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Molecular Properties of Hepatic Uptake Systems for Bile Acids and Organic Anions
... Uptake of these watersoluble derivatives was competitively inhibited by T 4 and T 3 , suggesting that they are alternative ligands for the iodothyronine transporters (197,204). Since the sulfonated compounds are organic anions, we tested the hypothesis that hepatic uptake of iodothyronine derivatives is mediated, at least in part, by organic anion transporters, in particular Na ϩ /taurocholate-cotransporting polypeptide (NTCP) and the (Na ϩ -independent) organic anion transporting polypeptides (OATPs) (208,209). ...
... This transporter is now also known as solute carrier family 10, member 1 (SLC10A1). NTCP is only expressed in hepatocytes, where it is localized selectively to the basolateral cell membrane (208,209). It is the major transporter of conjugated bile acids in liver, but it also mediates uptake of unconjugated bile acids and a number of non-bile acid amphipathic compounds, including estrogen conjugates such as estrone 3-sulfate (208,209). ...
... NTCP is only expressed in hepatocytes, where it is localized selectively to the basolateral cell membrane (208,209). It is the major transporter of conjugated bile acids in liver, but it also mediates uptake of unconjugated bile acids and a number of non-bile acid amphipathic compounds, including estrogen conjugates such as estrone 3-sulfate (208,209). A homologous bile acid transporter is expressed in ileum and kidney, where it is localized to the apical cell membrane (212)(213)(214)(215). ...
Although it was originally believed that thyroid hormones enter target cells by passive diffusion, it is now clear that cellular uptake is effected by carrier-mediated processes. Two stereospecific binding sites for each T4 and T3 have been detected in cell membranes and on intact cells from humans and other species. The apparent Michaelis-Menten values of the high-affinity, low-capacity binding sites for T4 and T3 are in the nanomolar range, whereas the apparent Michaelis- Menten values of the low-affinity, high-capacity binding sites are usually in the lower micromolar range. Cellular uptake of T4 and T3 by the high-affinity sites is energy, temperature, and often Na+ dependent and represents the translocation of thyroid hormone over the plasma membrane. Uptake by the low-affinity sites is not dependent on energy, temperature, and Na+ and represents binding of thyroid hormone to proteins associated with the plasma membrane. In rat erythrocytes and hepatocytes, T3 plasma membrane carriers have been tentatively identified as proteins with apparent molecular masses of 52 and 55 kDa. In different cells, such as rat erythrocytes, pituitary cells, astrocytes, and mouse neuroblastoma cells, uptake of T4 and T3 appears to be mediated largely by system L or T amino acid transporters. Efflux of T3 from different cell types is saturable, but saturable efflux of T4 has not yet been demonstrated. Saturable uptake of T4 and T3 in the brain occurs both via the blood-brain barrier and the choroid plexus-cerebrospinal fluid barrier. Thyroid hormone uptake in the intact rat and human liver is ATP dependent and rate limiting for subsequent iodothyronine metabolism. In starvation and nonthyroidal illness in man, T4 uptake in the liver is decreased, resulting in lowered plasma T3 production. Inhibition of liver T4 uptake in these conditions is explained by liver ATP depletion and increased concentrations of circulating inhibitors, such as 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, indoxyl sulfate, nonesterified fatty acids, and bilirubin. Recently, several organic anion transporters and L type amino acid transporters have been shown to facilitate plasma membrane transport of thyroid hormone. Future research should be directed to elucidate which of these and possible other transporters are of physiological significance, and how they are regulated at the molecular level.
... To identify and isolate the first mouse orthologue of the Oatp family [1,26], we first generated and cloned an RT-PCR fragment using mouse liver mRNA and OATP (OATP-A) primers. Sequence analysis revealed a 91 % identity of this fragment with the rat Oatp1 [2]. ...
... According to the TopPred2 program of Stockholm University [27], the protein is predicted to have 12 transmembrane domains. On the basis of the extracellular location of the two putative N-linked glycosylation sites at positions 124 and 135, which are conserved among the Oatp family [26], the N-and the C-terminal end of Oatp are proposed to be cytoplasmic ( Figure 1). The amino acid sequence of the mouse Oatp was compared with that of the other Oatp family members. ...
We have cloned a murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane-transport proteins from mouse liver. The cloned cDNA insert of 2783 bp with an open reading frame of 2011 bp codes for a 12-transmembrane 670-amino-acid protein with highest amino acid identity with the: rat Oatp1. When expressed in Xenopus laevis oocytes, the mouse Oatp exhibited the same substrate specificity as the rat Oatp1. Besides the common Oatp substrates bromosulphophthalein, taurocholate, oestrone 3-sulphate and ouabain, the new mouse Oatp also mediates transport of the Oatp1-specific magnetic-resonance-imaging agent gadoxetate. The Oatp2-specific cardiac glycoside digoxin, however, is not transported. Kinetic analyses performed for taurocholate and oestrone 3-sulphate revealed apparent K-m values of 12 mu M and 5 mu M respectively. Northern-blot analysis demonstrated a predominant expression in the liver with an additional moderate expression in the kidney. Taken together, the amino acid identity, the functional characteristics and the tissue distribution suggest that we have isolated the murine orthologue of the rat Oatp1, and consequently the identified protein will be called Oatp1. Using fluorescence in Situ hybridization, the murine Oatp1 gene was mapped to chromosome XA3-A5.
... The first 17 amino acids of NTCP correspond to TMD 1 of ASBT NM , resulting in an intracellular N-terminus. However, the N-terminus of NTCP is known to be extracellular Hagenbuch, 1997;Hallén, Mareninova, Branden, & Sachs, 2002). Therefore, these residues were excluded from the model. ...
... The human and rat NTCP/Ntcp protein bears five putative N-linked glycosylation sites but only the N-terminal located N-glycosylation sites (Asn5, Asn11) were demonstrated to be glycosylated and, therefore, located extracellular (Hagenbuch, 1997;Hallén, Mareninova, Branden, & Sachs, 2002). The native form of the rat Ntcp protein in the cell membrane shows a molecular weight of about 51 kDa in Western blots (Ananthanarayanan, Ng, Boyer, & Suchy, 1994;Stieger et al., 1994), whereas the molecular weight estimation for the deglycosylated form is 33 kDa. ...
The SLC10 family represents seven genes containing 1-12 exons that encode proteins in humans with sequence lengths of 348-477 amino acids. Although termed solute carriers (SLCs), only three out of seven (i.e. SLC10A1, SLC10A2, and SLC10A6) show sodium-dependent uptake of organic substrates across the cell membrane. These include the uptake of bile salts, sulfated steroids, sulfated thyroidal hormones, and certain statin drugs by SLC10A1 (Na(+)-taurocholate cotransporting polypeptide (NTCP)), the uptake of bile salts by SLC10A2 (apical sodium-dependent bile acid transporter (ASBT)), and uptake of sulfated steroids and sulfated taurolithocholate by SLC10A6 (sodium-dependent organic anion transporter (SOAT)). The other members of the family are orphan carriers not all localized in the cell membrane. The name "bile acid transporter family" arose because the first two SLC10 members (NTCP and ASBT) are carriers for bile salts that establish their enterohepatic circulation. In recent years, information has been obtained on their 2D and 3D membrane topology, structure-transport relationships, and on the ligand and sodium-binding sites. For SLC10A2, the putative 3D morphology was deduced from the crystal structure of a bacterial SLC10A2 analog, ASBT(NM). This information was used in this chapter to calculate the putative 3D structure of NTCP. This review provides first an introduction to recent knowledge about bile acid synthesis and newly found bile acid hormonal functions, and then describes step-by-step each individual member of the family in terms of expression, localization, substrate pattern, as well as protein topology with emphasis on the three functional SLC10 carrier members.
... To identify and isolate the first mouse orthologue of the Oatp family [1,26], we first generated and cloned an RT-PCR fragment using mouse liver mRNA and OATP (OATP-A) primers. Sequence analysis revealed a 91 % identity of this fragment with the rat Oatp1 [2]. ...
... According to the TopPred2 program of Stockholm University [27], the protein is predicted to have 12 transmembrane domains. On the basis of the extracellular location of the two putative N-linked glycosylation sites at positions 124 and 135, which are conserved among the Oatp family [26], the N-and the C-terminal end of Oatp are proposed to be cytoplasmic ( Figure 1). The amino acid sequence of the mouse Oatp was compared with that of the other Oatp family members. ...
We have cloned a murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane-transport proteins from mouse liver. The cloned cDNA insert of 2783 bp with an open reading frame of 2011 bp codes for a 12-transmembrane 670-amino-acid protein with highest amino acid identity with the rat Oatp1. When expressed in Xenopus laevis oocytes, the mouse Oatp exhibited the same substrate specificity as the rat Oatp1. Besides the common Oatp substrates bromosulphophthalein, taurocholate, oestrone 3-sulphate and ouabain, the new mouse Oatp also mediates transport of the Oatp1-specific magnetic-resonance-imaging agent gadoxetate. The Oatp2-specific cardiac glycoside digoxin, however, is not transported. Kinetic analyses performed for taurocholate and oestrone 3-sulphate revealed apparent K(m) values of 12 microM and 5 microM respectively. Northern-blot analysis demonstrated a predominant expression in the liver with an additional moderate expression in the kidney. Taken together, the amino acid identity, the functional characteristics and the tissue distribution suggest that we have isolated the murine orthologue of the rat Oatp1, and consequently the identified protein will be called Oatp1. Using fluorescence in situ hybridization, the murine Oatp1 gene was mapped to chromosome XA3-A5.
... The functionally characterized rat organic acid:Na + symporter exclusively functions by Na + symport for the uptake of bile acids and other organic compounds (Hagenbuch, 1997; Weinman et al., 1998). The specificities and mechanisms of the prokaryotic homologues have not been investigated, but a proton symport mechanism seems most likely for the uptake of organic acids. ...
... Although the family is small, members are found ubiquitously in bacteria, archaea and eukaryotes. The functionally characterized rat organic acid:Na + symporter exclusively functions by Na + symport for the uptake of bile acids and other organic compounds (Hagenbuch, 1997; Weinman et al., 1998 ). The specificities and mechanisms of the prokaryotic homologues have not been investigated, but a proton symport mechanism seems most likely for the uptake of organic acids. ...
We here describe all recognized established and putative transport proteins encoded within the genome of Bacillus subtilis. These fall into four classes of established transporter types: (1) channel proteins, (2) secondary active transporters, (3) primary active transporters, and (4) group translocators of the sugar-transporting phosphotransferase system (PTS). Additionally, some transporters are recognized that utilize an unknown mode of action or energy coupling mechanism. The secondary carriers (which represent the majority of Bacillus transporters) are subdivided according to substrate specificity and family association. Characteristics of the families as well as the individual transport systems are presented when sufficient information is available. The recognized transporters fall into 58 families including 4 channel types, 42 secondary carrier types, 3 primary carrier types, 4 PTS-types and 5 unknown types.
... The family of transport proteins usually referred to as organic anion transporting polypeptides (OATPs) is classified as 2.A.60 [the organic anion transporter (OAT) family] within the Transport Commission Protein Database (361), and also as the solute carrier gene family Slc21a/ SLC21A. Members of the OATP family generally share a common postulated secondary structure that includes 12 TMDs and several long extracellular and intracellular loops (147), although there is no primary sequence homology with members of the OCT family. The research on this group of transport proteins has focused on their well-documented role in mediating the Na ϩ -independent transport of bile salts in the liver, and the reader is directed to recent reviews that deal extensively with this subject (147,148,272,456,501). ...
... Members of the OATP family generally share a common postulated secondary structure that includes 12 TMDs and several long extracellular and intracellular loops (147), although there is no primary sequence homology with members of the OCT family. The research on this group of transport proteins has focused on their well-documented role in mediating the Na ϩ -independent transport of bile salts in the liver, and the reader is directed to recent reviews that deal extensively with this subject (147,148,272,456,501). Several members of the OATP family are, however, expressed in the kidney and have been suggested to play a role in the secretion/reabsorption of selected anionic substrates. ...
Organic cations and anions (OCs and OAs, respectively) constitute an extraordinarily diverse array of compounds of physiological, pharmacological, and toxicological importance. Renal secretion of these compounds, which occurs principally along the proximal portion of the nephron, plays a critical role in regulating their plasma concentrations and in clearing the body of potentially toxic xenobiotics agents. The transepithelial transport involves separate entry and exit steps at the basolateral and luminal aspects of renal tubular cells. It is increasingly apparent that basolateral and luminal OC and OA transport reflects the concerted activity of a suite of separate transport processes arranged in parallel in each pole of proximal tubule cells. The cloning of multiple members of several distinct transport families, the subsequent characterization of their activity, and their subcellular localization within distinct regions of the kidney now allows the development of models describing the molecular basis of the renal secretion of OCs and OAs. This review examines recent work on this issue, with particular emphasis on attempts to integrate information concerning the activity of cloned transporters in heterologous expression systems to that observed in studies of physiologically intact renal systems.
... NTCP is glycosylated at residues N5 and N11, and studies suggest that plasma membrane expression relies on glycosylation of at least one of these sites. 53,54 In 2015, Vaz et al. reported that the NTCP R252H variant showed insufficient glycosylation in vitro and lack of expression at the plasma membrane. 23 In 2017, Appelman and colleagues showed that glycosylation-deficient NTCP is rapidly degraded by lysosomes. ...
Na+-taurocholate co-transporting polypeptide (NTCP; SLC10A1) is a key hepatic uptake transporter for bile acids and drugs and is the main functional receptor for hepatitis B and D viruses. Next-generation sequencing (NGS) has revealed that a large number of rare SLC10A1 variants exist in the population. Little data exist regarding head-to-head comparison of in silico algorithms to predict functional effects of variants when compared to direct in vitro functional assessment. This study aimed to characterize rare SLC10A1 variants in vitro and to assess the performance of seven in silico algorithms to predict the observed functional impacts. Thirty-five previously uncharacterized, rare, missense SLC10A1 variants were transiently expressed in human embryonic kidney 293 type T (HEK293T) cells. Protein expression as well as uptake of NTCP substrates taurocholic acid (TCA) and rosuvastatin were assessed. Substrate-specific effects were observed for NTCP G191R, with TCA and rosuvastatin transport observed at 89% and 8% of wild type uptake, respectively. Significantly reduced transport of TCA and rosuvastatin was observed for 19 variants (p<0.05), with seven variants displaying decreased protein expression and marked reduction in transport of both substrates (<13% of wild type uptake, p<0.0001). Performance of in silico algorithms to predict in vitro uptake, assessed using area under the receiver operating characteristic curve (AUCROC), ranged from 0.69-0.97 and 0.72-0.84 for TCA and rosuvastatin uptake, respectively. In conclusion, we identified rare variants with significantly reduced NTCP expression and function. We demonstrated that no algorithm performed robustly enough to replace functional study in vitro, particularly given the broad substrate specificity of many pharmacogenes.
... One of the forms of oatp with putative thyroid hormone transport activity consists of 670 amino acids (M r , ϳ75 kDa) (16). Ntcp has an apparent M r of 51 kDa (40). There are many cellular proteins associated with thyroid hormones, and in only a few cases has the function been defined (18,19). ...
Export of l-T3 out of the cell is one factor governing the cellular T3 content and response. We previously observed in liver-derived cells that T3 export was inhibited by verapamil, suggesting that it is due to either ATP-binding cassette/multidrug resistance (MDR1/mdr1b) or multidrug resistance-related (MRP1/mrp1) proteins. To test this hypothesis we measured T3 export in FRTL-5, NIH-3T3, and rat hepatoma (HTC) cells that varied in expression of these proteins. FRTL-5 and NIH-3T3 cells were found to contain a T3 efflux mechanism that is verapamil inhibitable, saturable, and stereospecific. By contrast, T3 efflux in HTC cells was slow and unaffected by verapamil. Neither FRTL-5 nor NIH-3T3 cells express mdr1b, but all three cell types express mrp1, as assessed by immunoblotting. Overexpression of MDR1 in NIH-3T3 cells did not enhance verapamil-inhibitable T3 efflux. Photoaffinity labeling of FRTL-5 and NIH-3T3 cells with[ ¹²⁵I]l-T3 revealed a labeled 90- to 100-kDa protein that was not present in HTC cells. Verapamil and excess nonradioactive l-T3, but not d-T3, inhibited labeling of this protein. The lack of correlation between T3 efflux and MDR1 and mrp1 expression and the finding of a photoaffinity-labeled putative transport protein smaller than MDR1 or mrp1 protein (∼170 kDa) suggest that a novel protein is involved in the transport of T3 out of cells.
