verlukast and fluorexon

Accumulation and toxicity of cyanobacterial toxins, particularly microcystin-LR (MCLR) have been extensively studied in fish and aquatic invertebrates. However, MCLR excretion mechanisms, which could reduce this toxin's effects, have received little attention. The Patagonian silverside, Odontesthes hatcheri, is an omnivorous-planktivorous edible fish, which has been shown to digest cyanobacterial cells absorbing MCLR and eliminating the toxin within 48h without suffering significant toxic effects. We studied the effects of MCLR on glycoconjugate composition and the possible role of multidrug resistance associated proteins (Abcc) in MCLR export from the cells in O. hatcheri intestine. We treated O. hatcheri with 5μg MCLRg(-1) body mass administered with the food. Twenty four hours later, the intestines of treated and control fish were processed for lectin-histochemistry using concanavalin A (ConA), Triticum vulgaris agglutinin (WGA), and Dolichos biflorus agglutinin (DBA). MCLR affected the distribution of glycoconjugates by augmenting the proportion of ConA-positive at the expense of WGA-positive cells. We studied MCLR effects on the transport of the Abcc-like substrates 2,4-dinitrophenyl-S-glutathione (DNP-SG) and calcein in ex vivo intestine preparations (everted and no-everted sacs and strips). In treated preparations, CDNB together with MCLR (113μg MCLRg(-1) intestine, equivalent to 1.14μmolL(-1) when applied in the bath) or the Abcc inhibitor, MK571 was applied for one hour, during which DNP-SG was measured in the bath every 10min in order to calculate mass-specific DNP-SG transport rate. MCLR significantly inhibited DNP-SG transport (p<0.05), especially in middle intestine (47 and 24%, for luminal and serosal transport, respectively). In middle intestine strips, MCLR and MK571inhibited DNP-SG transport in a concentration dependent fashion (IC50 3.3 and 0.6μmolL(-1), respectively). In middle intestine strips incubated with calcein-AM (0.25μmolL(-1)), calcein efflux was inhibited by MCLR (2.3μmolL(-1)) and MK571 (3μmolL(-1)) by 38 and 27%, respectively (p<0.05). Finally, middle intestine segments were incubated with different concentrations of MCLR applied alone or together with 3μM MK571. After one hour, protein phosphatase 1 (PP1) activity, the main target of MCLR, was measured. 2.5μM MCLR did not produce any significant effect, while the same amount plus MK571 inhibited PP1 activity (p<0.05). This effect was similar to that of 5μM MCLR. Our results

Topics: Animals; Biological Transport; Concanavalin A; Fluoresceins; Glutathione; Glycosylation; Intestinal Mucosa; Marine Toxins; Microcystins; Microscopy, Fluorescence; Multidrug Resistance-Associated Proteins; Plant Lectins; Propionates; Quinolines; Smegmamorpha; Water Pollutants, Chemical

We studied Abcc mediated-transport in middle and posterior intestine of the rainbow trout, Oncorhynchus mykiss. Luminal and serosal transport were evaluated in everted and non-everted intestinal sacs, respectively, incubated with 1-chloro-2,4-dinitrobenzene (CDNB; 200 μM). CDNB enters the cells and is conjugated with glutathione via glutathione S-transferase (GST) to form 2,4-dinitrophenyl-S-glutathione (DNP-SG), a known Abcc substrate. DNP-SG concentration in the bath was recorded every 10 min, in order to calculate the mass-specific transport rate. For evaluating the possible involvement of Abcc proteins in microcystin-LR (MCLR) transport, 1.135 μM MCLR was added to the bath or inside the sacs, in everted or non-everted preparations, respectively. Both luminal and serosal DNP-SG efflux were significantly inhibited by MCLR. A concentration-response curve obtained using strips from middle intestine yielded an IC50 value of 1.33 μM MCLR. The Abcc inhibitor, MK571 produced concentration-dependent inhibition of DNP-SG similar to that produced by MCLR. Since competition of MCLR and CDNB as GST substrates could bias the DNP-SG transport results, we evaluated the effects of MCLR on calcein efflux, which does not depend on GST activity. We applied the non-fluorescent, cell-permeant compound calcein-AM (0.25 μM) to middle intestinal strips and recorded the efflux of its hydrolysis product, the fluorescent Abcc substrate calcein. 2.27 μM MCLR and 3 μM MK571 inhibited calcein efflux (17.39 and 20.2%, respectively). Finally, MCLR interaction with Abcc transporters was evaluated by measuring its toxic intracellular effects. Middle intestinal segments were incubated in saline solution with 1.135 μM MCLR (MC1), 2.27 μM MCLR (MC2), 3 μM MK571 (MK) or 1.135 μM MCLR+3 μM MK571 (MC1/MK). After 1h, GSH concentration, protein phosphatase 1 and 2A (PP1, PP2A) and GST activities were measured in each segment. MC1did not produce significant effect while MC1/MK and MC2 significantly inhibited PP1and PP2A in similar proportions (34-49%). MK alone significantly increased PP2A activity (40%) with no effect in any other variable. GST activity and GSH concentration were not affected by any treatment. Concentration-response curves for MCLR (1.135 to 13.62 μM) alone or plus 3 or 6 μM MK571 were obtained using PP1 activity as response variable. The IC50 values were 1.0, 0.52, and 0.37 μM, respectively. Our results suggest that O. mykiss enterocytes are capable of eliminating MCLR by GST

Topics: Animals; Biological Transport; Fluoresceins; Glutathione; Glutathione Transferase; Intestinal Mucosa; Intestines; Leukotriene Antagonists; Marine Toxins; Membrane Transport Proteins; Microcystins; Multidrug Resistance-Associated Proteins; Oncorhynchus mykiss; Propionates; Quinolines; Water Pollutants, Chemical

