calcein-am and verlukast

The aim of the study was to characterize ABC transport proteins gene expression and efflux activities in gills and hemocytes of the Mediterranean mussel and to evaluate their response to Cd. At basal level a higher expression of abcb-like gene was observed in gills than in hemocytes while abcc-like gene showed similar levels. Both P-gp and MRPs inhibitors (cyclosporine and MK571) blocked efflux activities in gills; hemocytes were sensitive only to MK571. After 120 min in vitro pre-exposure to CdCl2, the efflux activity increased significantly in gills and hemocytes. In vivo exposure to CdCl2 (0.4 μM) increased abcb-like gene expression in gills without affecting efflux activity. In hemocytes abcc-like gene resulted up-regulated and Ca-AM efflux resulted enhanced. An increased uptake of Cd in gills biopsies was observed in the presence of both P-gp and MRPs inhibitors. Our results indicate that ABC transporters seem involved in the first protective response to Cd and this response is tissue-specific.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cadmium; Cyclosporine; Fluoresceins; Fluorescent Dyes; Gene Expression Regulation; Gills; Hemocytes; Multidrug Resistance-Associated Proteins; Mytilus; Propionates; Quinolines; Water Pollutants, Chemical

P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP1) are membrane transporter proteins which function as efflux pumps at cell membranes and are considered to exert a protective function against the entry of xenobiotics. While evidence for Pgp and MRP transporter activity is reported for olfactory tissue, their possible interaction and participation in the olfactory response has not been investigated.. Functional activity of putative MDR transporters was assessed by means of the fluorometric calcein acetoxymethyl ester (calcein-AM) accumulation assay on acute rat and mouse olfactory tissue slices. Calcein-AM uptake was measured as fluorescence intensity changes in the presence of Pgp or MRP specific inhibitors. Epifluorescence microscopy measured time course analysis in the olfactory epithelium revealed significant inhibitor-dependent calcein uptake in the presence of each of the selected inhibitors. Furthermore, intracellular calcein accumulation in olfactory receptor neurons was also significantly increased in the presence of either one of the Pgp or MRP inhibitors. The presence of Pgp or MRP1 encoding genes in the olfactory mucosa of rat and mouse was confirmed by RT-PCR with appropriate pairs of species-specific primers. Both transporters were expressed in both newborn and adult olfactory mucosa of both species. To assess a possible involvement of MDR transporters in the olfactory response, we examined the electrophysiological response to odorants in the presence of the selected MDR inhibitors by recording electroolfactograms (EOG). In both animal species, MRPs inhibitors induced a marked reduction of the EOG magnitude, while Pgp inhibitors had only a minor or no measurable effect.. The findings suggest that both Pgp and MRP transporters are functional in the olfactory mucosa and in olfactory receptor neurons. Pgp and MRPs may be cellular constituents of olfactory receptor neurons and represent potential mechanisms for modulation of the olfactory response.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Biological Transport; Cyclosporine; Female; Fluoresceins; Gene Expression; Male; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Multidrug Resistance-Associated Proteins; Olfactory Mucosa; Olfactory Receptor Neurons; Probenecid; Propionates; Quinolines; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Verapamil

Resistance to antiepileptic drugs (AEDs) is one of the most serious problems in the treatment of epilepsy. Accumulating experimental evidence suggests that increased expression of the drug efflux transporter P-glycoprotein (Pgp) at the blood-brain barrier may be involved in the mechanisms leading to AED resistance. In addition to Pgp, increased expression of several multidrug resistance-associated proteins (MRPs) has been determined in epileptogenic brain regions of patients with pharmacoresistant epilepsy. However, it is not known whether AEDs are substrates for MRPs. In the present experiments, we evaluated whether common AEDs are transported by human MRPs (MRP1, 2 and 5) that are overexpressed in AED resistant epilepsy. For this purpose, we used a highly sensitive assay (concentration equilibrium transport assay; CETA) in polarized kidney cell lines (LLC, MDCKII) transfected with human MRPs. The assay was validated by known MRP substrates, including calcein-AM (MRP1), vinblastine (MRP2) and chloromethylfluorescein diacetate (CMFDA; MRP5). The directional transport determined with these drugs in MRP-transfected cell lines could be blocked with the MRP inhibitor MK571. However, in contrast to transport of known MRP substrates, none of the common AEDs (carbamazepine, valproate, levetiracetam, phenytoin, lamotrigine and phenobarbital) used in this study was transported by MRP1, MRP2 or MRP5. A basolateral-to-apical transport of valproate, which could be inhibited by MK571 and probenecid, was determined in LLC cells (both wildtype and transfected), but the specific transporter involved was not identified. The data indicate that common AEDs are not substrates for human MRP1, MRP2 or MRP5, at least in the in vitro models used in this study.

