Dual effect of 1-deoxymannojirimycin on the mannose uptake and on the N-glycan processing of the human colon cancer cell line HT-29

1-Deoxymannojirimycin (dMM), a specific alpha-mannosidase I inhibitor, completely blocks the conversion of Man9-8GlcNAc2 into Man7-5-GlcNAc2 in both differentiated and undifferentiated human adenocarcinoma HT-29 cells. Besides this well known effect on N-glycan trimming, we describe here a novel effect of this inhibitor on the D-[2-3H]mannose uptake that is exclusively observed in differentiated intestinal cells, i.e. cells that display a functional apical brush border membrane. This inhibition of D-[2-3H]mannose uptake was shown to be dose-dependent and reversible. Moreover, using microsomal fractions we showed that this effect depends only on the integrity of the brush border and is unrelated to the classical inhibitory effect of dMM on N-glycan processing. Furthermore, another N-glycan trimming inhibitor 1-deoxynojirimycin, an epimer of dMM, did not interfere with D-[2-3H]mannose uptake. This observation was in good agreement with the specificity of the effect induced by dMM. These results demonstrate a novel effect of dMM on highly differentiated intestinal cells and suggest that a carrier-mediated mannose transport could exist in those cells. Such an interaction between cell morphology and the biological effect of dMM should lead to a careful use of drugs acting on N-glycan processing.     

The effect of deoxymannojirimycin on the processing of the influenza viral glycoproteins

Deoxymannojirimycin (dMM) was tested as an inhibitor of the processing of the oligosaccharide portion of viral and cellular N-linked glycoproteins. The NWS strain of influenza virus was grown in MDCK cells in the presence of various amounts of dMM, and the glycoproteins were labeled by the addition of 2-[3H]mannose to the medium. At levels of 10 micrograms/ml dMM or higher, most of the viral glycopeptides became susceptible to digestion by endoglucosaminidase H, and the liberated oligosaccharide migrated mostly like a Hexose9GlcNAc on a calibrated column of Bio-Gel P-4. This oligosaccharide was characterized as a typical Man9GlcNAc by a variety of chemical and enzymatic procedures. Deoxymannojirimycin gave rise to similar oligosaccharide structures in the cellular glycoproteins. In both the viral and the cellular glycoproteins, this inhibitor caused a significant increase in the amount of [3H]mannose present in the glycoproteins. Deoxymannojirimycin did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it affect the yield or infectivity of NWS virus particles. However, its effect on mannose incorporation into lipid-linked saccharides depended on the incubation time, the virus strain, and the cell line. Thus, high concentrations of dMM showed some inhibition of mannose incorporation into lipid-linked oligosaccharides with the NWS strain in a 3-h incubation, but no inhibition was observed after 48 h of incubation. On the other hand, the PR8 strain was much more sensitive to dMM inhibition, and mannose incorporation into lipid-linked oligosaccharides was strongly inhibited when the virus was raised in chick embryo cells, but less inhibition was observed when this virus was grown in MDCK cells. Nevertheless, in these cases also, the major oligosaccharide structure in the glycoproteins was the Man9GlcNAc2 species.     

Inhibition of nucleoside transport by p38 MAPK inhibitors

While investigating the ability of p38 MAPK to regulate cytarabine (Ara C)-dependent differentiation of erythroleukemia K562 cells, we observed effects that indicated that the imidazoline class of p38 MAPK inhibitors prevented nucleoside transport. Incubation of K562 cells with SB203580, SB203580-iodo, or SB202474, an analogue of SB203580 that does not inhibit p38 MAPK activity, inhibited the uptake of [3H]Ara C or [3H]uridine and the differentiation of K562 cells. Consistent with the effects of these compounds on the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter (ENT1), incubation with SB203580 or SB203580-iodo eliminated the binding of [3H]NBMPR to K562 cells or membranes isolated from human erythrocytes. Furthermore, using a uridine-dependent cell type (G9c), we observed that SB203580 or SB203580-iodo efficiently inhibited the salvage synthesis of pyrimidine nucleotides in vivo. Thus these studies demonstrate that the NBMPR-sensitive equilibrative nucleoside transporters are novel and unexpected targets for the p38 MAPK inhibitors at concentrations typically used to inhibit protein kinases.     