Fertilization is an ordered sequence of cellular interactions that promotes gamete fusion to form a new individual. Since the pioneering work of Oskar Hertwig conducted on sea urchins, echinoderms have contributed to the understanding of cellular and molecular aspects of the fertilization processes. Studies on sea urchin spermatozoa reported the involvement of a plasma membrane protein that belongs to the ABC proteins superfamily in the acrosome reaction. ABC transporters are expressed in membranes of eukaryotic and prokaryotic cells, and are associated with the transport of several compounds or ions across biomembranes. We aimed to investigate ABCB1 and ABCC1 transporter activity in sea urchin spermatozoa and their involvement in fertilization. Our results indicate that Echinometra lucunter spermatozoa exhibit a low intracellular calcein accumulation (18.5% stained cells); however, the ABC blockers reversin205, verapamil, and MK571 increased dye accumulation (93.0-96.6% stained cells). We also demonstrated that pharmacologically blocking ABCB1 and ABCC1 decreased spermatozoa fertilizing capacity (70% inhibition), and this phenotype was independent of extracellular calcium. These data suggest that functional spermatozoa ABCB1 and ABCC1 transporters are crucial for a successful fertilization. Additional studies must be performed to investigate the involvement of membrane lipid homeostasis in the fertilization process.

Topics: Acrosome; Acrosome Reaction; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Calcium Channel Blockers; Fertilization; Fluoresceins; Leukotriene Antagonists; Male; Membrane Transport Proteins; Multidrug Resistance-Associated Proteins; Oligopeptides; Organic Anion Transporters; Propionates; Quinolines; Sea Urchins; Spermatozoa; Verapamil

The multidrug resistance proteins MRP1 and MRP2 are efflux transporters with broad substrate specificity, including glutathione, glucuronide, and sulfate conjugates. In the present study, the interaction of the dietary polyphenol curcumin with MRP1 and MRP2 and the interplay between curcumin-dependent MRP inhibition and its glutathione-dependent metabolism were investigated using two transport model systems. In isolated membrane vesicles of MRP1- and MRP2-expressing Sf9 cells, curcumin clearly inhibited both MRP1- and MRP2-mediated transport with IC(50) values of 15 and 5 microM, respectively. In intact monolayers of MRP1 overexpressing Madin-Darby canine kidney (MDCKII-MRP1) cells, curcumin also inhibited MRP1-mediated activity, although with a 3-fold higher IC(50) value than the one observed in the vesicle model. Interestingly, MRP2-mediated activity was hardly inhibited in intact monolayers of MRP2-overexpressing MDCKII (MDCKII-MRP2) cells upon exposure to curcumin, whereas the IC(50) value in the vesicle incubations was 5 microM. The difference in extent of inhibition of the MRPs by curcumin in isolated vesicles as compared to intact cells, observed especially for MRP2, was shown to be due to a swift metabolism of curcumin to two glutathione conjugates in the MDCKII cells. Formation of both glutathione conjugates was about six times higher in the MDCKII-MRP2 cells as compared with the MDCKII-MRP1 cells, a phenomenon that could be ascribed to the significantly lower glutathione levels in the cell line. The efflux of both conjugates, identified in the present study as monoglutathionyl curcumin conjugates, was demonstrated to be mediated by both MRP1 and MRP2. From dose-response curves with Sf9 membrane vesicles, glutathionylcurcumin conjugates appeared to be less potent inhibitors of MRP1 and MRP2 than their parent compound curcumin. In conclusion, curcumin clearly inhibits both MRP1- and MRP2-mediated transport, but the glutathione-dependent metabolism of curcumin plays a crucial role in the ultimate level of inhibition of MRP-mediated transport that can be achieved in a cellular system. This complex interplay between MRP inhibition and metabolism of MRP inhibitors, the latter affecting the ultimate potential of a compound for cellular MRP inhibition, may exist not only for a compound like curcumin but also for many other MRP inhibitors presently or previously developed on the basis of vesicle studies.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Baculoviridae; Cell Line; Curcumin; Cyclosporine; Dinitrochlorobenzene; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Ethacrynic Acid; Fluoresceins; Glutathione; Glutathione Transferase; Humans; Propionates; Quinolines; Recombinant Proteins; Spodoptera

Cellular and molecular mechanisms involved in the resistance to cytotoxic heavy metals remain largely to be characterized in mammalian cells. To this end, we have analyzed a metal-resistant variant of the human lung cancer GLC4 cell line that we have selected by a step-wise procedure in potassium antimony tartrate. Antimony-selected cells, termed GLC4/Sb30 cells, poorly accumulated antimony through an enhanced cellular efflux of metal, thus suggesting up-regulation of a membrane export system in these cells. Indeed, GLC4/Sb30 cells were found to display a functional overexpression of the multidrug resistance-associated protein MRP1, a drug export pump, as demonstrated by Western blotting, reverse transcriptase-polymerase chain reaction and calcein accumulation assays. Moreover, MK571, a potent inhibitor of MRP1 activity, was found to markedly down-modulate resistance of GLC4/Sb30 cells to antimony and to decrease cellular export of the metal. Taken together, our data support the conclusion that overexpression of functional MRP1 likely represents one major mechanism by which human cells can escape the cytotoxic effects of heavy metals.

Topics: Antimony; Antineoplastic Agents; Arsenites; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Blotting, Western; Cadmium Chloride; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fluoresceins; Gene Amplification; Humans; Inhibitory Concentration 50; Lung Neoplasms; Meglumine; Metallothionein; Metals, Heavy; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; RNA, Messenger; Tumor Cells, Cultured; Up-Regulation; Zinc Sulfate