The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: I. Kinetic properties of NBD-taurine transfer in symmetric conditions

Summary The molecular mechanism of anion exchange across the human red blood cell membrane was assessed with the fluorescent substrate analog NBD-taurine and the method of continuous monitoring of transport by fluorescence. The efflux of NBD-taurine was studied under a variety of experimental conditions such as temperature, pH and anion composition of cells and media. The temperature profile of NBD-taurine transfer from Cl-loaded cells into Cl media resembled that of Cl self-exchange, whereas that of NBD-taurine transfer from sulfate-loaded cells into sulfate media resembled that of sulfate self-exchange. Although the pH profiles of NBD-taurine transfer from Cl-loaded cells into Cl media and that of Cl self-exchange resembled each other, the analogous transfer with sulfate replacing Cl was markedly different. These and other data were analyzed and found to be consistent with a model which comprises the following: (a) a H⁺-titratable group in the carrier mechanism; (b) alteration of transport sites between the two sides of the membrane (i.e., ping-pong kinetics); and (c) transmembrane distribution of transport sites which is modulated by pH. It is shown that NBD-taurine transfer represents a tracer flux of a fluorescent substrate which gives a measure for the presence of monovalent transport sites at the inner surface of the membrane. The latter is markedly affected by the relative concentrations of anions and H⁺ on both sides of the red blood cell membrane.     

Oxidative damage by phenylhydrazine diminishes erythrocyte anion transport

Human erythrocytes were exposed to oxidative stress by treatment with the slowly hemolytic drug phenylhydrazine. Phenylhydrazine has been previously shown to trigger the production of toxic oxygen metabolites including O-2 and H2O2 and the formation of Heinz bodies. The concentration-dependent formation of Heinz bodies was confirmed using optical microscopy. Heinz body formation was accompanied by surface protuberances as shown by scanning electron microscopy. The formation of Heinz bodies was accompanied by inhibition of anion translocation. Anion translocation was measured using the anionic fluorescent substrate analog N-(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-1,3-diazole (NBD-taurine). The efflux of NBD-taurine was measured by continuous monitoring of transport by fluorescence (CMTF). The mean value of the kinetic rate constant for transport, k, was found to be -0.090 +/- 0.017 min-1. Phenylhydrazine was found to decrease k to less than one-half of control values in a dose-dependent fashion. The disruption of anion translocation may be related to the oxidative effects of phenylhydrazine and to the generation of Heinz bodies, which bind to the N-terminal domain of band 3.     

Reversal of p-glycoprotein-mediated multidrug resistance by CJX1, an amlodipine derivative, in doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells

P-glycoprotein-mediated drug efflux can yield a multidrug resistance (MDR) phenotype that is associated with a poor response to cancer chemotherapy. Development of safe and effective MDR reversing agents is an important approach in the clinic. The aim of this study was to observe the effects of CJX1, an amlodipine derivative, on the inhibition of P-gp function and P-gp-mediated MDR in K562/DOX cells and parental K562 cells. Based on the flow cytometric technology, the uptake, accumulation and efflux of rhodamine123 (Rh123) were detected in these cells by measuring Rh123-associated mean fluorescence intensity (MFI). The effects of CJX1 on the doxorubicin cytotoxicity were evaluated by assaying for MTT (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide) reduction and the reversal fold (RF) values. The DNA content, percentage of apoptosis and cell cycle analysis were monitored with flow cytometry. Intracellular accumulation of doxorubicin was also assessed by the determination of doxorubicin-associated MFI. Verapamil was employed as a comparative agent. Incubation of K562/DOX cells with CJX1 caused a marked increase in uptake and a notable decrease in efflux of Rh123, No such results were found in parental K562 cells. The inhibitory effect of the agent of P-gp function was reversible, but it persisted at least for 90 min after removal of 2.5 microM CJX1 from incubation medium. The doxorubicin-induced cytotoxicity, apoptosis and cell cycle perturbations were significantly potentiated by CJX1. The intracellular accumulation of doxorubicin was enhanced in the presence of various concentrations of CJX1. The CJX1 exhibited potent effects in vitro in the reversal of P-gp-mediated MDR, suggesting that the compound may become a candidate of effective MDR reversing agent in cancer chemotherapy.     

