Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export.
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The multidrug resistance proteins MRP1 and MRP2 are members of the same subfamily of ATP-binding cassette transporters. Besides organic molecules conjugated to negatively charged ligands, these proteins also transport cytotoxic drugs for which no negatively charged conjugates are known to exist. In polarized MDCKII cells, MRP1 routes to the lateral plasma membrane, and MRP2 to the apical plasma membrane. In these cells MRP1 transports daunorubicin, and MRP2 vinblastine; both transporters export reduced glutathione (GSH) into the medium. We demonstrate that glutathione transport in MDCKII-MRP1 cells is inhibited by the inhibitors of organic anion transporters sulfinpyrazone, indomethacin, probenecid and benzbromarone. In MDCKII-MRP2 cells, GSH export is stimulated by low concentrations of sulfinpyrazone or indomethacin, whereas export is inhibited down to control levels at high concentrations. We find that unmodified sulfinpyrazone is a substrate for MRP2, also at concentrations where GSH export is inhibited. We also show that GSH export in MDCKII-MRP2 cells increases in the presence of vinblastine, and that the stoichiometry between drug and GSH exported is between two and three. Our data indicate that transport of sulfinpyrazone and vinblastine is associated with GSH export. However, at high sulfinpyrazone concentrations this compound is transported without GSH. Models of MRP action are discussed that could explain these results. (+info)
Experimental arterial thromboembolism in baboons. Mechanism, quantitation, and pharmacologic prevention.
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A quantitative primate model of arterial thromboembolism has been characterized with respect to mechanism and usefulness in evaluating modifying variables. The model involved the kinetic measurements of (51)Cr-platelets and (125)I-fibrinogen consumption by femoral arteriovenous cannulae in chaired baboons. Cannula platelet consumption correlated directly with exposed cannular area for irradiated Silastic and polyurethane (correlation coefficients of 0.940 and 0.901, respectively; P < 0.001) and remained steady state for months. Nonirradiated Silastic was only minimally reactive with platelets. Despite increased rates of platelet consumption circulating fibrinogen was not measurably destroyed by any of the cannulae tested. Cannula platelet consumption was independent of cannula flow rate, platelet count, heparin anti-coagulation, and ancrod defibrinogenation.(111)In-platelet imaging of irradiated Silastic cannulae demonstrated luminal accumulation and subsequent embolization of irregular platelet masses. When irradiated Silastic cannulae were inserted as extension segments in the renal arteries of four animals the glomerular vessels became progressively occluded with nonfibrin-containing platelet thromboemboli. Nonirradiated Silastic cannulae in control arteries produced no significant vascular occlusion. Because the survival of platelets from animals with consumptive cannulae was not shortened in normal recipient animals we concluded that platelets were either irreversibly removed through thromboembolic consumption or unaffected in their viability. Oral administration of dipyridamole and sulfinpyrazone decreased cannula platelet consumption in a dose-dependent manner with complete interruption at 20 and 250 mumol/kg body wt per d (in three divided doses), respectively, whereas oral acetylsalicylic acid (10-330 mumol/kg per d) had no measurable effect on cannula platelet consumption. We conclude that this primate model simulates arterial thrombotic processes in man and that this model is suitable for the in vivo evaluation of biomaterials and of drugs that modify platelet behavior. (+info)
Metabolism of sulfinpyrazone sulfide and sulfinpyrazone by human liver microsomes and cDNA-expressed cytochrome P450s.
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Human liver microsomes catalyze the oxidation of sulfinpyrazone sulfide (SPZS) to a variable mixture of sulfinpyrazone (SPZ) enantiomers and two minor phenolic metabolites. In one, the thiophenyl ring is hydroxylated, whereas in the second an N-phenyl ring is hydroxylated. SPZ is further oxidized to sulfinpyrazone sulfone (SPZO) and a minor polar metabolite that also has an N-phenyl ring hydroxylated. Determination of the metabolism of SPZ and SPZS under modified incubation conditions of prior heat treatment, higher pH, and the presence of detergent indicated that the formation of SPZ was cytochrome P450 (P450)- but not flavin monooxygenase-dependent. Specific P450 inhibitors (sulfaphenazole, quinidine sulfate, coumarin, diethyldithiocarbamic acid, troleandomycin, and furafylline) and specific cDNA-expressed P450s were used to identify the major isoforms responsible for the oxidation of SPZS to SPZ and SPZ to SPZO. Both P450 2C9 and P450 3A4 were responsible for the oxidation of SPZS to SPZ, whereas P450 3A4 alone catalyzed the further oxidation of SPZ to SPZO. SPZS was found to be metabolized by P450 2C9 to SPZ with a high degree of enantiomeric selectivity (9:1) and a K(m) comparable with its previously determined K(i) for inhibition of the P450 2C9-dependent 7-hydroxylation of (S)-warfarin (WARF). In contrast, the P450 3A4-catalyzed oxidation of SPZS to SPZ proceeded with the same enantioselectivity but to a much lesser degree (58:42). These results provide evidence that the metabolism of both (S)-WARF and SPZS is mediated by a common enzyme, P450 2C9, which is central to understanding the WARF-SPZ interaction and SPZS-mediated drug interactions in general. (+info)
Resistance to mitoxantrone in multidrug-resistant MCF7 breast cancer cells: evaluation of mitoxantrone transport and the role of multidrug resistance protein family proteins.
