the two membrane surfaces. These results support the concept of a transcytotic process in which the blLPM is the sorting site for the hormone-induced system A carrier proteins that eventually reach the cLPM.

Shift from Biliary to Urinary Elimination of Acetaminophen-Glucuronide in Acetaminophen-Pretreated Rats

Despite its toxicity, acetaminophen (APAP) is used increasingly as an analgesic, antipyretic, and anti-inflammatory agent. We examined the effect of prior exposure to APAP on its biliary and urinary elimination. The biliary and urinary elimination of a test dose of APAP (150 mg/kg i.v.) was determined in male Wistar rats 24 h after pretreatment with vehicle, a single dose (1.0 g/kg i.p.), or increasing daily doses (0.2, 0.3, 0.6, and 1.0 g/kg/day i.p.) of APAP. Although elimination of the parent APAP was minimally affected, biliary excretion of APAP glucuronide was significantly decreased 70 and 80%, whereas urinary excretion was significantly increased 90 and 100% in the groups pretreated with single and repeated doses of APAP, respectively, relative to vehicle controls. Western analysis and confocal immunofluorescent microscopy indicated a marked increase in hepatic expression of multidrug resistance-associated protein 3 (Mrp3) in both groups pretreated with APAP, relative to expression of Mrp2. ATP-dependent transport of [3H]taurocholate, an Mrp3 substrate, was significantly increased in basolateral liver plasma membrane vesicles from rats pretreated with repeated doses of APAP relative to controls. Enterohepatic recirculation of APAP glucuronide after administration of the same test dose of the drug was significantly decreased in rats pretreated with repeated doses of APAP. These data indicate that APAP pretreatment induced a shift from biliary to urinary elimination of APAP glucuronide, consistent with the increased expression of Mrp3 in the basolateral domain of the hepatocyte. We postulate that decreased enterohepatic recirculation contributes to decreased APAP hepatotoxicity by reducing liver exposure.

The thiol sensitivity of glutathione transport in sidedness-sorted basolateral liver plasma membrane and in Oatp1-expressing HeLa cell membrane.

Sinusoidal efflux of hepatic reduced glutathione (GSH) is a key step in interorgan GSH/cysteine homeostasis and extracellular detoxification. Rat organic anion transporter polypeptide1 (Oatp1) is known to transport GSH but several features of sinusoidal GSH uptake, such as electrogenic property and asymmetric effects of uncharged thiols (increased efflux, decreased uptake), either cannot be accounted for by Oatp1 or have not been studied. The asymmetric effect of thiols has only been studied in intact cells and not directly in membrane vesicles. To accomplish the latter, we studied GSH uptake in inside-out-(IO) and rightside-out-(RO) oriented basolateral plasma membrane vesicles (bLPM). We also studied the kinetics and effect of thiols on GSH transport by Oatp1 stably expressed in HeLa cells. GSH uptake was approximately 2- to 3-fold higher in IO than RO bLPM. Dithiothreitol-stimulated GSH uptake in IO but inhibited uptake in RO bLPM, demonstrating that thiols exert direct asymmetric side-specific effects on GSH transport. Uptake in IO and RO bLPM was sigmoid (K(m) approximately 13 mM) with a 2-fold higher capacity in IO compared with RO bLPM. In both IO and RO bLPM, a component with a high affinity but low capacity for GSH (K(m) approximately 100 microM) was also present. Endogenous GSH transporter in HeLa cells was thiol-sensitive, electrogenic, and described by a single Michaelis-Menten component (K(m) approximately 15 mM). In contrast, GSH transport mediated by Oatp1 was insensitive to thiols and membrane potential, inhibited by cystine, and stimulated by an inward H(+) gradient. These findings identify novel functional asymmetries in sinusoidal efflux and uptake of GSH and further clarify the role of Oatp1 in GSH transport.