... These are the SoLute Carrier Superfamily (SLC), consisting of 55 families and at least 362 transporter genes coding for proteins that function primarily as facilitated influx pumps [8], and the ATP Binding Cassette Superfamily (ABC), which includes seven families and 48 members, most of which are active efflux pumps driven by ATP hydrolysis [9,10]. Given the importance of transporters as permeability barriers and selective carriers for nutrients, xenobiotics and clinically relevant drugs, a number of studies have explored their expression levels and cellular localization in tissues involved in drug absorption, distribution, metabolism and elimination (ADME) [9,[11][12][13][14][15][16][17][18]. Transporter expression has also been evaluated in organs that are protected by blood-tissue barriers, such as the brain and testes [19][20][21][22][23][24][25]. ...
The barrier epithelia of the cornea and retina control drug and nutrient access to various compartments of the human eye. While ocular transporters are likely to play a critical role in homeostasis and drug delivery, little is known about their expression, localization and function. In this study, the mRNA expression levels of 445 transporters, metabolic enzymes, transcription factors and nuclear receptors were profiled in five regions of the human eye: cornea, iris, ciliary body, choroid and retina. Through RNA expression profiling and immunohistochemistry, several transporters were identified as putative targets for drug transport in ocular tissues. Our analysis identified SLC22A7 (OAT2), a carrier for the anti-viral drug, acyclovir, in the corneal epithelium, in addition to ABCG2 (BCRP), an important xenobiotic efflux pump, in retinal nerve fibers and the retinal pigment epithelium. Collectively, our results provide an understanding of the transporters that serve to maintain ocular homeostasis and which may be potential targets for drug delivery to deep compartments of the eye.
... Majority of 1e33 are cholic acid derivatives (Fig. S8 in Supplementary material). It is known that various Na þ -dependent transporters [17,18] are responsible for the uptake of the bile acids (Na þ /taurocholate cotransporting polypeptide (Ntcp1), organic anion transporting polypeptide (Oatp1), microsomal epoxide hydrolase (mEH)) [19,20], and they can carry some of the functionalized cholic acid derivatives into the cell [21]. ...
An alignment-free 3D QSAR study on antiproliferative activity of the thirty-three 1,2,4,5-tetraoxane derivatives toward two human dedifferentiated cell lines was reported. GRIND methodology, where descriptors are derived from GRID molecular interaction fields (MIF), were used. It was found that pharmacophoric pattern attributed to the most potent derivatives include amido NH of the primary or secondary amide, and the acetoxy fragments at positions 7 and 12 of steroid core which are, along with the tetraoxane ring, common for all studied compounds. Independently, simple multiple regression model obtained by using the whole-molecular properties, confirmed that the hydrophobicity and the H-bond donor properties are the main parameters influencing potency of compounds toward human cervix carcinoma (HeLa) and human malignant melanoma (FemX) cell lines. Corollary, similar structural motifs are found to be important for the potency toward both examined cell lines.
... The mechanism by which the cells take up charged molecules such as steroid sulfates requires further investigation. Some mechanisms have been described for carrier-mediated uptake of organic anions by the liver that may be applicable but these have not been evaluated in mammary epithelium [25,26]. Similar to steroid sulfatase in MCF-7 and MDA-MB-231 cells [15], the normal breast cells also responded with increasing hydrolysis to concentrations of estrone sulfate up to at least 10 M. The K m was in the same range as that in breast cancer cell lines. ...
The study was designed to determine the process and limitations by which estrone sulfate may be a precursor of estradiol in the parenchymal cells of the normal breast. The concentration of estrone sulfate in breast nipple aspirate fluid was 1000-fold greater than that of estradiol. Concentrations of 3H-estrone sulfate in parenchymal cells were only 0.20-0.33 times that of the 1.0 nM concentration in the medium, while 3H-estrone achieved concentrations up to 24 times that in the medium at 37 degrees C. Nevertheless, estrone sulfate added to the medium was linearly converted within a 1000-fold concentration range to estrone in intact cells with a mean half-time of conversion of 628 min per 10(6) cells. Homogenized cells had a half-time of 246 min per 10(6) cells. Thus, the time for entry of estrone sulfate into cells reduced the rate by approximately 55%. In split samples, the Vmax values (+/- S.D.) for intact and homogenized cells were 12.6 +/- 1.4 and 18.3 nmol/h mg DNA, respectively (P<0.03). The corresponding Km values for intact and homogenized cells were 6.0 +/- 1.1 and 4.7 +/- 1.0 microM. Conversion of estrone sulfate to estradiol was more efficient in intact cells than in homogenates with mean half-times of 2173 and 7485 min per 10(6) cells, respectively. Conversion of estrone to estrone sulfate did not occur in these cells despite sulfonation of estrone by MCF-7 breast cancer cells under identical conditions. It is concluded that estrone sulfate can serve as a precursor for estradiol in normal breast tissue. Conversion of estrone to estradiol is a limiting step in the process.
... Several members of the oatp family, e.g., oatp1 and oatp2, have been found in the liver. 55 Both carriers transport some of the same substrates, however, with different affinities. To precisely determine the transport kinetics of bile acidconjugated amino acids and peptides, radiolabeling of the compounds is mandatory as well as individual expression of these carriers in a heterologous expression system. ...
Uptake of drugs by bile acid carriers could account for the selectivity of drug actions in the gut and liver. We have previously shown that conjugation of xenobiotics with bile acids facilitates their transfer to hepatocytes and ileal enterocytes. In this study l -alanine and 2 biooligomers, the tetrapeptidel -(ala)4 and a 15 mer oligodeoxynucleotide (ODN) were coupled covalently via linker molecules to the 3-position of bile acids. The l -alanine–coupled bile acid conjugates were rapidly taken up by the liver and efficiently eliminated into bile. These compounds mimicked hepatic transport of bile acids. Also in case of the tetrapeptide (ala)4, bile acid conjugation significantly improved hepatic and intestinal cell uptake and rendered the peptide conjugate resistant to peptidases. Because uptake by isolated hepatocytes was not dependent on sodium ions and was blocked by ochratoxin A, we assume basolateral transport by an oatp-type bile acid carrier. In the case of the 15 mer ODN, normal and bile acid–conjugated oligodeoxynucleotide appeared intact in bile but without marked improvement of hepatocellular uptake and biliary elimination. We conclude that bile acids can deliver small peptides to gut and parenchymal liver cells via bile acid transport pathways, whereas in the case of oligonucleotides an attached bile acid was not sufficient to shuttle them successfully into hepatocytes.
... Site-directed mutagenesis experiments with the rat orthologue demonstrated the first two putative N-linked glycosylation sites to be glycosylated, confirming that the N-terminus is extracellular. (Hagenbuch 1997). Additional posttranslational modifications of Ntcp include phosphorylation (Mukhopadhyay et al. 1998a, b) and ubiquitination . ...
Bile formation is an important function of the liver. Bile salts are a major constituent of bile and are secreted by hepatocytes into bile and delivered into the small intestine, where they assist in fat digestion. In the small intestine, bile salts are almost quantitatively reclaimed and transported back via the portal circulation to the liver. In the liver, hepatocytes take up bile salts and secrete them again into bile for ongoing enterohepatic circulation. Uptake of bile salts into hepatocytes occurs largely in a sodium-dependent manner by the sodium taurocholate cotransporting polypeptide NTCP. The transport properties of NTCP have been extensively characterized. It is an electrogenic member of the solute carrier family of transporters (SLC10A1) and transports predominantly bile salts and sulfated compounds, but is also able to mediate transport of additional substrates, such as thyroid hormones, drugs and toxins. It is highly regulated under physiologic and pathophysiologic conditions. Regulation of NTCP copes with changes of bile salt load to hepatocytes and prevents entry of cytotoxic bile salts during liver disease. Canalicular export of bile salts is mediated by the ATP-binding cassette transporter bile salt export pump BSEP (ABCB11). BSEP constitutes the rate limiting step of hepatocellular bile salt transport and drives enterohepatic circulation of bile salts. It is extensively regulated to keep intracellular bile salt levels low under normal and pathophysiologic situations. Mutations in the BSEP gene lead to severe progressive familial intrahepatic cholestasis. The substrates of BSEP are practically restricted to bile salts and their metabolites. It is, however, subject to inhibition by endogenous metabolites or by drugs. A sustained inhibition will lead to acquired cholestasis, which can end in liver injury.
... Man kann also davon ausgehen, dass NTCP in HEK293-Zellen ebenfalls glykosyliert ist. Im Ntcp der Ratte wurde bereits nachgewiesen, dass die Aminosäuren N 5 und N 12 N-glykosyliert vorliegen (Hagenbuch 1997 Immunpräzipitationen mit den generierten Mutanten müssen in weiteren Experimenten klären, an welcher potenziellen N-Glykosylierungsstelle tatsächlich Zuckerreste angehängt sind. ...
Die SLC10-Familie ist bekannt als die Familie der Na+-abhängigen Gallensäuretransporter. NTCP und ASBT sind die Gründungsmitglieder dieser Familie und verantwortlich für die Aufrechterhaltung des enterohepatischen Kreislaufs der Gallensäuren. Die Identifizierung weiterer Mitglieder führte zu einer Neubetrachtung dieser Familie. In der vorgelegten Arbeit wurde zum ersten Mal das sechste Familienmitglied, der humane Sodium-dependent Organic Anion Transporter SOAT (SLC10A6), molekular und funktionell charakterisiert. Der humane SOAT ist ein Protein mit 377 Aminosäuren und besitzt das SLC10-Signaturmotiv ALGMMPL. Seine höchste Verwandtschaft hat SOAT zum ASBT, mit welchem er eine Subfamilie bildet. SOAT ist ein transmembranäres Glykoprotein mit wahrscheinlich sieben TMD und einer Nex/Cin-Topologie. In stabil transfizierten Zellen wurde SOAT auf seine Transporteigenschaften untersucht. Im Gegensatz zu NTCP und ASBT zeigt SOAT keinen Transport der Gallensäuren Taurocholat, Cholat, Lithocholat, Deoxycholat und Chenodeoxycholat. Stattdessen transportiert er die sulfatierten Steroidhormone DHEAS, E1S und PREGS, welche auch Substrate des NTCP, aber nicht des ASBT sind. Weiterhin stellen die sulfatierte Gallensäure TLCS und die Xenobiotika 2-SMP und 4-SMP Substrate des SOAT dar. In Hemmstudien zeigten insbesondere Substanzen mit mindestens zwei Hydrocarbonringen und einer negativ geladenen Sulfatgruppe eine starke Interaktion mit dem SOAT-Transport. Der Transport über den SOAT ist Na+-abhängig und erreicht erst bei physiologischen Na+- Konzentrationen sein Transportmaximum. Weiterhin weist dieser bei pH-Werten <7,4 höhere Transportraten auf als bei pH≥7,4. NTCP besitzt für den Transport von Steroidsulfaten und Gallensäuren eine jeweils unterschiedliche Na+-Affinität und pH-Abhängigkeit. Daher vermuten wir im NTCP zwei Substratbindungsstellen, eine für Gallensäuren und eine für Steroidsulfate, welche im Laufe der Evolution jeweils nur im ASBT (Gallensäuren) bzw. SOAT (Steroidsulfate) funktionell erhalten geblieben sind. SOAT ist am höchsten im Hoden und relativ hoch in Plazenta und Brustdrüse exprimiert. Dieses Expressionsmuster steht im Gegensatz zur Expression von NTCP in Leber und ASBT in Ileum und Niere. Aufgrund der Transportfunktion von SOAT und der Expression in hormonabhängigen Geweben gehen wir davon aus, dass die physiologische Funktion des SOAT im Import sulfatierter Steroide liegt. Diese können intrazellulär durch die katalytische Aktivität der Steroidsulfatase in aktive Steroide überführt werden (Konzept der intrakrinen Hormonsynthese). Da SOAT, wie auch die Steroidsulfatase, in Brustkrebsgewebe nachweisbar ist, wird auch eine Beziehung zur Entstehung hormonabhängiger Mammakarzinome diskutiert. In diesem Zusammenhang gilt SOAT als neues Drug Target für die Therapie hormonabhängiger Mammakarzinome. The SLC10-family is well known as the family of sodium-dependent bile acid transporters SBAT. The founding members NTCP and ASBT are responsible for the maintenance of the enterohepatic circulation of bile acids. With the identification of new members (SLC10A4- SLC10A7) we got new insights into the relevance of this carrier family. The present work describes for the first time the molecular and functional characterization of the novel carrier SLC10A6, named Sodium-dependent Organic Anion Transporter SOAT. Human SOAT consists of 377 amino acids and contains the SLC10-signature motif ALGMMPL. SOAT and ASBT have the closest relationship within the SLC10-family and both derive from a common ancestor. SOAT is a transmembrane glycoprotein with a 7 predicted transmembrane topology and a Nex/Cin-topology. Transport measurements were performed in stably transfected SOATHEK293 cells to gain information about transport characteristics of human SOAT. In contrast to NTCP and ASBT, SOAT has no transport activity for the bile acids taurocholic acid, cholic acid, lithocholic acid, deoxycholic acid, and chenodeoxycholic acid. Instead the sulfated steroids DHEAS, E1S, and PREGS are transported. These compounds are also substrates of NTCP, but not ASBT. Furthermore the sulfated bile acid TLCS and the xenobiotics 2-SMP and 4-SMP are transported by SOAT. Strong inhibition of SOAT transport was demonstrated for compounds with at least two hydrocarbon rings and a negatively charged sulfate moiety including α-Naphthylsulfate, 2-SMP, 4-SMP, 1omega-SEP and sulfated bile acids. Transport by SOAT is Na+-dependent and reached maximal transport activity at physiological Na+- concentrations. Transport rates are higher at pH<7,4 than ≥7,4. The carrier NTCP showed different transport characteristics regarding Na+-affinity and pH-dependency for bile acids and steroid sulfates. For this we assume that two substrate binding pockets are present in NTCP, one for bile acids and one for steroid sulfates. During phylogenetic development ASBT and SOAT conserved only one of the functional properties of NTCP, bile acids and steroid sulfates, respectively. In man, SOAT is dominantly expressed in testis and showed also relatively high expression in placenta and mammary gland. This expression pattern is completely different to the expression of NTCP (liver) and ASBT (ileum, kidney). Because of the high expression in hormone-dependent tissues and its substrate pattern we assume the physiological function of SOAT in the cellular import of sulfated steroids. These circulating precursor steroids can be converted into active steroids after intracellular cleavage of the sulfate group by the steroid sulfatase (concept of intracrine hormone synthesis). As SOAT and steroid sulfatase were shown to be highly expressed in breast carcinoma, a role of SOAT for the pathophysiology of breast carcinoma is discussed.
... The basolateral membrane of a hepatocyte has both sodium-dependent and -independent bile acid transport systems. However, hepatic uptake of conjugated bile acids is mediated predominantly via a sodium co-transport mechanism (Anwer and Hegner, 1978;Meier, 1995 (Hagenbuch et al., 1991;Ananthanarayanan et al., 1994;Stieger et al., 1994;Hagenbuch, 1997;Love and Dawson, 1998), is the primary sodium-dependent transporter of the hepatocyte basolateral surface (Nathanson and Boyer, 1991;Anwer, 1993). NTCP belongs to the same transporter family as ASBT, and the human protein consists of 349 amino acids with a high affinity for TCA (Hagenbuch and Meier, 1994). ...
Bile acids are crucial to the solubilization and transport of dietary lipids. The hepatic synthesis and eventual excretion of these amphipathic molecules comprise the major pathway of cholesterol elimination in mammals. The reclamation and reuse of bile acids occurs via what is known as the enterohepatic circulation of bile acids. Crucial to this process is the tightly-regulated vectorial transport of bile acids in the ileocytes.
