Identification of cholate as a shared substrate for the unidirectional efflux systems for methotrexate in L1210 mouse cells

The bidirectional transport properties of cholate have been examined in leukemic L1210 mouse cells and compared with the transport of methotrexate. The cell entry of [3H]cholate was Na(+)-independent, linear with increasing concentrations of substrate, enhanced by decreasing pH, and uneffected by excess unlabeled cholate or by various anion-transport inhibitors and hence had the characteristics of passive diffusion or a pH-dependent mediated process with a high Kt for cholate. The efflux of [3H]cholate, however, could be attributed to carrier-mediated and energy-dependent transport. Efflux was rapid (t1/2 = 1.5 min) and could be increased with glucose and decreased with metabolic inhibitors, and it was inhibited by various compounds including bromosulfophthalein, probenecid, prostaglandin A1, reserpine, verapamil, quinidine, diamide, 1-methyl-3-isobutylxanthine and vincristine. The most potent inhibitor was prostaglandin A1, which reduced efflux by 50% at a concentration of 0.10 microM. Half-maximal inhibition by vincristine occurred at 4.8 microM. The maximum extent of inhibition with most of the inhibitors was 95%, although a lower value was observed with bromosulfophthalein (85%). When cholate efflux was compared with the efflux of methotrexate, both processes responded similarly to changes in the metabolic state of the cell. Moreover, the various inhibitors of cholate efflux also inhibited the efflux of methotrexate and the same concentration of each inhibitor was required for half-maximal inhibition of both processes. The efflux of folate and urate also proceeded via outwardly directed, unidirectional processes which were sensitive to bromosulfophthalein and probenecid. The results suggest that L1210 cells have the capacity for the unidirectional extrusion of cholate, methotrexate and probably other large, structurally dissimilar organic anions and that this efflux occurs via two or more very similar transport systems with a broad anion specificity. The function of an organic anion efflux system in vivo may be to facilitate the extrusion of cytotoxic metabolic anions which are too large to exit via the general anion-exchange carrier of these cells. Similarities in inhibitor specificity were also apparent between unidirectional anion efflux in L1210 cells and the drug efflux pump which is over-produced in cells with multidrug resistance.     

HCO3− and CO2 leakage from Synechococcus UTEX 625

The leakage of various inorganic carbon species from air-grown cells of Synechococcus UTEX 625 was investigated after a light to dark transition or during a light period using a mass spectrometer under a wide variety of experimental conditions. Total inorganic carbon efflux and CO₂ efflux during the initial period of darkness were measured with or without carbonic anhydrase in the reaction medium respectively. The HCO₃^? efflux after a light to dark transition was estimated by difference. Carbon dioxide efflux in the light was measured by inhibiting CO₂ transport with either Na₂S or COS₃ or quenching the ¹³C inorganic carbon transport by the addition of ¹²C inorganic carbon in excess. In cells in which CO₂ fixation was inhibited, when only the HCO₃^? transport system was fully operative, CO₂ effluxed continuously during the light period at a rate equal to about 25% of that in darkness. When only the CO₂ transport system was operative, HCO₃^? effluxed during the light period. The difference between the light and dark efflux rates was consistent with a 0.6 unit decrease in the intracellular pH upon darkening the cells. The permeabilities of the cell for CO₂ (2.94 ± 0.14 ± 10^?8ms^?1; mean ± SE, n=137) and HCO₃^? (1.4–1.7 ± 10^?9 ms^?1) were calculated.     

Localization of Hydrogen Ion and Chloride Ion Fluxes in Nitella

Alternating bands of acid and base formation have been detected along the length of the internodal cell of Nitella clavata when it is illuminated, while in the dark this phenomenon is minimal. Chloride influx occurs only or largely in the acid-extruding regions, and this is also a light-dependent ion movement. Chloride efflux is slightly dependent on illumination and is not localized as are H⁺ efflux and Cl^- influx. The results obtained support Kitasato's (1968) proposal that a large passive H⁺ influx is balanced by an active efflux of this ion. Transport mechanisms suggested by the correlations of Cl^- and HCO₃^- influxes with H⁺ extrusion are discussed.     

