Further studies on bilitranslocase,a plasma membrane protein involved in hepatic organic anion uptake

Bilitranslocase, a plasma membrane protein involved in bilirubin and other organic anion uptake by the liver, exhibits a high molecular weight (170 000) when isolated in the presence of deoxycholate. This value is decreased to approx. 100 000 if deoxycholate is not included in the isolation medium. Both preparations can be resolved into two kinds of subunit, α and β, of 37 000 and 35 500, respectively, by reduction with 2-mercaptoethanol and addition of sodium dodecyl sulfate. Under these conditions the two subunits are still capable of high-affinity sulfobromophthalein binding and, despite the presence of the detergent, may be isolated by preparative polyacrylamide gel electrophoresis still associated with the dye. It may be suggested that the physiological subunit composition of bilitranslocase is α₂-β.     

On the mechanism of bilitranslocase transport inactivation by phenylmethylsulphonyl fluoride

Bilitranslocase is a plasma membrane carrier involved in the uptake of bilirubin and other organic anions from the blood into the liver cell. In the membrane, the carrier occurs as two interchangeable metastable forms, with high and low affinity for the substrates, respectively. The latter form can be specifically produced by either cysteine- or arginine modification. In liver plasma membrane vesicles, the serine-specific reagent phenylmethylsulphonyl fluoride is a partial inhibitor of bilitranslocase-mediated BSP transport rate. In this work, phenylmethylsulphonyl fluoride is shown to reduce the carrier maximal transport rate, without affecting its affinity for that substrate. In addition, it is found that the chemical modification caused by this reagent neither influences the equilibrium between the high- and the low-affinity forms nor prevents their free interconversion. From the effects of combined derivatizations of cysteine(s), arginine(s) and serine(s), it is concluded that the functionally relevant aminoacid residues lie in a close spatial arrangement. Also, in this study, the PMSF-modified serine(s) is shown to be involved in bilirubin binding by bilitranslocase.     

Reconstitution of sulfobromophthalein transport in erythrocyte membranes induced by bilitranslocase

Bilitranslocase, the protein responsible for the anion translocation at the sinusoidal plasma membrane level in liver, was shown to be able to reconstitute the transport of sulfobromophthalein in liposomes in the past. The protein preparation used in those experiments consisted of two subunits of 35.5 and 37 kDa. The isolated 37 kDa protein, when inserted in erythrocyte membrane vesicles, confers to the particles the ability to carry out an electrogenic transport of sulfobromophthalein. The effect is specific and can be inhibited by monospecific polyclonal antibodies raised against the protein. In may be concluded that the 37 kDa protein band, present in previous preparations of bilitranslocase, is not only a necessary but also a sufficient component of the transport system for bilirubin and functional analogues.     

Inhibitory effect of cholesterol on the uptake of liposomes by liver and spleen

The effect of cholesterol content of small unilamellar (SUV) and reverse phase (REV) liposomes on blood clearance and tissue distribution has been studied. [¹⁴C]Inulin has been used as an aqueous marker of liposomes to represent the uptake of intact liposomes in tissues. The blood clearance of the intravenously-injected SUV and REV liposomes depends on the cholesterol content of liposomes. The cholesterol-free (0 mol%) liposomes are cleared more readily from the circulation than the cholesterol-poor liposomes (20 mol%) and the cholesterol-poor are cleared more rapidly than the cholesterol-rich (46.6 mol%) liposomes. This clearance pattern of liposomes from the circulation is not attributed to the change of size of liposomes due to the increase in cholesterol content of liposomes. However, poor stability of cholesterol-free or cholesterol-poor liposomes in the circulation is partly responsible, but the predominant factor responsible for the observed blood clearance pattern is the inhibitory effect of cholesterol on the uptake of liposomes by reticuloendothelial-rich tissues liver and spleen. Uptake of liposomes by these organs is decreased with increasing cholesterol content of vesicles. It is suggested that to produce liposome preparations with a long circulating half life in vivo it is necessary to inhibit their uptake by liver and spleen.     

