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Allan W. Wolkoff - One of the best experts on this subject based on the ideXlab platform.

  • Rat Organic Anion Transport Protein 1A1 Interacts Directly With Organic Anion Transport Protein 1A4 Facilitating Its Maturation and Trafficking to the Hepatocyte Plasma Membrane
    Hepatology (Baltimore Md.), 2019
    Co-Authors: Pijun Wang, John W. Murray, Wen Jun Wang, Jo Choi-nurvitadhi, Yaniuska Lescaille, Allan W. Wolkoff
    Abstract:

    Organic Anion Transport Proteins (OATPs) on the basolateral surface of hepatocytes mediate uptake of a number of drugs and endogenous compounds. Previous studies showed that rat OATP1A1 (rOATP1A1) has a postsynaptic density Protein, drosophila disc large tumor suppressor, zonula occludens-1 Protein (PDZ) consensus binding motif at its C-terminus and binds to PDZ domain containing 1 (PDZK1), which is required for its cell-surface localization. PDZK1 associates with rOATP1A1-containing endocytic vesicles within cells, mediating recruitment of motor Proteins required for microtubule-based trafficking to the plasma membrane. rOATP1A4 also traffics to the plasma membrane, although it lacks a PDZ binding consensus sequence. The current study was designed to test the hypothesis that trafficking of rOATP1A4 to the plasma membrane requires its direct interaction with rOATP1A1 resulting in a complex that traffics through the cell in common subcellular vesicles in which the cytosolic tail of rOATP1A1 is bound to PDZK1. We found that 74% of rOATP1A4-containing rat liver endocytic vesicles (n = 12,044) also contained rOATP1A1. Studies in transfected HEK293 cells showed surface localization of rOATP1A1 only when coexpressed with PDZK1 whereas rOATP1A4 required coexpression with rOATP1A1 and PDZK1. Studies in stably transfected HeLa cells that constitutively expressed PDZK1 showed that coexpression of rOATP1A4 with rOATP1A1 resulted in more rapid appearance of rOATP1A4 on the plasma membrane and faster maturation to its fully glycosylated form. Similar results were observed on immunofluorescence analysis of single cells. Immunoprecipitation of rat liver or transfected HeLa cell lysates with rOATP1A1 antibody specifically co-immunoprecipitated rOATP1A4 as determined by western blotting. Conclusion: These studies indicate that optimal rOATP1A4 trafficking to the cell surface is dependent upon coexpression and interaction with rOATP1A1. As rOATP1A1 binds to the chaperone Protein, PDZK1, rOATP1A4 functionally hitchhikes through the cell with this complex.

  • PDZK1 binding and serine phosphorylation regulate subcellular trafficking of organic Anion Transport Protein 1a1
    American Journal of Physiology-gastrointestinal and Liver Physiology, 2010
    Co-Authors: Jo H. Choi, John W. Murray, Allan W. Wolkoff
    Abstract:

    Although perturbation of organic Anion Transport Protein (oatp) cell surface expression can result in drug toxicity, little is known regarding mechanisms regulating its subcellular distribution. Many members of the oatp family, including oatp1a1, have a COOH-terminal PDZ consensus binding motif that interacts with PDZK1, while serines upstream of this site (S634 and S635) can be phosphorylated. Using oatp1a1 as a prototypical member of the oatp family, we prepared plasmids in which these serines were mutated to glutamic acid [E634E635 (oatp1a1EE), phosphomimetic] or alanine [A634A635 (oatp1a1AA), nonphosphorylatable]. Distribution of oatp1a1AA and oatp1a1EE was largely intracellular in transfected human embryonic kidney (HEK) 293T cells. Cotransfection with a plasmid encoding PDZK1 revealed that oatp1a1AA was now expressed largely on the cell surface, while oatp1a1EE remained intracellular. To quantify these changes, studies were performed in HuH7 cells stably transfected with these oatp1a1 plasmids. These cells endogenously express PDZK1. Surface biotinylation at 4°C followed by shift to 37°C showed that oatp1a1EE internalizes quickly compared with oatp1a1AA. To examine a physiological role for phosphorylation in oatp1a1 subcellular distribution, studies were performed in rat hepatocytes exposed to extracellular ATP, a condition that stimulates serine phosphorylation of oatp1a1 via activity of a purinergic receptor. Internalization of oatp1a1 under these conditions was rapid. Thus, although PDZK1 binding is required for optimal cell surface expression of oatp1a1, phosphorylation provides a mechanism for fast regulation of the distribution of oatp1a1 between the cell surface and intracellular vesicular pools. Identification of the Proteins and motor molecules that mediate these trafficking events represents an important area for future study.

  • Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane Protein with three N-linked carbohydrate chains
    American journal of physiology. Gastrointestinal and liver physiology, 2008
    Co-Authors: Pijun Wang, John W. Murray, Soichiro Hata, Yansen Xiao, Allan W. Wolkoff
    Abstract:

    Organic Anion Transport Protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous Transport Proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocy...

  • The Human Organic Anion Transport Protein SLC21A6 Is Not Sufficient for Bilirubin Transport
    The Journal of biological chemistry, 2003
    Co-Authors: Pijun Wang, Richard B. Kim, J. Roy Chowdhury, Allan W. Wolkoff
    Abstract:

    Abstract A recent study (Cui, Y., Konig, J., Leier, I., Buchholz, U., and Keppler, D. (2001) J. Biol. Chem. 276, 9626–9630) suggests that human OATP2 (SLC21A6), also known as OATP-C and LST1, mediates hepatic bilirubin Transport. Because of methodologic concerns, this study was designed to examine this issue using a bilirubin Transport assay that was validated in overnight cultured rat hepatocytes. These studies showed that cultured rat hepatocytes Transported bilirubin with kinetics virtually identical to the Transport of sulfobromophthalein. This assay was then used to quantify bilirubin Transport by HeLa cells that had been stably transfected with OATP2 under regulation of a metallothionein promoter. Immunoblot analysis revealed abundant expression of OATP2 after incubation of cells for 48 h in zinc, whereas uninduced cells had no expression of this Protein. In OATP2-expressing (zinc-induced) HeLa cells at 37 °C, the uptake of [35S]sulfobromophthalein was substantial (51.6 ± 16.5 pmol/15 min/mg Protein, n = 5) with little cell-associated ligand in non-expressing (uninduced) cells (0.54 ± 0.16 pmol/15 min/mg Protein, n = 5, p 0.2) in cell-associated [3H]bilirubin in induced (OATP2-expressing) as compared with uninduced cells (11.25 ± 3.02 pmol/15 min/mg Protein versus 9.15 ± 1.68 pmol/min/mg Protein, respectively, n = 5) We obtained similar results in OATP2-transfected HEK293 cells that were used in the original report. The existence of a bilirubin Transporter has been an important field of investigation for many years. Although the current study indicates that a role for OATP2 in hepatocyte bilirubin Transport is unlikely, it provides new and sensitive tools that can be adapted to examine the function of putative bilirubin Transporters in the future.

  • Dichotomous development of the organic Anion Transport Protein in liver and choroid plexus
    The American journal of physiology, 1998
    Co-Authors: Ruth Hogue Angeletti, Ari J. Bergwerk, Phyllis M. Novikoff, Allan W. Wolkoff
    Abstract:

    Both adult liver and choroid plexus express the organic Anion Transport Protein (oatp1) and Transport [35S]bromosulfophthalein (BSP). Studies of the developing rat liver reveal that oatp1 mRNA and Protein do not begin to be expressed until 15 days postnatal and are at adult levels by 30 days. Uptake of [35S]BSP follows the same time course. In contrast, neonatal rat choroid plexus expresses oatp1 mRNA and Protein. When quantified on a weight basis, the uptake of [35S]BSP in choroid plexus is lower in the adult than at earlier stages of development. Although fluorescence confocal microscopy of adult rat choroid plexus shows that oatp is localized to the apical surface, facing the cerebrospinal fluid, this method reveals an intracellular localization of oatp1 in the neonate. Approximately 12 wk are required for the appearance of the adult pattern of distribution. Changes in the localization and activity of oatp1 during development could play an important role in the pathobiology of maturation of the liver and the central nervous system.