The ileal lipid-binding protein (ILBP), a member of the intracellular lipid-binding family of proteins, is a cytosolic protein that is thought to be involved in intracellular trafficking of bile acids. The overall goal of this thesis was to characterize the role of ILBP in bile acid binding and in the modulation of the enterohepatic circulation. In order to study bile acids, a quantitative method for their analysis using a high performance liquid chromatograph coupled to an evaporative light scattering detector was developed. An isocratic solvent system was used to resolve in a single run conjugated and unconjugated bile acid species relevant to human and rodent physiology. The detection of various bile acids was linear over a range of 0.08 to 10 nmol of injected molecules. This convenient and cost-effective method for the routine analysis of bile acids was used to characterize the rank order of binding preference of recombinant murine ILBP (mILBP) for bile acids. Among the bile acids, mILBP showed the greatest preference for conjugated species that contained a doubly hydroxylated steroid moiety. Among fatty acids, mILBP preferred species that had longer chain length and increased saturation, similar to other members of the intracellular lipid binding protein family.
To define the role of ILBP in the preservation of bile acids in the enterohepatic circulation, the phenotype of mice deficient in the ilbp gene was studied. While the enterohepatic cycling of bile acids was unaffected in these animals, the overall bile acid content of the small intestine was decreased. Cholesterol levels in male ilbp null mice have decreased plasma cholesterol and increased hepatic HMG CoA reductase activity coupled with increased neutral sterol excretion. These results suggest a decrease in cholesterol absorption in male ILBP-deficient mice and demonstrate a gender disparity in bile acid metabolism. In conclusion, ILBP might play a subtle role in cellular bile acid transport that may involve the intracellular perception of these molecules.
... Ntcp is a member of the Na + -dependent organic anion transporter family, which also comprises the apical Na + -dependent bile acid transporter (ASBT, Slc10a2) and the Na + -dependent organic anion transporter (SOAT, Slc10a6). The Ntcp gene, Slc10a1, is expressed in the basolateral membrane of mammalian differentiated hepatocytes (Boyer et al. 1993;Hagenbuch 1997). In rat, it was recently shown to be expressed also in pancreatic acinar cells . ...
Schilddrüsenhormone sind wichtig für die Entwicklung des Gehirns. Ein zu geringer Thyroxinspiegel der Mutter ist die Hauptursache für Fehlbildungen des fötalen Gehirns. Bei Erwachsenen gelangt das Hormon Thyroxin über den Thyroxintransporter SLCO1C1, der auf Endothelzellen der Blut-Hirn-Schranke und Epithelzellen des Plexus choroideus exprimiert wird, ins Gehirn. SLCO1C1 ist bisher der einzig bekannte Transporter, der die Aufnahme von Thyroxin ins adulte Gehirn vermittelt. Das Expressionsprofil von SLCO1C1 während der Entwicklung war bisher allerdings unbekannt. Um die Expression von SLCO1C1 während der Entwicklung zu untersuchen, wurde ein transgener Ansatz mittels BAC Rekombination gewählt. Die Nukleotidsequenz der Cre Rekombinase (konstitutiv) oder der Cre Rekombinase, die an die mutierte Ligandenbindungsstelle des Östrogenrezeptors fusioniert wurde (induzierbar), wurde auf dem BAC mittels homologer Rekombination in den Genlokus von Slco1c1 eingefügt. Die modifizierten BACS wurden anschließend in den Pronukleus von Mäusen injiziert um transgene Tiere zu generieren, welche die Cre Rekombinase im Slco1c1 Locus entweder konstitutiv oder induzierbar exprimieren. In diesen transgenen Mäusen konnten sowohl die Transkripte der Cre also auch von Slco1c1 exklusiv im Gehirn mittels RT-PCR nachgewiesen werden. In der Immunhistochemie wiesen Mäuse aus der konstitutiven Linie Cre Expression in den Endothelzellen der Blut-Hirn-Schranke und im Plexus choroideus auf. Dieses Expressionsmuster wurde durch die Verpaarung der Cre-Linien mit einer Reportermauslinie bestätigt. Außerdem konnte gezeigt werden, dass Cre während der Entwicklung in Neuronen vieler Hirnregionen (wie in der kortikalen Ebene 2/3, dem Hippokampus und dem Bulbus olfactorius) aktiv ist. Cre-vermittelte Rekombination wurde in den neurogenen Zonen an Tag E14 und E18 nachgewiesen, während keine Cre Aktivität in adulten Neuronen vorhanden war. In der induzierbaren Cre Linie konnte nach Tamoxifen-Injektion keine Cre Aktivität in adulten Neuronen gefunden werden passend zu einer embryonalen transkriptionellen Aktivität des Slco1c1-Locus in neuronalen Progenitorzellen. Die neuronale Expression von Cre während der Entwicklung lässt vermuten, dass Thyroxin eine Rolle in der Expression von SLCO1C1 spielt. Hypothyroxinämie, die durch die Gabe von Propylthiouracil (PTU) induziert wurde, reduzierte die Anzahl der Neuronen in den kortikalen Schichten 2/3, die von SLCO1C1 exprimierenden Progenitorzellen abstammen. Weiterhin wurde die Rolle von SLCO1C1 in der adulten Neurogenese untersucht. Durch RT-PCR konnten Slco1c1 Transkripte in kultivierten neuronalen Stammzellen (NSC) nachgewiesen werden. In adulten Slco1c1-Cre Tieren waren Cre und der neuronale Stammzellmarker GFAP in der Subventrikulärzone kolokalisiert. Sowohl unter Kontrollbedingungen als auch nach einer zerebralen Ischämie konnte gezeigt werden, dass die Lokalisation von Cre mit dem Marker für unreife Neuronen Dcx nahe der Subventrikulärzone übereinstimmt . Zusammenfassend wurde die Slco1c1-Cre transgene Mauslinie generiert und genutzt, um Areale des Gehirns zu identifizieren, in denen Slco1c1 während der Entwicklung exprimiert wird. SLCO1C1 ist möglicherweise an den Effekten von Schilddrüsenhormonen bei der Neurogenese beteiligt. Thyroid hormones (THs) are important for brain development. Maternal hypothyroxinemia, low maternal thyroxine levels, is a major cause of defects in fetal brain development. In adults, thyroxine enters brain through the thyroxine transporter SLCO1C1, which is expressed in endothelial cells of the blood-brain barrier (BBB) and in epithelial cells of the choroid plexus. Till now, SLCO1C1 is the only known transporter that mediates the entry of thyroxine into the adult brain. However, the expression pattern of SLCO1C1 during development was unknown. To characterize the expression of SLCO1C1 in development, a transgenic approach was employed by bacterial artificial chromosome (BAC) recombineering. Sequences of Cre recombinase (constitutive) or Cre recombinase fused to a mutated estrogen receptor ligand binding domain (inducible) were inserted into the Slco1c1 locus on a BAC by homologous recombination. The modified BACs were subsequently injected into pronuclei to generate mice carrying the Slco1c1-driven Cre, which is expressed either constitutively or in an inducible manner. In the transgenic mice, both Cre and Slco1c1 transcripts could be detected by RT-PCR only in the brain. Mice from the constitutive line showed Cre expression in BBB and choroid plexus according to immunohistochemistry. This localization was confirmed by crossing mice with a reporter mouse line. In addition, neurons in many brain regions (such as cortical layer 2/3, the hippocampus and olfactory bulbs) expressed Cre during brain development, since Cre activity could not be detected in adult neurons but Cre-mediated recombination was found in the neurogenic zones at E14 and E18. In the inducible line, no Cre activity in the adult neurons was found, which also confirmed an early neuronal expression of Slco1c1-Cre. The neuronal expression of Cre in development indicates a role of thyroxine in the SLCO1C1-expressing neurons. Hypothyroxinemia induced by propyl-thio-uracil (PTU) decreased the number of cortical layer 2/3 SLCO1C1-expressing neurons. Furthermore, a role of SLCO1C1 in adult neurogenesis was also investigated. By RT-PCR, Slco1c1 transcripts could be detected in cultured neural stem cells (NSC). In adults, Cre colocalized with a neural stem cell marker, GFAP, in the subventricular zone. Both under normal conditions and in a stroke model, Cre was found to colocalize with an immature neuronal marker, Dcx, near the subventricular zone. In conclusion, Slco1c1-Cre transgenic mouse lines were generated and were used to identify Slco1c1-expressing brain structures during development. SLCO1C1 may be involved in the effects of thyroid hormones on neurogenesis.
... The Rat Ntcp gene is located on chromosome 6q24 and encodes for a 363 amino acid protein and has an apparent mass of 51kDa Meier and Stieger 2002). It is N-glycosylated at two sites at amino acids N5 and N11 (Hagenbuch 1997). The mouse Ntcp gene is located on chromosome 12q D1 (Cohn et al. 1995), and similar to the rat Ntcp protein, mouse Ntcp is a 362 amino acid protein but exists in 2 isoforms, Ntcp1 and Ntcp2, differing in the C-terminal sequence (Cattori et al. 1999). ...
In recent years the discovery of a number of major transporter proteins expressed in the liver and intestine specifically involved in bile acid transport has led to improved understanding of bile acid homeostasis and the enterohepatic circulation. Sodium (Na(+))-dependent bile acid uptake from portal blood into the liver is mediated primarily by the Na(+) taurocholate co-transporting polypeptide (NTCP), while secretion across the canalicular membrane into the bile is carried out by the bile salt export pump (BSEP). In the ileum, absorption of bile acids from the lumen into epithelial cells is mediated by the apical Na(+) bile salt transporter (ASBT), whereas exit into portal blood across the basolateral membrane is mediated by the organic solute transporter alpha/beta (OSTalpha/beta) heterodimer. Regulation of transporter gene expression and function occurs at several different levels: in the nucleus, members of the nuclear receptor superfamily, regulated by bile acids and other ligands are primarily involved in controlling gene expression, while cell signalling events directly affect transporter function, and subcellular localization. Polymorphisms, dysfunction, and impaired adaptive responses of several of the bile acid transporters, e.g. BSEP and ASBT, results in liver and intestinal disease. Bile acid transporters are now understood to play central roles in driving bile flow, as well as adaptation to various pathological conditions, with complex regulation of activity and function in the nucleus, cytoplasm, and membrane.
... One of the forms of oatp with putative thyroid hormone transport activity consists of 670 amino acids (M r , ϳ75 kDa) (16). Ntcp has an apparent M r of 51 kDa (40). There are many cellular proteins associated with thyroid hormones, and in only a few cases has the function been defined (18,19). ...
Export of L-T3 out of the cell is one factor governing the cellular T3 content and response. We previously observed in liver-derived cells that T3 export was inhibited by verapamil, suggesting that it is due to either ATP-binding cassette/multidrug resistance (MDR1/mdr1b) or multidrug resistance-related (MRP1/mrp1) proteins. To test this hypothesis we measured T3 export in FRTL-5, NIH-3T3, and rat hepatoma (HTC) cells that varied in expression of these proteins. FRTL-5 and NIH-3T3 cells were found to contain a T3 efflux mechanism that is verapamil inhibitable, saturable, and stereospecific. By contrast, T3 efflux in HTC cells was slow and unaffected by verapamil. Neither FRTL-5 nor NIH-3T3 cells express mdrlb, but all three cell types express mrpl, as assessed by immunoblotting. Overexpression of MDR1 in NIH-3T3 cells did not enhance verapamil-inhibitable T3 efflux. Photoaffinity labeling of FRTL-5 and NIH-3T3 cells with [125I]L-T3 revealed a labeled 90- to 100-kDa protein that was not present in HTC cells. Verapamil and excess nonradioactive L-T3, but not D-T3, inhibited labeling of this protein. The lack of correlation between T3 efflux and MDR1 and mrpl expression and the finding of a photoaffinity-labeled putative transport protein smaller than MDR1 or mrp1 protein (approximately 170 kDa) suggest that a novel protein is involved in the transport of T3 out of cells.
... Table 8 provides the current protein members of this family. Proteins of the OAT family catalyze the Na +independent facilitated transport of organic anions such as bromosulfobromophthalein and prostaglandins as well as conjugated and unconjugated bile acids (taurocholate and cholate, respectively) (Hakes and Berezney, 1991;Jacquemin et al., 1994;Kanai et al., 1995;Hagenbuch, 1997;Abe et al., 1998;Chan et al., 1998;Schuster, 1998). These transporters are found exclusively in animals. ...
In 1998 we updated earlier descriptions of the largest family of secondary transport carriers found in living organisms, the major facilitator superfamily (MFS). Seventeen families of transport proteins were shown to comprise this superfamily. We here report expansion of the MFS to include 29 established families as well as five probable families. Structural, functional, and mechanistic features of the constituent permeases are described, and each newly identified family is shown to exhibit specificity for a single class of substrates. Phylogenetic analyses define the evolutionary relationships of the members of each family to each other, and multiple alignments allow definition of family-specific signature sequences as well as all well-conserved sequence motifs. The work described serves to update previous publications and allows extrapolation of structural, functional and mechanistic information obtained with any one member of the superfamily to other members with limitations determined by the degrees of sequence divergence.
... Such drugs tend to be ionized (either anionic or cationic) with molecular weights > 400 and contain further polar (H-bonding) groups (Meijer et al. 1990). A wide variety of active carrier systems are known to be present at the sinusoidal membrane that facilitate the active uptake of a diverse range of drugs (Hagenbuch 1997, Kullak-Ublick 1999. Once inside the hepatocyte the second step of hepatobiliary excretion involves transfer of drugs to metabolic sites and/or the biliary canalicular membrane, which is mediated by intracellular transfer proteins and passive di usion (LeBlanc 1994). ...
1. The molecular and functional characterization of transport proteins is emerging rapidly and significant numbers of drugs have been shown to be substrates or inhibitors. The purpose of this review is to highlight the in vivo preclinical and clinical evidence that supports a role for transport proteins in attenuating the absorption, distribution and excretion (ADE) of drugs. 2. For absorption, a clear role has emerged for P-glycoprotein in limiting permeability across the gastrointestinal tract. As a result, a wide variety of drugs suffer from incomplete, variable and non-linear absorption. Similarly, at the blood-brain barrier a range of drugs has limited brain penetration due to P-glycoprotein-mediated efflux, which can limit therapeutic effectiveness of CNS agents. In the liver, transport proteins are present on the sinusoidal membrane that can be the rate-limiting step in hepatic clearance for some drugs. Mechanistic studies clearly suggest a key role and broad substrate specificity for the OATP family of sinusoidal transporters. Mainly ATP-dependent transport proteins such as P-glycoprotein and MRP2 govern active biliary excretion. 3. Drug-drug interactions have been demonstrated involving inhibition or induction of transport proteins. Clinically significant interactions in the gastrointestinal tract and kidney have been observed with inhibitors such as ketoconazole, erythromycin, verapamil, quinidine, probenecid and cimetidine. Clinically significant inhibition at the blood-brain barrier is more difficult to demonstrate, relying on pharmacodynamic and toxicodynamic changes, but an example is quinidine increasing loperamide-induced central effects in humans. 4. This review highlights the emerging role of transport proteins in ADE of drugs and suggests these need to be considered, in drug discovery and development, with respect to variability in drug disposition and response.
... In hepatocytes, both sodium-dependent and sodium-independent transport systems on the sinusoidal membranes extract bile acids from portal blood. Of these, the sodium/bile acid co-transporting polypeptide (ntcp) is considered to be quantitatively the most important transporter of conjugated bile acids in these cells [7,8]. The transport of bile acids from the basolateral to the canalicular membrane of liver cells is a poorly understood process. ...
The liver-derived McNtcp.24 cells transport bile acids and show distinctive responses to the two classes of conjugated bile acids. Whereas taurine-conjugated bile acids are non-toxic, glycine-conjugated bile acids efficiently induce apoptosis. The aim of this study was to determine if the differential sensitivity is limited to cells that normally transport bile acids and if bile acid binding proteins could reduce bile acid-mediated apoptosis. The apical sodium/bile acid co-transporter (asbt) was expressed in Chinese hamster ovary (CHO) cells to establish active bile acid transport in a non-liver-derived cell model (CHO.asbt). A high-affinity bile acid binder was expressed in McNtcp.24 cells.
The tolerance of McNtcp.24 cells to taurine-conjugated bile acids was associated with the stimulation of phosphatidylinositol 3-kinase (PI3K) activity. Treatment of CHO.asbt cells with taurine- and glycine-conjugated bile acids resulted in apoptosis. Unlike in McNtcp.24 cells, PI3K activity was not increased in CHO.asbt cells treated with taurine-conjugated bile acids. High level expression of a bile acid binder did not attenuate bile acid-induced cytotoxicity in McNtcp.24 cells.