Analysis of the inhibition potential of zosuquidar derivatives on selected bacterial and fungal ABC transporters

Abstract

The increasing number of multidrug-resistant pathogenic microorganisms is a serious public health issue. Among the multitude of mechanisms that lead to multidrug resistance, the active extrusion of toxic compounds, mediated by MDR efflux pumps, plays an important role. In our study we analyzed the inhibitory capability of 26 synthesized zosuquidar derivatives on three ABC-type MDR efflux pumps, namely Saccharomyces cerevisiae Pdr5 as well as Lactococcus lactis LmrA and LmrCD. For Pdr5, five compounds could be identified that inhibited rhodamine 6G transport more efficiently than zosuquidar. One of these is a compound with a new catechol acetal structure that might represent a new lead compound. Furthermore, the determination of IC₅₀ values for rhodamine 6G transport of Pdr5 with representative compounds reveals values between 0.3 and 0.9 μM. Thus the identified compounds are among the most potent inhibitors known for Pdr5. For the ABC-type efflux pumps LmrA and LmrCD from L. lactis, seven and three compounds, which inhibit the transport activity more than the lead compound zosuquidar, were found. Interestingly, transport inhibition for LmrCD was very specific, with a drastic reduction by one compound while its diastereomers showed hardly an effect. Thus, the present study reveals new potent inhibitors for the ABC-type MDR efflux pumps studied with the inhibitors of Pdr5 and LmrCD being of particular interest as these proteins are well known model systems for their homologs in pathogenic fungi and Gram-positive bacteria.     

Light-dependent bicarbonate uptake and CO2 efflux in the marine microalga Nannochloropsis gaditana

 Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO₂ fluxes in cell suspensions using mass spectrometry. Addition of H¹³CO₃ ⁻ to cell suspensions in the dark caused a transient increase in the CO₂ concentration in the medium far in excess of the equilibrium CO₂ concentration. The magnitude of this release was dependent on the length of time the cells had been kept in the dark. Once equilibrium between the Ci species had been achieved, a CO₂ efflux was observed after saturating light intensity was applied to the cells. External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO₂ by active transport. Photosynthetic O₂ evolution and the release CO₂ in the dark depend on HCO₃ ⁻ uptake since both were inhibited by the anion exchange inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark. Ethoxyzolamide, a CA inhibitor, markedly inhibited CO₂ efflux in the dark, indicating that CO₂ efflux was dependent upon the intracellular dehydration of HCO₃ ⁻. These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO₂ and HCO₃ ⁻ and thus causes a subsequent release of CO₂ to the external medium. Received: 20 September 1999 / Accepted: 25 October 1999     

A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition

Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC₅₀ value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC₅₀ values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.     

Control of the induction of ion transport through mitochondrial membranes by the enzymes of the oxidative phosphorylation system

It has been shown that the induction of earlier described system of potassium-dependent transport of hydrogen ions in mitochondria at low pH values of the incubation medium is inhibited by the inhibitors of mitochondria respiratory chain and ATPase. It has been found that antimycin and oligomycin suppress the efflux of potassium ions from mitochondria in the presence of succinic acid. The uncoupler (FCCP) turns the effect of ATPase inhibitors to the efflux of potassium ions and acceleration of mitochondria respiration under experimental conditions. At the same time TMPD removes the effect of antimycin on potassium ion efflux from uncoupled FCCP of mitochondria. The data obtained are explained in terms of the postulate that under experimental conditions along with the system of potassium-dependent ion transport there appears leakage of protons through the ATPase channel. A conclusion is made concerning the control of ion transport induction in mitochondria by the enzymes of oxidative phosphorylation system.     

Searching for mechanisms of N-methyl-D-aspartate-induced glutathione efflux in organotypic hippocampal cultures

N-Methyl-d-aspartate (NMDA)-receptor stimulation evoked a selective and partly delayed elevated efflux of glutathione, phosphoethanolamine, and taurine from organotypic rat hippocampus slice cultures. The protein kinase inhibitors H9 and staurosporine had no effect on the efflux. The phospholipase A₂ inhibitors quinacrine and 4-bromophenacyl bromide, as well as arachidonic acid, a product of phospholipase A₂ activity, did not affect the stimulated efflux. Polymyxin B, an antimicrobal agent that inhibits protein kinase C, and quinacrine in high concentration (500 µM), blocked efflux completely. The stimulated efflux after but not during NMDA incubation was attenuated by a calmodulin antagonist (W7) and an anion transport inhibitor (DNDS). Omission of calcium increased the spontaneous efflux with no or small additional effects by NMDA. In conclusion, NMDA receptor stimulation cause an increased selective efflux of glutathione, phosphoethanolamine and taurine in organotypic cultures of rat hippocampus. The efflux may partly be regulated by calmodulin and DNDS sensitive channels.     