Role of rat organic anion transporter 3 (Oat3) in the renal basolateral transport of glutathione

The tripeptide GSH is important in maintenance of renal redox status and defense against reactive electrophiles and oxidants. Previous studies showed that GSH is transported across the basolateral plasma membrane (BLM) into the renal proximal tubule by both sodium-coupled and sodium-independent pathways. Substrate specificity and inhibitor studies suggested the function of several carriers, including organic anion transporter 3 (Oat3). To test the hypothesis that rat Oat3 can function in renal GSH transport, the cDNA for rat Oat3 was expressed as a His6-tagged protein in E. coli, purified from inclusion bodies and by Ni2+-affinity chromatography, and reconstituted into proteoliposomes. cDNA-expressed and reconstituted Oat3 transported both GSH and p-aminohippurate (PAH) in exchange for 2-oxoglutarate (2-OG) and 2-OG and PAH in exchange for GSH, and PAH uptake was inhibited by both probenecid and furosemide, consistent with function of Oat3. mRNA expression of Oat3 and several other potential carriers was detected by RT-PCR in rat kidney cortex but was absent from NRK-52E cells, a rat proximal tubular cell line. Basolateral uptake of GSH in NRK-52E cells showed little PAH- or 2-OG-stimulated uptake. We conclude that Oat3 can function in GSH uptake and that NRK-52E cells possess a low background rate of GSH uptake, making these cells a good model for overexpression of specific, putative GSH carriers.     

Flow cytofluorometric investigation of the uptake by hepatocytes and spleen cells of targeted and untargeted liposomes injected intravenously into mice

Untargeted liposomes (composition: PC-PS-cholesterol) and targeted liposomes (composition: PC-PS-cholesterol-lactosylceramide) having encapsulated concentration-quenched carboxyfluorescein were injected intravenously into mice. 1 h after injection, the mice livers were perfused, excised and the hepatocytes were separated from nonparenchymal cells and analysed in a fluorescence-activated cell sorter analyzer. The result was that hepatocytes took up significantly more liposomes when lactosylceramide was inserted in the liposome bilayers, which was in good agreement with observations made on the in vivo uptake of liposome-encapsulated insulin gene (Soriano, P. et al. (1983) Proc. Natl. Acad. Sci. USA, 80, 7128–7133). Cytofluorimetric analysis of the spleen cells showed that approx. 10% of the splenic lymphocytes take up high amounts of lactosylceramide liposomes, whereas most of the phospholipid liposomes are taken up by the phagocytic cells. The flow cytofluorimetric analysis shows, moreover, the internalization of the liposomes by the target cells and allows a quantitation of this uptake. Thus, in vivo targeting of the liposomes to specific liver and splenic cells, by means of glycolipid insertion in the liposome bilayer, is shown to take place with delivery of the liposomal aqueous space marker to these cells.     

Stabilization of large multilamellar liposomes by human serum in vitro

The leakage of 5,6-carboxyfluorescein from large multilamellar liposomes prepared from dipalmitoylphosphatidylcholine (without or with cholesterol) was investigated in vitro in the presence of human serum. Below the phospholipid phase transition temperature, the rate of dye release is retarted 3–8-fold in the presence of up to 25% human serum in the incubation medium, as compared to the release in isotonic phosphate-buffered saline. This effect is significantly augmented by incorporation of 50 mol% cholesterol into the lipid bilayer. At and above the phase transition temperature, the initial rapid dye leakage in the presence of serum is followed by a slow long-term release. Incubation of the liposomes with serum is assumed to result in the association of serum proteins with the outermost lipid bilayer which in turn will lead to their stabilization, while the inner lamellae are not immediately accessible to the serum proteins. The permeability of the outer protein-rich lipid bilayer appears to be restricted, as concluded from the decreased dye release in the presence of serum. Massive leakage from multilamellar liposomes appears to be primarily due to bilayer defects occurring in the lipid transition region rather than being caused by protein-lipid interactions. The results of our in vitro experiments are discussed in terms of the potential usefulness of multilamellar liposomes as drug carriers in vivo for local and topical applications.     

The glucose transport activity of human erythrocyte membranes. Reconstitution in phospholipid liposomes and fractionation by molecular sieve and ion exchange chromatography