The data suggest that McNtcp.24 cells possess a mechanism that can elaborate distinctive responses to the different classes of bile acids. Additionally, activation of a signaling pathway involving PI3K appears to be the dominant mechanism responsible for the tolerance of McNtcp.24 cells to taurine-conjugated bile acids.
... Members of SBATs are located either in apical membranes of ileum enterocytes, kidney tubule cells and bile duct cells where they perform bile acid reabsorption, or in the basolateral membrane of hepatocytes where they initiate bile acid secretion [1][2][3][4][5][6][7][8][9]. SBATs constitute a subgroup of the superfamily of sodium-dependent cotransporters with about 35% homology among the clones from different species, e.g. from rat, mouse, rabbit, hamster, and human [10]. Their molecular mass is about 50 kDa and the predicted structure which is derived from hydrophobicity analysis contains either seven or nine transmembrane domains; all SBATs are glycosylated at the extracellular N-terminus and contain a cytoplasmic C-terminus. ...
The relevance of functional amino acids for taurocholate transport by the sodium-dependent taurocholate cotransporting polypeptide Ntcp was determined by site-directed mutagenesis. cRNA from 28 single-points mutants of the rat liver Ntcp clone was expressed in Xenopus laevis oocytes. Mutations were generated in five conserved negatively charged amino acids (aspartates and glutamates) which were present in nine members of the SBAT-family, in two nonconserved negatively charged amino acids, in all eight Ntcp-cysteines, and in two threonines from a protein kinase C consensus region of the Ntcp C-terminus. Functional amino acids were Asp115, Glu257, and Cys266, which were found to be essential for the maintenance of taurocholic acid transport. Asp115 is located in the large intracellular loop III, whereas Glu257 and Cys266 are located in the large extracellular loop VI. Four mutations of threonines from the C-terminus of the Ntcp by alanines or tyrosines showed no effects on sodium-dependent taurocholate transport. Introduction of the FLAG(R) motif into several transport negative point mutations demonstrated that all mutated proteins besides one were present within the cell membrane of the oocytes and provided proof that an insertion defect has not caused transport deficiency by these Ntcp mutants. The latter was observed only with the transport negative mutant Asp24Asn. In conclusion, loop amino acids are required for sodium-dependent substrate translocation by the Ntcp.
... Based on bioinformatic predictions and experimental data, it has been shown that NTCP and ASBT have an extracellular N-terminus, an odd number of transmembrane helices (seven or nine) and a cytoplasmic Cterminus (Hagenbuch et al. 1991; Hagenbuch and Meier 1994; Stieger et al. 1994; Dawson and Oelkers 1995; Hallén et al. 1999; Hallén et al. 2002b; Zhang et al. 2004). Several potential N-glycosylation sites are present in NTCP, ASBT and SOAT proteins, and site-directed mutagenesis has revealed that only N 5 and N 11 in rat Ntcp (Hagenbuch 1997), and N 10 in human ASBT (Zhang et al. 2004) are glycosylated. These findings clearly placed the N-terminus of these proteins to the extracellular surface of the plasma membrane. ...
The solute carrier family 10 (SLC10) comprises two sodium-dependent bile acid transporters, i.e. the Na(+)/taurocholate cotransporting polypeptide (NTCP; SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT; SLC10A2). These carriers are essentially involved in the maintenance of the enterohepatic circulation of bile acids mediating the first step of active bile acid transport through the membrane barriers in the liver (NTCP) and intestine (ASBT). Recently, four new members of the SLC10 family were described and referred to as P3 (SLC10A3), P4 (SLC10A4), P5 (SLC10A5) and sodium-dependent organic anion transporter (SOAT; SLC10A6). Experimental data supporting carrier function of P3, P4, and P5 is currently not available. However, as demonstrated for SOAT, not all members of the SLC10 family are bile acid transporters. SOAT specifically transports steroid sulfates such as oestrone-3-sulfate and dehydroepiandrosterone sulfate in a sodium-dependent manner, and is considered to play an important role for the cellular delivery of these prohormones in testes, placenta, adrenal gland and probably other peripheral tissues. ASBT and SOAT are the most homologous members of the SLC10 family, with high sequence similarity ( approximately 70%) and almost identical gene structures. Phylogenetic analyses of the SLC10 family revealed that ASBT and SOAT genes emerged from a common ancestor gene. Structure-activity relationships of NTCP, ASBT and SOAT are discussed at the amino acid sequence level. Based on the high structural homology between ASBT and SOAT, pharmacological inhibitors of the ASBT, which are currently being tested in clinical trials for cholesterol-lowering therapy, should be evaluated for their cross-reactivity with SOAT.
... cations with high hydrogen bonding potential) suggest that passive diffusion across membranes is relatively slow and the rapid uptake of UK-191,005 into the liver is therefore more likely to be an active process, facilitated by carrier proteins on the sinusoidal membrane of the hepatocyte. A wide variety of active carrier systems are known to be present at the sinusoidal membrane facilitating the uptake of a diverse range of drugs (Hagenbuch, 1997;Meijer et al., 1997). The involvement of active transport processes in the hepatic uptake of UK-191,005 was confirmed in isolated rat hepatocytes at 4°C and 37°C, where uptake of UK-191,005 was found to be temperature-dependent and saturable (K m 6.5 M; Fig. 4). ...
A rational drug discovery process was initiated to design a potent and prostate-selective alpha1(L)-adrenoceptor antagonist with pharmacokinetic properties suitable for once a day administration after oral dosing, for the treatment of benign prostatic hyperplasia. Two series of compounds based on a quinoline or quinazoline template were identified with appropriate pharmacology. A series of high molecular weight cations with high hydrogen-bonding potential had extensive in vivo clearance, despite demonstrating metabolic stability. Studies in the isolated perfused rat liver and fresh rat hepatocytes indicated that active transport protein-mediated hepatobiliary elimination is an efficient clearance process for these compounds. A reduction in molecular weight and hydrogen-bonding potential resulted in a second series of compounds with in vivo hepatic clearance predictable from in vitro metabolic clearance. Initially, lipophilicity was reduced within this second series to reduce metabolic clearance and increase elimination half-life. However, this strategy also resulted in a concomitant reduction in volume of distribution and a negligible effect on prolonging half-life. An alternative strategy was to increase the intrinsic metabolic stability of the molecule by careful structural modifications while maintaining lipophilicity. Replacement of the metabolically vulnerable morpholine side chain resulted in identification of UK-338,003, (N-[2-(4-amino-6,7-dimethoxy-5-pyridin-2-yl-quinazolin-2-yl)-1,2,3,4-tetrahydro-isoquinolin-5-yl]-methanesulfonamide), which fulfilled the objectives of the discovery program with suitable pharmacology (human prostate alpha1(L) pA(2) of 9.2 with 25-fold selectivity over rat aorta alpha1(D)) and sufficiently long elimination half-life in human volunteers (11-17 h) for once a day administration.
Drug-induced liver injury (DILI) remains a major cause of drug failures during preclinical development and withdrawals from the market. Clinically, drug-induced cholestasis (DIC) represents one of the most severe manifestations of DILI. Dysfunction of hepatic bile acid transporters is known to play a pivotal role in the development of DIC, via drug-mediated inhibition and regulation of transporter proteins. Significant advances have been achieved in our understanding of the molecular mechanisms underlying DIC. This chapter aims to provide a systemic review of bile acid synthesis and transport in the body, and a focused discussion of hepatic bile acid transporters and their association with the development of DIC.
Purpose of review: Action and metabolism of thyroid hormones are intracellular events, which require their uptake across the plasma membrane. Cellular uptake of thyroid hormone is mediated by transporters, and recently, some of these transporters have been identified at the molecular level. Here we review the biochemistry, molecular biology, and clinical relevance of these transporters.
Recent findings: Iodothyronine transporters have been identified among the members of different transporter families, including Na/taurocholate cotransporting polypeptide, different (Na-independent) organic anion transporting polypeptides, the L-type amino acid transporters, and monocarboxylate transporter-8. Two transporters are particularly interesting: organic anion transporting polypeptide-1C1, which shows a high affinity for T4 and T3 and is almost exclusively expressed in brain, and monocarboxylate transporter-8, which is a very active iodothyronine transporter with a slight preference for T3 and is abundantly expressed in different tissues, including brain. Organic anion transporting polypeptide-1C1 is probably important for T4 transport across the blood-brain barrier and that monocarboxylate transporter-8 for T3 uptake in neurons. Recently, mutations in MCT8, which is located on the X chromosome, were identified in male patients with severe psychomotor retardation and elevated serum levels of T3.
Summary: Recent studies have greatly increased our knowledge about the identity of thyroid hormone transporters and in particular their physiologic relevance. The dramatic consequences of mutations in monocarboxylate transporter-8 represent a novel mechanism of thyroid hormone resistance due to inhibited cellular entry of the hormone.
Hepatobiliary transport of endogenous and exogenous compounds is mediated by the coordinated action of multiple transport systems present at the sinusoidal (basolateral) and canalicular (apical) membrane domains of hepatocytes. During the last few years many of these transporters have been cloned and functionally characterized. In addition, the molecular bases of several forms of cholestatic liver disease have been defined. Combined, this has greatly expanded our understanding of the normal physiology of bile formation, the pathophysiology of intrahepatic cholestasis, as well as of drug elimination and disposition processes. In this review recent advances, with respect to function and regulation of ATP binding cassette transport proteins expressed in liver, are summarized and discussed.
The aim of this project was to investigate pharmacological methods of overcoming resistance in cancer. Novel compounds, targeted therapies and non-steroidal anti-inflammatory drugs (NSAIDs) were examined for their potential to modulate and manipulate specific forms of drug resistance.
Sixty one novel compounds were tested in combination with chemotherapeutic drugs in proliferation assays for their ability to overcome MRP1 and P-gp-mediated drug resistance. Two compounds were successful P-gp modulators in the DLKP-A cell line; the ditrifluoroacetyl resveratrol derivative, RBM15, and the macrocycle derivative, KG104.
A panel of nine therapeutic agents were evaluated for their potential to down-regulate multidrug resistant protein expression and thus, overcome P-gp, MRP1 or BCRP-mediated drug resistance, at and below pharmacologically-relevant concentrations. Two of these agents (indomethacin and 17-AAG) partially down-regulated the expression of P-gp in the A549-Taxol cell line but did not overcome P-gp-mediated resistance when combined with docetaxel simultaneously or in pre-treated proliferation assays. Three agents (lapatinib, sulindac sulphide and 17-AAG) reduced the expression of MRP1 in the A549 cell line. Only sulindac sulphide overcame MRP1-mediated resistance in the combination proliferations assays; however, this was due to the inhibitory mechanism of sulindac sulphide and not due to the down-regulation of the MRP1 protein. Five agents (17-AAG, lapatinib, indomethacin, elacridar and gefitinib) down-regulated the expression of BCRP in the DLKP-SQ/mitox cell line. Lapatinib, gefitinib, elacridar and 17-AAG overcame BCRP-mediated resistance in both the combination and pre-treatment proliferation assays. The data indicates that the amount of down-regulation resulting from treatment with these drugs was insufficient to overcome drug resistance. Up-regulation of the three MDR transporter proteins was observed with a variety of agents tested. This suggests that, long-term treatment with such agents could lead to the development and amplification of multidrug resistance, and therefore, reduce the effectiveness of substrate chemotherapies in patients.
Targeted therapies, including tyrosine kinase inhibitors (TKIs, such as lapatinib), are the latest significant development in the treatment of cancer. Lapatinib sensitised HER2-expressing cell lines to chemotherapeutic agents in the presence or absence of EGFR expression. This agent was also found to be a more active sensitiser in P-gp-expressing cell lines, while erlotinib was more active in BCRP-expressing cell lines. Gefitinib was the least active of three TKIs at modulating P-gp, MRP1 or BCRP. Following a 48 hours treatment, lapatinib up-regulated the expression and function of COX-2. It also stimulated COX-2 activity directly. This lapatinib-mediated COX-2 induction was independent of its TKI action on EGFR, and HER2 and could have serious therapeutic effects as COX-2 is known to increase cell growth, inhibit apoptosis, and enhance metastasis and angiogenesis.
A COX-2-specific inhibitor, celecoxib, overcame P-gp, MRP1 and BCRP-mediated resistance. At pharmacologically relevant concentrations, celecoxib significantly overcame MRP1 and BCRP-mediated resistance. And to a much lesser extent celecoxib overcame P-gp mediated resistance above pharmacologically relevant concentrations. The combination of lapatinib with celecoxib could be of therapeutic benefit, as the combination of these agents could collectively inhibit COX-2, P-gp, MRP1, BCRP, HER2 and EGFR activity in tumours expressing multiple oncoproteins resistant pathways and enhanced signalling pathways.
It is hoped that a novel treatment regimens, using these agents and TKI drugs with traditional chemotherapeutic agents, could improve current treatment strategies resulting in increased survival rates and decreased mortality.
The aim of this thesis was to identify and characterize the membrane proteins responsible for the carrier-mediated uptake of endogenous and exogenous organic cations into the liver. Zie: Summary
The pharmacokinetics of a number of synthetic peptidomimetic thrombin inhibitors is determined by extensive hepatic elimination. The objective was to further characterize the disposition in vivo of Pefa 1023, a novel 3-amidinophenylalanine piperazide-type thrombin inhibitor, by influencing the hepatic handling with indocyanine green (ICG), which is actively taken up by the liver.
Pefa 1023 was administered intravenously to bile duct-cannulated rats, either alone or in combination with ICG. The concentrations of Pefa 1023 in blood plasma and bile were measured by a bioassay (thrombin clotting time), concentrations of indocyanine green were measured spectrophotometrically.
ICG (10 mg/kg i.v. 15 min prior to or simultaneously with Pefa 1023) markedly influenced the plasma level and biliary excretion rate of the thrombin inhibitor Pefa 1023 given in a dose of 1 mg/kg i.v. The plasma level was more than twice that of the control, the maximum biliary excretion rate about one third and the fraction of dose excreted in bile about two thirds.
The anionic dye ICG is able to interfere with the hepatic handling of a cationic, amidinophenylalanine piperazide-type thrombin inhibitor with the consequence of reduced hepatic clearance leading to higher plasma levels and lower biliary excretion of the latter.
Thyroid hormone action and metabolism are intracellular events that require transport of the hormone across the plasma membrane. We tested the possible involvement of the Na+/taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide (oatp1) in the hepatic uptake of the prohormone T4, the active hormone T3, and the metabolites rT3 and 3,3'-T2. Xenopus laevis oocytes were injected with 2.3 ng Ntcp or oatp1 cRNA and, after 2-3 days, incubated for 1 h at 25 degrees C with usually 0.1 microM 125I-labeled ligand. Uninjected oocytes showed marked uptake of iodothyronines and this was further increased by Ntcp and oatp1 cRNA, i.e., 1.9- and 2.8-fold for T4, 1.7- and 1.7-fold for T3, 1.8- and 6.0-fold for rT3, and 1.3- and 1.4-fold for 3,3'-T2, respectively. Mostly due to much lower uptake by uninjected oocytes, Ntcp and oatp1 cRNA induced larger, 12- to 76-fold increases in uptake of iodothyronine sulfates. The Ntcp cRNA-induced iodothyronine uptake was completely inhibited in Na+-deplete medium, whereas the oatp1 cRNA-induced uptake was not affected. These results suggest that hepatic uptake of thyroid hormones and their metabolites is mediated at least in part by Ntcp and oatp1.
To isolate the murine Na+/taurocholate cotransporting polypeptide (Ntcp), we screened a mouse liver cDNA library and identified Ntcp1, encoding a 362 amino acid protein and Ntcp2, encoding a 317 amino acid protein which had a shorter C-terminal end. Both isoforms mediated saturable Na+-dependent transport of taurocholate when expressed in Xenopus laevis oocytes. Analysis of the gene revealed that Ntcp2 is produced by alternative splicing where the last intron is retained.
