المؤلفون: A, Van Aubel R, B, Koenderink J, G, Peters J, H, Van Os C, G, Russel F

المصدر: Molecular Pharmacology; October 1999, Vol. 56 Issue: 4 p714-9, 6p

مستخلص: The present study examined how the multidrug resistance protein (MRP) 2, which is an ATP-dependent anionic conjugate transporter, also mediates transport of the chemotherapeutic cationic drug vinblastine (VBL). We show that ATP-dependent [(3)H]VBL (0.2 microM) uptake into membrane vesicles from Sf9 cells infected with a baculovirus encoding rabbit Mrp2 (Sf9-Mrp2) was similar to vesicles from mock-infected Sf9 cells (Sf9-mock) but could be stimulated by reduced glutathione (GSH) with a half-maximum stimulation of 1.9 +/- 0.1 mM. At 5 mM GSH, initial ATP-dependent [(3)H]VBL uptake rates were saturable with an apparent K(m) of 1.5 +/- 0.3 microM. The inhibitory effect of VBL on Mrp2-mediated ATP-dependent transport of the anionic conjugate [(3)H]leukotriene C(4) was potentiated by increasing GSH concentrations. Membrane vesicles from Sf9-Mrp2 cells exhibited a approximately 7-fold increase in initial GSH uptake rates compared with membrane vesicles from Sf9-mock cells. Uptake of [(3)H]GSH was osmotically sensitive, independent of ATP, and was trans-inhibited by GSH. The anionic conjugates estradiol-17beta-D-glucuronide and leukotriene C(4) cis-inhibited [(3)H]GSH uptake but only in the presence of ATP. Whereas ATP-dependent [(3)H]VBL uptake was stimulated by GSH, VBL did not affect [(3)H]GSH uptake. Our results show that GSH is required for Mrp2-mediated ATP-dependent VBL transport and that Mrp2 transports GSH independent of VBL.

المؤلفون: A, van Aubel R, A, van Kuijck M, B, Koenderink J, M, Deen P, H, van Os C, G, Russel F

المصدر: Molecular Pharmacology; June 1998, Vol. 53 Issue: 6 p1062-7, 6p

مستخلص: The multidrug resistance-associated protein Mrp2 is expressed in liver, kidney, and small intestine and mediates ATP-dependent transport of conjugated organic anions across the apical membrane of epithelial cells. We recently cloned a rabbit cDNA encoding a protein that on basis of highest amino acid homology and tissue distribution was considered to be the rabbit homolog of rat Mrp2. To investigate whether rabbit Mrp2 mediates ATP-dependent transport similar to rat Mrp2, we expressed rabbit Mrp2 in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. Mrp2 was expressed as an underglycosylated protein in Sf9 cells and to a higher level compared with rabbit liver and renal proximal tubules. Both 17beta-estradiol-17-beta-D-glucuronide ([3H]E217betaG, 50 nM) and [3H]leukotriene C4 (3 nM) were taken up by Sf9-Mrp2 membrane vesicles in an ATP-dependent fashion. Uptake of [3H]E217betaG was dependent on the osmolarity of the medium and saturable for ATP (Km = 623 microM). Leukotriene C4, MK571, phenolphthalein glucuronide, and fluorescein-methotrexate were good inhibitors of [3H]E217betaG transport. The inhibitory potency of cyclosporin A and methotrexate was moderate, whereas fluorescein, alpha-naphthyl-beta-D-glucuronide, and p-nitrophenyl-beta-D-glucuronide did not inhibit transport. In conclusion, we show direct ATP-dependent transport by recombinant rabbit Mrp2 and provide new data on Mrp2 inhibitor specificity.

المؤلفون: Van Aubel RA; Department of Pharmacology and Toxicology, University of Nijmegen, The Netherlands., Koenderink JB, Peters JG, Van Os CH, Russel FG

المصدر: Molecular pharmacology [Mol Pharmacol] 1999 Oct; Vol. 56 (4), pp. 714-9.