Permselectivity changes in malaria (Plasmodium falciparum) infected human red blood cell membranes

The development of the malaria parasite Plasmodium falciparum in human red blood cells induces parasite-dependent perturbations in the permselectivity properties of the host cell membrane. The changes appear as parasites develop from ring to the trophozoite stage and persist during schizogony. In the present work we assessed the permeability changes of the infected cells to anionic substances by the use of radioactive and fluorescent probes. Our data show that i) covalent binding probes, such as diisothiocyano ditritiostilbene disulfonic acid [3H]H2DIDS, which are virtually impermeant to normal red blood cells, became markedly permeant to trophozoites and schizonts, as evidenced by high labeling of intracellular hemoglobin; ii) permeation of the fluorescent anion transport substrate NBD-taurine, measured in the efflux mode, was very rapid and substantially enhanced in parasitized erythrocytes, as compared with noninfected cells; iii) this efflux could not be blocked by H2DIDS, which is a specific inhibitor of anion transport in normal red blood cells; iv) permeation of anionic probes was temperature dependent (Ea:11 +/- 1 kcal/mole); and v) could be blocked by nonspecific transport inhibitors that are known to interact with membrane lipids. The appearance of a new permeation pathway in the host cell membrane of trophozoites is associated with structural modification of the host cell membrane matrix.     

Energy-dependent accumulation of daunorubicin into subcellular compartments of human leukemia cells and cytoplasts.

Anthracycline accumulation was evaluated by flow cytometry or radiolabeled drug assays in cells and cytoplasts (enucleated cells) prepared from parental and multidrug-resistant human K562 leukemia cells. Treatment with energy inhibitors, such as dinitrophenol (DNP) or sodium azide/deoxyglucose, led to a marked decrease in daunorubicin accumulation in parental cells and cytoplasts. Another ionophore, monensin, also caused a significant decrease in daunorubicin accumulation; however, ATPase inhibitors ouabain, vanadate, and N-ethylamaleimide had little or no effect. The lysosomatropic agents chloroquine and methylamine caused a moderate decrease in anthracycline accumulation. Fluorescence microscopy showed that the DNP-sensitive daunorubicin uptake occurred in a nonnuclear subcellular compartment. Studies using increasing daunorubicin concentrations demonstrated fluorescence quenching that occurred in the nonnuclear, DNP-sensitive compartment. The effect of inhibitors on the accumulation of rhodamine 123 and acridine orange strongly implicated lysosomes as the principal compartment of this inhibitable daunorubicin accumulation. Cytoplasts from P-glycoprotein containing multidrug-resistant K562 cells demonstrated a verapamil-reversible, decreased daunorubicin accumulation that was observed in resistant whole cells. Verapamil pretreatment of cytoplasts from resistant cells revealed the subcellular DNP-sensitive uptake present in parental cytoplasts. These studies demonstrate that cytoplasts are an effective means to study drug transport in mammalian cells without nuclear drug binding. Parental K562 cells and cytoplasts exhibit an energy-dependent accumulation of daunorubicin into cytoplasmic organelles that is also present in resistant cells and cytoplasts when P-glycoprotein mediated efflux is inhibited.     

Characterisation of three pathways for osmolyte efflux in human erythroleukemia cells

Cell volume decrease is a key step during differentiation of erythroid cells. This could arise from membrane transporter activation leading to a loss of cell osmolytes; however, the pathways involved are poorly understood. We have characterised Cl(-)-independent K(+) and (3)H-taurine efflux from the erythroleukemia cell line, K562. K(+) efflux (measured using (86)Rb(+)) from pre-loaded cells subjected to hypo-osmotic challenge demonstrated two phases, a rapid increase in K(+) efflux followed by a smaller slower increase. Swelling-activated taurine efflux only demonstrated a single phase. Both phases of K(+) efflux were significantly (P<0.05) blocked by anion channel inhibitor 5-nitro-2-(3-phenypropylamino)-benzoic acid (NPPB). However the antiestrogen, tamoxifen, only inhibited the slow late phase. The initial rapid phase had a higher IC(50) for NPPB inhibition than the slow phase, and was insensitive to protein kinases inhibitors KN-62, wortmannin and PD98059. For the slow K(+) efflux phase, the IC(50) for NPPB inhibition and the inhibition by KN-62, wortmannin, genistein or PD98059, were very similar to those measured for the hypo-osmotically-activated taurine efflux. With NPPB (100 microM) present, the slow K(+) efflux phase was further significantly decreased by the Ca(2+) chelator BAPTA-AM or by the Ca(2+)-activated K(+) channel blockers clotrimazole and charybdotoxin but not by apamin. Thus, at least 3 Cl(-)-independent pathways are involved: (a) a tamoxifen-sensitive and taurine-permeable anion channel; (b) a tamoxifen-insensitive and taurine-impermeable K(+) efflux pathway; and (c) a subtype of Ca(2+)-activated K(+) channel. Any or all of these could be involved in the cell volume decrease associated with differentiation in K562 cells.     