A comprehensive classification system for transmembrane molecular transporters has been proposed. This system is based on (i) mode of transport and energy-coupling mechanism, (ii) protein phylogenetic family, (iii) phylogenetic cluster, and (iv) substrate specificity. The proposed "Transport Commission" (TC) system is superficially similar to that implemented decades ago by the Enzyme Commission for enzymes, but it differs from the latter system in that it uses phylogenetic and functional data for classification purposes. Very few families of transporters include members that do not function exclusively in transport. Analyses reported reveal that channels, primary carriers, secondary carriers (uni-, sym-, and antiporters), and group translocators comprise distinct categories of transporters, and that transport mode and energy coupling are relatively immutable characteristics. By contrast, substrate specificity and polarity of transport are often readily mutable. Thus, with very few exceptions, a unified family of transporters includes members that function by a single transport mode and energy-coupling mechanism although a variety of substrates may be transported with either inwardly or outwardly directed polarity. The TC system allows cross-referencing according to substrates transported and protein sequence database accession numbers. Thus, familial assignments of newly sequenced transport proteins are facilitated. In this article I examine families of transporters that are eukaryotic specific. These families include (i) channel proteins, mostly from animals; (ii) facilitators and secondary active transport carriers; (iii) a few ATP-dependent primary active transporters; and (iv) transporters of unknown mode of action or energy-coupling mechanism. None of the several ATP-independent primary active transport energy-coupling mechanisms found in prokaryotes is represented within the eukaryotic-specific families. The analyses reported provide insight into transporter families that may have arisen in eukaryotes after the separation of eukaryotes from archaea and bacteria. On the basis of the reported analyses, it is suggested that the horizontal transfer of genes encoding transport proteins between eukaryotes and members of the other two domains of life occurred very infrequently during evolutionary history.
Progressive familial intrahepatic cholestasis (PFIC) is a group of severe genetic cholestatic liver diseases of early life. PFIC types 1 and 2 are characterized by cholestasis and a low to normal serum gamma-glutamyltransferase (GGT) activity, whereas in PFIC type 3, the serum GGT activity is elevated. PFIC types 1 and 2 occur due to mutations in loci at chromosome 18 and chromosome 2, respectively. The pathophysiology of PFIC type 1 is not well understood. PFIC types 2 and 3 are caused by transport defects in the liver affecting the hepatobiliary secretion of bile acids and phospholipids, respectively. Benign recurrent intrahepatic cholestasis (BRIC) is linked to a mutation in the same familial intrahepatic cholestasis 1 locus at chromosome 18. Defects of bile acid synthesis may be difficult to differentiate from these transport defects. Intrahepatic cholestasis of pregnancy (ICP) appears to be related to these cholestatic diseases. For example, heterozygosity in families with PFIC type 3 is associated with ICP, but ICP has also been reported in families with BRIC. In Dubin-Johnson syndrome there is no cholestasis; only the hepatobiliary transport of conjugated bilirubin is affected. This, therefore, is a mild disease, and patients have a normal lifespan.
We have cloned two complementary DNAs (cDNAs), RL-Mtx-1 and RL-Mtx-2, corresponding to the bile acid- sensitive methotrexate carrier from rat liver by direct full-length rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) using degenerated primers that were deduced from published sequences of tumor cell methotrexate transporters. When expressed in Xenopus laevis oocytes and cosM6 cells, both clones mediate methotrexate and bumetanide transport. RL-Mtx-1 consists of 2,445 bp with an open reading frame of 1,536 bp. The corresponding protein with 512 amino acids has a molecular weight of 58 kd. RL-Mtx-2 (2,654 bp) differs by an additional insert of 203 bp. This insert is located in frame at position 1,196 of the RL-Mtx-1 and contains the typical splice junction sites at the 5' and 3' end, indicating that the RL-Mtx-2 messenger RNA (mRNA) is generated by alternative splicing. The insert contains a stop codon that shortens the RL-Mtx-2 protein to 330 amino acids (38 kd). Both cDNAs contain the binding site sequence for the dioxin/nuclear translocator responsive element (Ah/Arnt-receptor) in conjunction with a barbiturate recognition sequence (Barbie box). Preliminary results show that the Barbie box acts as a negative regulatory element. The two liver cDNA clones show homologies to the published sequences of folate and the reduced folate carriers, but no homology is found to the transport systems for organic anions like the Ntcp1, oatp1, OAT-K1, and OAT1. Expression of the mRNA for the methotrexate carrier is found in liver, kidney, heart, brain, spleen, lung, and skeletal muscle, but not in the testis as revealed by Northern blot analysis. The highest abundance of the mRNA is found in the kidney.
Mdr2 P-glycoprotein deficiency in mice (Mdr2(-/-) leads to formation of cholesterol/cholesterol-depleted bile and reduced plasma HDL cholesterol. We addressed the questions: (1) does HDL in Mdr2(-/-) mice normalize upon phospholipid and/or cholesterol feeding, and (2): is the Mdr2(-/-) liver capable of handling excess dietary cholesterol.
Male and female Mdr2(-/-) and Mdr2(+/+) mice were fed diets with or without additional phosphatidylcholine and/or cholesterol. Plasma, hepatic and biliary lipids as well as liver function parameters and expression of transport proteins involved in bile formation were analyzed.
Feeding excess phospholipids and/or cholesterol did not affect lipoprotein levels in Mdr2(+/+) or Mdr2(-/+) mice. Dietary cholesterol caused hyperbilirubinemia (male +100%; female +500%) and elevated plasma bile salts (male +200%; female +1250%) in Mdr2(-/-) mice only, independent of phospholipids. Bile flow nor biliary bile salt and bilirubin secretion were affected in cholesterol-fed Mdr2(-/-) mice. Elevated plasma bile salts may be related to cholesterol-induced reduction of hepatic Na+-taurocholate cotransporting protein expression in Mdr2(-/-) mice.
Excess dietary phospholipids and cholesterol do not normalize low HDL associated with Mdr2 P-glycoprotein-deficiency. Induction of hyperbilirubinemia and hypercholanemia by dietary cholesterol in Mdr2(-/-) mice delineates the important role of biliary lipid secretion in normal hepatic functioning.
The thyroid hormone (TH; 3,3',5,5'-tetra-iodothyronine and 3,3',5'-triiodothyronine) regulates growth, development, and critical metabolic functions. Thyroid diseases are among the most prevalent group of metabolic disorders in the Western world. TH exerts effects through complex biological pathways, which offer a wealth of opportunities to pharmacologically intervene in TH signalling at numerous steps. These include biosynthesis, cell-specific uptake or export (involving L-type amino acid transporter, organic anion transporter, organic cation transporter, or multidrug resistance transporter), as well as nuclear targeting and actions (the latter including TH receptor binding and histone acetylation/deacetylation). Such processes represent potentially important pharmacological targets for the design of novel or improved therapies for TH disorders, obesity, and cardiovascular diseases.
The aim of these investigations was to establish the secretion of ubiquinone Q10 (UQ10) in bile of sheep under glucose-induced cholestasis. Experiments were performed on 9 cannulated sheep divided into three groups: I-infused with sodium taurocholate, II-with Na-taurocholate plus glucose, III-with Na-taurocholate and glucose plus propranolol, phentolamine and atropine. Infusion of glucose increased plasma glucose concentration from 3.89 +/- 0.593 mM/l to 12.69 +/- 0.852 mM/l in 90 min and produced elevation of plasma insulin from 124.68 +/- 1.984 to 839.54 +/- 29.212 pM/l. Employment of blocking agents reduced insulin release to maximum 685.71 +/- 50.087 pM/l in 90 min. Under infusion of Na-taurocholate, bile flow averaged 14.016 +/- 0.706 microl/min/kg b wt. In the second group, bile flow decreased to 7.08 +/- 0.59 microl/min/kg b wt. in 90 min, and reached 11.25 +/- 0.25 microl/min/kg b wt in 240 min. Addition of the blocking agents in the third group, resulted in a significant (p < 0.05) decrease in bile flow to 3.733 +/- 0.680 microl/min/kg b wt in 105 min. This reduction of bile flow occurred with significant (p < 0.05) reduction of bile acids secretion that averaged 0.032 +/- 0.087 mM/min/kg in the first hour after glucose infusion and was maintained to the end of the experiment. Marked (p < 0.05) increase in UQ10 secretion was observed in both experimental groups. Maximum values of UQ10 secretion were obtained during the second hour of the experiment and averaged 0.449 +/- 0.196ng/min/kg b wt in the second, and 0.338 +/- 0.184ng/min/kg b wt in the third group of animals. Because at the end of the experiment UQ10 secretion gradually decreased we have concluded that free radicals generated during cholestasis lead to reduction of endogenous antioxidant capacity.
Thyroid hormone is essential for the development of the brain and the nervous system. Cellular entry is required for conversion of thyroid hormones by the intracellular deiodinases and for binding of T(3) to its nuclear receptors. Several transporters capable of thyroid hormone transport have been identified. Functional expression studies using Xenopus laevis oocytes have so far identified two categories of transporters involved in thyroid hormone uptake (i.e., organic anion transporters and amino acid transporters). Among the organic anion transporters, both Na(+) taurocholate cotransporting polypeptide (NTCP) and various members of the organic anion transporting polypeptide (OATP) family mediate transport of iodothyronines. Because iodothyronines are a particular class of amino acids derived from tyrosine residues, it is no surprise that some amino acid transporters have been shown to be involved in thyroid hormone transport. We have characterized monocarboxylate transporter 8 (MCT8) as a very active and specific thyroid hormone transporter, the gene of which is located on the X chromosome. MCT8 is highly expressed in liver and brain but is also widely distributed in other tissues. MCT8 shows 50% amino acid identity with a system T amino acid transporter 1 (TAT1). TAT1, also called MCT10, has been characterized to transport aromatic amino acids but no iodothyronines. We have also found that mutations in MCT8 are associated with severe X-linked psychomotor retardation and strongly elevated serum T(3) levels in young boys.
Cellular entry is required for conversion of thyroid hormone by the intracellular deiodinases and for binding of 3,3',5-triiodothyronine (T(3)) to its nuclear receptors. Recently, several transporters capable of thyroid hormone transport have been identified. Functional expression studies using Xenopus laevis oocytes have demonstrated that organic anion transporters (e.g., OATPs), and L-type amino acid transporters (LATs) facilitate thyroid hormone uptake. Among these, OATP1C1 has a high affinity and specificity for thyroxine (T(4)). OATP1C1 is expressed in capillaries throughout the brain, suggesting it is critical for transport of T(4) over the blood-brain barrier. We have also characterized a member of the monocarboxylate transporter family, MCT8, as a very active and specific thyroid hormone transporter. Human MCT8 shows preference for T(3) as the ligand. MCT8 is highly expressed in liver and brain but is also widely distributed in other tissues. The MCT8 gene is located on the X chromosome. Recently, mutations in MCT8 have been found to be associated with severe X-linked psychomotor retardation and strongly elevated serum T(3) levels.
The hepatobiliary system and the kidneys are the main routes by which drugs and their metabolites leave the body. Compounds that are mainly excreted into bile in general have relatively high molecular weights, are amphipathic and highly bound to plasma proteins. In contrast, compounds that are predominantly excreted into urine have relatively low molecular weights, are more hydrophilic and generally less protein bound. The first step in drug elimination in liver and kidney is uptake into hepatocytes or into proximal tubular cells. The substrate specificity and affinity of the uptake carriers expressed at the basolateral membranes of hepatocytes and proximal tubular cells could therefore play an important role for the determination of the main elimination route of a compound. This review discusses the tissue distribution, substrate specificity, transport mechanism, and regulation of the members of the organic anion transporting polypeptide (Oatp/OATP) superfamily (solute carrier family SLC21A) and the SLC22A family containing transporters for organic cations (OCTs) and organic anions (OATs). The Oatps/OATPs are mainly important for the hepatic uptake of large amphipathic organic anions, organic cations and uncharged substrates, whereas OCTs and OATs mediate uptake of predominantly small organic cations and anions in liver and kidney.
Secondary transmembrane transport carriers fall into families and superfamilies allowing prediction of structure and function. Here we describe hundreds of sequenced homologues that belong to six families within a novel superfamily, the bile/arsenite/riboflavin transporter (BART) superfamily, of transport systems and putative signalling proteins. Functional data for members of three of these families are available, and they transport bile salts and other organic anions, the bile acid:Na(+) symporter (BASS) family, inorganic anions such as arsenite and antimonite, the arsenical resistance-3 (Acr3) family, and the riboflavin transporter (RFT) family. The first two of these families, as well as one more family with no functionally characterized members, exhibit a probable 10 transmembrane spanner (TMS) topology that arose from a tandemly duplicated 5 TMS unit. Members of the RFT family have a 5 TMS topology, and are homologous to each of the repeat units in the 10 TMS proteins. The other two families [sensor histidine kinase (SHK) and kinase/phosphatase/synthetase/hydrolase (KPSH)] have a single 5 TMS unit preceded by an N-terminal TMS and followed by a hydrophilic sensor histidine kinase domain (the SHK family) or catalytic domains resembling sensor kinase, phosphatase, cyclic di-GMP synthetase and cyclic di-GMP hydrolase catalytic domains, as well as various noncatalytic domains (the KPSH family). Because functional data are not available for members of the SHK and KPSH families, it is not known if the transporter domains retain transport activity or have evolved exclusive functions in molecular reception and signal transmission. This report presents characteristics of a unique protein superfamily and provides guides for future studies concerning structural, functional and mechanistic properties of its constituent members.
We have isolated a cDNA clone by screening a rabbit kidney cortex cDNA library for expression of sodium-independent transport of L-arginine and L-alanine in Xenopus laevis oocytes. Expressed uptake relates to a single component of sodium-independent transport for dibasic and neutral amino acids. This transport activity resembles the functionally defined system b0,+ and carries cystine and dibasic amino acids with high affinity. The rBAT (b0,+ amino acid transporter-related) mRNA is found mainly in kidney and intestinal mucosa. It encodes a predicted 77.8-kDa protein with only one putative transmembrane domain and seven potential N-glycosylation sites. This protein could either be a constitutive element or a specific activator of system b0,+.
The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.
In order to characterize the driving forces for the concentrative uptake of unconjugated bile acids by the hepatocyte, the effects of pH gradients on the uptake of [3H]cholate by rat basolateral liver plasma membrane vesicles were studied. In the presence of an outwardly directed hydroxyl gradient (pH 6.0 outside and pH 7.5 inside the vesicle), cholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than at equilibrium ("overshoot"). In the absence of a pH gradient (pH 6.0 or 7.5 both inside and outside the vesicle), uptake was relatively slower and no overshoot was seen. Reductions in the magnitude of the transmembrane pH gradient were associated with slower initial uptake rates and smaller overshoots. Cholate uptake under pH gradient conditions was inhibited by furosemide and bumetanide but not by 4, 4'-diisothiocyano-2,2'-disulfonic stilbene (SITS), 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (DIDS), or probenecid. In the absence of a pH gradient, an inside-positive valinomycin-induced K+ diffusion potential caused a slight increase in cholate uptake which was insensitive to furosemide. Moreover, in the presence of an outwardly directed hydroxyl gradient, uphill cholate transport was observed even under voltage clamped conditions. These findings suggest that pH gradient-driven cholate uptake was not due to associated electrical potentials. Despite an identical pKa to that of cholate, an outwardly directed hydroxyl gradient did not drive uphill transport of three other unconjugated bile acids (deoxycholate, chenodeoxycholate, ursodeoxycholate), suggesting that a non-ionic diffusion mechanism cannot account for uphill cholate transport. In canalicular vesicles, although cholate uptake was relatively faster in the presence of a pH gradient than in the absence of a gradient, peak uptake was only slightly above that found at equilibrium under voltage clamped conditions. These findings suggest a specific carrier on the basolateral membrane of the hepatocyte which mediates hydroxyl/cholate exchange (or H+-cholate co-transport). A model for uphill cholate transport is discussed in which the Na+ pump would ultimately drive Na+/H+ exchange which in turn would drive hydroxyl/cholate exchange.
Unmethylated CpG rich islands are a feature of vertebrate DNA: they are associated with housekeeping and many tissue specific
genes. CpG islands on the active X chromosome of mammals are also unmethylated. However, islands on the inactive X chromosome
are heavily methylated. We have identified a CpG island in the 5' region of the G6PD gene, and two islands forty Kb 3' from
the G6PD gene, on the human X chromosome. Expression of the G6PD gene is associated with concordant demethylation of all three
CpG islands. We have shown that one of the two islands is in the promoter region of a housekeeping gene, GdX. In this paper
we show that the second CpG island is also associated with a gene, P3. The P3 gene has no homology to previously described
genes. It is a single copy, 4 kb gene, conserved in evolution, and it has the features of a housekeepin two genes is with
in the CpG island and that sequences in the islands have promoter function.
