Edward M. Zevely

Structural requirements for anion substrates of the methotrexate transport system in L1210 cells

A broad spectrum of structurally diverse anions reversibly inhibits the influx of methotrexate in L1210 cells. Several of the more effective anions and their respective inhibition constants (Ki values) were: 5-methyltetrahydrofolate (0.3 microM), bromosulfophthalein (2 microM), thiamine pyrophosphate (3 microM), 8-anilino-1-naphthalene sulfonate (7 microM), phthalate (20 microM), and AMP (50 microM). Moderate inhibition was observed with Pi (Ki = 400 microM) and other divalent inorganic anions, while small monovalent anions such as Cl- (Ki = 30 mM) were the least effective. When these same anions were tested for an effect on methotrexate efflux, stimulation was observed with some anions, while others had no effect. Enhancement was produced by folate compounds and p-amino-benzoylglutamate, small monovalent (e.g., Cl-, acetate, and lactate) and divalent (e.g., phosphate and succinate) anions, a few nucleotides (e.g., AMP), and thiamine pyrophosphate, while little or no effect was associated with trivalent anions (e.g., citrate), most nucleotides, and large organic anions (e.g., bromosulfophthalein, NAD, and NADP). Anions with the ability to promote methotrexate efflux in control cells lost this capacity upon exposure of the cells to an irreversible inhibitor of methotrexate influx. These results support the hypothesis that methotrexate transport proceeds via an anion-exchange mechanism and moreover provide evidence that anion substrates for this system can be identified by their ability to promote methotrexate efflux. Anions which appear most likely to participate in this exchange cycle in vivo are Pi and AMP.

Anion exchange mechanism for transport of methotrexate in L1210 cells

Abstract Structurally diverse anions (folate, 5-formyltetrahydrofolate, AMP, ADP, thiamine pyrophosphate, phosphate, sulfate, and chloride) that are competitive inhibitors of methotrexate influx in L1210 cells also enhance the efflux of methotrexate from these cells. The increase in efflux reaches a maximum of 2- to 4-fold depending upon the anion employed, and the anion concentrations required for half-maximal stimulation of efflux are similar to their K i values for inhibition of methotrexate influx. A competitive inhibitor of methotrexate uptake (fluorescein-diaminopentane-methotrexate) that is not transported by this system, does not increase methotrexate efflux. These results suggest that the efflux of intracellular methotrexate is coupled to the concomitant uptake of an extracellular anion.

Transport of methotrexate in l1210 cells: effect of ions on the rate and extent of uptake.

Abstract Transport of methotrexate (MTX) in L1210 cells is highly dependent upon the ionic composition of the external medium. Half-maximal rates of MTX transport ( K t values) vary from 0.9 μ m in cells suspended in potassium-Hepes buffer containing Mg 2+ (Hepes-Mg), to 10 μ m in phosphate-buffered saline (PBS). At saturating levels of substrate, however, transport rates approach the same maximum velocity ( V ) regardless of the buffering medium. The increased K t value for MTX in PBS is due to the presence of the competitive inhibitors, phosphate ( K i = 0.87 mM) and Cl − ( K i = 46 mM). Concentration gradients for MTX at the steady state are also much lower (about 20-fold) in PBS than in Hepes-Mg; the components of PBS that reduce this uptake parameter are phosphate, Cl − , Ca 2+ , and Na + . Ions that decrease the influx rate or the steady-state level also produce an increase in MTX efflux. Glucose (which increases ATP levels) reduces influx rates and steady-state levels of MTX, and induces efflux in both PBS and Hepes-Mg. Conversely, the combination of azide plus iodoacetate (which reduces ATP levels) stimulates MTX uptake in PBS, but has little effect on MTX transport parameters in Hepes-Mg. The unusually high sensitivity of MTX transport to various anions is consistent with the hypothesis that this system catalyzes the exchange of external MTX for an intracellular anion, and that efflux of the anion down a concentration gradient provides the driving force for active transport of MTX.

Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells☆

Abstract A binding component with a high affinity for 5-methyltetrahydrofolate ( K D = 0.11μ m ) is present on the external surface of L1210 cells. The amount of binder (1 pmol/mg protein) corresponds to 8 × 10 4 sites per cell. The participation of this component in the high-affinity 5-methyltetrahydrofolate/methotrexate transport system is supported by similarities in the K D values for 5-methyltetrahydrofolate and methotrexate binding and the K t values of these compounds for transport. Relative affinities for other folate substrates (aminopterin, 5-formyltetrahydrofolate, and folate) and various competitive inhibitors (thiamine pyrophosphate, ADP, AMP, arsenate, and phosphate) are also similar for both the binding component and the transport system. The measured binding activity does not represent low-temperature transport of substrate into cells, since it is readily saturable with time and is eliminated by either washing the cells with buffer or by the addition of excess unlabeled substrate.

Inhibitory effects of probenecid on the individual transport routes which mediate the influx and efflux of methotrexate in L1210 cells.

L1210 cells contain a single transport system which mediates the influx of methotrexate and at least three routes for drug efflux [G. B. Henderson and E. M. Zevely, J. biol. Chem. 259, 1526 (1984)]; each of these processes is sensitive to probenecid. The influx carrier was inhibited reversibly and completely by probenecid with a Ki of 0.25 mM, while efflux via the same system was relatively unaffected by this compound (50% inhibition above 2.0 mM). The two remaining efflux routes (which do not contribute to methotrexate influx) showed a much higher sensitivity to probenecid. Efflux via these components was reduced half-maximally at probenecid concentrations of 0.08 and 0.22 mM, respectively, and a complete block was achieved with excess amounts (2.0 mM) of the inhibitor. Intracellular levels of ATP, glucose metabolism, and the membrane potential were also reduced by probenecid, indicating that the mechanism for inhibiting methotrexate efflux may involve the ability of probenecid to act as a metabolic inhibitor. Probenecid may have a broad capacity for inhibiting anion transport processes since it also reduced sulfate influx and efflux via the general anion carrier system.

Intracellular phosphate and its possible role as an exchange anion for active transport of methotrexate in L1210 cells

Abstract L1210 cells transport P i in the absence of added Na + . Uptake shows saturation kinetics (K t = 1.7 mM), is temperature-dependent, and can be reduced 80% by high levels of unlabeled P i , and thus has the characteristics of a carrier-mediated process. This transport process is also inhibited by methotrexate. The methotrexate-sensitive component constitutes half of total P i uptake, and is reduced by 50% at a concentration of methotrexate (2 μM) that is comparable to its K t (1.5 μM) for transport into the cells. An impermeable fluorescent analog of methotrexate and an irreversible inhibitor of the methotrexate transport system (carbodiimide-activated methotrexate) also inhibit this same P i uptake component. It is concluded that methotrexate and P i can be transported by the same carrier system. The basis for this shared uptake is suggested to be that the methotrexate carrier protein facilitates the obligatory exchange of extracellular folate compounds for intracellular divalent anions, and that a primary exchange anion is P i . A principal energy source for active transport of methotrexate might then be the concentration gradient for P i that is maintained by the Na + -dependent, P i transport system of these cells.

Folate transport in Lactobacillus casei: Solubilization and general properties of the binding protein☆

Abstract Intact cells of Lactobacillus casei grown in a medium containing a low level (5 nM) of folate have the capacity to bind (at 4°) appreciable quantities (0.35 nmoles/10 10 cells) of the vitamin. Folate binding is rapid, saturable (K d = 36 nM), insensitive to sulfhydryl reagents, and has a broad pH optimum. A folate-binding protein has been solubilized in high yield by sonic disruption of lysozyme-treated cells in the presence of [ 3 H]folate and Triton X-100. The protein-folate-Triton complex (MW 230,000 by filtration through Sephadex G-150) is stable to dialysis but dissociates upon heating (50% loss of bound folate after 5 min at 49°). Evidence is presented to suggest that the binding protein functions as the carrier of folate during its transport into the cells.