Single-cell flux measurement by continuous fluorescence microphotolysis

Continuous fluorescence microphotolysis (CFM) was adapted to flux measurements in single cells. The principle of the method is simple: Cells are equilibrated with a fluorescent solute, an individual cell is continuously irradiated by a laser beam focussed down to approximately the diameter of the cell, and fluorescence originating from the irradiated cell is monitored. In this procedure irradiation irreversibly photolyzes chromophores in the cell while fresh chromophores enter the cell by membrane transport (flux). The resulting fluorescence decay can be analyzed for the rate constants of both membrane transport and photolysis. As an experimental test of the new method the band-3 mediated transport of the fluorescent anion N-(7-nitrobenzofuranzan-4-yl)-taurine (NBD-taurine) across the erythrocyte membrane was measured. For various experimental conditions good agreement between values obtained by CFM and by fluorescence microphotolysis (FM) was observed. By measurements on single ghosts it was furthermore found that photolysis of NBD-taurine is first-order with respect to the power of irradiation. On this basis a stepped-intensity procedure was worked out that facilitates data evaluation in flux measurements. Also, by analysing the relations between CFM and FM flux measurements a method was devised by which FM data can be corrected for (inevitable) photolysis.     

Peroxyl-oxidized Erythrocyte Membrane Band 3 Protein with Anion Transport Capacity is Degraded by Membrane-bound Proteinase

Human red blood cells anion exchange protein (band 3) exposed to peroxyl radicals produced by thermolysis of 2,2′-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through band 3 was smaller indicating that removal achieved by proteinases includes oxidized band 3 which still retain transport ability. Proteinases recognize band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that band 3 aggregates removal may prevent macrophage recognition of the senescent condition which would lead to cell disposal.     

Peroxyl-oxidized erythrocyte membrane band 3 protein with anion transport capacity is degraded by membrane-bound proteinase

Human red blood cells anion exchange protein (band 3) exposed to peroxyl radicals produced by thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through band 3 was smaller indicating that removal achieved by proteinases includes oxidized band 3 which still retain transport ability. Proteinases recognize band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that band 3 aggregates removal may prevent macrophage recognition of the senescent condition which would lead to cell disposal.     

Volume-activated taurine permeability in cells of the human erythroleukemic cell line K562

The effects of hypotonic shock on cell volume, taurine influx and efflux were examined in the human erythroleukemic cell line K562. Cells exposed to hypotonic solutions exhibited a regulatory volume decrease (RVD) following rapid increases in cell volume. Cell swelling was associated with a increased taurine influx and efflux. The volume-activated taurine pathway was Na⁺-independent, and increased in parallel with increasing cell volume. The chloride channel blocker, 2,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), completely blocked the volume-activated taurine influx and efflux, while [dihydroin-denyl]oxy]alkanoic acids (DIOA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an anion exchanger and anion channel blocker, respectively, also inhibited significantly. These results suggest that taurine transport is increased in response to hypotonic stress, which may be mediated via a volume-activated, DCDPC-sensitive anion channel. © 1996 Wiley-Liss, Inc.     

New insights into the P-glycoprotein-mediated effluxes of rhodamines.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma drug transporter P-glycoprotein (P-gp). This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P-gp transports such a wide diversity of compounds across the membrane. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. The aim of the present study was to further characterize the transport of several rhodamine analogues, either positively charged or zwitterionic. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow-cytometric analysis of dye accumulation in the wild-type drug sensitive K562 that do not express P-gp and its MDR subline that display high levels of MDR. The measurements were made in real time using intact cells. The kinetic parameter, ka = VM/km, which is a measure of the efficiency of the P-gp-mediated efflux of a substrate was similar for almost all the rhodamine analogues tested. In addition these values were compared with those determined previously for the P-gp-mediated efflux of anthracycline. Our conclusion is that the compounds of these two classes of molecules, anthracyclines and rhodamines, are substrates of P-gp and that their pumping rates at limiting low substrate concentration are similar. The findings presented here are the first to show quantitative information about the kinetic parameters for P-gp-mediated efflux of rhodamine analogues in intact cells.     