Philip S. Low - One of the best experts on this subject based on the ideXlab platform.

  • Role of band 3 in regulating metabolic flux of red blood cells
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Ian A. Lewis, M. Estela Campanella, John L Markley, Philip S. Low
    Abstract:

    Deoxygenation elevates glycolytic flux and lowers pentose phosphate pathway (PPP) activity in mammalian erythrocytes. The membrane Anion Transport Protein (band 3 or AE1) is thought to facilitate this process by binding glycolytic enzymes (GEs) and inhibiting their activity in an oxygen-dependent manner. However, this regulatory mechanism has not been demonstrated under physiological conditions. In this study, we introduce a 1H-13C NMR technique for measuring metabolic fluxes in intact cells. The role of band 3 in mediating the oxygenated/deoxygenated metabolic transition was examined by treating cells with pervanadate, a reagent that prevents the GE–band 3 complex from forming. We report that pervanadate suppresses oxygen-dependent changes in glycolytic and PPP fluxes. Moreover, these metabolic alterations were not attributable to modulation of bisphosphoglycerate mutase, direct inhibition of GEs by pervanadate, or oxidation, which are the major side effects of pervanadate treatment. These data provide direct evidence supporting the role of band 3 in mediating oxygen-regulated metabolic transitions.

  • Role of band 3 in regulating metabolic flux of red blood cells
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Ian A. Lewis, M. Estela Campanella, John L Markley, Philip S. Low
    Abstract:

    Deoxygenation elevates glycolytic flux and lowers pentose phosphate pathway (PPP) activity in mammalian erythrocytes. The membrane Anion Transport Protein (band 3 or AE1) is thought to facilitate this process by binding glycolytic enzymes (GEs) and inhibiting their activity in an oxygen-dependent manner. However, this regulatory mechanism has not been demonstrated under physiological conditions. In this study, we introduce a 1H-13C NMR technique for measuring metabolic fluxes in intact cells. The role of band 3 in mediating the oxygenated/deoxygenated metabolic transition was examined by treating cells with pervanadate, a reagent that prevents the GE–band 3 complex from forming. We report that pervanadate suppresses oxygen-dependent changes in glycolytic and PPP fluxes. Moreover, these metabolic alterations were not attributable to modulation of bisphosphoglycerate mutase, direct inhibition of GEs by pervanadate, or oxidation, which are the major side effects of pervanadate treatment. These data provide direct evidence supporting the role of band 3 in mediating oxygen-regulated metabolic transitions.

Peter J. Meier - One of the best experts on this subject based on the ideXlab platform.

  • localization of organic Anion Transport Protein 2 in the apical region of rat retinal pigment epithelium
    Investigative Ophthalmology & Visual Science, 2002
    Co-Authors: Andreas Wenzel, Peter J. Meier, Christian Grimm, Stephan R Vavricka, Dietmar Benke, Charlotte E Reme
    Abstract:

    PURPOSE: The organic Anion Transporting Protein (Oatp)-2 has been cloned from brain and retina. It mediates Transport of many endogenous and exogenous amphiphilic compounds across the plasma membrane in a sodium-independent manner. In the brain it resides at the luminal and abluminal membrane of the capillary endothelium and at the basolateral membrane of the choroid plexus epithelium. In the liver, it is expressed at the basolateral membrane of hepatocytes. Its exact localization and function in the retina are unknown. Therefore, the purposes of the present study were to determine the cellular and subcellular localization and the potential functional aspects of Oatp2 in the retina. METHODS: Oatp2 was detected in rat retinal tissue by immunofluorescence confocal microscopy and by Western blot analysis, with a specific antibody. A Xenopus laevis oocyte expression system was used for functional Transport studies. RESULTS: Oatp2 immunoreactivity was abundantly present at the apical microvilli of the rat retinal pigment epithelium and to a lesser degree in small retinal vessels. In the oocyte expression system, N-retinyl-N-retinylidene ethanolamine (A2E), an unusual cationic, amphiphilic retinoid, exhibited competitive Cis inhibition of Oatp2-mediated digoxin Transport with an estimated K(i) of approximately 37 microM. CONCLUSIONS: In rat retina, Oatp2 is localized at the interface between the pigment epithelium and the photoreceptor outer segments. A2E is a competitive inhibitor of Oatp2-mediated substrate Transport, suggesting that A2E or A2E-like compounds and some retinoids may be substrates for Oatp2 Transport.

  • The choroid plexus epithelium is the site of the organic Anion Transport Protein in the brain
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Ruth Hogue Angeletti, Phyllis M. Novikoff, Peter J. Meier, Shailaja Rao Juvvadi, Jean-marc Fritschy, Allan W. Wolkoff
    Abstract:

    The mRNA for organic Anion Transport Protein (oatp) was previously shown to be present in abundance in liver and kidney, and in small amounts in brain. Data obtained from experiments with reverse transcriptase–PCR techniques and in situ hybridization analysis showed that the oatp mRNA is present within the brain, localized to the choroid plexus. A sequence-specific antibody to the oatp polypeptide demonstrated the presence of the expected polypeptide with a molecular weight of 80,000 plus an immunoreactive species with a higher molecular weight in preparations of choroid plexus membranes. Examination of the choroid plexus by fluorescence confocal microscopy revealed that immunoreactive oatp polypeptide is localized to the apical surface of the choroid plexus epithelial cells, which contacts the cerebrospinal fluid. This localization of oatp is consistent with previous experiments showing vectorial Transport of organic Anions between the choroid plexus and the cerebrospinal fluid.

  • Immunologic distribution of an organic Anion Transport Protein in rat liver and kidney
    American Journal of Physiology-Gastrointestinal and Liver Physiology, 1996
    Co-Authors: Ari J. Bergwerk, Phyllis M. Novikoff, Xiaoying Shi, Shuang Bai, Allison C. Ford, Robert D. Burk, Emmanuel Jacquemin, Naoaki Kanai, Bruno Stieger, Peter J. Meier
    Abstract:

    A Na(+)-independent organic Anion Transport Protein was recently cloned from rat liver using a Xenopus laevis oocyte expression system [E. Jacquemin, B. Hagenbuch, B. Stieger, A.W. Wolkoff, and P.J. Meier, Proc. Natl. Acad. Sci. USA 91: 133-137, 1994]. Although expression of this Protein is sufficient for cells to Transport the organic Anion bromosulfophthalein, little is known about its cell biology or biochemical characteristics. Northern blot analysis performed under high-stringency conditions revealed hybridization with RNA only from liver and kidney; transcripts appeared the same in these two organs. Within kidney, hybridization was greatest when RNA extracted from the outer medulla was used. Immunoblot analysis revealed that in liver, the Transporter was enriched in 0.1 M Na2CO3-extracted membranes and sinusoidal plasma membrane preparations, consistent with its being an integral membrane Protein. This 80-kDa Protein migrated as a 65-kDa Protein after treatment with N-glycanase. Immunomorphological examination of liver revealed basolateral plasma membrane localization. In 0.1 M Na2CO3-extracted membranes of kidney, the Transporter migrated as an 83-kDa Protein on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On reduction, it resolved into peptides of 33 and 37 kDa. SDS-PAGE migration of the liver Protein was unaffected by reduction. Immunomorphological examination of kidney revealed apical plasma membrane localization in the S3 segment of the proximal tubule of the outer medulla. Differential processing and trafficking of this Transporter in liver and kidney may have important functional and regulatory consequences.

