Initial Efflux Rate Research Articles

Effects of the chemosensitizer verapamil on P-glycoprotein substrate efflux in rainbow trout hepatocytes

The ATP-dependent membrane transporter P-glycoprotein (P-gp) is associated with resistance to a wide variety of chemical substrates, as well as the multi-drug resistance (MDR) phenotype in mammals. Less is known regarding P-gp's function and relevance in teleosts; this study expanded the range of known substrates and the inhibitory effects of a model chemosensitizer verapamil. The P-gp-mediated uptake and efflux dynamics of 5 known mammalian substrates (berberine, cortisol, doxorubicin, rhodamine 123 [R123], and vinorelbine) were examined in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes with and without co-exposure to varying doses of verapamil. Initial substrate uptake rates (pmol/106 cells/min) varied widely and were in order: berberine (482 ± 94) > R123 (364 ± 67) > doxorubicin (158 ± 41) > cortisol (20.3 ± 5.9) > vinorelbine (15.3 ± 3.5). Initial efflux rates (pmol/106 cells/min) were highest in berberine (464 ± 110) > doxorubicin (341 ± 57) > R123 (106 ± 33) > cortisol (26.6 ± 6.1) > vinorelbine (9.0 ± 2.4). Transport of vinorelbine and R123 is verapamil sensitive, but verapamil had no effect on transport of berberine, cortisol, or doxorubicin. Cortisol and doxorubicin showed evidence of high P-gp affinity, thus displacing verapamil from their shared P-gp binding site. Cortisol, doxorubicin, R123, and vinorelbine transport by rainbow trout P-gp was confirmed, while berberine could not be confirmed or excluded as a substrate. Binding sites and affinities were similar between mammalian and trout P-gp for doxorubicin, R123, and vinorelbine, while fish P-gp had a higher affinity for cortisol than mammalian P-gp. This study demonstrated that the range of substrates, as well as binding sites and affinities, of fish P-gp are well-aligned with those in mammals.

Kinetic Effects of Inside‐Positive Membrane Potential on the Lactose Efflux of LacY

LacY, the lactose permease of E. coli., utilizes the free energy released from the downhill movement of H+ in response of an electrochemical H+ gradient (Δμ̃H+, interior negative and/or alkaline) to catalyze the accumulation of lactose against a concentration gradient. In the absence of Δμ̃H+, the downhill transport of lactose in response of a concentration gradient drives uphill translocation of H+ to generate a H+ gradient, and the polarity of the H+ gradient depends on the direction of lactose gradient. In other words, LacY is a lactose/H+ symporter, in which the lactose transport and H+ translocation are obligatorily coupled. LacY functions as a monomer with a total of 12 transmembrane helices arranged into two 6‐helix bundles tethered by a long loop between helix VI and VII. The sugar‐ and H+‐binding sites are located at approximate the center of the permease and can be accessed from either side of the membrane via an alternating access conformational change driven by the binding and dissociation of sugar. In LacY, Glu325 plays an essential role in H+ translocation since replacing Glu325 with neutral amino residues abolishes transport activities involving H+ translocation, but the mutants still bind sugar with normal affinity and catalyze equilibrium exchange. Previous studies have shown that the presence of Δμ̃H+ (interior negative) significantly accelerates lactose influx by decreasing the Km 50‐100‐fold with no change in Kd. The goal of this study is to investigate kinetic effects of Δμ̃H+ on lactose efflux in right‐side‐out membrane vesicles. We measured the initial rate of efflux at different pHs in the absence or presence of an imposed membrane potential (ΔΨ, generated by K+ influx in response of a concentration gradient in the presence of valinomycin, interior positive). The results revealed that the lactose efflux of WT LacY exhibits an apparent pK value near 7.2, which is not affected by the imposition of ΔΨ (interior positive). The initial rate of lactose efflux increases in the presence of imposed ΔΨ (interior positive) at all tested pHs, although enhancement is about two orders of magnitude less than that observed in lactose influx. Moreover, the effect of ΔΨ (interior positive) on the Km of lactose efflux is almost negligible compared to the dramatic change of the Km of lactose influx caused by ΔΨ (interior negative). In contrast to WT LacY, the E325A mutant does not carry out lactose efflux in the absence or presence of imposed ΔΨ (interior positive) at all tested pHs.Support or Funding InformationNSF Eager Grant MCB‐1547801 to H.R.K., NIH Grant 1R01 GM120043 to H.R.K., and a grant from Ruth and Bucky Stein to H.R.K..