ATP/Mg2+-dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells

To study the mechanism of active drug efflux in multidrug-resistant cells, the interaction between [3H] vincristine (VCR) and plasma membrane prepared from an adriamycin (ADM)-resistant variant (K562/ADM) of human myelogenous leukemia K562 cells was examined by filtration method. [3H]VCR bound to the plasma membrane prepared from K562/ADM cells, but not from parental K562 cells, depending on the concentrations of ATP and Mg2+. Adenosine 5'-O-(3-thio)triphosphate was not effective in the binding of [3H]VCR, indicating that ATP hydrolysis is required for this binding. Dissociation constant (Kd) of VCR binding was 0.24 +/- 0.04 microM in the presence of 3 mM ATP. In the absence of ATP, specific binding of VCR to K562/ADM membrane was also observed; however, the affinity (Kd = 9.7 +/- 3.1 microM) was 40 times lower than that observed in the presence of ATP. The high affinity VCR binding to K562/ADM membrane was dependent on temperature. The bound [3H]VCR molecules were rapidly released by unlabeled VCR added to the reaction mixture at 25 degrees C. The high affinity binding of [3H]VCR to K562/ADM membrane was inhibited by VCR, vinblastine, actinomycin D, and ADM, to which K562/ADM cells exhibit cross-resistance, whereas 5-fluorouracil and camptothecin, to which K562/ADM cells are equally sensitive as K562 cells, did not inhibit the [3H]VCR binding. Furthermore, verapamil and other agents, which are known to circumvent drug resistance by inhibiting the active efflux of antitumor agents from resistant cells, could also inhibit the high affinity [3H]VCR binding. These results indicate that ATP/Mg2+-dependent high affinity VCR binding to the membrane of resistant cells closely correlates with the active drug efflux of this resistant cell line.     

Intracellular transport of the transmembrane glycoprotein G of vesicular stomatitis virus through the Golgi apparatus as visualized by electron microscope radioautography

The intracellular migration of G protein in vesicular stomatitis virus-infected cells was visualized by light and electron microscope radioautography after a 2-min pulse with [3H]mannose followed by nonradioactive chase for various intervals. The radioactivity initially (at 5-10 min) appeared predominantly in the endoplasmic reticulum, and the [3H]mannose-labeled G protein produced was sensitive to endoglycosidase H. Silver grains were subsequently (at 30-40 min) observed over the Golgi apparatus, and the [3H]mannose-labeled G protein became resistant to endoglycosidase H digestion. Our data directly demonstrate the intracellular transport of a plasmalemma-destined transmembrane glycoprotein through the Golgi apparatus.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [3H]leucine or [3H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [3H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [3H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [3H]mannose and [14C]leucine confirmed this. The decreased incorporation of [3H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Nucleic acid synthesis in cancerous cells under the effect of gnidilatimonoein from Daphne mucronata

Cytotoxicity evaluation of gnidilatimonoein, the most active isolated diterpene ester from Daphne mucronata [Sadeghi H, Mianabadi M, Yazdanparast R, (2002) Journal of Tropical. Medicinal Plant1 3: 169-173], revealed the strong antiproliferative activity among several different human cancer cell lines (K562, CCRF-CEM, HL-60 and MOLT-4 leukemia cell lines, LNCaP-FGC-10 a prostate cancer cell line) and a mouse BALB/C fibrosarcoma cell line (WEHI-164). Using flow cytometry technique, it was found that treatment of the most responsive cells (K562) with gnidilatimonoein inhibited the progression of cells through G1 phase by almost 15% compared to the untreated cells. The population of the treated cells in the S and G2 phases also reduced by 8.3% and 5.4%, respectively. Based on the extent of [3H]-thymidine and [3H]-uridine incorporation into DNA and RNA, respectively, the major metabolic effects of gnidilatimonoein were found to be mainly on DNA and to a less extent on RNA synthesis. Additionally, the activity of inosine-5'-monophosphate dehydrogenase (IMPDH), under the effects of genidilatimonoein, was reduced in the treated cells by 44%. These data strongly suggest that the purine biosynthetic pathway is significantly affected by gnidilatimonoein.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates; comparison with phosphomannoisomerase