Anion transport in single erythrocyte ghosts measured by fluorescence microphotolysis

Fluorescence microphotolysis was used to measure in single resealed human erythrocyte ghosts the band 3-mediated transport of the fluorescent anion N-(7-nitrobenzofurazan-4-yl)-taurine (NBD-taurine). Transport was reduced to less than 5% of the control by the specific inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). The accuracy of the determination of the rate constant for NBD-taurine influx was approximately +/- 15% as calculated from repetitive measurements in individual ghosts. The sample population distribution of the rate constant was slightly skewed towards values larger than the mean. The rate of NBD-taurine transport showed an optimum near pH 7. The Arrhenius plot was linear in the range from 28.5 degrees C to 51 degrees C with an apparent activation enthalpy of 21.4 kcal/mol.     

Effect of phenols on natural killer (NK) cell-mediated death in the K562 human leukemic cell line

Cancer disease is a major cause of death in Western societies. Epidemiologically, antioxidant phenols have been associated with diminished incidence of cancer, while experimentally, they have cytotoxic effects on cancer cells. The aim of this study was to clarify whether natural antioxidant phenols render K562 human leukemic cells more susceptible to natural killer (NK) cell apoptosis and/or necrosis. K562 cells were pre-incubated with 7 different phenols (p-hydroxy benzoic acid, syringic acid, ferulic acid, p-coumaric acid, o-coumaric acid, gallic acid, and rutin) individually and afterwards targeted with NK cells at a ratio 1/5. Percentages of apoptotic and necrotic cells were assayed via flow cytometric analysis of annexin V and PI-stained cells. For the morphological assessment, cells were stained with acridine orange and ethidium bromide and were examined under a fluorescence microscope. Pre-treatment with gallic acid significantly rendered K562 cells more susceptible to NK cell-mediated necrosis, while pre-treatment with rutin significantly rendered K562 cells more susceptible to apoptosis. Gallic acid and rutin exert anticarcinogenic activity via the enhancement of K562 cell susceptibility to NK cell-mediated necrosis and apoptosis, respectively.     

The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: II. Kinetic properties of NBD-taurine transfer in asymmetric conditions

The transport of inorganic anions across human red blood cell membranes is accomplished by a carrier-like mechanism which involves an electroneutral and obligatory one-for-one anion exchange. The transport kinetics were described by models that involve alternation of single transport sites between the two membrane surfaces. These models predict that each carrier shows either an inward-facing Ei or an outward-facing Eo, conformation, each capable of binding either a monovalent anion or a divalent anion + a proton, to yield an electroneutral translocating complex. Unidirectional transport rates provide, therefore, a measure for the relative concentration of carriers at a given membrane surface. In the present work we assessed how modulation of the transmembrane distribution of carriers by the anion composition of cells and media, and by pH, affect the anion transport system. We have set the system in asymmetric conditions with respect to anions, so that a fast transportable anion (e.g., chloride) was present in one side of the membrane and slow transportable anions (e.g., sulfate, phosphate, oxalate, isethionate, gluconate, HEPES) were present on the other side of the membrane. The skewed distribution of carriers induced in these conditions were assessed by two methods: 1) NBD-taurine transfer which provided a measure for [Ei], the monovalent inward-facing form of the carrier, and 2) inhibition of NBD-taurine transfer by the specific impermeant and competitive inhibitor 4,4'-dinitro-2,2'-stilbene disulfonic acid (DNDS), which provided a measure for the availability of the carrier at the outer membrane surface. In the various symmetric and asymmetric conditions, we found marked differences in transport rates and transport profiles as well as in the susceptibility of the system to inhibition by DNDS. Direct binding studies of DNDS to cells in the various asymmetric conditions supported the conclusion derived from transport studies that transport sites can be recruited towards the membrane surface facing the slow transportable anions.     

Spontaneous mitochondrial membrane potential change during apoptotic induction by quercetin in K562 and K562/adr cells

Natural products from plants such as flavonoids are potential drugs to overcome multidrug resistance (MDR) in cancer treatments. However, their modes of action are still unclear. In this study, the effects of quercetin on mitochondrial membrane potential (DeltaPsim) change as well as quercetin's ability to induce apoptosis and inhibit Pgp-mediated efflux of 99mTc-MIBI in K562/adr cells were investigated. Quercetin exhibits cytotoxicity against erythroleukemic cells: IC50 are 11.0 +/- 2.0 micromol/L and 5.0 +/- 0.4 micromol/L for K562 and K562/adr, respectively. Quercetin induces cell death via apoptosis in both K562 and K562/adr cells and does not inhibit Pgp-mediated efflux of 99mTc-MIBI. Quercetin (10 micromol/L, 3 h) and etoposide (100 micromol/L, 24 h) induce similar levels of apoptosis in K562 and K562/adr cells. Quercetin induces an increase followed by a decrease in |DeltaPsim| value depending on its concentration. A decrease in the |DeltaPsim| value is associated with an increase in the percentage of early apoptotic cells. It is clearly shown that quercetin results in a spontaneous DeltaPsim change during apoptotic induction. Therefore, quercetin is potentially an apoptotic-inducing agent, which reacts at the mitochondrial level.     