Dopamine transporter phosphorylation site threonine 53 is stimulated by amphetamines and regulates dopamine transport, efflux, and cocaine analog binding

The dopamine transporter (DAT) controls the spatial and temporal dynamics of dopamine neurotransmission through reuptake of extracellular transmitter and is a target for addictive compounds such as cocaine, amphetamine (AMPH), and methamphetamine (METH). Reuptake is regulated by kinase pathways and drug exposure, allowing for fine-tuning of clearance in response to specific conditions, and here we examine the impact of transporter ligands on DAT residue Thr-53, a proline-directed phosphorylation site previously implicated in AMPH-stimulated efflux mechanisms. Our findings show that Thr-53 phosphorylation is stimulated in a transporter-dependent manner by AMPH and METH in model cells and rat striatal synaptosomes, and in striatum of rats given subcutaneous injection of METH. Rotating disc electrode voltammetry revealed that initial rates of uptake and AMPH-induced efflux were elevated in phosphorylation-null T53A DAT relative to WT and charge-substituted T53D DATs, consistent with functions related to charge or polarity. These effects occurred without alterations of surface transporter levels, and mutants also showed reduced cocaine analog binding affinity that was not rescued by Zn2+ Together these findings support a role for Thr-53 phosphorylation in regulation of transporter kinetic properties that could impact DAT responses to amphetamines and cocaine.

INJECTION PROPAGATION MODEL IN SANDY SOIL

This articles focuses on the process of injection solution in the sand as physical phenomena. During theoretical studies of the propagation of the fluid phase in a porous medium, actual is the definition of the parameters of the jet, necessary for layer material discontinuity in the contact zone of the jet to the surface. It can be achieved through the injection process modeling in a dispersion medium by constructing geometrical similarity of injection jet. It is found that the propagation of the fluid phase in the solid body pores suggests that as they reach the point at the distance from the injector to the «end» of the jet in pores pre-saturated by water, gradual change in injection rate depending on the initial rate of efflux and viscosity occurs.

Folates provoke cellular efflux and drug resistance of substrates of the multidrug resistance protein 1 (MRP1)

Cellular folate concentration was earlier reported to be a critical factor in the activity and expression of the multidrug resistance protein MRP1 (ABCC1). Since MRP1 mediates resistance to a variety of therapeutic drugs, we investigated whether the cellular folate concentration influences the MRP1-mediated cellular resistance against drugs. As a model system, we used the human ovarian carcinoma cell line 2008wt, and its stably MRP1/ABCC1-transfected subline 2008/MRP1. These cell types have a moderate and high expression of MRP1, respectively. In folate-deprived 2008/MRP1 cells, the MRP1-mediated efflux of its model substrate calcein decreased to ~55 % of the initial efflux rate under folate-rich conditions. In 2008wt cells, only a small decrease in efflux was observed. Folate depletion for 5-10 days markedly increased (~500 %) cellular steady-state accumulation of calcein in 2008/MRP1 cells and moderately in 2008wt cells. A subsequent short (24 h) exposure to 2.3 μM L-leucovorin decreased calcein levels again in MRP1-overexpressing cells. Folate deprivation markedly increased growth inhibitory effects of the established MRP1 substrates daunorubicin (~twofold), doxorubicin (~fivefold), and methotrexate (~83-fold) in MRP1-overexpressing cells, proportional to MRP1 expression. In conclusion, this study demonstrates that increased cellular folate concentrations induce MRP1/ABCC1-related drug efflux and drug resistance. These results have important implications in the understanding of the role of MRP1 and its homologs in clinical drug resistance.

