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Effects of 2,4-Dinitrophenol and Other Metabolic Inhibitors on The (CANCER RESEARCH 40, 3669-3673. October 1980] 0008-5472/80-0040-OOOOS02.00 Effects of 2,4-Dinitrophenol and Other Metabolic Inhibitors on the Bidirectional Carrier Fluxes, Net Transport, and Intracellular Binding of Methotrexate in Ehrlich Ascites Tumor Cells1 David W. Fry, J. Courtland White, and I. David Goldman2 Department ol Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 ABSTRACT edly influenced by (a) the concentration of the inhibitor, (b) whether measurements are taken during influx, net transport, 2,4-Dinitrophenol (DNP), an uncoupler of oxidative phos- or the steady state, (c) whether total intracellular or freely phorylation, has been frequently used to evaluate the effects exchangeable MTX is determined, and (d) whether or not of energy depletion on methotrexate (MTX) transport. The glucose or other energy-producing substrates are present. results from these studies, however, have shown a multiplicity of effects that suggest a more complicated interaction with the MTX transport system than adenosine 5'-triphosphate deple INTRODUCTION tion alone. Accordingly, studies were undertaken to compare Membrane transport of MTX3 is a complex process. While the effects of DNP with a variety of other metabolic inhibitors this system conforms in many respects to other carrier-me on influx, efflux, net uptake, and intracellular binding of MTX in diated processes with the demonstration of temperature and Ehrlich ascites tumor cells. Low concentrations of DNP (0.1 sulfhydryl dependence (10), heteroexchange diffusion (9, 25), mw) inhibited efflux and increased the intracellular steady-state and inhibition by structural analogs (8, 10), some properties of concentration of MTX, both of which were totally reversed by the transport system are poorly understood. For instance, influx glucose. These alterations were similar to those caused by and net transport are highly sensitive to changes in the anionic inhibitors of the electron transport system (azide, rotenone, (8, 13) and cationic (5) composition of the extracellular com antimycin A, cyanide, and oligomycin) and anaerobic glycolysis partment. Further, metabolic poisons enhance, rather than (2-deoxyglucose) and are compatible with inhibition of an en inhibit, influx and net transport of MTX in some systems, ergy-dependent exit pump. As the concentration of DNP was presumably related to inhibition of an energy-dependent exit increased, influx was competitively inhibited with a K¡of 336 pump (7). The effects of DNP on MTX transport are of particular UM. Inhibition was instantaneous and was reversed by removal interest. This agent has been commonly used as an uncoupler of DNP but not by addition of glucose. The inhibition of MTX of oxidative phosphorylation to evaluate the effects of ATP influx by DNP was different from the effects of other metabolic depletion on MTX transport. However, a wide variety of effects inhibitors which consistantly stimulated influx, an effect totally of DNP on MTX uptake have been reported, ranging from reversed by glucose. Likewise, as DNP concentrations were inhibition (15, 22), to stimulation (11 ), and to no effect at all (4, increased, inhibition of efflux was enhanced but was only 16). Although some discrepancies may be explained by differ partially reversed by glucose, which suggests that at high ences in cell lines, the data suggest that the effects of DNP concentrations of DNP inhibition was due to both energy de may be more complex than energy depletion alone and that pletion and an additional mechanism not associated with en this agent may interact in more than one way with the MTX ergy metabolism. DNP, as well as all other metabolic inhibitors, transport system. Accordingly, studies were undertaken to increased the apparent nonexchangeable fraction of MTX; further clarify the effects of DNP on MTX transport. The results evidently, this is due to energy depletion, since this effect was indicate that, whereas DNP produces changes similar to other totally reversed by glucose. The additional nonexchangeable metabolic inhibitors, there is a second effect that appears to MTX remained unchanged over at least 2 hr of incubation and be based upon a direct interaction between DNP and the MTX was not diminished by repeated