Relationship between Photosynthesis and Respiration: The Effect of Carbohydrate Status on the Rate of CO(2) Production by Respiration in Darkened and Illuminated Wheat Leaves

The rate of dark CO₂ efflux from mature wheat (Triticum aestivum cv Gabo) leaves at the end of the night is less than that found after a period of photosynthesis. After photosynthesis, the dark CO₂ efflux shows complex dependence on time and temperature. For about 30 minutes after darkening, CO₂ efflux includes a large component which can be abolished by transferring illuminated leaves to 3% O₂ and 330 microbar CO₂ before darkening. After 30 minutes of darkness, a relatively steady rate of CO₂ efflux was obtained. The temperature dependence of steady-state dark CO₂ efflux at the end of the night differs from that after a period of photosynthesis. The higher rate of dark CO₂ efflux following photosynthesis is correlated with accumulated net CO₂ assimilation and with an increase in several carbohydrate fractions in the leaf. It is also correlated with an increase in the CO₂ compensation point in 21% O₂, and an increase in the light compensation point. The interactions between CO₂ efflux from carbohydrate oxidation and photorespiration are discussed. It is concluded that the rate of CO₂ efflux by respiration is comparable in darkened and illuminated wheat leaves.     

Ascorbic acid efflux and re-uptake in endothelial cells: maintenance of intracellular ascorbate

Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70–80% of ascorbate to the medium over several hours at 37°C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel.     

Dim-Red-Light-Induced Increase in Polar Auxin Transport in Cucumber Seedlings : I. Development of Altered Capacity, Velocity, and Response to Inhibitors

We have developed and characterized a system to analyze light effects on auxin transport independent of photosynthetic effects. Polar transport of [³H]indole-3-acetic acid through hypocotyl segments from etiolated cucumber (Cucumis sativus L.) seedlings was increased in seedlings grown in dim-red light (DRL) (0.5 μmol m⁻² s⁻¹) relative to seedlings grown in darkness. Both transport velocity and transport intensity (export rate) were increased by at least a factor of 2. Tissue formed in DRL completely acquired the higher transport capacity within 50 h, but tissue already differentiated in darkness acquired only a partial increase in transport capacity within 50 h of DRL, indicating a developmental window for light induction of commitment to changes in auxin transport. This light-induced change probably manifests itself by alteration of function of the auxin efflux carrier, as revealed using specific transport inhibitors. Relative to dark controls, DRL-grown seedlings were differentially less sensitive to two inhibitors of polar auxin transport, N-(naphth-1-yl) phthalamic acid and 2,3,5-triiodobenzoic acid. On the basis of these data, we propose that the auxin efflux carrier is a key target of light regulation during photomorphogenesis.     

Uptake and efflux of sulfate in Neurospora crassa

Sulfate efflux from an intracellular pool was observed with both wild-type and cys-11 cells of Neurospora and apparently occurs by way of the sulfate transport system. Efflux requires the presence of external sulfate or the related ions, chromate, selenate, or thiosulfate, and probably occurs by an exchange reaction. The sulfur amino acids, cysteine or methionine, do not promote sulfate efflux. The K_m for efflux is much greater than the K_m for sulfate uptake, which permits the accumulation of a considerable intracellular pool before efflux becomes significant. Substantial transmembrane movement of sulfate both influx and exit, was found to occur in azidetreated cells, although the net uptake of sulfate was abolished by this inhibitor. Both sulfate uptake and efflux are inhibited by p-chloromercuribenzoate which suggests that the sulfate permease possesses an essential sulfhydryl group.     

Correlations between proton-efflux patterns and growth patterns during geotropism and phototropism in maize and sunflower

By placing seedlings of sunflower (Helianthus annuus L.) or maize (Zea mays L.) on agar plates containing a pH indicator dye it is possible to observe surface pH patterns along the growing seedling by observing color changes of the indicator dye. Using this method we find that in geotropically stimulated sunflower hypocotyls or maize coleoptiles there is enhanced proton efflux on the lower surface of the organ prior to the initiation of curvature. As curvature develops the pattern of differential acid efflux becomes more intense. A similar phenomenon is observed when these organs are exposed to unilateral illumination, i.e. enhanced acid efflux occurs on the dark side of the organ prior to the initiation of phototropic curvature and the pattern of differential acid efflux intensifies as phototropic curvature develops. These observations indicate that differential acid efflux occurs in response to tropistic stimuli and that the acid efflux pattern may mediate the development of tropistic curvatures.     