Abstract 061: Interstitial Matrix Flow-Regulation:Myocardial Fibroblasts Respond to Colloidosmotic Pressure

Ischemia-reperfusion injury induces large differences in hydration potential between ischemic and nonischemic areas of the myocardium. They influence the rate and volume of fluid transfer in myocardial explants; temperature-dependent hydration-potential differences of approximately 100 mmHg in at-risk areas, as opposed to contiguous areas that are not at risk, suggest prompt interstitial fluid-transfer control mechanisms (Circ. Res . 2012;11:A235) . Aim: We adapted osmotic-stress techniques to determine whether myocardial fibroblasts, which are known to respond to mechanical and flow signals, also respond to hydration-potential changes. Methods: Fibroblasts were isolated from midwall regions of the left ventricles of healthy pigs using standard procedures. They were incorporated into 3-dimensional collagen gels of 500 mm 3 volume at 150 cells/mm 3 and equilibrated overnight in cultures using the nontoxic, inert polymer polyethylene glycol 8000 (molecular radius ~26.5 Å; concentration range 0-10% w/w) to adjust colloidosmotic pressure from approximately 5 to 205 mmHg . After the gels were detached from the dish, fluid flux was derived from any time-dependent changes in their dimensions. Results: The volume of gels without fibroblasts did not change significantly at any coloidosmotic pressure. In those with fibroblasts, the volume decreased, more slowly in those subjected to higher-than-plasma pressure levels. Progression curves conformed well to a two-exponential term model (R 2 >0.9) suggesting that two parallel processes contribute to matrix efflux, one relatively fast, with decay constant 0.274 ± 0.014 (n = 3) , and another ~50-fold slower. Initial rates were calculated from the fitted curves, and linear regression analysis used to examine their dependence on colloidosmotic pressure. Initial efflux rates decreased with pressure, mean slope, - 0.29 ± 0.08 µl/h/mmHg (R 2 = 0.7; P-value = 0.006). Conclusion: In vitro , fibroblasts in collagen matrices regulate fluid efflux in response to colloidosmotic stresses within the range of hydration-potential differences measured in myocardial explants after ischemia-reperfusion injury.

Activity of tonoplast proton pumps and Na+/H+ exchange in potato cell cultures is modulated by salt

The efficient exclusion of excess Na from the cytoplasm and vacuolar Na(+) accumulation are the main mechanisms for the adaptation of plants to salt stress. This is typically carried out by transmembrane transport proteins that exclude Na(+) from the cytosol in exchange for H(+), a secondary transport process which is energy-dependent and driven by the proton-motive force generated by plasma-membrane and tonoplast proton pumps. Tonoplast enriched-vesicles from control and 150 mM NaCl-tolerant calli lines were used as a model system to study the activity of V-H(+)-PPase and V-H(+)-ATPase and the involvement of Na(+) compartmentalization into the vacuole as a mechanism of salt tolerance in Solanum tuberosum. Both ATP- and pyrophosphate (PP(i))-dependent H(+)-transport were higher in tonoplast vesicles from the salt-tolerant line than in vesicles from control cells. Western blotting of tonoplast proteins confirmed that changes in V-H(+)-PPase activity are correlated with increased protein amount. Conversely, immunodetection of the A-subunit of V-H(+)-ATPase revealed that a mechanism of post-translational regulation is probably involved. Na(+)-dependent dissipation of a pre-established pH gradient was used to measure Na(+)/H(+) exchange in tonoplast vesicles. The initial rates of proton efflux followed Michaelis-Menten kinetics and the V(max) of proton dissipation was 2-fold higher in NaCl-tolerant calli when compared to the control. H(+)-coupled exchange was specific for Na(+) and Li(+) and not for K(+). The increase of both the pH gradient across the tonoplast and the Na(+)/H(+) antiport activity in response to salt strongly suggests that Na(+) sequestration into the vacuole contributes to salt tolerance in potato.