Hepatocellular transport proteins and their role in liver disease

TRANSPORT PROTEINS Basolateral transport systems Na-dependent bile salt uptake Uptake of bile salts into the liver was first characterized in experimental models such as the isolated perfused rat liver, isolated hepatocyte cultures and basolateral plasma membrane vesicles. These studies indicated that more than 80% of taurocholate uptake but less than 50% of cholate uptake into hepatocytes is sodiumdependent. Whereas unconjugated bile salts are uncharged molecules that can traverse membranes by passive nonionic diffusion, conjugation with glycine or taurine decreases their pKa values and necessitates the presence of a specific carrier protein for hepatocellular uptake. The chief uptake system for conjugated bile salts in mammalian liver was isolated by expression and molecular cloning strategies and has been called the Na + taurocholate cotransporting polypeptide (gene symbol: SLC10A1). Rat Ntcp consists of 362 amino acids with an apparent molecular mass of 51 kD and is expressed exclusively at the basolateral membrane of hepatocytes (Figure 1). Ntcp mediates sodium-dependent uptake of taurocholate and other bile salts when expressed in stably transfected COS-7, Chinese hamster ovary (CHO) and hepatoblastoma (HepG2) cells or in cRNA injected Xenopus laevis oocytes, with apparent Km values between 17-42 μmol/L. The only non-bile salt substrates that are transported by Ntcp are selected sulfated steroid conjugates such as estrone-3sulfate and dehydroepiandrosterone sulfate (DHEAS) . In human liver, NTCP represents a 349-amino acid protein. NTCP is structurally related to the intestinal bile salt transporter (IBAT), that also mediates the Nadependent uptake of bile saltsand that is expressed not only in ileum, but also in the kidney and in cholangiocytes. Na-dependent taurocholate uptake is reduced in experimental models of cholestasis such as bile duct l igat ion , endotoxinemia and par t ia l hepatectomy, is reduced in primary hepatocyte cultures and is absent in various hepatoma cell lines . These changes in hepatic Na dependent bile salt uptake correlate with expression levels of Ntcp. Thus, Ntcp mRNA and protein levels are decreased in bile duct ligation, endotoxinemia and ethinyl estradiol induced cholestasis. In patients with a diagnosis of extrahepatic biliary atresia and clinical evidence of cholestasis, NTCP mRNA levels are also decreased.

Effects of sea water and stanniectomy on branchial Ca(2+) handling and drinking rate in eel (Anguilla anguilla L.).

We examined the effects of seawater adaptation and extirpation of the Stannius corpuscles on branchial Ca(2+) flows, gill plasma membrane Ca(2+) transporters and drinking rate of European eels, Anguilla anguilla. Transepithelial Ca(2+) inflow in the gills increased 2 weeks after transfer of the eels from fresh water to sea water and after stanniectomy. Neither of these treatments changed the membrane density or the affinity of the Ca(2+)-extrusion mechanisms (Ca(2+)-ATPase and Na(+)/Ca(2+)-exchanger) in the gill cells, as measured in basolateral plasma membrane vesicles. We conclude that the increase in the Ca(2+)-transporting capacity observed in the gills of fish exposed to the larger transepithelial Ca(2+) fluxes, resulting from exposure to sea water or stanniectomy, involves an increase in number and/or size of the Ca(2+)-transporting cells, but not in the membrane density of Ca(2+) transporters. Branchial Ca(2+) outflow was higher in stanniectomised than in sham-operated fish. Changes in electrochemical driving forces as well as plasma stanniocalcin or teleocalcin levels may be the basis for the observed differences. Stanniectomy enhanced drinking in stanniectomised eels. Drinking was not further affected by transfer to sea water. These observations suggest that the corpuscles of Stannius are involved in the water balance.

Molecular Mechanisms for the Hepatic Uptake of Magnetic Resonance Imaging Contrast Agents

The mechanisms were investigated for the hepatic transport of 4 different gadolinium complexes used as contrast agents for magnetic resonance imaging (MRI). In basolateral rat hepatocyte plasma membrane vesicles, Gd-DTPA uptake was indistinguishable from non-specific binding to vesicles; Gd-BOPTA and Gd-EOB-DTPA entered plasma membrane vesicles following a linear, concentration-dependent mechanism up to 1.5 mM of substrate. By contrast, Gd-B 20790 uptake followed a saturative kinetic with an apparent Kmof 92 ± 15 μM and a Vmaxof 143 ± 42 pmol/mg prot/15 sec, and it occurred into an osmotic-sensitive space. Sulfobromophthalein ant taurocholate, but not unconjugated bilirubin inhibited the uptake rate of Gd-B 20790 but not that of the other three compounds. Injection intoXenopus laevisoocytes of 5 ng of human OATP cRNA resulted, after 3 days, in a ≥2-fold stimulation (p < 0.001) of transport of Gd-B 20790 but not of Gd-BOPTA or Gd-EOB-DTPA. Collectively, these data indicate that the hepatic uptake of the MRI contrast agent Gd-B 20790 is a carrier-mediated mechanism operated by OATP while MRI compounds with other chemical structures enter the hepatocyte by other mechanisms.

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

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. 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. 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. 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.

Characteristics of a low affinity passive Ca2+ influx component in rat parotid gland basolateral plasma membrane vesicles.

