Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion ...channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 μM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.
The seminal vesicles are the major source of prostaglandins in seminal fluid. For prostanoid action on cell surfaces they must be released from synthesizing cells. MRP4/ABCC4 (multidrug resistance ...protein 4 adenosine triphosphate-binding cassette, subfamily C, member 4) is an adenosine triphosphate dependent export pump for organic anions that may mediate prostanoid transport across the plasma membranes. Therefore, we analyzed whether MRP4 is expressed in the seminal vesicles and other tissues of the human urogenital tract, whether MRP4 and prostanoid synthesizing enzymes are co-expressed in the same cell type and whether MRP4 functions as a prostanoid export pump.
The expression and localization of MRP4 and prostanoid synthesizing enzymes were investigated in several tissues of the male human urogenital tract by immunoblot and immunofluorescence analyses. Prostanoid transport was measured into inside-out membrane vesicles from cells expressing recombinant human MRP4.
MRP4 and prostanoid synthesizing enzymes were co-expressed in the epithelial cells of human seminal vesicles. Moreover, MRP4 was localized in the plasma membrane of epithelial cells of the ureter, in the basolateral membrane of glandular epithelial cells of the prostate, and in smooth muscle cells of the bladder and corpus cavernosum. Transport studies established MRP4 as an efflux pump for prostaglandin E
2 (Michaelis constant K
m 3.5 μM), thromboxane B
2 (K
m 9.9 μM) and prostaglandin F
2α (K
m 12.6 μM).
The co-expression of prostanoid synthesizing enzymes and MRP4 in epithelial cells of the human seminal vesicles and the function of MRP4 as a prostanoid efflux pump indicate that MRP4 mediates prostanoid transport from these cells, which are the main prostanoid synthesizing cells in the male urogenital tract.
Human organic anion transporter 7 (OAT7, SLC22A9) is a hepatic transport protein poorly characterized so far. We therefore sought to identify novel OAT7 substrates and factors contributing to ...variable hepatic OAT7 expression. Using OAT7-expressing cells, pravastatin was identified as a substrate. Hepatic SLC22A9/OAT7 mRNA and protein expression varied 28-fold and 15-fold, respectively, in 126 Caucasian liver samples. Twenty-four variants in SLC22A9 were genotyped, including three rare missense variants (rs377211288, rs61742518, rs146027075), which occurred only heterozygously. No variant significantly affected hepatic SLC22A9/OAT7 expression. The three missense variants, however, showed functional consequences when expressed in vitro. Hepatic nuclear factor 4-alpha (HNF4α) emerged as a major transcriptional regulator of SLC22A9 by a series of in silico and in vitro analyses. In conclusion, pravastatin is the first identified OAT7 drug substrate. Substantial inter-individual variability in hepatic OAT7 expression, majorly driven by HNF4α, may contribute to pravastatin drug disposition and might affect response.The Pharmacogenomics Journal advance online publication, 4 August 2015; doi:10.1038/tpj.2015.55.
We cloned and expressed a new organic anion transporting polypeptide (OATP), termed human OATP2, (OATP-C, LST-1; symbol SLC21A6), involved in the uptake of various lipophilic anions into human liver. ...The cDNA encoding OATP2 comprised 2073 base pairs, corresponding to a protein of 691 amino acids, which were 44% identical to the known human OATP. An antibody directed against the carboxy terminus localized OATP2 to the basolateral membrane of human hepatocytes. Northern blot analysis indicated a strong expression of OATP2 only in human liver. Transport mediated by recombinant OATP2 and its localization were studied in stably transfected Madin-Darby canine kidney strain II (MDCKII) and HEK293 cells. Confocal microscopy localized recombinant OATP2 protein to the lateral membrane of MDCKII cells. Substrates included 17beta-glucuronosyl estradiol, monoglucuronosyl bilirubin, dehydroepiandrosterone sulfate, and cholyltaurine. 17beta-Glucuronosyl estradiol was a preferred substrate, with a Michaelis-Menten constant value of 8.2 microM; its uptake was Na(+) independent and was inhibited by sulfobromophthalein, with a inhibition constant value of 44 nM. Our results indicate that OATP2 is important for the uptake of organic anions, including bilirubin conjugates and sulfobromophthalein, in human liver.
The membrane proteins mediating the ATP-dependent transport of lipophilic substances conjugated to glutathione, glucuronate, or sulfate have been identified as members of the multidrug resistance ...protein (MRP) family. Several isoforms of these conjugate export pumps with different kinetic properties and domain-specific localization in polarized human cells have been cloned and characterized. Orthologs of the human MRP isoforms have been detected in many different organisms. Studies in mutant rats lacking the apical isoform MRP2 (symbol ABCC2) indicate that anionic conjugates of endogenous and exogenous substances cannot exit from cells at a sufficient rate unless an export pump of the MRP family is present in the plasma membrane. Several mutations in the human
MRP2 gene have been identified which lead to the absence of the MRP2 protein from the hepatocyte canalicular membrane and to the conjugated hyperbilirubinemia of Dubin–Johnson syndrome. Overexpression of recombinant MRP2 confers resistance to multiple chemotherapeutic agents. Because of its function in the terminal excretion of cytotoxic and carcinogenic substances, MRP2 as well as other members of the MRP family, play an important role in detoxification and chemoprevention.