Differences in trans‐stimulated chloroquine efflux kinetics are linked to PfCRT in Plasmodium falciparum

The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum has remained controversial. Currently discussed models include a carrier or a channel for chloroquine, the former actively expelling the drug, the latter facilitating its passive diffusion, out of the parasite's food vacuole, where chloroquine accumulates and inhibits haem detoxification. Here we have challenged both models using an established trans-stimulation efflux protocol. While carriers may demonstrate trans-stimulation, channels do not. Our data reveal that extracellular chloroquine stimulates chloroquine efflux in the presence and absence of metabolic energy in both chloroquine-sensitive and -resistant parasites, resulting in a hyperbolic increase in the apparent initial efflux rates as the concentration of external chloroquine increases. In the absence of metabolic energy, the apparent initial efflux rates were comparable in both parasites. Significant differences were only observed in the presence of metabolic energy, where consistently higher apparent initial efflux rates were found in chloroquine-resistant parasites. As trans-stimulation is characteristic of a carrier, and not a channel, we interpret our data in favour of a carrier for chloroquine being present in both chloroquine-sensitive and -resistant parasites, however, with different transport modalities.

Protein-protein interactions and membrane localization of the human organic solute transporter

Two proteins that mediate bile acid export from the ileal enterocyte, organic solute transporter (OST)-alpha and -beta, have recently been identified. It is unclear whether these two proteins associate directly and how they interact to mediate transport function and membrane localization. In this study, the protein-protein interactions, transport functions, and membrane localization of human (h)OST-alpha and -beta proteins were examined. The results demonstrated that coexpression of hOST-alpha and -beta in transfected cells resulted in a three- to fivefold increase of the initial rate of taurocholate influx or efflux compared with cells expressing each protein individually and nontransfected cells. Confocal microscopy demonstrated plasma membrane colocalization of hOST-alpha and -beta proteins in cells cotransfected with hOST-alpha and -beta cDNAs. Protein-protein interactions between hOST-alpha and -beta were demonstrated by mammalian two-hybrid and coimmunoprecipitation analyses. Truncation of the amino-terminal 50 amino acid extracellular residues of hOST-alpha abolished its interaction with hOST-beta and led to an intracellular accumulation of the two proteins and to only background levels of taurocholate transport. In contrast, carboxyl-terminal 28 amino acid truncated hOST-alpha still interacted with hOST-beta, and majority of this cytoplasmic tail-truncated protein was expressed on the basolateral membrane when it was stably cotransfected with hOST-beta protein in Madin-Darby canine kidney cells. In summary, hOST-alpha and -beta proteins are physically associated. The intracellular carboxyl-terminal domain of hOST-alpha is not essential for this interaction with hOST-beta. The extracellular amino-terminal fragment of hOST-alpha may contain important information for the assembly of the heterodimer and trafficking to the plasma membrane.

Ion Specificity and Ionic Strength Dependence of the Osmoregulatory ABC Transporter OpuA

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624-10629). C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuADelta12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was approximately 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg(2+) and Ba(2+)) were more effective in activating OpuA than NH(4)(+), K(+), Na(+), or Li(+), consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb(+) and Cs(+) were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb(+) and Cs(+) were no longer inhibitory in OpuADelta12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuADelta12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.

Alterations in respiration rate of isolated rainbow trout hepatocytes exposed to the P-glycoprotein substrate rhodamine 123