Monoclonal antibodies have been utilized to characterize the hepatocyte Na+-dependent bile acid transport system. Sinusoidal plasma membrane proteins in the 49-54-kDa range, which are thought to be components of this transport system, based on photo-affinity labeling and reconstitution studies, have been partially purified by affinity chromatography and utilized as an immunogen for the production of a panel of monoclonal antibodies (mAb). One of these mAbs, 25A-3, recognized both a 49- and a 54-kDa protein as assessed by immunoprecipitation. In addition, it was shown to protect the bile acid transport system from inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a dose-dependent manner. DIDS covalently labeled membrane proteins of 49 and 54 kDa, and this process could be significantly inhibited when performed in the presence of mAb 25A-3. Furthermore, the DIDS-labeled membrane proteins were immunoprecipitated by 25A-3. These results establish that one of these membrane components is the bile acid carrier protein. Another mAb (25D-1) which immunoprecipitated only a 49-kDa protein was shown to block the protective effect of 25A-3 on DIDS inhibition of bile acid transport. In addition both antibodies effected each other's binding capacity to hepatocytes and reacted with the same 49-kDa protein as established by sequential immunoprecipitation. Binding studies indicated that there are approximately 3.3 X 10(6) 49-kDa transport molecules/hepatocyte. These results firmly establish that the 49-kDa protein is the Na+-dependent hepatocyte bile acid transporter.
When [14C]oleate bound to bovine serum albumin was incubated at 37 degrees C for 7 min with isolated rat hepatocytes in the absence of glucose, the cumulative oleate uptake curve had two components: a rapid, linear segment over the first 30 sec, followed by a slower, curvilinear component. At 173 microM [14C]oleate/albumin (1:1, mol/mol), the initial component had a slope (Vo) of 118 +/- 18 pmol per min per 5 X 10(4) hepatocytes (mean +/- SD). During this initial 30 sec, virtually no oleate was oxidized, and less than 11% was esterified. By 5 min, 79% was esterified; oxidation never exceeded 4%. Addition of 2 mM glucose significantly increased oleate esterification and thereby available oleate binding sites on cytosolic fatty acid binding protein but had no influence on Vo. Pretreatment with trypsin reduced Vo by 49 +/- 15%. These data indicate that the initial component of the oleate uptake curve reflects predominantly influx, whereas the subsequent component reflects a balance between influx, efflux, and intracellular metabolism. Influx is independent of intracellular binding, oxidation, and esterification and may reflect a membrane-associated carrier-mediated process.
Turnover of hepatic glutathione in vivo in the rat is almost entirely accounted for by cellular efflux, of which 80-90% is sinusoidal. Thus, sinusoidal efflux play a major quantitative role in homeostasis of hepatic glutathione. Som preliminary observations from our laboratory (1983. J. Pharmacol. Exp. Ther. 224:141-147.) and circumstantial evidence in the literature seemed to imply that the raising of the hepatic glutathione concentration above normal was not accompanied by a rise in the rate of sinusoidal efflux. Based on these observations, we hypothesized that the sinusoidal efflux was probably a saturable process and that at normal levels of hepatic glutathione the efflux behaved as a zero-order process (near-saturation). We tested our hypothesis by the use of isolated rat livers perfused in situ, single pass, with hemoglobin-free, oxygenated buffer medium at pH 7.4 and 37 degrees C. Preliminary experiments established a range of perfusion rates (3-4 ml/min per g) for adequacy of oxygenation, lack of cell injury, and minimization of variability contributed by perfusion rates. Hepatic glutathione was lowered to below normal by a 48-h fast, diethylmaleate (0.1-1.0 ml/kg i.p.), and buthionine sulfoximine (8 mmol/kg i.p.), and raised to above normal by 3-methylcholanthrene (20 mg/kg x 3 d i.p.) and cobalt chloride (0.05-0.27 g/kg-1 subcutaneously). Steady state sinusoidal efflux from each liver was measured over a 1-h perfusion, during which the coefficient of variation of glutathione in perfusates stayed within 10%. Hepatic glutathione efflux as a function of hepatic concentration was characterized by saturable kinetics with sigmoidal (non-hyperbolic) features. The data were fitted best with the Hill model and the following parameter values were estimated: Vmax = 20 nmol/min per g, Km = 3.2 mumol/g, and n = 3 binding/transport sites. The efflux could be inhibited reversibly by sulfobromophthalein-glutathione conjugate but was not affected by the addition of glutathione to the perfusion medium. The results support our hypothesis that sinusoidal efflux of glutathione is near saturation (approximately equal to 80% of Vmax) at normal (fed and fasted) liver glutathione concentrations. The phenomenon of saturability coupled with the ability to inhibit the efflux leads us to propose that sinusoidal efflux from hepatocytes appears to be a carrier-mediated process. Some recent studies by others, using sinusoidal membrane-enriched vesicles, also support these conclusions.
Basolateral plasma membrane vesicles were prepared from rat liver by a new technique using self-generating Percoll gradients. The method is rapid (total spin time of 2.5 h) and protein yields were high (0.64 mg/g of liver). Transmission electron microscopy studies and measurements of marker enzyme activities indicated that the preparation was highly enriched in basolateral membranes and substantially free of contamination by canalicular membranes or subcellular organelles. High total recoveries for protein yield and marker enzyme activities during the fractionation procedure indicated that enzymatic activity was neither lost (inactivation) nor increased (activation). Thus, the pattern of marker enzyme activities found in the membrane preparation truly reflected substantial enrichment in membranes from the basolateral surface. Analysis of freeze-fracture electron micrographs suggested that approximately 75% of the vesicles were oriented "right-side-out." In order to assess the functional properties of the vesicles, the uptake of [3H]taurocholate was studied. In the presence of a Na+ gradient, taurocholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot"). In the absence of a gradient but in the presence of equimolar Na+ inside and outside of the vesicle, taurocholate uptake was faster than in the absence of Na+. These findings support a direct co-transport mechanism for the uptake of taurocholate and Na+. Kinetic studies demonstrated that Na+-dependent taurocholate uptake was saturable with a Km of 36.5 microM and a Vmax of 5.36 nmol mg-1 protein min-1. The high yield, enzymatic profile and retention of transport properties suggest that this membrane preparation is well suited for studies of basolateral transport.
The taurocholate transport system in normal and transformed hepatocytes has been characterized using transport kinetics and photoaffinity labeling procedures. A photoreactive diazirine derivative of taurocholate, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino [ 1,2-3H ]ethanesulfonic acid (7-ADTC), which has been shown to be a substrate for the bile acid carrier system, was photolyzed in the presence of intact hepatocytes, hepatoma tissue culture (HTC) cells, and plasma membranes derived from the hepatocyte sinusoidal surface. Irradiation of membranes in the presence of 7-ADTC resulted in the incorporation of the photoprobe into two proteins with Mr = 68,000 and 54,000. The specificity of labeling was confirmed by the significant inhibition of labeling observed when photolysis was carried out in the presence of taurocholate. The 68,000-Da protein was easily extracted with water and was shown to exhibit electrophoretic properties identical with rat serum albumin. The 54,000-Da protein required Triton X-100 for solubilization, indicating a strong association with the plasma membrane. Labeling of intact hepatocytes also resulted in specific labeling of the 54,000-Da protein. In contrast to hepatocytes, HTC cells derived from Morris hepatoma 7288C as well as H4-II-E cells derived from Reuber hepatoma H-35 exhibited a total loss of mediated bile acid uptake. Photolysis of 7-ADTC in the presence of HTC cells did not result in the labeling of any proteins, a result consistent with the loss of transport activity, and further supporting the specificity of the labeling reaction. The anion transport inhibitor N-(4-azido-2-nitrophenyl)-2-aminoethyl-[ 35S ]sulfonate, which has been shown to be a substrate for the bile acid carrier system also labeled the 54,000-Da plasma membrane protein when photolyzed in the presence of intact hepatocytes. These results suggest that the 54,000-Da protein is a component of the hepatocyte bile acid transport system and that the activity of this system is greatly reduced in several hepatoma cell lines.
Expression cloning in Xenopus laevis oocytes was used to clone the Na+/taurocholate cotransporting polypeptide (Ntcp) and a Na+-independent organic anion transporting polypeptide (oatp) from rat liver. Ntcp is a glycoprotein of 362 amino acids, with a calculated molecular mass of 39 kD. It is predicted to span the membrane 7 times and is only expressed in differentiated hepatocytes of mammalian liver. Functionally, it mediates selective Na+-dependent bile acid uptake, oatp is a glycoprotein of 670 amino acids with a calculated molecular mass of 74 kD which is predicted to have 10 transmembrane domains. Functionally, oatp can mediate chloride-dependent and -independent bromosulfophthalein (BSP) uptake as well as Na+-independent bile acid transport. Neither Ntcp nor oatp demonstrate any significant homologies between each other and in comparison to other so far cloned transport proteins.Copyright © 1994 S. Karger AG, Basel
A computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence has been devised. For this purpose, a hydropathy scale has been composed wherein the hydrophilic and hydrophobic properties of each of the 20 amino acid side-chains is taken into consideration. The scale is based on an amalgam of experimental observations derived from the literature. The program uses a moving-segment approach that continuously determines the average hydropathy within a segment of predetermined length as it advances through the sequence. The consecutive scores are plotted from the amino to the carboxy terminus. At the same time, a midpoint line is printed that corresponds to the grand average of the hydropathy of the amino acid compositions found in most of the sequenced proteins. In the case of soluble, globular proteins there is a remarkable correspondence between the interior portions of their sequence and the regions appearing on the hydrophobic side of the midpoint line, as well as the exterior portions and the regions on the hydrophilic side. The correlation was demonstrated by comparisons between the plotted values and known structures determined by crystallography. In the case of membrane-bound proteins, the portions of their sequences that are located within the lipid bilayer are also clearly delineated by large uninterrupted areas on the hydrophobic side of the midpoint line. As such, the membrane-spanning segments of these proteins can be identified by this procedure. Although the method is not unique and embodies principles that have long been appreciated, its simplicity and its graphic nature make it a very useful tool for the evaluation of protein structures.
At [Na+]o = 118 mM the concentrative transfer of cholic and taurocholic acid from the perfusate into the isolated rat liver displays saturation kinetics (taurocholate: V = 299 nmol-min-1-g-1, Km = 61 muM; Cholate: V=327 nmol-min-1-g-1, Km = 436 muM). Perfusion with an isotonic sodium-free medium did not change the feature of a carrier-mediated transport but did markedly reduce V without affecting Km (taurocholate: V = 65 nmol-min-1-g-1, Km = 78 muM; cholate: V = 104 nmol-min-1-g-1, Km = 354 muM). It was experimentally assured that the observed reduction of bile salt uptake was not a consequence of regurgitation of bile salts or due to an excessive intracellular accumulation during cholestasis in the sodium-free state. The rate of taurocholate efflux is very low when compared with the rapid rate of the uptake. A stimulatory action of extracellular sodium on this pathway was also observed. Inhibition of the (Na+ + K+)-ATPase by 1 mM ouabain resulted in a decrease of bile salt uptake. Activation of the enzyme by potassium readmission to a K+-deprived liver enhanced bile salt uptake. The immediate response to alteration of the enzyme activity suggests a close association of a fraction of bile acid active transport with the sodium pump.
Binding and transport characteristics for uptake of taurocholic acid by isolated rat liver cells were studied.
An adsorption of taurocholate to the cell surface is terminated in less than 15 s. A K s of 0.55 mM and a total binding capacity of 3.8 nmol/mg cell protein is determined.
The rate of uptake of taurocholate follows Michaelis‐Menten kinetics with K m = 19 μM and V = 1.7 nmol/mg protein min.
There is a broad pH optimum for uptake between pH 6.5–8.0.
The activation energy amounts to 29 kcal/mol. At high taurocholate concentration an unusual upward bend is observed in the Arrhenius plot.
Taurocholate uptake is competitively inhibited by taurochenodeoxycholate ( K i = 9 μM). It is noncompetitively inhibited by bromosulfophthalein ( K i = 3 μM).
At physiological taurocholate concentrations a 200‐fold intracellular accumulation of taurocholate is observed.
Uptake is inhibited by about 75% by either antimycin A, carbonylcyanide m ‐chlorophenyl‐hydrazone, ouabain.
Replacement of extracellular Na ⁺ by either K ⁺ or sucrose results in a 75% decrease of uptake.
It is concluded that taurocholate uptake is a carrier‐mediated process, and suggested that the energy for intracellular accumulation is made available by cotransport of Na ⁺ .
The influence of bromosulfophthalein and bilirubin on the Na independent and Na dependent uptake of cholate and taurocholate was examined in isolated rat parenchyma cells. BSP and bilirubin inhibited the Na dependent uptake of cholate and taurocholate competitively and the Na dependent uptake non competitively. It is postulated that the Na independent uptake system for bile acid represents a shared carrier system for other organic anions and that the Na dependent uptake system is specific for bile acid. The exchange mechanisms between bilirubin and BSP and the Na dependent uptake system appear to be different. The investigations presented are discussed together with the clinical picture of hemolytic icterus and bile acid metabolism in neonates.
Glutamate transport across plasma membranes of neurons, glial cells and epithelial cells of the small intestine and kidney proceeds by high- and low-affinity transport systems. High-affinity (Km 2-50 microM) transport systems have been described that are dependent on Na+ but not Cl- ions and have a preference for L-glutamate and D- and L-aspartate. In neurons high-affinity glutamate transporters are essential for terminating the postsynaptic action of glutamate by rapidly removing released glutamate from the synaptic cleft. We have isolated a complementary DNA encoding an electrogenic Na(+)- but not Cl(-)-dependent high-affinity glutamate transporter (named EAAC1) from rabbit small intestine by expression in Xenopus oocytes. We find EAAC1 transcripts in specific neuronal structures in the central nervous system as well as in the small intestine, kidney, liver and heart. The function and pharmacology of the expressed protein are characteristic of the high-affinity glutamate transporter already identified in neuronal tissues. The abnormal glutamate transport that is associated with certain neurodegenerative diseases and which occurs during ischaemia and anoxia could be due to abnormalities in the function of this protein.
The mechanisms involved in the hepatocellular uptake of Krebs-cycle intermediates were investigated in isolated basolateral (sinusoidal and lateral) rat liver plasma membrane (blLPM) vesicles. An inwardly directed Na+ gradient markedly stimulated uptake of 2-oxoglutarate and succinate into voltage- and pH-clamped blLPM vesicles. This Na+-dependent portion of the dicarboxylate uptake was characterized by (a) saturability with increasing substrate concentrations (K
m= 6.4–10 mM; V
max≈0.2 nmol min−1 mg protein−1), (b) cisinhibition by lithium (10 mM), other Krebs-cycle dicarboxylates (1 mM) and DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; 1 mM) but not by sulphate, monocarboxylates, oxalate, acidic amino acids, bile salts and probenecid, (c) stimulation by an intravesicular negative K+-diffusion potential indicating electrogenic [(Na+)n>2-succinate] cotransport, and (d) a pH optimum for transport between 7.0 and 7.5. In the absence of Na+, an inside alkaline pH gradient also markedly stimulated 2-oxoglutarate uptake. This pH-gradient-driven 2-oxoglutarate uptake was insensitive to lithium, but could also be inhibited by DIDS and succinate. Furthermore, saturation kinetics demonstrated K
m (≈ 34 mM) and V
max (≈ 0.8 nmol min−1 mg protein−1) values that were clearly different from those of the Na+-dependent uptake system. These results indicate the occurrence of two separate dicarboxylate transport systems along the sinusoidal border of hepatocytes, one being a Na+-dicarboxylate symporter and the other representing an anion-exchange system.