The isotopic discrimination, diastereotopic specificity and intramolecular hydrogen transfer characterizing the reaction catalyzed by phosphomannoisomerase are examined. During the monodirectional conversion of D-[2-3H]mannose 6-phosphate to D-fructose 6-phosphate and D-fructose 1,6-bisphosphate, the reaction velocity is one order of magnitude lower than with D-[U-14C]mannose 6-phosphate and little tritium (less than 6%) is transferred intramolecularly. Inorganic phosphate decreases the reaction velocity but favours the intramolecular transfer of tritium. Likewise, when D-[1-3H]fructose 6-phosphate prepared from D-[1-3H]glucose is exposed solely to phosphomannoisomerase, the generation of tritiated metabolites is virtually restricted to 3H2O and occurs at a much lower rate than the production of D-[U-14C]mannose 6-phosphate from D-[U-14C]fructose 6-phosphate. However, no 3H2O is formed when D-[1-3H]fructose 6-phosphate generated from D-[2-3H]glucose is exposed to phosphomannoisomerase, indicating that the diastereotopic specificity of the latter enzyme represents a mirror image of that of phosphoglucoisomerase. Advantage is taken of such a contrasting enzymic behaviour to assess the back-and-forth flow through the reaction catalyzed by phosphomannoisomerase in intact cells exposed to D-[1-3H]glucose, D-[5-3H]glucose or D-[6-3H]glucose. Relative to the rate of glycolysis, this back-and-forth flow amounted to approx. 4% in human erythrocytes and rat parotid cells, 9% in tumoral cells of the RINm5F line and 47% in rat pancreatic islets.     

Mannose analog 1-deoxymannojirimycin inhibits the Golgi-mediated processing of bean storage glycoproteins

The asparagine-linked oligosaccharide chains of glycoproteins can be processed to form a wide variety of structures. The Golgi complex is the main compartment involved in this processing. In mammalian cells the first enzyme acting along the Golgi processing pathway is mannosidase I, whose action is a prerequisite for any further processing and which is inhibited by the mannose analog 1-deoxymannojirimycin (dMM). To have insights into the processing pathway in plant cells, we have studied the in vivo effect of dMM on the processing of the bean (Phaseolus vulgaris) storage proteins phaseolin and phytohemagglutinin, two well characterized plant glycoproteins. Cotyledons obtained from developing seeds were labeled with radioactive leucine, glucosamine, or fucose in the presence or absence of dMM. Treatment with dMM fully inhibited the acquisition of resistance to endo-β-N-acetylglucosaminidase H by phaseolin and phytohemagglutinin and the incorporation of fucose into protein. Furthermore, the apparent molecular weight of the polypeptides of phaseolin and phytohemagglutinin synthesized in dMM-treated cotyledons was consistent with the exclusive presence of oligommanose oligosaccharide chains which had not been processed in the Golgi complex. The inhibition of processing did not prevent exit from the Golgi complex, and most probably the storage proteins were correctly targeted to the protein bodies as indicated by the post-translational polypeptide cleavage of phaseolin. These results indicate that the action of a mannosidase is the first obligatory step of Golgi-mediated processing also in a plant cell and, together with data obtained in other laboratories on the in vitro specificity of glycosidases and glycosyltransferases present in the Golgi complex of plant cells, support the hypothesis that the key early reactions in Golgi-mediated processing are similar if not identical in plants and mammals.     

The uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptors.

It is not yet clear if the endocannabinoid 2-arachidonoylglycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2-AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 +/- 2.0 and 15.3 +/- 3.1 microM, Bmax 1.70 +/- 0.30 and 0.24 +/- 0.04 nmol.min-1.mg protein-1, respectively). Despite these similar Km values, 2-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 +/- 3.9 microM) significantly higher than those required to either 2-AG or AEA to inhibit [3H]2-AG uptake (Ki = 18.9 +/- 1.8 and 20.5 +/- 3.2 microM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2-AG alone; (b) the uptake of [14C]AEA and [3H]2-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 +/- 0.7 and 10.2 +/- 1.7 microM, respectively) and linvanil (Ki = 9.5 +/- 0.7 and 6.4 +/- 1.2 microM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2-AG, thus suggesting that 2-AG action can be regulated through NO release; (d) AEA and 2-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.     