Human erythrocyte membranes, at a protein concentration of 1–2 g/l, were solubilized with 0.12 M cholate in the presence of 0.06 M phospholipid (egg yolk phospholipids or phosphatidylcholine). More than 40% of the protein was solubilized. Cholate was removed by molecular sieve chromatography, whereby liposomes formed. These liposomes exchanged D-glucose faster than L-glucose. The recovery of glucose transport activity in the reconstituted system was estimated to be higher than 16%.The liposomes were heterogeneous in size, as shown by molecular sieve chromatography on Sepharose 4B, and small liposomes predominated. In liposomes formed with phosphatidylcholine, the distribution of glucose transport activity did not parallel the distribution of protein or phospholipid, and the activity was found mainly in the smallest liposomes. The proteins were incorporated mainly into the liposomes that eluted at the lowest ionic strength upon ion exchange chromatography.The glucose transport activity separated into three main peaks upon ion exchange chromatography of egg yolk phospholipid liposomes. The activity eluted at low ionic strength. The liposomes contained proteins mainly from the 3- and 4.5-regions (nomenclature according to Steck, T.L. (1974) J. Cell Biol. 62, 1–19). The activity peaks were highest in the first part of the chromatogram. The protein distribution did not coincide with the variation in activity over each peak. Therefore, it cannot be excluded that a minor component not seen in the electrophoretic analyses might be responsible for the glucose transport activity.     

Interactions of liposomes with the reticuloendothelial system: II. Nonspecific and receptor-mediated uptake of liposomes by mouse peritoneal macrophages

Liposomes are taken up as intact vesicles by mouse peritoneal macrophages in a process which is temperature sensitive and is affected by inhibitors of glycolytic metabolism and of microfilament activity. Macrophages take up negatively charged vesicles more readily than positively charged vesicles (2-fold) or neutral vesicles (4-fold). Macrophages take up similar amounts of multilamellar liposomes, reversed phase liposomes and small unilamellar liposomes in terms of lipid, however this corresponds to vastly different numbers of particles and amounts of trapped volume. Coating the liposomes with macromolecular ligands capable of interacting with macrophage surface receptors can markedly promote liposome uptake. Thus, formation of an IgG-antigen complex on the liposome surface results in a 10²-fold enhancement of liposome uptake, while coating the vesicles with fibronectin results in a 10-fold augmentation of uptake. Uptake via IgG-mediated and fibronectin-mediated processes seem to be independent since excess unlabelled, IgG-coated liposomes will inhibit the uptake of radioactively-labelled IgG-coated liposomes much more effectively than the uptake of radioactively-labelled fibronectin-coated liposomes. Cell-bound liposomes can readily be visualized on and inside of the macrophages using fluorescence microscopy techniques.     

Plasma membrane transport systems in higher plants: From black boxes to molecular physiology

Considerable progress in identifying transport systems of the plant plasma membrane has been made recently. The putative systems cloned to date comprise H⁺-ATPases, potassium, chloride and water channels, and carriers involved in the transport of glucose, sucrose, amino acids, peptides, potassium, nitrate, ammonium, phosphate, sulfate, iron and copper. Most of these systems were identified first in Arabidopsis thaliana . Successful cloning strategies have involved the following variety of techniques: complementation of yeast mutants, screening of Arabidopsis mutants, immunoscreening of a cDNA expression library expressed in mammalian cells, screening of genomic and cDNA libraries with probes (or degenerate oligonucleotides) derived from yeast and/or animal genes, or database screening for sequence similarity to eukaryotic counterparts. Many related transport systems have subsequently been identified either by screening libraries directly, or by systematic cDNA sequencing programs. Surprisingly large gene families have been revealed. Heterologous expression systems, such as yeast, Xenopus oocytes or insect cells, provide tools for studying the transport activities, biochemical properties and structure-function relationships of these systems. Their expression and functions in planta are investigated using northern blot analysis, in situ hybridization, and transgenic approaches. Individual systems encoded by the same gene family can differ in their transport properties and have distinct tissue expression patterns. Such diversity might be central to the integration of solute transport at the whole plant level, allowing the differential expression of sets of transport systems specifically tailored to the requirements of each tissue.     

Divalent cation enhancement of the agglutinability by soyabean lectin of liposomes prepared from total lipid of erythrocytes and of erythrocyte membranes

CaCl₂ or MgCl₂ but not NaCl enhances the soyabean lectin-induced agglutination of liposomes prepared from total lipids of erythrocyte membranes. The addition of purified phosphatidylserine to the total lipids of erythrocyte membranes before the formation of liposomes inhibits lectin-induced agglutinability of the preparation in the absence of CaCl₂, but not in its presence. When preformed phosphatidylserine liposomes are added to liposomes of total lipids of erythrocyte ghosts, they do not inhibit agglutination, indicating that phosphatidylserine does not inhibit the lectin directly. CaCl₂ or MgCl₂ but not NaCl also stimulates the soyabean lectin-induced agglutination of human erythrocyte membranes.Electron micrographs indicate that the liposome preparations are multilamellar and separate even in the presence of CaCl₂. When such liposomes are treated with lectin with or without CaCl₂, the electron micrographs show significant agglutination without apparent fusion. The reversal of the agglutination of liposomes by specific sugars followed by turbidimetric and electron microscopic techniques supports the conclusion that CaCl₂ stimulated lectin-induced agglutination is unaccompanied by fusion.The stimulation by divalent cations of lectin-induced agglutination of erythrocyte ghosts or of our liposomes may be due to a decrease in apparent surface charge of these membrane systems.     

Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes

Liposomes of defined size and homogeneity have been prepared by sequential extrusion of the usual multilamellar vesicles through polycarbonate membranes. The process is easy, reproducible, produces no detectable degradation of the phospholipids, and can double the encapsulation efficiency of the liposome preparation. Multilamellar vesicles extruded by this technique are shown by both negative stain and freeze-fracture electron microscopy to have mean diameters approaching the pore diameter of the polycarbonate membrane through which they were extruded. When sequentially extruded down through a 0.2 μm membrane, the resulting vesicles exhibit a very homogeneous size distribution with a mean diameter of 0.27 μm while maintaining an acceptable level of encapsulation of the aqueous phase.     

Relative contribution of OAT1 and OAT3 transport activities in isolated perfused rabbit renal proximal tubules

The expression of both OAT1 and OAT3 along the isolated rabbit renal proximal tubule (RPT) was determined using RT-PCR. They were found to be very strong in S2 segment and weak in S1 and S3 segments. We further examined the relative transport activity of these transporters in isolated perfused rabbit RPT using [³H]para-aminohippurate ([³H]PAH), and estrone sulfate ([³H]ES) as specific substrates for rbOAT1 and rbOAT3, respectively. The transport activity of OAT1 was in the order S2 > S1 = S3 segments and that of OAT3 was in the order S1 = S2>>S3 segments. The addition of α-ketoglutarate (100 μM) in the bathing medium increased both OAT1 and OAT3 transport activities in all segments of proximal tubule. The kinetics of [³H]succinic acid transport, used to measure the activity of sodium dicarboxylate transporter 3 (NaDC3), were examined. The J_max for succinic acid was in the order S2 > S3 and unmeasurable in the S1 segment. Our data indicate that both OAT1 and OAT3 play quantitatively significant roles in the renal transport of organic anions along the proximal tubule but predominately in S2 segment. The relative contribution of both transporters depends on their relative expression levels and may possibly be affected by the activity of NaDC3 in RPT.     

Mechanism of arginine transport in Chlorella

The incubation of Chlorella cells with glucose causes the induction of an uptake system, which is specific for the basic amino acids arginine and lysine. Both amino acids are taken up in the positively charged form and with high affinity (K _m values 2 M and 7 M, respectively). The transport of arginine depolarizes the membrane by 20–30 mV. The charge compensation is achieved within a few seconds after arginine addition by the proton pump, later on K⁺ efflux serves for charge compensation. No evidence for arginine-proton symport was found, neither by inhibitor studies nor by use of other Chlorella strains which have a slower-responding proton pump. The accumulation of arginine is appreciably higher than it should be according to the thermodynamic force of the membrane potential. There is, however, some evidence that a large proportion of arginine is trapped by intracellular compartments and is therefore not in equilibrium with the outside arginine.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - FCCP p-trifluoromethoxycarbonylcyanide phenylhydrazone     

Proton-coupled organic cation transport in renal brush-border membrane vesicles

We had previously proposed that organic cations are transported across the brush-border membrane in the canine kidney by a H⁺ exchange (or antiport) system (Holohan, P.D. and Ross, C.R. (1981) J. Pharmacol. Exp. Ther. 216, 294–298). In the present report, we demonstrate that in brush-border membrane vesicles the transport of organic cations is chemically coupled to the countertransport of protons, by showing that the uphill or concentrative transport of a prototypic organic cation, N¹-methylnicotinamide (NMN), is chemically coupled to the flow of protons down their chemical gradient. In a reciprocal manner, the concentrative transport of protons is coupled to the counterflow of organic cations down their concentration gradient. The transport of organic cations is monitored by measuring [³H]NMN while the transport of protons is monitored by measuring changes in acridine orange absorbance. The functional significance of the coupling is that a proton gradient lowers the K_m and increases the V_max for NMN transport.     