Transport of the antifolate cancer drug methotrexate was studied in vesicles isolated from the basolateral membrane of rat liver. Transport of methotrexate by basolateral membrane vesicles (BLMVs) was mostly via uptake into an osmotically active intravesicular space, with some binding (approximately 9%), as shown by initial uptake studies and by varying medium osmolarity with increasing concentrations of sucrose. Methotrexate transport was linear for the first 20 s of incubation. Transport was not affected by imposition of a Na+ gradient across the vesicular membrane. Transport of methotrexate displayed a broad pH optimum: at an intravesicular pH of 7.5, the initial rate of uptake was not significantly different at extravesicular pH values ranging from 5.5 to 7.5, but uptake was less at extravesicular pH of 5.0 or 8.0. Methotrexate transport was saturable: Km = 0.15 +/- 0.05 microM and Vmax = 11.4 +/- 1.1 pmol 10 s-1 mg-1 protein. Methotrexate uptake into BLMVs was not inhibited by 5-methyltetrahydrofolate nor by 5-formyltetrahydrofolate but was weakly inhibited by folic acid in a concentration-dependent manner. Uptake was also inhibited by anion-exchange inhibitor 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid (DIDS), and by the structurally unrelated anions ATP, ADP, Cl-, SO4(2-), and oxalate2-. Adenosine (no negative charge) had no effect on transport. When vesicles were preloaded with anions (ADP, SO4(2-), oxalate2-) such that an anion gradient existed from the intra- to the extravesicular compartment, and methotrexate uptake was measured, no stimulation of uptake was seen. Methotrexate uptake into rat liver BLMVs was electrogenic as shown by stimulation of the initial rate of uptake by a valinomycin-imposed K+ diffusion potential across the vesicular membrane. These results suggest that methotrexate is transported into the hepatocyte across the basolateral membrane by an electrogenic, multispecific anion carrier system.
A complementary DNA clone encoding a transporter for glycine has been isolated from rat brain, and its functional properties have been examined in mammalian cells. The transporter displays high affinity for glycine (KM approximately 100 microM) and is dependent on external Na+ and Cl-. Northern blot analysis indicates that the distribution of the mRNA encoding the glycine transporter is restricted to the nervous system. In situ hybridization data are consistent with roles for the transporter in both glycine neurotransmission and glycine modulation of N-methyl-D-aspartate (NMDA) receptors in the hippocampus. The identification of this transporter therefore opens the study of the molecular mechanisms underlying both inhibitory glycinergic transmission and NMDA-mediated excitatory transmission.
Susceptibility of rodent cells to infection by ecotropic murine leukaemia viruses (MuLV) is determined by binding of the virus envelope to a membrane receptor that has multiple membrane-spanning domains. Cells infected by ecotropic MuLV synthesize envelope protein, gp70, which binds to this receptor, thereby preventing additional infections. The consequences of envelope-MuLV receptor binding for the infected host cell have not been directly determined, partly because the cellular function of the MuLV receptor protein is unknown. Here we report a coincidence in the positions of the first eight putative membrane-spanning domains found in the virus receptor and in two related proteins, the arginine and histidine permeases of Saccharomyces cerevisiae (Fig. 1), but not in any other proteins identified by computer-based sequence comparison of the GenBank data base. Xenopus oocytes injected with receptor-encoding messenger RNA show increased uptake of L-arginine, L-lysine and L-ornithine. The transport properties and the expression pattern of the virus receptor behave in ways previously attributed to y+, the principal transporter of cationic L-amino acids in mammalian cells.
Modulation of hepatic cholate transport by transmembrane pH-gradients and during interferences with the homeostatic regulation of intracellular pH and K+ was studied in the isolated perfused rat liver. Within the concentration range studied uptake into the liver was saturable and appeared to be associated with release of OH- and uptake of K+. Perfusate acidification ineffectually stimulated uptake. Application of NH4Cl caused intracellular alkalinization, release of K+ and stimulation of cholate uptake, withdrawal of NH4Cl resulted in intracellular acidification, regain of K+ and inhibition of cholate uptake. Inhibition of Na+/H(+)-exchange with amiloride reduced basal release of acid equivalents into the perfusate, initiated K(+)-release, and inhibited both, control cholate uptake and its recovery following intracellular acidification. K(+)-free perfusion caused K(+)-release and inhibited cholate uptake. K(+)-readmission resulted in brisk K(+)-uptake and recovery of cholate transport. Both effects were inhibited by amiloride. Interference with cholate transport through modulation of pH homeostasis by diisothiocyanostilbenedisulfonate (DIDS) could not be demonstrated because DIDS affected bile acid transport directly. Biliary bile acid secretion was stimulated by intracellular alkalinization and by activation of K(+)-transport. Uncoupling of the mutual interference between pH-dependent cholate uptake and K(+)-transport by amiloride indicates tertiary active transport of cholate. In this, Na+/K(+)-ATPase provides the transmembrane Na(+)-gradient to sustain Na+/H(+)-exchange which maintains the transmembrane pH-gradient and thus supports cholate uptake. Effects of canalicular bile acid secretion are consistent with a saturable, electrogenic transport.
In the present study the transport of glutamine across rat liver basolateral membrane was examined with special emphasis on the existence of an Na(+)-independent system and on the characteristics of the Na(+)-dependent system with respect to stoichiometry of glutamine to Na+. Well-validated and purified liver basolateral membrane vesicles were used in the study. Results of studies on the effect of incubation medium osmolarity and incubation temperature indicated that glutamine uptake by liver basolateral membrane vesicles is largely the result of transport of the substrate into the intravesicular compartment with little binding to basolateral membrane vesicles. Transport of glutamine with time was Na+ gradient dependent (out greater than in) with a distinct "overshoot" phenomenon. Replacing Na+ with an equivalent concentration of K+, NH4+, choline, or mannitol caused significant inhibition of the initial rate of glutamine transport; on the other hand, Li+ could partially substitute for Na+. The initial rate of transport of glutamine as a function of concentration (0.05-12 mmol/L) was saturable both in the presence and in the absence of an inwardly directed Na+ gradient. Apparent Km values of 2.95 and 3.35 mmol/L and Vmax values of 11,565 and 6663 pmol.mg protein-1.10s-1 were calculated in the presence and absence of a Na+ gradient, respectively. Both in the presence and absence of an Na+ gradient (out greater than in), transport of [3H]glutamine was significantly inhibited by the addition to the incubation medium of unlabeled glutamine as well as histidine, asparagine, and serine. Transport of glutamine by the Na(+)-dependent process was significantly inhibited or stimulated, respectively, by inducing a relatively positive or negative intravesicular space. On the other hand, glutamine transport by the Na(+)-independent process was not affected by changes in transmembrane electrical potential. Using the "activation method," the stoichiometry of glutamine Na+ transport was found to be 1:1. These results show that glutamine transport in rat liver basolateral membrane vesicles is carrier mediated both in the presence and absence of an Na+ gradient. Furthermore, the Na(+)-dependent process is electrogenic in nature (net positive) and cotransports one glutamine molecule with one Na+. Transport of glutamine by the Na(+)-independent system, on the other hand, is electroneutral in nature.
The complementary DNA sequence encoding the cell-surface receptor for ecotropic host-range murine retroviruses (ecoR) shows that it contains 622 amino acids and 14 hydrophobic potentially membrane-spanning sequences. Because this receptor occurs on many or all murine cells and is probably essential for viability of cultured fibroblasts, its normal function might be to transport an essential metabolite. We expressed ecoR in Xenopus laevis oocytes by injecting RNA transcribed from the cloned cDNA. These oocytes specifically bound the gp70 envelope glycoprotein from an ecotropic murine leukaemia virus. An inward current was recorded electrophysiologically when a mixture of amino-acids was applied: this resulted from a stereoselective, saturable uptake of lysine, arginine and ornithine; it was independent of sodium and not substantially altered by gp70. Cysteine and homoserine were also taken up, but sodium was necessary for their transport. These properties of ecoR correspond to those of the y+ amino-acid transporter. Our results demonstrate the subversion of a ubiquitous cell membrane protein, in this case a basic amino acid transporter, for use as a retroviral receptor.
Liver plasma membrane ecto-ATPase activity is largely restricted to the bile canalicular membrane. To determine whether a transport process is also selectively present on this membrane surface to reclaim adenosine derived from the intracanalicular degradation of ATP, the characteristics of hepatic nucleoside transport were examined in canalicular (cLPM) and basolateral (blLPM) rat liver plasma membrane vesicles. In the presence of the adenosine deaminase inhibitor, deoxycoformycin, an inwardly directed Na+ gradient markedly stimulated [3H]adenosine uptake in cLPM vesicles. Canalicular Na(+)-dependent [3H]adenosine uptake was enhanced by an intravesicular-negative membrane potential and inhibited by dissipation of the Na+ gradient with gramicidin D. Both purine and pyrimidine nucleosides inhibited canalicular adenosine transport. 6-[(4-Nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, an inhibitor of nucleoside transport in erythrocytes and nonepithelial cells, had no effect on canalicular adenosine transport. Canalicular Na(+)-dependent [3H]adenosine uptake exhibited saturability with a Michaelis-Menten constant of 8.3 microM and a maximum transport rate of 7.6 pmol.5 s-1.mg protein-1. In contrast, [3H]adenosine uptake in blLPM vesicles was not stimulated by an inwardly directed Na+ gradient. These findings demonstrate asymmetric distribution of hepatic Na(+)-dependent nucleoside transport. Reclamation of intracanalicular adenosine resulting from ecto-ATPase activity may explain the presence of this transport process selectively on the bile canalicular membrane.
Liver parenchymal cells continuously extract high amounts of bile acids from portal blood plasma. This uptake process is mediated by a Na+/bile acid cotransport system. A cDNA encoding the rat liver bile acid uptake system has been isolated by expression cloning in Xenopus laevis oocytes. The cloned transporter is strictly sodium-dependent and can be inhibited by various non-bile-acid organic compounds. Sequence analysis of the cDNA revealed an open reading frame of 1086 nucleotides coding for a protein of 362 amino acids (calculated molecular mass 39 kDa) with five possible N-linked glycosylation sites and seven putative transmembrane domains. Translation experiments in vitro and in oocytes indicate that the transporter is indeed glycosylated and that its polypeptide backbone has an apparent molecular mass of 33-35 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with mRNA species from rat kidney and intestine as well as from liver tissues of mouse, guinea pig, rabbit, and man.
Members of the G protein-linked receptor superfamily have not yet yielded to X-ray crystallography. However, diffraction data from other membrane-bound receptors - the photosynthetic reaction centre and bacteriorhodopsin - have provided some information that may also apply to the G protein family. John Findlay and Elias Eliopoulos integrate this information together with analysis of amino acid sequences from cloned receptors, to derive workable three-dimensional models of these proteins. Such models identify ligand-binding and G protein-associating domains.
Lambda G28, a mouse genomic clone homologous to the human P3 gene and associated with a CpG island, also hybridizes to human probes for the neighboring GdX gene. The two genes, P3 and GdX (DXS253E and DXS254E), physically linked on the human X chromosome, lie within a similar physical distance on the mouse X chromosome. The CpG island corresponds to that at the 5' of the human GdX gene. The relative orientation of the two genes is the same. The DNA sequence in coding and noncoding regions is very conserved.
We have isolated a 2.7-kilobase rat liver cDNA clone that contains the entire 544-amino acid coding sequence for matrin F/G. This protein has previously been localized to the internal, fibrogranular areas of the nuclear matrix and shown to bind to DNA on nitrocellulose blots. The predicted amino acid sequence from the coding region of this cDNA shows that this protein contains approximately 50% hydrophobic amino acids with secondary structure predictions suggesting a large percentage of beta-sheet regions. No significant homologies were found with any other known proteins, including the nuclear lamins. The predicted amino acid sequence was also searched for DNA binding motifs. Two putative zinc finger motifs were found. In addition, a 7-mer palindromic sequence (Ser-Ser-Thr-Asn-Thr-Ser-Ser) was discovered within one of these zinc finger DNA binding regions. A possible regulatory role for this element is discussed.
The ontogenesis of Na+-K+-ATPase activity and Na+-taurocholate cotransport was studied in basolateral plasma membrane vesicles from fetal and neonatal rat liver. Membrane vesicles from each age group were 30-fold enriched in the basolateral marker enzyme Na+-K+-ATPase, 4- to 7-fold enriched in the bile canalicular membrane marker enzymes alkaline phosphatase and Mg2+ ATPase, and not significantly enriched in activities of marker enzymes for intracellular organelles. Na+-K+-ATPase activity was significantly lower in basolateral membranes from late fetal (day 21-22) and neonatal (day 1) rat liver. Kinetic analysis of Na+-K+-ATPase activity at various concentrations of ATP revealed that the maximum velocity of enzyme reaction (Vmax) for Na+-K+-ATPase was 70 and 90% of adult activity in the fetus and the neonate, respectively. The ATP Km was significantly lower in the neonate than the adult, suggesting a higher affinity of the neonatal enzyme for ATP. In contrast to the early maturation of Na+-K+-ATPase, transport of taurocholate was markedly lower in both fetal and neonatal vesicles compared with the adult. Taurocholate uptake on day 19 of gestation did not differ in the presence of a Na+ or K+ gradient, and uphill transport, as indicated by an overshoot, did not occur. On day 20 taurocholate uptake was stimulated by a Na+ compared with a K+ gradient, and accumulation of isotope above equilibrium was demonstrated. Na+-dependent transport of taurocholate by late fetal (day 22) and neonatal vesicles was saturable but the Vmax at each age was significantly lower and the apparent Km higher in developing compared with adult membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Transport of 35S-labeled sulfobromophthalein [35S]BSP was studied in short-term cultured rat hepatocytes incubated in bovine serum albumin. At 37 degrees C, initial uptake of [35S]BSP was 5-10-fold that at 4 degrees C, linear for at least 15 min, saturable, inhibited by bilirubin, and reduced by greater than 70% after ATP depletion or isosmotic substitution of sucrose for NaCl in medium. Replacement of Na+ by K+ or Li+ did not alter uptake, whereas replacement of Cl- by HCO-3 or gluconate- reduced uptake by approximately 40%. Substitution of Cl- by the more permeant NO-3 enhanced initial BSP uptake by 30%. Efflux of [35S]BSP from cells to media was inhibited by 40% after ATP depletion or sucrose substitution. To confirm these results in a more physiologic system, transport of [3H]bilirubin was studied in isolated livers perfused with control medium or medium in which Cl- was replaced by gluconate-. Perfusion data analyzed by the model of Goresky, revealed 40-50% reductions in influx and efflux with gluconate- substitution. These results are consistent with existence of a Cl-/organic anion-exchange mechanism similar to that described by others in renal tubules.
The well-characterized erythrocyte glucose transporter is also expressed in brain, adipocytes, kidney, muscle, and certain transformed cells, but not in liver, intestine, or the islets of Langerhans. Using as probe a cDNA encoding the rat brain glucose transporter, we isolated from a rat liver cDNA library a clone encoding a protein 55% identical in sequence to the rat brain transporter, and with a superimpossible hydropathy plot. We expressed this protein in an E. coli mutant defective in glucose uptake; the protein was incorporated into the bacterial membrane and functioned as a glucose transporter. This new transporter is expressed in liver, intestine, kidney, and the islets of Langerhans; immunofluorescence analysis showed that it is present in the plasma membrane of the insulin-producing beta cells. Insulinoma cells express, inappropriately, the erythrocyte glucose transporter, and we suggest that this may be related to their inability to secrete insulin in response to elevations in glucose.
cDNA clones encoding a glucose transporter-like protein have been isolated from adult human liver and kidney cDNA libraries by cross-hybridization with the human HepG2/erythrocyte glucose transporter cDNA. Analysis of the sequence of this 524-amino acid glucose transporter-like protein indicates that it has 55.5% identity with the HepG2/erythrocyte glucose transporter as well as a similar structural organization. Studies of the tissue distribution of the mRNA coding for this glucose transporter-like protein in adult human tissues indicate that the highest amounts are present in liver with lower amounts in kidney and small intestine. The amounts of glucose transporter-like mRNA in other tissues, including colon, stomach, cerebrum, skeletal muscle, and adipose tissue, were below the level of sensitivity of our assay. The single-copy gene encoding this glucose transporter-like protein has been localized to the q26.1----q26.3 region of chromosome 3.