Oleanolic acid derivatives induce apoptosis in human leukemia K562 cell involved in inhibition of both Akt1 translocation and pAkt1 expression

Oleanolic acid (OA) derivatives exhibit numerous pleiotropic effects in many cancers. The present study aimed to investigate the molecular mechanisms of 5′-amino-oleana-2,12-dieno[3,2-d]pyrimidin-28-oic acid (compound 4) and oleana-2,12-dieno[2,3-d]isoxazol-28-oic acid (compound 5) inducing apoptosis in human leukemia K562 cell. We investigated the effects of the compounds on K562 cell growth, apoptosis and cell cycle. The compounds showed strong inhibitory effects on K562 cell viability in a dose-dependent manner determined by the 3-(4,5-dimethylthiazoyl)-2,5-diphenyltetrazolium bromide assay and significantly increased chromatin condensation and apoptotic bodies in K562 cells. Flow cytometry assay suggested that the compounds induced inhibition of K562 cell proliferation associated with G1 phase arrest. In addition, the compounds inhibited Akt1 recruiting to membrane in CHO cells which express Akt1-EGFP constitutively and down-regulated the expression of pAkt1 in K562 cell. These results suggested that the compounds can efficiently inhibit proliferation and induce apoptosis perhaps involved in inactivation of Akt1. The OA derivatives may be potential chemotherapeutic agents for the treatment of human cancer.     

A novel tubulin polymerization inhibitor,MPT0B206, downregulates Bcr-Abl expression and induces apoptosis in imatinib-sensitive and imatinib-resistant CML cells

Imatinib, a Bcr-Abl-specific inhibitor, is effective for treating chronic myeloid leukemia (CML), but drug resistance has emerged for this disease. In this study, we synthesized a novel tubulin polymerization inhibitor, MPT0B206 (N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-formamide), and demonstrated its apoptotic effect and mechanism in imatinib-sensitive K562 and imatinib-resistant K562R CML cells. Western blotting and immunofluorescence microscopy showed that MPT0B206 induced microtubule depolymerization in K562 and K562R cells. MPT0B206 inhibited the growth of these cells in a concentration- and time-dependent manner. It did not affect the viability of normal human umbilical vein endothelial cells. MPT0B206 induced G2/M cell cycle arrest and the appearance of the mitotic marker MPM-2 in K562 and K562R cells, which is associated with the upregulation of cyclin B1 and the dephosphorylation of Cdc2. Treatment of K562 and K562R cells with MPT0B206 induced apoptosis and reduced the protein levels of procaspase-9 and procaspase-3 and increased caspase-3 activity and PARP cleavage. MPT0B206 also reduced the levels of the antiapoptotic proteins Mcl-1 and Bcl-2 and increased the level of the apoptotic protein Bax. Additional experiments showed that MPT0B206 markedly downregulated Bcr-Abl mRNA expression and total and phosphorylated Bcr-Abl protein levels and inhibited the phosphorylation of its downstream proteins STAT5, MAPK, and AKT, and the protein level of c-Myc in K562 and K562R cells. Furthermore, MPT0B206 triggered viability reduction and apoptosis in CML cells carrying T315I-mutated Bcr-Abl. Together, these results suggest that MPT0B206 is a promising alternative for treating imatinib-resistant CML.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast.

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-14C]hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-14C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-14C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-14C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-14C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-3H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the 'salvage' pathways and de novo synthesis of purines and pyrimidines.     

Recycling glycoproteins do not return to the cis-Golgi

Recycling of a number of glycoproteins along the site of action of mannosidase I (the distal endoplasmic reticulum/cis-Golgi) was followed in several different cell lines. Treatment of cells with 1-deoxymannojirimycin (dMM) produced high mannose oligosaccharides at positions otherwise occupied by complex-type oligosaccharides in these glycoproteins. Conversion of high-mannose-type oligosaccharides to complex oligosaccharides of proteins initially synthesized in the presence of dMM was used as a marker for recycling of glycoproteins along the site of action of dMM. In contrast to findings reported by Snider and Rogers (Snider, M. D., and O. C. Rogers. 1986. J. Cell Biol. 103:265-275), removal of dMM did not result in reconversion of high-mannose oligosaccharides to complex-type sugars, even after prolonged periods of culture. We conclude that surface glycoproteins do not recycle through the cis-medial Golgi elements.     