Reducing intracellular xenobiotic concentration is an important defence strategy used by cells challenged with foreign chemicals. One mechanism used to achieve this goal is via the use of P-glycoproteins (P-gps), ATP-dependent transporters that mediate the removal of hydrophobic compounds from cells. The energetic costs of this mechanism are unknown, therefore, the activity and respiratory costs associated with the P-gp-mediated efflux of rhodamine 123 (R123) was measured in isolated rainbow trout hepatocytes. The accumulation of R123 was rapid and concentration-dependent. Initial accumulation rates were 1.79 ± 0.41, 7.29 ± 1.06 and 15.30 ± 1.74 ng R123/min/10 6 cells when exposed to 1, 5 and 10 μM R123, respectively. Efflux was measured in cells ‘pre-loaded’ with R123 at each concentration, resulting in initial efflux rates of 0.77 ± 0.12, 2.02 ± 0.35 and 3.51 ± 0.84 ng R123/min/10 6 cells, respectively. The baseline oxygen consumption rate of hepatocytes was 33.21 ± 1.09 ng O 2/min/10 6 cells. Respiration rates were significantly higher in cells exposed to 5 and 10 μM R123 (39.08 ± 0.80 and 41.72 ± 0.61 ng O 2/min/10 6 cells), representing increases over basal rates of 18.5 and 25.7%, respectively. Measurements of isolated mitochondrial respiration established that changes in hepatocyte oxygen consumption were not through the direct effects of R123 on mitochondria. The P-gp inhibitor, XR9576 significantly inhibited R123 efflux from cells with a concomitant return of respiration rates to baseline values. This study demonstrates that increased P-gp transport of xenobiotics can significantly raise cellular respiration rates and may result in higher energy costs for organisms living in P-gp-substrate contaminated environments.

Evaluation of brain mitochondrial glutamate and α‐ketoglutarate transport under physiologic conditions

Some models of brain energy metabolism used to interpret in vivo (13)C nuclear magnetic resonance spectroscopic data assume that intramitochondrial alpha-ketoglutarate is in rapid isotopic equilibrium with total brain glutamate, most of which is cytosolic. If so, the kinetics of changes in (13)C-glutamate can be used to predict citric acid cycle flux. For this to be a valid assumption, the brain mitochondrial transporters of glutamate and alpha-ketoglutarate must operate under physiologic conditions at rates much faster than that of the citric acid cycle. To test the assumption, we incubated brain mitochondria under physiologic conditions, metabolizing both pyruvate and glutamate and measured rates of glutamate, aspartate, and alpha-ketoglutarate transport. Under the conditions employed (66% of maximal O(2) consumption), the rate of synthesis of intramitochondrial alpha-ketoglutarate was 142 nmol/min.mg and the combined initial rate of alpha-ketoglutarate plus glutamate efflux from the mitochondria was 95 nmol/min.mg. It thus seems that much of the alpha-ketoglutarate synthesized within the mitochondria proceeds around the citric acid cycle without equilibrating with cytosolic glutamate. Unless the two pools are in such rapid exchange that they maintain the same percent (13)C enrichment at all points, (13)C enrichment of glutamate alone cannot be used to determine tricarboxylic acid cycle flux. The alpha-ketoglutarate pool is far smaller than the glutamate pool and will therefore approach steady state faster than will glutamate at the metabolite transport rates measured.

An apical K+-dependent HCO3− transport pathway opposes transepithelial HCO3− absorption in rat medullary thick ascending limb