Topics: Anticonvulsants; Biological Transport; Cell Line; Central Nervous System Agents; Epilepsy; Fluoresceins; Humans; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; Reproducibility of Results; Transfection; Vinblastine

The ATP binding cassette (ABC)-transporters are energy dependent efflux pumps which regulate the pharmacokinetics of both anti-cancer chemotherapeutic agents, e.g. taxol, and of human immunodeficiency virus-1 (HIV-1) protease inhibitors (HPIs), e.g. saquinavir. Increased expression of several ABC-transporters, especially P-glycoprotein (P-gp) and multidrug resistance protein 2 (MRP2), are observed in multidrug resistant (MDR) tumor cells and on HIV-1 infected lymphocytes. In addition, due to their apical expression on vascular endothelial barriers, both P-gp and MRP2 are of crucial importance towards dictating drug access into sequestered tissues. However, although a number of P-gp inhibitors are currently in clinical trials, possible inhibitors of MRP2 are not being thoroughly investigated. The experimental leukotriene receptor antagonist (LTRA), MK-571 is known to be a potent inhibitor of MRP transporters. Using the MRP2 over-expressing Madin-Darby canine kidney cell line, MDCKII-MRP2, we evaluated whether the clinically approved LTRAs, e.g. montelukast (Singulair) and zafirlukast (Accolate), can similarly suppress MRP2-mediated efflux. We compared the efficacy of increasing concentrations (20-100 microM) of MK-571, montelukast, and zafirlukast, in suppressing the efflux of calcein-AM, a fluorescent MRP substrate, and the radiolabeled [(3)H-] drugs, taxol and saquinavir. Montelukast was the most potent inhibitor (p<0.01) of MRP2-mediated efflux of all three substrates. Montelukast also increased (p<0.01) the duration of intracellular retention of both taxol and saquinavir. More than 50% of the drugs were retained in cells even after 90 min post removal of montelukast from the medium. Our findings implicate that montelukast, a relatively safe anti-asthmatic agent, may be used as an adjunct therapy to suppress the efflux of taxol and saquinavir from MRP2 overexpressing cells.

Topics: Acetates; Animals; Anti-Asthmatic Agents; Antineoplastic Agents, Phytogenic; Biological Transport; Cell Line; Chemotherapy, Adjuvant; Cyclopropanes; Dogs; Drug Resistance, Neoplasm; Drug Resistance, Viral; Fluoresceins; HIV Infections; HIV Protease Inhibitors; Indoles; Leukotriene Antagonists; Multidrug Resistance-Associated Proteins; Neoplasms; Paclitaxel; Phenylcarbamates; Propionates; Quinolines; Saquinavir; Sulfides; Sulfonamides; Time Factors; Tosyl Compounds

We describe the use of the sea urchin as a model for studying efflux transporters and estimating energy cost for the cytotoxin protective system provided by these transporters. The unfertilized egg has low transport activity, which increases to a new steady state shortly after fertilization. Activity results from p-glycoprotein (p-gp) and MRP type transporters which protect the embryo from cytotoxic drugs that can disrupt cell division or induce apoptosis. The energy cost is estimated from a novel use of calcein-AM as a substrate; keeping 0.25 microM substrate levels out of the cell utilizes only 0.023% of steady state respiration.