  • Stable inducible expression of a functional rat liver organic Anion Transport Protein in HeLa cells.
    The Journal of biological chemistry, 1995
    Co-Authors: Xiaoying Shi, Shuang Bai, Allison C. Ford, Robert D. Burk, Emmanuel Jacquemin, Bruno Hagenbuch, Peter J. Meier, Allan W. Wolkoff
    Abstract:

    Recently we expression cloned a rat liver organic Anion Transport Protein in Xenopus laevis oocytes (Jacquemin, E. Hagenbuch, B, Stieger, B., Wolkoff, A.W., and Meier, P.J.,(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 133-137). In the present study, we have stably transfected the cDNA encoding this Protein into HeLa cells by using a vector containing a zinc-inducible promoter. The parent cells have virtually no baseline Transport of [35S]sulfobromophthalein, whereas the induced transfected cells express a novel 74-kDa Protein and avidly Transport this ligand. Transport by these cells is saturable (Km = 3.3 microM, Vmax = 257 pmol/min/mg Protein), bidirectional, and highly temperature-dependent. In the presence of albumin, uptake of [35S]sulfobromophthalein requires the presence of extracellular Cl, whereas in the absence of albumin, this C1- dependence is not seen. These studies indicate that cellular uptake of sulfobromophthalein does not result from direct interaction with the plasma membrane lipid bilayer but rather requires the presence of a specific plasma membrane Transporter.

Ian A. Lewis - One of the best experts on this subject based on the ideXlab platform.

  • Role of band 3 in regulating metabolic flux of red blood cells
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Ian A. Lewis, M. Estela Campanella, John L Markley, Philip S. Low
    Abstract:

    Deoxygenation elevates glycolytic flux and lowers pentose phosphate pathway (PPP) activity in mammalian erythrocytes. The membrane Anion Transport Protein (band 3 or AE1) is thought to facilitate this process by binding glycolytic enzymes (GEs) and inhibiting their activity in an oxygen-dependent manner. However, this regulatory mechanism has not been demonstrated under physiological conditions. In this study, we introduce a 1H-13C NMR technique for measuring metabolic fluxes in intact cells. The role of band 3 in mediating the oxygenated/deoxygenated metabolic transition was examined by treating cells with pervanadate, a reagent that prevents the GE–band 3 complex from forming. We report that pervanadate suppresses oxygen-dependent changes in glycolytic and PPP fluxes. Moreover, these metabolic alterations were not attributable to modulation of bisphosphoglycerate mutase, direct inhibition of GEs by pervanadate, or oxidation, which are the major side effects of pervanadate treatment. These data provide direct evidence supporting the role of band 3 in mediating oxygen-regulated metabolic transitions.

  • Role of band 3 in regulating metabolic flux of red blood cells
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Ian A. Lewis, M. Estela Campanella, John L Markley, Philip S. Low
    Abstract:

    Deoxygenation elevates glycolytic flux and lowers pentose phosphate pathway (PPP) activity in mammalian erythrocytes. The membrane Anion Transport Protein (band 3 or AE1) is thought to facilitate this process by binding glycolytic enzymes (GEs) and inhibiting their activity in an oxygen-dependent manner. However, this regulatory mechanism has not been demonstrated under physiological conditions. In this study, we introduce a 1H-13C NMR technique for measuring metabolic fluxes in intact cells. The role of band 3 in mediating the oxygenated/deoxygenated metabolic transition was examined by treating cells with pervanadate, a reagent that prevents the GE–band 3 complex from forming. We report that pervanadate suppresses oxygen-dependent changes in glycolytic and PPP fluxes. Moreover, these metabolic alterations were not attributable to modulation of bisphosphoglycerate mutase, direct inhibition of GEs by pervanadate, or oxidation, which are the major side effects of pervanadate treatment. These data provide direct evidence supporting the role of band 3 in mediating oxygen-regulated metabolic transitions.

David J. Cutler - One of the best experts on this subject based on the ideXlab platform.