Highly purified rat basolateral liver plasma membrane vesicles were used to examine the mechanism and the driving forces for hepatic uptake of the beta-amino acid, taurine. An inwardly directed 100 mM NaCl gradient stimulated the initial rate of taurine uptake and energized a transient twofold accumulation of taurine above equilibrium ("overshoot"). In contrast, uptake was slower and no overshoot was detected in the presence of a KCl gradient. A negative intravesicular electrical potential generated by the presence of permeant anions or an outwardly directed K+ gradient with valinomycin increased Na+-stimulated taurine uptake. External Cl- stimulated Na+-dependent taurine uptake independent of effects on the transmembrane electrical potential difference. Na+-dependent taurine uptake showed a sigmoidal dependence on extravesicular Na+ concentration, suggesting multiple Na+ ions are involved in the translocation of each taurine molecule. Na+-dependent taurine uptake demonstrated Michaelis-Menten kinetics with a maximum velocity of 0.537 nmol.mg protein-1.min-1 and an apparent Km of 174 microM. [3H]taurine uptake was inhibited by the presence of excess unlabeled taurine, beta-alanine, or hypotaurine but not by L-glutamine or L-alanine. In summary, using basolateral liver plasma membrane vesicles, we have shown that hepatic uptake of taurine occurs by a carrier-mediated, secondary active transport process specific for beta-amino acids. Uptake is electrogenic, stimulated by external Cl-, and requires multiple Na+ ions for the translocation of each taurine molecule.
The mechanisms and driving forces for hepatic uptake of sulfate were investigated in basolateral (sinusoidal) rat liver plasma membrane vesicles. A transmembrane pH difference (pH 8.0 inside, 6.0 outside) stimulated sulfate uptake above equilibrium ("overshoot"). This pH gradient-stimulated sulfate uptake was saturable with increasing concentrations of sulfate and could be inhibited by probenecid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone, and nigericin. At low buffer concentrations and pH 6.0 an inwardly directed sodium gradient also stimulated sulfate uptake. This sodium-dependent sulfate uptake could be inhibited by amiloride and DIDS, indicating indirect coupling of sodium and sulfate flux through concomitant sodium-proton and sulfate-hydroxyl exchange. Cisinhibition of initial pH gradient-stimulated sulfate uptake, as well as transstimulation of sulfate uptake under pH-equilibrated conditions (pH 7.5 inside and outside), were observed with sulfate, thiosulfate, oxalate, and succinate, but not with chloride, bicarbonate, acetate, lactate, pyruvate, p-aminohippurate, citrate, glutamate, aspartate, and taurocholate. Furthermore, cholate and sulfobromophthalein exhibited competitive inhibition of pH gradient-stimulated sulfate uptake. In addition, an inside-to-outside hydroxyl gradient also stimulated uptake of cholate and this pH gradient-sensitive portion of cholate uptake was inhibited by extravesicular sulfate. In contrast to basolateral membranes, no evidence for multispecific sulfate-hydroxyl exchange was found in canalicular plasma membrane vesicles.
Discriminant analysis can be used to precisely classify membrane proteins as integral or peripheral and to estimate the odds that the classification is correct. Specifically, using 102 membrane proteins from the National Biomedical Research Foundation (NBRF) database we find that discrimination between integral and peripheral membrane proteins can be achieved with 99% reliability. Hydrophobic segments of integral membrane proteins can also be distinguished from interior segments of globular soluble proteins with better than 95% reliability. We also propose a procedure for determining boundaries of membrane-spanning segments and apply it to several integral membrane proteins. For the limited data available (such as on transplantation antigens), the residues at the boundaries of a membrane-spanning segment are predictable to within the error inherent in the concept of boundary. As a specific indication of resolution, seven membrane-spanning segments of bacteriorhodopsin are resolved with no information other than sequence, and the predicted boundary residues agree with the experimental data on proteolytic cleavage sites. Several definitive but yet to be tested predictions are also made, and the relation to other predictive methods is briefly discussed. A computer program in FORTRAN for prediction of membrane-spanning segments is available from the authors.
The amino acid sequence of the glucose transport protein from human HepG2 hepatoma cells was deduced from analysis of a complementary DNA clone. Structural analysis of the purified human erythrocyte glucose transporter by fast atom bombardment mapping and gas phase Edman degradation confirmed the identity of the clone and demonstrated that the HepG2 and erythrocyte transporters are highly homologous and may be identical. The protein lacks a cleavable amino-terminal signal sequence. Analysis of the primary structure suggests the presence of 12 membrane-spanning domains. Several of these may form amphipathic alpha helices and contain abundant hydroxyl and amide side chains that could participate in glucose binding or line a transmembrane pore through which the sugar moves. The amino terminus, carboxyl terminus, and a highly hydrophilic domain in the center of the protein are all predicted to lie on the cytoplasmic face. Messenger RNA species homologous to HepG2 glucose transporter messenger RNA were detected in K562 leukemic cells, HT29 colon adenocarcinoma cells, and human kidney tissue.
Taurocholate (TC) uptake by adult rat hepatocytes co-cultured with other rat liver epithelial cells (RLEC) was studied comparatively to hepatocytes in primary culture. Cells were cultured on Petri dishes for desired times prior to measuring their ability to transport TC. TC uptake was linear for 150 sec in both culture conditions. In hepatocytes cultured alone, the initial rate of TC uptake at an extracellular concentration of 100 μ M was 0.19 ± 0.02 nmole per min per 106 cells after 48 hr of culture and decreased by 75% after 4 to 6 days. In hepatocytes co-cultured with RLEC, the rate of uptake at 48 hr (0.31 ± 0.01 nmole per min per 106 cells) was significantly higher than in hepatocytes cultured alone (p < 0.01); in addition, TC uptake remained stable at an average rate of 0.17 ± 0.01 nmole per min per 106 cells for up to 56 days. No detectable uptake was found in RLEC cultured alone. TC uptake exhibited both saturable (Vmax = 0.30 ± 0.03 nmole per min per 106 cells and Km = 42.6 ± 4.4 μM) and nonsaturable components. These kinetic parameters were similar to those previously reported in isolated hepatocytes and in short-term cultured hepatocytes. TC uptake exhibited sodium dependence and was significantly reduced when extracellular sodium was replaced by lithium and sucrose, or in the presence of 1 mM ouabain. After 18 days of co-culture, TC uptake had qualitatively the same characteristics as at 48 hr, with a saturable and a nonsaturable component.
These findings strongly support the view that the sodium-dependent bile acid uptake system present in normal hepatocytes was maintained in the co-culture system as previously reported for other liver-specific functions.
The high-affinity component of glycine uptake by the hepatoma cell line HTC and by the ordinary rat hepatocyte corresponds to System Gly, the agency serving for glycine uptake by pigeon red blood cells and rabbit reticulocytes, and at most to only a minor extent to System ASC. This component was identified in HTC by its sensitivity to inhibition by sarcosine but scarcely by 2-(methylamino) isobutyric acid, by its insensitivity to lowering of the pH, and by the unique relation of its rate to the square of the Na+ concentration. The identity of the low-affinity component with System A was confirmed by opposite properties, and by its stimulation by insulin or amino acid starvation. Both components differed sharply from the System ASC uptake as measured with threonine.
An algorithm has been developed which identifies alpha-helices involved in the interactions of membrane proteins with lipid bilayers and which distinguishes them from helices in soluble proteins. The membrane-associated helices are then classified with the aid of the hydrophobic moment plot, on which the hydrophobic moment of each helix is plotted as a function of its hydrophobicity. The magnitude of hydrophobic moment measures the amphiphilicity of the helix (and hence its tendency to seek a surface between hydrophobic and hydrophilic phases), and the hydrophobicity measures its affinity for the membrane interior. Segments of membrane proteins in alpha-helices tend to fall in one of three regions of a hydrophobic moment plot: (1) monomeric transmembrane anchors (class I HLA transmembrane sequences) lie in the region of highest hydrophobicity and smallest hydrophobic moment; (2) helices presumed to be paired (such as the transmembrane M segments of surface immunoglobulins) and helices which are bundled together in membranes (such as bacteriorhodopsin) fall in the adjacent region with higher hydrophobic moment and smaller hydrophobicity; and (3) helices from surface-seeking proteins (such as melittin) fall in the region with still higher hydrophobic moment. alpha-Helices from globular proteins mainly fall in a region of lower mean hydrophobicity and hydrophobic moment. Application of these methods to the sequence of diphtheria toxin suggests four transmembrane helices and a surface-seeking helix in fragment B, the moiety known to have transmembrane function.
Transport of alprostadil (prostaglandin E1) and dinoprost (prostaglandin F2 alpha) was studied in enzymatically dispersed normal and streptozocin-treated rat hepatocytes prepared by collagenase perfusion. Cell suspensions incubated at 37 degrees were sampled at time intervals for a period of 5 min and the supernatant analyzed for prostaglandins after centrifugation. The data analysis employed a theory and a model for solute transfer at the cell membrane-water interphase. Biophysical parameters such as the effective partition and the apparent permeability constants were used to define the transport mechanism. The apparent permeability coefficient of alprostadil and dinoprost transfer through normal hepatocytes was calculated to be 5 X 10(-3) and 3 X 10(-3) cm/sec with a mean partition coefficient of 1345 and 764 for both solutes, respectively. The permeability coefficient of alprostadil and dinoprost transfer through diabetic hepatocytes were 3 X 10(-3) and 2 X 10(-3) cm/sec with partition coefficient of 572 and 206, respectively. The results showed differences in prostaglandin transport between normal and diabetic hepatocytes, resulting from morphological and lipid alteration in the cytoplasmic membrane.
Hepatocytes isolated from fed rats were used to investigate glutamine transport. Glutamine transport appears as a composite process involving at least two saturable components. The Na+-dependent component probably represents the entry through the N system. The Na+-independent component was also inhibited by histidine and exhibited trans-stimulation, suggestive of a facilitated diffusion process. Kinetic parameters for both systems suggest that facilitated diffusion only plays a minor role in glutamine influx. In contrast, the Km for glutamine efflux was consistent with a physiological role of the facilitated-diffusion component in glutamine release. In Na+ medium, relatively constant distribution ratios (about 8) between intra- and extra-cellular concentrations were observed, with external glutamine ranging from 0.5 to 5 mM. The present observations suggest that glutamine influx might largely be mediated by the N system, whereas facilitated diffusion allows hepatocytes to release glutamine when intracellular concentrations are elevated. The physiological consequences of this bidirectional transfer of glutamine across the liver cell membrane is discussed.
The hepatic transport of three different drugs, the organic anion dibromosulphophthalein, the organic cation d-tubocurarine and the uncharged compound ouabain was studied in vivo in the isolated perfused rat liver and isolated hepatocytes. The respective clearances by uptake were determined for the various substrates and corrected for differences in hepatic blood flow and extracellular protein binding in the three liver preparations. The corrected uptake values in the intact organ, in vivo and in the isolated perfused liver were highly comparable; for dibromosulphophthalein a clearance of 2.1 ml/min per 10(6) hepatocytes was found in vivo, whereas in perfusion a value of 2.4 ml/min per 10(6) cells was calculated. For d-tubocurarine, the values were 34 x 10 (-4) and 55 x 10(-4) ml/min per 10(6) cells obtained in vivo and in the isolated perfused organ, respectively. With ouabain as the substrate, the in vivo clearance amounted to 5.1 x 10(-2), whereas in the isolated perfused liver a value of 4.8 x 10(-2) ml/min per 10(6) cells was calculated. The clearance by uptake obtained for dibromosulphophthalein was ouabain in the isolated hepatocytes appeared to be a factor of 2-3 lower than in the intact organ. In the case of d-tubocurarine however the clearance was identical to that in vivo and the isolated perfused liver. The rate of secretion from isolated hepatocytes was, for dibromosulphophthalein identical to, and for d-tubocurarine and ouabain lower than that in the intact organ, especially as compared with the in vivo preparation. It is concluded that transport function is well preserved in the isolated perfused liver and isolated hepatocytes. For certain substrates freshly isolated hepatocytes may exhibit a somewhat lower uptake and/or secretion rate, in spite of a good cell quality as judged by generally accepted criteria for cell viability. Whether this is due to changes in membrane composition (not detected by our viability tests) or a selection of a subpopulation of hepatocytes, is discussed.U
Uptake of bilirubin, sulfobromophthalein (BSP), and other organic anions by the liver is a process with kinetics consistent with carrier mediation. The molecular basis of this transport mechanism is unknown. In the search for the putative organic anion carrier or receptor, the interaction of BSP with rat liver cell plasma membrane (LPM) has been studied. Specific binding of [(35)S]BSP to LPM was determined using a filtration assay. Results revealed high affinity (K(a) = 0.27 muM(-1)), saturable (6.3 nmol/mg protein) binding, which was eliminated after preincubation with trypsin. Although [(35)S]BSP was strongly bound to LPM, the binding was rapidly reversible, preventing direct identification and study of a specific binding site(s). To avoid this problem, a photoaffinity probe was devised, in which [(35)S]BSP is covalently bound to LPM after exposure to ultraviolet light. Subsequent sodium dodecyl sulfate gel electrophoresis and fluorography revealed radioactivity predominantly associated with a single 55,000-mol wt protein. A protein with identical electrophoretic mobility was purified from deoxycholate solubilized LPM after affinity chromatography on glutathione-BSP-agarose gel. This protein migrated as a single band on sodium dodecyl sulfate gel electrophoresis and on urea gel isoelectric focusing. It contained 1-2 residues of sialic acid per 55,000-dalton protein, and was immunologically distinct from rat albumin and ligandin. It bound bilirubin with a K(d) of 20 muM, as determined by tryptophan fluorescence quenching. Although the high affinity of this LPM protein for organic anions suggests that it may function as a hepatocellular organic anion receptor, its role in transport of these compounds is unknown.
Hepatic taurine concentration is 30--100 times that of plasma, suggesting an efficient taurine uptake mechanism in the hepatocyte. The characteristics of hepatic taurine transport were studied in primary cultures of adult rat hepatocytes. Taurine uptake was concentrative and linear for over 4 h. At taurine concentrations 2.5--100 microM, uptake was saturable with constants Km = 44 microM, V = 0.28 nmol/mg protein per min and EA = 13.2 kcal/mol. Uptake was inhibited 41% by incubation under N2 and was competitively inhibited by beta-alanine (Ki = 94 microM) and hypotaurine (Ki = 14 microM). Uptake was linearly dependent upon Na+ concentration from 0 to 140 mM. A second nonsaturable uptake process was identifiable only at taurine concentrations greater than 1 mM. This process was presumed to represent passive diffusion. At taurine concentrations existing in plasma, taurine enters the hepatocyte primarily by a single, Na+-dependent, carrier-mediated, oxygen-requiring transport process.
Based on a recently cloned rat liver organic anion transporter, we attempted to clone the corresponding human liver organic anion transporting polypeptide.
A human liver complementary DNA library was screened with a specific rat liver complementary DNA probe. The human liver transporter was cloned by homology with the rat protein and functionally characterized in Xenopus laevis oocytes.
The cloned human liver organic anion transporting polypeptide consists of 670 amino acids and shows a 67% amino acid identity with the corresponding rat liver protein. Injection of in vitro transcribed complementary RNA into frog oocytes resulted in the expression of sodium-independent uptake of [35S]bromosulfophthalein (Michaelis constant [Km], approximately 20 mumol/L), [3H]cholate (Km, approximately 93 mumol/L), [3H]taurocholate (Km, approximately 60 mumol/L), [14C]glycocholate, [3H]taurochenodeoxycholate, and [3H]tauroursodeoxycholate (Km, approximately 19 mumol/L). Northern blot analysis showed cross-reactivity with messenger RNA species from human liver, brain, lung, kidney, and testes. Polymerase chain reaction analysis of genomic DNA from a panel of human-rodent somatic cell hybrids mapped the cloned human organic anion transporter to chromosome 12.
These studies show that the cloned human liver organic anion transporter is closely related to, but probably not identical to, the previously cloned rat liver transporter. Furthermore, its additional localization in a variety of extrahepatic tissues suggests that it plays a fundamental role in overall transepithelial organic anion transport of the human body.
Carrier-mediated prostaglandin transport has been postulated to occur in many tissues. On the basis of sequence homology,
the protein of unknown function encoded by the rat matrin F/G complementary DNA was predicted to be an organic anion transporter.
Expression of the matrin F/G complementary DNA in HeLa cells or Xenopus oocytes conferred the property of specific transport
of prostaglandins. The tissue distribution of matrin F/G messenger RNA and the sensitivity of matrin F/G-induced prostaglandin
transport to inhibitors were similar to those of endogenous prostaglandin transport. The protein encoded by the matrin F/G
complementary DNA is thus preferably called PGT because it is likely to function as a prostaglandin transporter.