Probenicid inhibition of fluorescence extrusion after MCB-staining of rat-1 fibroblasts

The intracellular fluorescence level of cells stained continuously with monochlorobimane was monitored by flow cytometry in order to assess the initial rate of glutatione to monochlorobimane conjugation as a measure of glutathione S-transferase activity. In addition to a rapid initial increase and a plateau level, a decline in fluorescence intensity was found upon prolonged flow cytometric monitoring. Exposure to probenicid, an inhibitor of an ATP-dependent organic anion pump, prevented this decrease. Incubation with vanadate and verapamil was without effect. Thus, extrusion of fluorescentglutathione-conjugate perturbs the proportionality between initial glutathione level and monochlorobimane-dependent fluorescence intensity. Monitoring by flow cytometry the decrease in monochlorobimane-dependent fluorescence may be useful to detect multidrug resistant cells.     

Altered structure and anion transport properties of band 3 (AE1, SLC4A1) in human red cells lacking glycophorin A

We have studied the properties of band 3 in different glycophorin A (GPA)-deficient red cells. These red cells lack either both GPA and glycophorin B (GPB) (M(k)M(k) cells) or GPA (En(a-) cells) or contain a hybrid of GPA and GPB (MiV cells). Sulfate transport was reduced in all three red cell types to approximately 60% of that in normal control red cells as a result of an increased apparent K(m) for sulfate. Transport of the monovalent anions iodide and chloride was also reduced. The reduced iodide transport resulted from a reduction in the V(max) for iodide transport. The anion transport site was investigated by measuring iodide fluorescence quenching of eosin-5-maleimide (EMA)-labeled band 3. The GPA-deficient cells had a normal K(d) for iodide binding, in agreement with the unchanged K(m) found in transport studies. However, the apparent diffusion quenching constant (K(q)) was increased, and the fluorescence polarization of band 3-bound EMA decreased in the variant cells, suggesting increased flexibility of the protein in the region of the EMA-binding site. This increased flexibility is probably associated with the decrease in V(max) observed for iodide transport. Our results suggest that band 3 in the red cell can take up two different structures: one with high anion transport activity when GPA is present and one with lower anion transport activity when GPA is absent.     

Characterization of regulatory volume decrease in the THP-1 and HL-60 human myelocytic cell linesn

Exposure to hypotonic stress produces a transient increase in cell volume followed by a regulatory volume decrease (RVD) in both THP-1 and HL-60 cells. In contrast, cells exposed to hypotonic stress in a high K/low Na Hanks' solution not only failed to volume regulate, but displayed a secondary swelling. Thus, while an outward K gradient was required ful KVD, the secondary swelling indicated that hypotonic stress increased permeability in the absence of a negative membrane potential. The K channel blocker quinine (1–4 mM) blocked RVD in both cell types. Gramicidin's ability to overcome the quinine block of RVD indicated that RVD is mediated by a quinine-sensitive cation transport mechanism that is independent of the swelling-induced anion transport mechanism. Barium (1–4 mM), another K channel blocker, slowed the rate of RVD, while 4-aminopyridine, charybdotoxin, tetraethylammonium chloride, tetrabutylammonium chloride, and gadolinium had no effect on RVD. Furthermore, RVD was not mediated by calcium-activated conductances, since it occurred normally in Ca-free medium, in medium containing cadmium, and in BAPTA-loaded cells. Gramicidin produced little or no volume change in isotonic medium, suggesting that basal C1 permeability of both THP-1 and HL-60 cells is low. However, swelling induced an anion efflux pathway that is permeable to both chloride and bromide, but is impermeable to methanesulfonate and glutamate. The anion channel blocker 3,5-diiodosalicylic acid (DISA) antagonized RVD in both cell types. In conclusion, RVD in THP-1 and HL-60 cells is mediated by independent anion and cation transport mechanisms that involve both a DISA-sensitive anion pathway and a quinine-inhibitable K efflux pathway, neither of which requires increases in intra-cellular calcium to be activated. © 1994 wiley-Liss, Inc.