Absorption of HCO(3)(-) in the medullary thick ascending limb (MTAL) is mediated by apical membrane Na(+)/H(+) exchange. The identity and function of other apical acid-base transporters in this segment have not been defined. The present study was designed to examine apical membrane HCO(3)(-)/OH(-)/H(+) transport pathways in the rat MTAL and to determine their role in transepithelial HCO(3)(-) absorption. MTALs were perfused in vitro in Na(+)- and Cl(-)-free solutions containing 25 mM HCO(3)(-), 5% CO(2). Lumen addition of either 120 mM Cl(-) or 50 mM Na(+) (50 microM EIPA present) had no effect on intracellular pH (pH(i)). Lumen Cl(-) addition also had no effect on pH(i) in the presence of 145 mM Na(+) or in the nominal absence of HCO(3)(-)/CO(2). Thus there was no evidence for apical Cl(-)/HCO(3)(-) (OH(-)) exchange, Na(+)-dependent Cl(-)/HCO(3)(-) exchange, or Na(+)-HCO(3)(-) cotransport. In contrast, in tubules studied in Na(+)- and Cl(-)-free solutions containing 25 mM HCO(3)(-), 5% CO(2) and 120 mM K(+), removal of luminal K(+) induced a rapid and pronounced decrease in pH(i) (DeltapH(i) = 0.56 +/- 0.06 pH U). pH(i) recovered following lumen K(+) readdition. The initial rate of net base efflux induced by lumen K(+) removal was decreased 85% at the same pH(i) in the nominal absence of HCO(3)(-)/CO(2), indicating a dependence on HCO(3)(-)/CO(2) and arguing against apical K(+)/H(+) exchange. A combination of the apical K(+) channel blockers quinidine (0.1 mM) and glybenclamide (0.25 mM) had no effect on the lumen K(+)-induced pH(i) changes, arguing against electrically coupled K(+) and HCO(3)(-) conductances. The effect of lumen K(+) on pH(i) was inhibited by 1 mM H(2)DIDS. In addition, lumen addition of DIDS increased transepithelial HCO(3)(-) absorption from 10.7 +/- 0.7 to 14.9 +/- 0.7 pmol x min(-1) x mm(-1) (P < 0.001) and increased pH(i) slightly in MTAL studied in physiological solutions (25 mM HCO(3)(-) and 4 mM K(+)). Lumen DIDS stimulated HCO(3)(-) absorption in the absence and presence of furosemide. These results are consistent with an apical membrane K(+)-dependent HCO(3)(-) transport pathway that mediates coupled transfer of K(+) and HCO(3)(-) from cell to lumen in the MTAL. This mechanism, possibly an apical K(+)-HCO(3)(-) cotransporter, functions in parallel with apical Na(+)/H(+) exchange and opposes transepithelial HCO(3)(-) absorption.

Folate concentration dependent transport activity of the Multidrug Resistance Protein 1 (ABCC1)

The Multidrug Resistance Protein MRP1 (ABCC1) can confer resistance to a variety of therapeutic drugs. In addition, MRP1/ABCC1 mediates cellular export of natural folates, such as folic acid and l-leucovorin. In this study we determined whether cellular folate status affected the functional activity of MRP1/ABCC1 mediated efflux of an established substrate, the anthracycline daunorubicin (DNR). As a model system we used the human ovarian carcinoma cell line 2008wt, and its MRP1/ABCC1 transfected subline 2008/MRP1. Both types of these moderate- and high-MRP1/ABCC1 expressing cells displayed efflux of DNR when maintained in standard culture media (2.3 μM folic acid). The initial total cellular DNR efflux rate in 2008/MRP1 cells was ∼2-fold higher compared to 2008wt cells. This efflux consisted of MRP1/ABCC1 mediated transport, possibly non-MRP1 mediated transport, as well as passive diffusion. Benzbromarone, a specific MRP1 inhibitor, decreased the initial efflux rate in 2008/MRP1 cells (4-fold) and in 2008wt cells (2-fold). When 2008/MRP1 cells were challenged for 2 days in folate-free medium, total cellular DNR efflux was decreased to 43% of the initial efflux rate under folate-rich conditions. In 2008wt cells DNR efflux was decreased to 84% of the folate-rich conditions. Benzbromarone did not inhibit DNR efflux after the folate-free period in both cell lines. Repletion of folate by a 2–24 hr exposure to 2.5 μM l-leucovorin or folic acid resulted in a complete restoration of DNR efflux. In contrast, expression of MRP1/ABCC1 protein was not changed significantly during the folate-free period or the repletion-period, nor were cellular ATP or ADP pools. In conclusion, this study demonstrates that the cellular folate status can influence the transport activity of MRP1/ABCC1. These results have potentially important implications in the understanding of the (patho-)physiological roles of MRP1/ABCC1, and possibly other ABC transporter proteins in cellular folate homeostasis and drug resistance.

Cross-resistance to cisplatin in cells with acquired resistance to copper.