نوع المنشور: Journal Article; Research Support, Non-U.S. Gov't

بيانات الدورية: Publisher: American Society for Pharmacology and Experimental Therapeutics Country of Publication: United States NLM ID: 0035623 Publication Model: Print Cited Medium: Print ISSN: 0026-895X (Print) Linking ISSN: 0026895X NLM ISO Abbreviation: Mol Pharmacol Subsets: MEDLINE

مواضيع طبية MeSH: Mitochondrial Proteins* , Saccharomyces cerevisiae Proteins*, Antineoplastic Agents, Phytogenic/*metabolism , Glutathione/*metabolism , Ribosomal Proteins/*metabolism , Vinblastine/*metabolism, Animals ; Biological Transport ; Cell Line ; Insecta ; Leukotriene C4/pharmacokinetics ; Rabbits ; Ribosomal Proteins/genetics ; Transfection ; Tritium

مستخلص: The present study examined how the multidrug resistance protein (MRP) 2, which is an ATP-dependent anionic conjugate transporter, also mediates transport of the chemotherapeutic cationic drug vinblastine (VBL). We show that ATP-dependent [(3)H]VBL (0.2 microM) uptake into membrane vesicles from Sf9 cells infected with a baculovirus encoding rabbit Mrp2 (Sf9-Mrp2) was similar to vesicles from mock-infected Sf9 cells (Sf9-mock) but could be stimulated by reduced glutathione (GSH) with a half-maximum stimulation of 1.9 +/- 0.1 mM. At 5 mM GSH, initial ATP-dependent [(3)H]VBL uptake rates were saturable with an apparent K(m) of 1.5 +/- 0.3 microM. The inhibitory effect of VBL on Mrp2-mediated ATP-dependent transport of the anionic conjugate [(3)H]leukotriene C(4) was potentiated by increasing GSH concentrations. Membrane vesicles from Sf9-Mrp2 cells exhibited a approximately 7-fold increase in initial GSH uptake rates compared with membrane vesicles from Sf9-mock cells. Uptake of [(3)H]GSH was osmotically sensitive, independent of ATP, and was trans-inhibited by GSH. The anionic conjugates estradiol-17beta-D-glucuronide and leukotriene C(4) cis-inhibited [(3)H]GSH uptake but only in the presence of ATP. Whereas ATP-dependent [(3)H]VBL uptake was stimulated by GSH, VBL did not affect [(3)H]GSH uptake. Our results show that GSH is required for Mrp2-mediated ATP-dependent VBL transport and that Mrp2 transports GSH independent of VBL.

المؤلفون: van Aubel RA; Department of Pharmacology, University of Nijmegen, 6500 HB Nijmegen, The Netherlands., van Kuijck MA, Koenderink JB, Deen PM, van Os CH, Russel FG

المصدر: Molecular pharmacology [Mol Pharmacol] 1998 Jun; Vol. 53 (6), pp. 1062-7.

نوع المنشور: Journal Article; Research Support, Non-U.S. Gov't

بيانات الدورية: Publisher: American Society for Pharmacology and Experimental Therapeutics Country of Publication: United States NLM ID: 0035623 Publication Model: Print Cited Medium: Print ISSN: 0026-895X (Print) Linking ISSN: 0026895X NLM ISO Abbreviation: Mol Pharmacol Subsets: MEDLINE

مواضيع طبية MeSH: Drug Resistance, Multiple*, Adenosine Triphosphate/*pharmacology , Carrier Proteins/*physiology, Animals ; Anion Transport Proteins ; Biological Transport ; Carrier Proteins/antagonists & inhibitors ; Carrier Proteins/biosynthesis ; Rabbits ; Rats ; Recombinant Proteins/biosynthesis ; Spodoptera

مستخلص: The multidrug resistance-associated protein Mrp2 is expressed in liver, kidney, and small intestine and mediates ATP-dependent transport of conjugated organic anions across the apical membrane of epithelial cells. We recently cloned a rabbit cDNA encoding a protein that on basis of highest amino acid homology and tissue distribution was considered to be the rabbit homolog of rat Mrp2. To investigate whether rabbit Mrp2 mediates ATP-dependent transport similar to rat Mrp2, we expressed rabbit Mrp2 in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. Mrp2 was expressed as an underglycosylated protein in Sf9 cells and to a higher level compared with rabbit liver and renal proximal tubules. Both 17beta-estradiol-17-beta-D-glucuronide ([3H]E217betaG, 50 nM) and [3H]leukotriene C4 (3 nM) were taken up by Sf9-Mrp2 membrane vesicles in an ATP-dependent fashion. Uptake of [3H]E217betaG was dependent on the osmolarity of the medium and saturable for ATP (Km = 623 microM). Leukotriene C4, MK571, phenolphthalein glucuronide, and fluorescein-methotrexate were good inhibitors of [3H]E217betaG transport. The inhibitory potency of cyclosporin A and methotrexate was moderate, whereas fluorescein, alpha-naphthyl-beta-D-glucuronide, and p-nitrophenyl-beta-D-glucuronide did not inhibit transport. In conclusion, we show direct ATP-dependent transport by recombinant rabbit Mrp2 and provide new data on Mrp2 inhibitor specificity.