An investigation into the roles of photosynthesis and respiration in h efflux from aerated suspensions of asparagus mesophyll cells

Aerated and stirred suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the roles of respiration and photosynthesis in net H⁺ efflux. Rates varied between 0.12 and 1.99 nanomoles H⁺ per 10⁶ cells per minute or 3 and 40 nanomoles H⁺ per milligram chlorophyll per minute. The mean rate of H⁺ efflux was 10% greater in the dark. 3-(3,4-Dichlorophenyl)-l,l-dimethylurea, an inhibitor of noncyclic photophosphorylation, did not inhibit H⁺ efflux from illuminated cells. Bubbling with N₂ or addition of oligomycin, an inhibitor of mitochondrial ATP production, resulted in rapid and virtually complete inhibition of H⁺ efflux in light or dark. In the absence of aeration, H⁺ efflux came to a halt but resumed with aeration or illumination. When aeration was switched to CO₂-free air, rates of H⁺ efflux were reduced 43% in the dark and 57% in the light. Oligomycin eliminated dark CO₂ fixation but not photosynthetic CO₂ fixation. It is suggested that H⁺ efflux is dependent on respiration and dark CO₂ fixation, but independent of photosynthesis.     

Ureidosuccinic acid permeation in Saccharomyces cerevisiae

Some strains of Saccharomyces cerevisiae exhibit a specific transport system for ureidosuccinic acid, which is regulated by nitrogen metabolism. Ureidosuccinic acid uptake occurs with proline but with ammonium sulfate as nitrogen source it is inhibited. The V for transport is 20–25 μmol/ml cell water per min. The apparent K_m is 3 · 10^-5. For the urep1 mutant (ureidosuccinic acid permease less) the internal concentration never exceeds the external one.In the permease plus strain ureidosuccinic acid can be concentrated up to 10 000 fold and the accumulated compound remains unchanged in the cells. Energy poisons such as dinitrophenol, carbonyl cyanide-m-chlorophenyl-drazone (CCCP) or NaN₃ inhibit the uptake. No significant efflux of the accumulated compound occurs even in the presence of these drugs.The specificity of the permease is very strict, only amino acids carrying an α-N-carbamyl group are strongly competitive inhibitors.The high concentration capacity of the cells and the lack of active exit of the accumulated compound support the hypothesis of a carrier mediated active transport system.     

Transport in halobacterium halobium: Light-induced cation-gradients,amino acid transport kinetics,and properties of transport carriers

Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na⁺. Measurements of ²²Na flux, exterior pH change, and membrane potential, ΔΨ (with the dye 3,3′-dipentyloxadicarbocyanine) indicate that the means of Na⁺ transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H⁺/Na⁺ > 1). The resulting large chemical gradient for Na⁺ (outside > inside), as well as the membrane potential, will drive the transport of 18 amino acids. The 19th, glutamate, is unique in that its accumulation is indifferent to ΔΨ: this amino acid is transported only when a chemical gradient for Na⁺ is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H⁺ collapses within 1 min, while the large Na⁺ gradient and glutamate transporting activity persists for 10–15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na⁺, arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V_max and K_m comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na⁺, in an electrically neutral fashion, so that only the chemical component of the Na⁺ gradient is a driving force. The transport of all amino acids but glutamate is bidirectional. Actively driven efflux can be obtained with reversed Na⁺ gradients (inside > outside), and passive efflux is considerably enhanced by intravesicle Na⁺. These results suggest that the transport carriers are functionally symmetrical. On the other hand, noncompetitive inhibition of transport by cysteine (a specific inhibitor of several of the carriers) is only obtained from the vesicle exterior and only for influx: these results suggest that in some respects the carriers are asymmetrical. A protein fraction which binds glutamate has been found in cholate-solubilized H. halobium membranes, with an apparent molecular weight of 50,000. When this fraction (but not the others eluted from an Agarose column) is reconstituted with soybean lipids to yield lipoprotein vesicles, facilitated transport activity is regained. Neither binding nor reconstituted transport depend on the presence of Na⁺. The kinetics of the transport and of the competitive inhibition by glutamate analogs suggest that the protein fraction responsible is derived from the intact transport system.     

Functional Characteristics of Pyruvate Transport inPhycomyces blakesleeanus

Jesús A. Marcos, Dolores de Arriaga, Félix Busto, and Joaquín Soler¹1997. Functional Characteristics of Pyruvate Transport inPhycomyces blakesleeanus. Fungal Genetics Biology25, 204-215. A saturable and accumulative transport system for pyruvate has been detected inPhycomyces blakesleeanusNRRL 1555(−) mycelium. It was strongly inhibited by α-cyano-4-hydroxycinnamate. -Lactate and acetate were competitive inhibitors of pyruvate transport. The initial pyruvate uptake velocity and accumulation ratio was dependent on the external pH. TheV_maxof transport greatly decreased with increasing pH, whereas the affinity of the carrier for pyruvate was not affected. The pyruvate transport system mediated its homologous exchange, which was essentially pH independent, and efflux, which increased with increasing external pH. The uptake of pyruvate was energy dependent and was strongly inhibited by inhibitors of oxidative phosphorylation and of the formation of proton gradients. Glucose counteracted the inhibitory effect of the pyruvate transport produced by inhibitors of mitochondrial ATP synthesis. Our results are consistent with a pyruvate/proton cotransport inP. blakesleeanusprobably driven by an electrochemical gradient of H⁺generated by a plasma membrane H⁺-ATPase.     