Based on sequence homology to the human organic anion transporting polypeptide 2 (OATP2; SLC21A6), we cloned a new member of the SLC21A superfamily of solute carriers, termed OATP8 (SLC21A8). The ...protein of 702 amino acids showed an amino acid identity of 80% with human OATP2. Based on Northern blotting, the expression of OATP8 was restricted to human liver. Cosmid clones containing the genes encoding human OATP1 (SLC21A3), OATP2 (SLC21A6), and OATP8 (SLC21A8) served to establish their genomic organization. All three genes contained 14 exons with 13 identical splice sites when transferred to the amino acid sequence. An antibody raised against the carboxyl terminus localized OATP8 to the basolateral membrane of human hepatocytes and the recombinant glycoprotein, expressed in MDCKII cells, to the lateral membrane. Transport properties of OATP8 were studied in stably transfected MDCKII and HEK293 cells. Organic anions transported by human OATP8 included sulfobromophthalein, with aKm of 3.3 μm, and 17β-glucuronosyl estradiol, with a Km of 5.4 μm. Several bile salts were not substrates. Thus, human OATP8 is a new uptake transporter in the basolateral hepatocyte membrane with an overlapping but distinct substrate specificity as compared with OATP2, which is localized to the same membrane domain.
Delivery of therapeutic agents to the brain and its neoplasms depends on the presence of membrane transport proteins in the blood-brain barrier and in the target cells. The cellular and subcellular ...localization of these membrane transporters determines the drug accessibility to the brain and its tumors. We therefore analyzed the expression and localization of six members of the multidrug resistance protein family of ATP-dependent efflux pumps (ABCC1-ABCC6, formerly MRP1-MRP6) and of six organic anion uptake transporters (OATP1A2, OATP1B1, OATP1B3, OATP1C1, OATP2B1, and OATP4A1) in 61 human glioma specimens of different histologic subtypes. Real-time PCRs indicated expressions of ABCC1, ABCC3, ABCC4, and ABCC5. In addition, we detected expressions of the OATP uptake transporter genes SLCO1A2, SLCO1C1, SLCO2B1, and SLCO4A1. At the protein level, however, only OATP1A2 and OATP2B1 were detectable by immunofluorescence microscopy in the luminal membrane of endothelial cells forming the blood-brain barrier and the blood-tumor barrier, but not in the glioma cells. ABCC4 and ABCC5 proteins were the major ABCC subfamily members in gliomas, localized both at the luminal side of the endothelial cells and in the glioma cells of astrocytic tumors and in the astrocytic portions of oligoastrocytomas. These results indicate that expression of ABCC4 and ABCC5 is associated with an astrocytic phenotype, in accordance with their expression in astrocytes and with the higher chemoresistance of astrocytic tumors as compared with oligodendrogliomas. Our data provide a basis for the assessment of the role of uptake transporters and efflux pumps in the accessibility of human gliomas for chemotherapeutic agents.
Membrane transporters play an essential role in the transport of endogenous and exogenous compounds, and consequently they mediate the uptake, distribution, and excretion of many drugs. The clinical ...relevance of transporters in drug disposition and their effect in adults have been shown in drug–drug interaction and pharmacogenomic studies. Little is known, however, about the ontogeny of human membrane transporters and their roles in pediatric pharmacotherapy. As they are involved in the transport of endogenous substrates, growth and development may be important determinants of their expression and activity. This review presents an overview of our current knowledge on human membrane transporters in pediatric drug disposition and effect. Existing pharmacokinetic and pharmacogenetic data on membrane substrate drugs frequently used in children are presented and related, where possible, to existing ex vivo data, providing a basis for developmental patterns for individual human membrane transporters. As data for individual transporters are currently still scarce, there is a striking information gap regarding the role of human membrane transporters in drug therapy in children.
Abstract
JOREK is a massively parallel fully implicit non-linear extended magneto-hydrodynamic (MHD) code for realistic tokamak X-point plasmas. It has become a widely used versatile simulation code ...for studying large-scale plasma instabilities and their control and is continuously developed in an international community with strong involvements in the European fusion research programme and ITER organization. This article gives a comprehensive overview of the physics models implemented, numerical methods applied for solving the equations and physics studies performed with the code. A dedicated section highlights some of the verification work done for the code. A hierarchy of different physics models is available including a free boundary and resistive wall extension and hybrid kinetic-fluid models. The code allows for flux-surface aligned iso-parametric finite element grids in single and double X-point plasmas which can be extended to the true physical walls and uses a robust fully implicit time stepping. Particular focus is laid on plasma edge and scrape-off layer (SOL) physics as well as disruption related phenomena. Among the key results obtained with JOREK regarding plasma edge and SOL, are deep insights into the dynamics of edge localized modes (ELMs), ELM cycles, and ELM control by resonant magnetic perturbations, pellet injection, as well as by vertical magnetic kicks. Also ELM free regimes, detachment physics, the generation and transport of impurities during an ELM, and electrostatic turbulence in the pedestal region are investigated. Regarding disruptions, the focus is on the dynamics of the thermal quench (TQ) and current quench triggered by massive gas injection and shattered pellet injection, runaway electron (RE) dynamics as well as the RE interaction with MHD modes, and vertical displacement events. Also the seeding and suppression of tearing modes (TMs), the dynamics of naturally occurring TQs triggered by locked modes, and radiative collapses are being studied.
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