Cells selected for resistance to cisplatin (DDP) demonstrate cross-resistance to copper (Cu) suggesting one or more common mechanisms of cellular defense. We sought to determine whether cells selected for resistance to Cu are cross-resistant to DDP. Parental HuH7 human hepatoma cells and the CuR27 Cu-resistant subline were compared for sensitivity to Cu and DDP by clonogenic assay, and with respect to drug uptake and efflux by measuring cellular Cu and Pt content. CuR27 cells were found to be 1.8-fold resistant to Cu and 8.6-fold cross-resistant to DDP. Changes in the cellular pharmacokinetics of Cu in the CuR27 cells were paralleled by changes in the kinetics of DDP. The accumulations of Cu and DDP measured at 1 min were, respectively, 36% and 26% of those in the parental HuH7 cells. The initial rate of efflux from the CuR27 cells was 6.2-fold faster for Cu and 2.5-fold faster for DDP than from the HuH7 cells. Cu reduced the accumulation of DDP in the HuH7 cells in a concentration-dependent manner and vice versa. DDP also reduced the efflux of Cu. Western blot analysis demonstrated that expression of the Cu exporter ATP7B was increased 3.9-fold in the CuR27 cells. In this model system, cross-resistance between Cu and DDP was bidirectional and accompanied by parallel changes in the cellular pharmacokinetics of both compounds. The results are consistent with the idea that transporters and chaperones that normally mediate Cu homeostasis also directly or indirectly modulate the accumulation of DDP.

Kinetic analysis about the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) by isolated rat hepatocytes.

The purpose of the present study was to investigate the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) using isolated rat hepatocytes. The initial uptake rate of ANS by isolated hepatocytes was determined. The uptake process of ANS was saturable, with a Km of 29.1 +/- 3.2 microM and Vmax of 2.9 +/- 0.1 mmol/min/mg protein. Subsequently, the initial efflux rate of ANS from isolated hepatocytes was determined by resuspending preloaded cells to 3.0% (w/v) BSA buffer. The efflux process for total ANS revealed a little saturability. The mean value of the efflux clearance was 2.2 +/- 0.1 microL/min/mg protein. The efflux rate of ANS from hepatocytes was markedly decreased at 4 degrees C, indicating that the apparent efflux of ANS might not be attributed to the release of ANS bound to the cell surface, but to the efflux of ANS from intracellular space. The efflux clearance was furthermore corrected for the unbound intracellular ANS concentration on the basis of its binding parameters to cytosol. The relation between efflux rate and unbound ANS concentration was fitted well to the Michaelis-Menten equation with a saturable and a nonsaturable components. The Vmax and Km values were 0.54 mmol/min/mg protein, and 10.0 microM, respectively. Based on the comparison of the ratios of Vmax to Km (Vmax/Km) corresponding to the transport clearance, the influx clearance was two times higher than the efflux clearance. Together with our preliminary studies that ATP suppression in hepatocytes substantially inhibited ANS influx rate, we concluded that the hepatic uptake of ANS is actively taken up into hepatocytes via the carrier mediated transport system.

Regulatory volume decrease in Trypanosoma cruzi involves amino acid efflux and changes in intracellular calcium.

A regulatory volume decrease (RVD) in response to hyposmotic stress has been characterized in different life-cycle stages of Trypanosoma cruzi. Hyposmotic stress initially caused swelling, but this was rapidly reversed by a compensatory volume reversal that was essentially complete by 5 min. Volume recovery was associated with an amino acid efflux that accounted for approximately 50% of the regulatory volume decrease in all three life-cycle stages. The amino acid efflux was selective for neutral and anionic amino acids, but excluded cationic amino acids. Acidocalcisomes contained an amino acid pool over four times more concentrated than whole-cell levels, but about 90% of this was composed of Arg and Lys, so involvement of this pool in amino acid efflux was ruled out. Hyposmotic stress induced a rise in intracellular calcium that was dependent on influx of calcium across the plasma membrane, since chelation of extracellular calcium abolished the response. Influx of calcium was confirmed by demonstration of manganese-mediated quenching of intracellular fura-2 fluorescence and partial inhibition of the rise in calcium by calcium channel blockers. Manipulation of intra- and extracellular calcium levels had minor effects on the initial rate of amino acid efflux and no effect on the rate of volume recovery.