The isolation of dihydrofolate reductases by affinity chromatography on folate-sepharose.

Abstract Folate-Sepharose affinity column material has been prepared and used for the isolation of dihydrofolate reductases from three sources: (a) amethopterin-resistant L1210 cells in culture; (b) amethopterin-resistant Lactobacillus casei ; and (c) human leukemic leukocytes. The column material was synthesized in two steps: (i) condensation of folate (1 part) and diaminohexane (10 parts) promoted by 1-ethyl-3(3′-dimethylaminopropyl) carbodiimide (5 parts) in dimethylsulfoxide; and (ii) combination of the product, 1-aminohexyl-6-amidofolate, with cyanogen bromide-activated Sepharose. Application of a crude cellular homogenate from each source to the affinity matrix led to preferential enzyme retention. After an initial wash with solutions of increased ionic strength, recovery of the two mammalian enzymes was achieved in a greater than 65% yield by elevation of the pH and addition of folate to the elution buffer. The L1210 enzyme was homogeneous by polyacrylamide electrophoresis ( R f = 0.20). The L. casei enzyme (specific activity ∼9 unit/mg) was recovered by merely increasing the eluting buffer strength. However, preliminary passage of the cell extract through DEAE-cellulose followed by affinity chromatography gave improved purification (specific activity ∼16 unit/mg). The reductases from both the L1210 and L. casei sources were recovered as a single form of the enzyme after affinity chromatography, that with no bound NADPH.

Characterization of the multiple transport routes for methotrexate in L1210 cells using phthalate as a model anion substrate

Summaryo-Phthalate is actively transported into L1210 cells and the primary route for cell entry is the same transport system which mediates the influx of methotrexate and other folate compounds. The identity of the influx route has been established by the following observations: (A) Phthalate influx is competitively inhibited by methotrexate and the inhibition constant (Ki) is comparable to theKi for half-maximal influx of methotrexate; (B) Various anions inhibit the influx of phthalate and methotrexate with comparableKi values; (C) The influx of phthalate and methotrexate both fluctuate in parallel with changes in the anionic composition of the external medium; and (D) A specific covalent inhibitor of the methotrexate transport system (NHS-methotrexate) also blocks the transport of phthalate. In contrast, the efflux of phthalate does not occur via the methotrexate influx carrier, but rather by two separate processes which can be distinguished by their sensitivities to bromosulfophthalein. Efflux via the bromosulfophthalein-sensitive route constitutes 75% of total efflux and is enhanced by glucose and inhibited by oligomycin. The inability of phthalate to exit via the methotrexate influx carrier is due to competing intracellular anions which prevent phthalate from interacting with the methotrexate binding site at the inner membrane surface.

Transport of methotrexate in L1210 cells: Mechanism for inhibition by p-chloromercuriphenylsulfonate and N-ethylmaleimide

Methotrexate transport in L1210 cells is highly sensitive to inhibition by p-chloromercuriphenylsulfonate (CMPS) and, to a lesser extent, by N-ethylmaleimide. A 50% reduction in the methotrexate influx rate occurred upon exposure of cells to 3 microM CMPS or 175 microM N-ethylmaleimide, while complete inhibition was achieved at higher levels of these agents. Dithiothreitol reversed the inhibition by CMPS, suggesting that a sulfhydryl residue is involved. This residue is apparently not located at the substrate binding site of the transport protein, since methotrexate failed to protect the system from inactivation by either CMPS or N-ethylmaleimide, and the transport protein retained the ability to bind substrate (at 4 degrees C) after exposure to these inhibitors (at 37 degrees C). Methotrexate efflux was also inhibition by CMPS (50% at 4 microM), indicating that both the uptake and efflux of methotrexate in L1210 cells occur via the same transport system. High concentrations of CMPS (greater than 20 microM) increased the efflux rate, apparently by damaging the cell membrane and allowing the passive diffusion of methotrexate out of the cell.