Acid phosphatases bind to the main high density lipoprotein apolipoprotein A-I

Light-dependent Ca²⁺ efflux via the Ca²⁺/H⁺ antiport in the photosynthetic purple sulfur bacterium Chromatium vinosum was inhibited by three phenothiazines: chlorpromazine; trifluoperazine and phenothiazine. The inhibitors had no effect on Ca²⁺ uptake by C. vinosum in the dark nor any effect on the light-dependent efflux of either Na⁺ or Tl⁺ catalyzed, respectively, by the C. vinosum Na⁺/H⁺ or K⁺/H⁺ antiports. Ruthenium red and LaCl₃, neither of which inhibited light-dependent Ca²⁺ efflux in C. vinosum, markedly inhibited Ca²⁺ uptake in the dark by C. vinosum cells. Ruthenium red had no effect on the uptake of either Na⁺or the K⁺ analog T1⁺ by C. vinosum cells in the dark. These results have been interpreted in terms of two separate Ca²⁺ transport systems in C. vinosum: (i) a phenothiazine-sensitive and ruthenium red, La³⁺-insensitive Ca²⁺/H⁺ antiport responsible for Ca²⁺ efflux in the light; and (ii) a ruthenium red and La³⁺-sensitive but phenothiazine-insensitive Ca²⁺ uptake system.     

Active transport of chloride by Anacystis nidulans

Chloride uptake by the cyanobacterium Anacystis nidulans at 38°C is energy dependent showing maximum rate (around 5.10^-7 mol Cl^-xml cell water^-1xmin^-1) and accumulation (up to 160 fold) in light and air. The respective values in air and darkness were 40–70% lower. In the dark under N₂ no uptake was found. Chloride transport had an optimum at pH 6.7 and a K _M of 2.10^-5 M which was pH-independent. It was inhibited by carbonyl cyanide m-chlorophenylhydrazone and N,N′-dicyclohexylcarbodiimide in the light and in the dark, and also to a lesser extent by valinomycin. 3-(3,4-dichlorophenyl)-1,1-dimethylurea in the light caused a moderate stimulation. To obtain information about the energy source of active chloride transport the action of the four inhibitors on membrane potential (determined through the distribution of triphenylmethylphosphonium) and ATP level (determined by the firefly method) was examined. It was found that a high negative membrane potential was unfavorable for chloride accumulation probably by stimulating passive efflux. On the other hand a good correlation between ATP level and chloride transport activity was obtained. Attempts to induce chloride uptake by sudden acidification of the external medium in presence of N,N′-dicyclohexyl-carbodiimide or during anaerobiosis were not successful. Two mechanisms of chloride uptake are discussed:

1. primary active transport by an ATP-dependent pump, and
2. “chemiosmotic” secondary active transport linked to a proton gradient, the present data favoring mechanism a.

     

Separation and inhibitor specificity of a second unidirectional efflux route for methotrexate in L1210 cells.

L1210 cells mediate the unidirectional and energy-dependent efflux of methotrexate. Efflux occurs primarily via a system which has a high sensitivity to prostaglandin A1, vincristine, reserpine, verapamil, and bromosulfophthalein, but evidence has also been obtained for a second efflux component with a lower response to these inhibitors. Pretreatment of L1210 cells with low concentrations of vincristine reduces methotrexate efflux by three fold and uncovers a second efflux component with an inhibitor specificity which is distinctly different from the primary efflux route. Vincristine treatment increased by 8-20-fold the concentration required for half-maximal efflux inhibition by prostaglandin A1, reserpine, bromosulfophthalein, and verapamil but had no effect on inhibition by probenecid, quinidine, or carbonylcyanide m-chlorophenylhydrazone. A selective block in the primary efflux system and retention of the second component was also achieved in cells exposed to low concentrations of prostaglandin A1 or bromosulfophthalein. These results support prior conclusions that L1210 cells contain both a primary and secondary unidirectional efflux route for methotrexate. The second system has been difficult to detect and quantitate since it comprises only 25% of total unidirectional efflux and shows a relatively low response to various efflux inhibitors.