Studies on the mechanism of enkephalin receptor down regulation

The association of [3H] [D-Ala2, D-Leu5] enkephalin ([3H]DADLE]) with mouse neuroblastoma cells (N4TG1) was investigated. Under identical conditions the time course, dose response curve and temperature dependence for ligand uptake were similar to those for ligand-induced receptor loss (down regulation). Uptake of [3H]DADLE was inhibited by opiate ligands as well as by the metabolic inhibitors sodium azide and 2,4 dinitrophenol. Comparison of the effects of these inhibitors on receptor binding, ligand uptake and receptor loss indicated that these cells accumulate [3H]DADLE in excess of their surface receptor number. The data suggest that receptor recycling occurs and that ligand is internalized via receptor mediated endocytosis.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Human transferrin receptor contains O-linked oligosaccharides

We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types.     

Amine uptake into intact mast cell granules in vitro

Histamine, the principal amine of rat peritoneal mast cells, is taken up into isolated granules with intact membranes. Uptake is pH- and concentration-dependent and is not stimulated by the addition of Mg2+-ATP. The saturable uptake has a Km of 91.1 microM and a Vmax of 95.4 pmol (mg of protein)-1 min-1. Uptake is abolished by 5 mM ammonium ion. 5-HT, the other endogenous amine of the granules, and dopamine and tyramine, which do not occur naturally in rat mast cells, each competitively inhibits [3H]-histamine uptake with Ki's close to 1 microM. Reserpine, a putative amine carrier blocker, inhibits uptake at nanomolar concentrations. At high concentrations, uptake of [3H]-5-HT is nonsaturable; at low concentrations, a saturable component is observed with a Km of 1.6 microM. Uptake of [3H]-5-HT is not enhanced by Mg2+-ATP. It is pH-dependent but with a lower apparent pKa than that of histamine. [3H]-5-HT uptake can be completely inhibited by ammonium ions. Amine inhibition of [3H]-5-HT gives nonlinear Dixon plots, and high concentrations of the competing amines or reserpine cannot completely block uptake. We propose a model consistent with these results in which amine uptake occurs by several distinct saturable transport systems. According to the model, histamine is transported by a single system, which also transports 5-HT and dopamine. 5-HT and dopamine are transported by one or more other systems.     

Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions

Studies are presented of the equilibrium binding of [3H]-d-tubocurarine (dTC) and [3H]acetylcholine (AcCh) to Torpedo postsynaptic membranes. The saturable binding of [3H]dTC is characterized by two affinities: Kd1 = 33 +/- 6 nM and Kd2 = 7.7 +/- 4.6 microM, with equal numbers of binding sites. Both components are completely inhibited by pretreatment with excess alpha-bungarotoxin or 100 microM nonradioactive dTC and competitively inhibited by carbamylcholine with a KI = 100 nM, but not affected by the local anesthetics dimethisoquin, proadifen, and meproadifen. The biphasic nature of [3H]dTC binding was unaltered in solutions of low ionic strength and by preparation of Torpedo membranes in the presence of N-ethylmaleimide, a treatment which yields dimeric AcCJ receptors. dTC competitively inhibits the binding of [3H]AcCH and decreases the fluorescence of 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane-2-trimethylammonium (Dns-Chol) in a manner quantitatively consistent with its directly measured binding properties. It decreases the initial rate of 3H-labeled Naja nigricollis alpha-toxin binding by 50% at 60 nM with an apparent Hill coefficient of 0.58. The stoichiometry of total dTC, AcCh, and alpha-neurotoxin binding sites in Torpedo membranes was determined by radiochemical techniques and by a novel fluorescence assay utilizing Dns-Chol as an indicator, yielding ratios of 0.9 +/- 0.1:0.9 +/- 0.2:1, respectively. The biphasic equilibrium binding function is not unique to dTC since other ligands inhibited [3h]acCh binding in a biphasic manner with apparent inhibition constants as follows: gallamine triethiodide (K11 = 2 microM, K12 = 1 mM); Me2dTC (K11 = 500 nM, K12 = 10 microM); decamethonium (K11 = 100 nM, K12 = 1.6 microM). Carbamylcholine, however, inhibited [3H]AcCh binding with a single KI = 100 nM. The observed competition between those ligands and [3H] AcCh cannot be completely accounted for by competitive interaction with two different affinities, and the deviations are discussed in terms of the positive cooperativity of the [3H] AcCh binding function itself. It is concluded that dTC binds only to the AcCh sites in Torpedo membranes and that those sites display two affinities for dTC but only one for AcCh.     

Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.