Topics: Animals; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Division; Cyclosporins; Cytotoxins; Embryo, Nonmammalian; Energy Metabolism; Etoposide; Fluoresceins; Membrane Transport Proteins; Models, Animal; Propionates; Quinolines; Sea Urchins; Verapamil; Vinblastine

MRP1 activity was evaluated and compared in 11 cell lines with different levels of MRP1 expression using functional assays of calcein acetoxymethyl ester (calcein-AM), carboxyfluorescein diacetate (CFDA) and Rhodamine 123 (Rh123) in combination with the modulators cyclosporin A (CsA), probenecid and MK571. A good correlation was found between MRP1 expression and the modulatory effect of MK571 on calcein-AM uptake (P = 0.01 and probenecid effect on CFDA uptake (P = 0.02). Additionally, the combined modulatory effect of MK571 and probenecid on CFDA uptake (P < 0.0001) and on calcein-AM uptake (P = 0.0001) were highly significant. No correlation was found between MRP1 expression and the effects of three modulators on Rh123 uptake or efflux. In conclusion, calcein-AM and CFDA uptake assays are the best choices to probe MRP1 activity and combination of two modulators may improve the efficiency of these assays.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Bronchodilator Agents; Cyclosporine; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Humans; Leukemia; Multidrug Resistance-Associated Proteins; Probenecid; Propionates; Quinolines; Rhodamine 123; Tumor Cells, Cultured

Multidrug resistance protein-6 (MRP6) is a membrane transporter whose deficiency leads to the connective tissue disorder Pseudoxanthoma elasticum (PXE). In vitro dermal fibroblasts from normal and PXE subjects, homozygous for the R1141X mutation, were compared for their ability to accumulate and to release fluorescent calcein, in the absence and in the presence of inhibitors and competitors of the MDR-multidrug resistance protein (MRP) systems, such as 3-(3-(2-(7-choro-2 quinolinyl) ethenyl)phenyl ((3-dimethyl amino-3-oxo-propyl)thio) methyl) propanoic acid (MK571), verapamil (VPL), vinblastine (VBL), chlorambucil (CHB), benzbromarone (BNZ) and indomethacin (IDM). In the absence of chemicals, calcein accumulation was significantly higher and the release significantly slower in PXE cells compared to controls. VBL and CHB reduced calcein release in both cell strains, without affecting the differences between PXE and control fibroblasts. VPL, BNZ and IDM consistently delayed calcein release from both control and PXE cells; moreover, they abolished the differences between normal and MRP6-deficient fibroblasts observed in the absence of chemicals. These findings suggest that VPL, BNZ and IDM interfere with MRP6-dependent calcein extrusion in in vitro human normal fibroblasts. Interestingly, MK571 almost completely abolished calcein release from PXE cells, whereas it induced a strong but less complete inhibition in control fibroblasts, suggesting that MRP6 is not inhibited by MK571. Data show that MRP6 is active in human fibroblasts, and that its sensitivity to inhibitors and competitors of MDR-MRPs' membrane transporters is different from that of other translocators, namely, MRP1. It could be suggested that MRP1 and MRP6 transport different physiological substances and that MRP6 deficiency cannot be overcome by other membrane transporters, at least in fibroblasts. These data further support the hypothesis that MRP6 deficiency may be relevant for fibroblast metabolism and responsible for the metabolic alterations of these cells at the basis of connective tissue clinical manifestations of PXE.

Topics: Adult; Benzbromarone; Cell Count; Cell Division; Chlorambucil; Female; Fibroblasts; Fluoresceins; Fluorescent Dyes; Humans; Indomethacin; Lysosomes; Male; Membrane Transport Proteins; Microscopy, Fluorescence; Middle Aged; Multidrug Resistance-Associated Proteins; Propionates; Pseudoxanthoma Elasticum; Quinolines; Skin; Verapamil; Vinblastine