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We examined the role of multidrug resistance protein (MRP) 1 (ABCC1) in the emergence of mitoxantrone (MX) cross-resistance in a MCF7 breast cancer cell line selected for resistance to etoposide. The resistant cell line, MCF7/VP, expresses high levels of MRP1, whereas the parental cell line, MCF7/WT, does not. MCF7/VP cells are 6-10-fold cross-resistant to MX when compared with MCF7/WT cells. Drug transport studies in intact MCF7/VP cells revealed that MX resistance is associated with reduced MX accumulation due to enhanced MX efflux. MX efflux is ATP dependent and inhibited by sulfinpyrazone and cyclosporin A. Inhibition of MX efflux with these agents sensitizes cells to MX cytotoxicity and partially reverses MX resistance in MCF7/VP cells. Whereas resistance is partially attributable to increased MX efflux in MRP1-expressing MCF7/VP cells, we found no evidence for glutathione or other conjugates of MX in these cells. Moreover, glutathione depletion with buthionine sulfoximine had no effect on MX transport or sensitivity in MCF7/VP cells. MRP1 substrates are generally amphiphilic anions such as glutathione conjugates or require the presence of physiological levels of glutathione for MRP1-mediated transport. Therefore we conclude that MRP1 overexpression is unlikely to be responsible for increased MX efflux and resistance in MCF7/VP cells. In considering the potential involvement of other MRP family isoforms, a 3-fold increase in the expression of MRP5 was observed in MCF7/VP cells. However, stable expression of a transduced MRP5 expression vector in MCF7/WT cells failed to confer MX resistance. Because other transporters known to be associated with MX resistance, including P-glycoprotein and BCRP/MXR (ABCG2), are not expressed in MCF7/VP cells, we conclude that increased MX efflux and resistance in MCF7/VP cells is attributable to a novel transport mechanism or that MX represents a novel class of cationic, glutathione-independent MRP1 substrates. (+info)
Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity.
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The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX. (+info)
Increased platelet aggregates in patients with transient ischemic attacks.
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In order to evaluate the pathogenetic importance of platelet aggregates in cerebrovascular disease, a platelet count ratio method was used to study 66 patients with transient ischemic attacks (TIAs). Thirty normal subjects and 22 patients without thromboembolic disorders were also included as controls. The mean platelet aggregate ratio of the TIA group was 0.75 +/- 0.03 SEM which was significantly lower than that of normal subjects (0.90 +/- 0.02) or patients controls (0.88 +/- 0.01) (P less than 0.01). Seventeen patients with TIA were then treated with aspirin (1,200 mg) and dipyridamole (200 mg) daily. The platelet aggregate ratios were normalized in 13 patients. Of four patients who did not respond to this regimen, one did respond to sulfinpyrazone. When sulfinpyrazone was discontinued, recurrence of symptoms was preceded by an increase in platelet aggregates. These findings suggest that platelet aggregates may play an important role in the pathogenesis of cerebrovascular insufficiency. The determination of platelet aggregates appears useful in selecting patients for antiplatelet therapy. (+info)
THE EFFECT OF SULFINPYRAZONE (ANTURAN) ON PLATELET ECONOMY AND BLOOD COAGULATION IN MAN.
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Sulfinpyrazone (Anturan) administered in therapeutic doses over a period of several weeks produced prolongation of platelet survival and reduced turnover but with little change in blood coagulation. The changes in platelet survival and turnover were associated with reduced platelet adhesiveness. It is therefore possible to affect platelet economy in man significantly, without producing corresponding effects on blood coagulation. (+info)
Contribution of multidrug resistance protein 2 (MRP2/ABCC2) to the renal excretion of p-aminohippurate (PAH) and identification of MRP4 (ABCC4) as a novel PAH transporter.
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p-Aminohippurate (PAH) is the classical substrate used in the characterization of organic anion transport in renal proximal tubular cells. Although basolateral transporters for PAH uptake from blood into the cell have been well characterized, there is still little knowledge on the apical urinary efflux transporters. The multidrug resistance protein 2 (MRP2/ABCC2) is localized to the apical membrane and mediates ATP-dependent PAH transport, but its contribution to urinary PAH excretion is not known. In this report, we show that renal excretion of PAH in isolated perfused kidneys from wild-type and Mrp2-deficient (TR(-)) rats is not significantly different. Uptake of [(14)C]PAH in membrane vesicles expressing two different MRP2 clones isolated from Sf9 and MDCKII cells exhibited a low affinity for PAH (Sf9, 5 +/- 2 mM; MDCKII, 2.1 +/- 0.6 mM). Human MRP4 (ABCC4), which has recently been localized to the apical membrane, expressed in Sf9 cells had a much higher affinity for PAH (K(m) = 160 +/- 50 microM). Various inhibitors of MRP2-mediated PAH transport also inhibited MRP4. Probenecid stimulated MRP2 at low concentrations but had no effect on MRP4; but at high probenecid concentrations, both MRP2 and MRP4 were inhibited. Sulfinpyrazone only stimulated MRP2, but inhibited MRP4. Real-time PCR and Western blot analysis showed that renal cortical expression of MRP4 is approximately fivefold higher as compared with MRP2. MRP4 is a novel PAH transporter that has higher affinity for PAH and is expressed more highly in kidney than MRP2, and may therefore be more important in renal PAH excretion. (+info)