Lactosylceramide-induced stimulation of liposome uptake by Kupffer cells in vivo

Incorporation of 8 mol percent lactosylceramide into small unilamellar vesicles consisting of cholesterol and sphingomyelin in an equimolar ratio and containing [³H]inulin as a marker resulted in an increase in total liver uptake and a drastic change in intrahepatic distribution of the liposomes after intravenous injection into rats. The control vesicles without glycolipid accumulated predominantly in the hepatocytes, but incorporation of the glycolipid resulted in a larger stimulation of Kupffer-cell uptake (3.2-fold) than of hepatocyte uptake (1.2-fold). Liposome preparations both with and without lactosylceramide in which part of the sphingomyelin was replaced by phosphatidylserine, resulting in a net negative charge of the vesicles, were cleared much more rapidly from the blood and taken up by the liver to higher extents. The negative charge had, however, no influence on the intrahepatic distributions. The fast hepatic uptake of the negatively charged liposomes allowed competition experiments with substrates for the galactose receptors on liver cells. Inhibition of blood clearance and liver uptake of lactosylceramide-containing liposomes by $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ indicated the involvement of specific recognition sites for the liposomal galactose residues. This inhibitory effect of $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ was shown to be mainly the result of a decreased liposome uptake by the Kupffer cells, compatible with the reported presence of a galactose specific receptor on this cell type (Kolb-Bachofen et al. (1982) Cell 29, 859–866). The difference between the results on sphingomyelin-based liposomes as described in this paper and those on phosphatidylcholine-based liposomes as published previously (Spanjer and Scherphof (1983) Biochim. Biophys. Acta 734, 40–47) are discussed.     

Sodium-dependentl-serine transport in plasma membrane vesicles isolated from Ehrlich cells by two-phase compartmentation

Summary Plasma membrane vesicles were prepared from Ehrlich cells using two-phase system compartmentation. The highly pure plasma membrane vesicles obtained presented a negligible mitochondrial contamination and were suitable for studies of amino acid transport.l-Serine transport showed a clear ionic specificity, maximum incorporation being observed when an inwardly directed NaSCN gradient was used. Na⁺-dependentl-serine transport was dependent on assay temperature and membrane potential, and it seemed to be carried out by two different transport systems. An essential sulfhydryl group seemed to be involved in the transport process.     

In vivo and in vitro sugar transport in frog intestine

In the frog intestine, both in vitro and in vivo, experiments were carried out in order to increase knowledge of the mechanism of sugar exit across the basolateral membrane of the enterocyte. The frog intestine was chosen because it lacks crypt cells and, consequently, any external fluid circuit mechanism during sugar transport can be avoided. Therefore, the sugar concentration in the absorbate collected on the serosal side is likely to be similar to that present underneath the basolateral membrane of the enterocyte. Under this condition, cell and absorbate sugar concentrations are similar; yet there is a concomitant net transintestinal sugar transport. Moreover, in in vivo experiments a net transintestinal sugar transport takes place even against a concentration difference. These results suggest that sugar exit across the basolateral membrane is not simply due to a chemically facilitated diffusion.     

Amino-acid transport in suspension-cultured plant cells

The carrier system which transports L-leucine (L-leu) into suspension-culturedNicotiana tabacum L. cv. Wisconsin 38 cells appeared to be constitutive since it was always present and was not induced by L-leu even in nitrogen-starved cells. However, L-leu uptake rates for cells grown in medium containing L-leu were transiently reduced as a result of either transinhibition or repression. Growth-phase cells appeared to have more L-leu carriers per unit area of membrane than stationary-phase cells, and for this reason growing-phase cells exhibit higher L-leu uptake rates. These higher rates reflect a physiological or developmental condition since growth-phase cells did not dramatically change their L-leu uptake rates when subcultured, while stationary-phase cells doubled their rates within 6 h after being subcultured. Cells grown in a medium lacking a useable carbon souce had uptake rates higher than control rates for several days. These higher rates peaked after about 1 d and then decreased over the next several days. Cells grown in a medium lacking a nitrogen souce responded similarly except that the increased rates peaked after about 3 d and persisted longer. Kinetic analysis of uptake rates in cells grown without a carbon souce for 1 d or without a nitrogen souce for 3 d indicated that the L-leu carrier had K_ms similar to those of untreated cells. These results indicate that cultured tobacco cells respond to their environment by increasing or decreasing the number or activity of kinetically similar L-leu carriers.Abbreviations L and S medium Linsmaier and Skoog (1965) medium with additions - L-leu L-leucine IV=McDaniel et al. 1981