We have previously reported the presence of two Ca2+ influx components with relatively high (KCa = 152 +/- 79 microM) and low (KCa = 2.4 +/- 0.9 mM) affinities for Ca2+ in internal Ca2+ pool-depleted rat parotid acinar cells [Chauthaiwale et al. (1996) Pfluegers Arch. 432: 105-111]. We have also reported the presence of a high affinity Ca2+ influx component with KCa = 279 +/- 43 microM in rat parotid gland basolateral plasma membrane vesicles (BLMV). [Lockwich, Kim & Ambudkar (1994) J. Membrane Biol. 141:289-296]. The present studies show that a low affinity Ca2+ influx component is also present in BLMV with KCa = 2.3 +/- 0.41 mM (Vmax = 16.36 +/- 4.11 nmoles of Ca2+/mg protein/min). Our data demonstrate that this low affinity component is similar to the low affinity Ca2+ influx component that is activated by internal Ca2+ store depletion in dispersed parotid gland acini by the following criteria: (i) similar KCa for calcium flux, (ii) similar IC50 for inhibition by Ni2+ and Zn2+; (iii) increase in KCa at high external K+, (iv) similar effects of external pH. The high affinity Ca2+ influx in cells is different from the low affinity Ca2+ influx component cells in its sensitivity to pH, KCl, Zn2+ and Ni2+. The low and high affinity Ca2+ influx components in BLMV can also be distinguished from each other based on the effects of Zn2+, Ni2+, KCl, and dicyclohexylcarbodiimide. In aggregate, these data demonstrate the presence of a low affinity passive Ca2+ influx pathway in BLMV which displays characteristics similar to the low affinity Ca2+ influx component detected in parotid acinar cells following internal Ca2+ store depletion.

Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells.

Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the 'epithelial-specific isoform' called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization.

Characterization of Apical and Basolateral Plasma Membrane Domains Derived from Cultured Rat Cholangiocytes

Cholangiocytes, the epithelial cells that line intrahepatic bile ducts, are composed of plasma membranes with discrete apical (lumenal) and basolateral domains that contain different channels, transporters, and receptors. In recent work, we developed a long-term, primary culture system of normal rat cholangiocytes (NRC). Our aims here were to prepare and characterize apical and basolateral plasma membrane vesicles from NRC. Using serial isopycnic centrifugation on sucrose gradients, we generated separate apical and basolateral plasma membrane vesicles. We characterized these vesicles by transmission electron microscopy, specific marker enzyme assays, and immunoblotting; we also determined the percentage of sealed vesicles and their intravesicular volume and sidedness using biochemical approaches. Our results showed that vesicles derived from the apical and basolateral plasma membrane domains of NRC were highly purified, predominately sealed, right-side-out vesicles that differed in size and lipid and protein composition. This experimental model represents a novel tool that will be useful for additional functional studies on the channels, transporters, and receptors differentially distributed in the plasma membrane of biliary epithelia.

Hepatic bile salt flux does not modulate level and activity of the sinusoidal Na +-taurocholate cotransporter (ntcp) in rats

Background/Aims: Efficient uptake at the basolateral plasma membrane of hepatocytes is required for maintenance of the enterohepatic circulation of bile salts. Uptake occurs mainly via a Na +-dependent process mediated by ntcp, a recently cloned and characterized 51 kDa glycoprotein. The aim of this study was to evaluate the role of variations in hepatic bile salt flux through the liver in the regulation of ntcp activity and expression under non-cholestatic conditions. Methods: We determnied the kinetics of Na +-dependent taurocholate transport in isolated basolateral plasma membrane vesicles as well as hepatic ntcp protein and ntcp mRNA levels in long-term (8 days) bile-diverted rats, with a transhepatic bile salt flux of 0, and in streptozotocin-induced diabetic rats with a 2.5-fold increased bile salt flux. Results: We found no changes in the kinetics of taurocholate transport in the absence of transhepatic bile salt flux due to bile diversion. Ntcp protein and ntcp mRNA levels were also unaffected in bile-diverted rats. Likewise, no changes in taurocholate transport kinetics, ntcp protein or ntcp mRNA levels were detected in streptozotocin-diabetic rats when compared to non-diabetic controls. Thus, variation in hepatic bile salt flux from 0 to 250% of normal values had no effect on hepatic ntcp expression or taurocholate transport activity in basolateral plasma membrane vesicles in rats. In contrast, 4 days of bile duct ligation resulted in a strong decrease in ntcp mRNA and protein levels, as recently also reported by others. Conclusions: Our data indicate that ntcp is not regulated by the transhepatic flux of bile acids under non-cholestatic conditions.

Regulation of hepatocyte bile salt transporters during hepatic regeneration.