Efflux system for pyridoxine in Schizosaccharomyces pombe.

Pyridoxine-charged Schizosaccharomyces pombe released pyridoxine rapidly at 30 degrees C: very low amounts of three other B6 vitamers were also released. The rate of efflux was temperature-dependent. The initial rate of efflux was dependent on the concentration of pyridoxine in the cells: the rate was almost zero at lower than 0.02 mM and became saturated at higher than 0.2 mM. Na+, sodium azide, and dinitrophenol increased the rate in both the presence and absence of D-glucose. Mg++, thiamine, and menadione inhibited the efflux. The intracellular concentration of ATP did not significantly affect the efflux rate. The system may be dependent on a membrane potential of the yeast cells. It was found that the fission yeast cells have a gate or carrier system for efflux of pyridoxine, which was distinct from that in Saccharomyces cerevisiae.

Monitoring drug efflux from sensitive and multidrug-resistant single cancer cells with microvoltammetry.

Multidrug resistance (MDR) is the eventual cross-resistance of certain cancer cells to a series of chemically unrelated drugs. It is attributed to a number of possible biophysical processes, one of them being increased drug efflux from resistant cells which leads to a decreased intracellular drug accumulation and retention. In this work, a carbon fiber microdisk electrode was used to monitor directly doxorubicin efflux from single preloaded cancer cells. Electrochemical cleaning, adsorptive preconcentration, and an electrocatalytic effect due to ambient oxygen made it possible to detect eventually very low drug concentrations (down to 1 nM) at good temporal resolution (down to 30 s/measurement) very close (< or = 1 micron) to single cancer cells for the first time. The results from a sensitive (AUXB1) and a drug-resistant (CHRC5) version of Chinese hamster ovarian cancer cells show that resistant cells exhibit a much higher initial efflux rate and shorter efflux time constant when both cell lines are preloaded up to the same intracellular drug concentration. These observations are consistent with results obtained from populations of the same cells by conventional techniques, proving that microvoltammetry can be used to monitor doxorubicin efflux at the single-cell level. Compared with existing methodologies, however, whose data represent only average cell behavior at typically low temporal resolution, the technique described here can provide information on the microheterogeneity of cancer cell populations in terms of drug efflux at high temporal resolution. The actual driving force of efflux is obtained since concentrations are measured directly at individual cells. This approach may lead to important new information on the mechanisms and prospective treatments of MDR.

Copper resistant human hepatoblastoma mutant cell lines without metallothionein induction overexpress ATP7B.

Mutant human hepatoblastoma cell lines resistant to copper toxicity were isolated from mutagenized HuH7. Two copper resistant cell lines (CuR), CuR 23 and CuR 27, had reduced basal expression of metallothionein (MT) messenger RNA (mRNA) and exhibited minimal or no increase in resistance to cadmium or zinc toxicity. Copper uptake, efflux of newly transported copper, glutathione content, and efflux rate were comparable with HuH7, whereas holoceruloplasmin synthesis and secretion were slightly decreased. Subcellular distribution of copper at steady-state showed an increase in organelle and membrane fractions with a reduction in cytosol. Expression of ATP7B mRNA was fivefold increased, and ATP7B protein approximately threefold increased in both CuR 23 and 27. Another cell line, CuR 41, showed increased basal expression of MT and ATP7B mRNA but not ATP7B protein, and resistance to cadmium and zinc toxicity. Copper uptake in CuR 41 was comparable with HuH7, but initial rates of efflux of copper and glutathione were reduced. The synthesis of holoceruloplasmin but not ceruloplasmin peptide was markedly diminished in CuR 41. Subcellular distribution of copper showed an increase in cytosolic and decreased organelle and membrane-associated copper. These data suggest that cellular resistance to copper toxicity was achieved in two independent cell lines without MT induction and that the induction of ATP7B may lead to the enhanced intracellular sequestration of copper by organelles.