Bile formation is an essential liver-specific function, and the hepatic regeneration that occurs in response to hepatocellular injury is often associated with cholestasis. We have employed a partial hepatectomy model to examine the effect of hepatic regeneration on tissue-specific bile salt transporters and on Na(+)-K(+)-adenosinetriphosphatase (ATPase). Liver-specific sodium-dependent taurocholate uptake by basolateral plasma membrane vesicles was undetectable 24 h after hepatectomy. Basolateral membrane protein expression of the sodium-taurocholate cotransporter and gene expression of Ntcp were decreased by > 90% 24 h after partial hepatectomy. In vitro transcription assays demonstrated that Ntcp gene transcription was also markedly reduced. In contrast, hepatic Na(+)-K(+)-ATPase activity, protein expression, and gene expression were unaffected by partial hepatectomy. Similarly, protein and gene expression of the ectoATPase, a putative canalicular bile salt transporter, and canalicular ATP-dependent taurocholate uptake remained unchanged. Partial hepatectomy results in a marked reduction in the gene transcription and expression of the liver-specific Ntcp, as well as a decrease in protein expression and loss of transport activity. These changes provide a potential mechanism for the decrease in hepatocellular bile salt transport that is associated with hepatic regeneration.

Binding proteins for cyclic and linear oligopeptides in plasma membranes and the cytosol of rat hepatocytes

Using a cyclolinopeptide A analogue, the Hydrophobic cyclic peptide c(-Ala-Lys-Pro-Phe-Phe-Ala-Lys-Pro-Phe-Phe-), termed CDP (cyclodecapeptide), as ligand in affinity chromatography, hepatocellular peptide binding proteins were isolated from the integral part of plasma membranes and the cytosol. The sequence of the isolated protein with MW of 50 kDa from the integral part of the plasma membrane fraction was identical to cytochrome P450 II C13 and cytochrome P450 II C22, whereas the sequence of the 54 kDa protein was identical to 3-hydroxyandrogen-UDP-glucuronosyltransferase. These proteins have also been described as binding proteins for bile acids. As shown in earlier studies, bile acids and CDP also compete for uptake into hepatocytes. In the cytosol, a further known bile acid binding protein, the glutathione-S-transferase (G-S-T) subunit Yb1, was isolated and sequenced as binding protein for CDP and also for a further cyclopeptide, the somatostatin analogue OO8, and a linear peptide with renin-inhibiting activity, EMD 55068. As shown in uptake studies using isolated basolateral plasma membrane vesicles, G-S-T was able to increase the uptake of EMD 51921, a linear peptide with renin-inhibiting potency, into the vesicles when the latter were preloaded with G-S-T. The binding of the substrate to the outside of the preloaded vesicles was not different than binding to unloaded vesicles. The maximal transport rate of the carrier-mediated/facilitated diffusion and the rate of permeation, however, were doubled in the presence of G-S-T, pointing to the involvement of intracellular binding proteins such as G-S-T in the unloading of the carrier protein and in the reduction of the free substrate concentration.

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.

Hepatocyte transport of bile acids and organic anions in endotoxemic rats: Impaired uptake and secretion

BACKGROUND & AIMS: In sepsis, intrahepatic cholestasis occurs frequently, suggesting impaired hepatocyte transport of bile acids and organic anions. The aim of the study was to define the magnitude, time course, and the site of impaired biliary secretion in a rat sepsis model. METHODS: Maximal transport for two bile acids (cholyltaurine and chenodeoxycholyltaurine) and two organic anions (sulfobromophthalein and sulfolithocholyltaurine) was measured in isolated perfused livers at various times after lipopolysaccharide injection. Basolateral and canalicular liver plasma membrane vesicles were used to characterize the impairment in hepatocyte transport. RESULTS: Maximal hepatocyte transport was reduced for all compounds by 60%-81% compared with controls. Bile acid-independent bile flow was reduced by 51%. Impairment was maximal 12 hours after endotoxin injection and recovered thereafter. In basolateral plasma membrane vesicles, sodium-dependent transport for bile acids was reduced by 36%-47%. Sodium-independent transport of organic anions was reduced by 40%-55%. Adenosine triphosphate-stimulated transport was greatly decreased in canalicular vesicles prepared from endotoxemic animals for all four compounds probably because of a reduced number of transport molecules, based on kinetic studies. CONCLUSIONS: Basolateral and canalicular bile acid and organic anion transport are markedly impaired in endotoxemia. These mechanisms may contribute to the cholestasis of sepsis. (Gastroenterology 1997 Jan;112(1):214-25)