changes of the extracellular carrier or its microenvironment which inhibits the bidirectional fluid; this suggests that it was tightly bound. Dicumarol, another flows of this antifolate. These observations provide useful in uncoupler of oxidative phosphorylation, produced effects sim formation on (a) the application of DNP as an agent to evaluate ilar to those of DNP; however, arsenale had little effect on MTX the energetics of MTX transport, (D) the nature of the bidirec transport, which suggests that these alterations were not due tional flows of MTX across the Ehrlich ascites tumor membrane, to uncoupling alone. The results indicate that although most and (c) the different effects of energy inhibitors on the unidi metabolic inhibitors affect MTX transport by inhibition of energy rectional fluxes and net transport of MTX in this cell system. metabolism, DNP affects the system in at least two ways: (a) by energy depletion similar to that of other metabolic inhibitors; and (£>)byan apparent interaction with the carrier at the cell MATERIALS AND METHODS membrane which inhibits bidirectional fluxes. The results may Chemicals. [3',5',9-3H]MTX was obtained from Moravek resolve some of the variation in the literature on the effects of (City of Industry, Calif.) and purified by DEAE-cellulose chro- DNP on MTX transport by showing that observations are mark matography (10). [carboxy-14C]lnulin was obtained from New 1 Supported in part by Grants AM-07150 and CA-16906 from the NIH. 2 To whom requests for reprints should be addressed. ' The abbreviations used are: MTX, methotrexate (4-amino-N'°-methylptero- Received December 3, 1979; accepted July 14, 1980. lyglutamic acid); DNP. 2,4-dinitrophenol OCTOBER 1980 3669 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1980 American Association for Cancer Research. D. W. Fry et al. England Nuclear (Boston, Mass.). Antimycin A, sodium arse Table 1 nale, 2-deoxyglucose, dicumarol, oligomycin, and rotenone Effects of DNP and other metabolic inhibitors on MTX influx were from Sigma Chemical Company (St. Louis, Mo.). Sodium Cells were exposed to 2-deoxyglucose for 10 min, or to other inhibitors for 5 min before MTX. Influx was measured over 100 sec. All values were compared azide and DNP were from Fisher Chemical Company (Fair with the control. Lawn, N. J.), potassium cyanide was from Allied Chemical InhibitorDNP influx96.1ofcontrol (Morristown, N. J.), and d/-5-formyltetrahydrofolate was from "73.4 ± 2.3a- mM)DNP(0.1 3.1C48.1± Lederle Laboratories (Pearl River, N. Y.). mM)DNP(0.25 1.8o31± Cells, Media, and Incubation Techniques. Ehrlich ascites mM)DNP(0.5 mM)DNP(1.0 3.6d16.4.5± tumor cells were grown in male CF-1 male (Sprague-Dawley, 0.33d87.9± mM)Glucose(1 0 mM) + glucose (10 2.7C87.0± Madison, Wis.) and passed weekly by i.p. inoculation of 0.2 ml mM)Arsenale(10.0 1.9C38.5± mM)Dicumarol(1.0 of undiluted ascitic fluid. Cells were suspended in buffer com 5.1"196.3+ /IM)Rotenone(50.0 4.2d83.2± posed of 136 mM NaCI, 4.4 mM KCI, 16 mw NaHCO3, 1.1 mM /JM)Rotenone(1 .0 mM)Antimycin(1 .0 fiM) + glucose (to 12.16178.1± KHjPCv 1.0 mM MgCI?, and 1.9 mM CaCI?. The pH was 12.1°175.9±± (IM)PotassiumA (0.5 maintained at 7.4 by passing warmed and humidified 95% O2/ 7.9d150.0± mM)Oligomycincyanide (1 .0 4.6"149.3± 5% CO? over the cell suspension. The suspension was stirred fig/ml)Azide (0.3 5.2d80.0 mM)Azide(10.0 by a Teflon paddle in specifically designed flasks inserted into 1.9C134.8± a 37°water bath. Unidirectional fluxes, net uptake, and intra- mM)2-Deoxyglucose(10.0 mM) + glucose (10 (50.0 mM)n33332844524441044% ± 2.8o cellular binding of [3H]MTX were measured as previously de 1Mean ±S.E. of n experiments performed on separate dates. scribed (7, 10). Transport fluxes were stopped by injection of * p>0.2 (paired f test). the cell suspension into 10 volumes of 0°0.85% NaCI solution cp<0.01 (paired (test). a p < 0.001 (paired ( test). (pH 7.4). The cell fraction was separated by centrifugaron (2000 x g for 30 to 60 sec) and washed twice with the 0° 0.85% NaCI solution. The washed pellet was aspirated into the tip of a Pasteur pipet, extruded onto a polyethylene tare, and dried overnight at 70°. The dried pellets were weighed on a Cahn electrobalance (Cahn Instruments, Paramount, Calif.), placed in a scintillation vial, and dissolved in 0.2 ml of 1 N KOH for 1 hr at 70°.The digest was neutralized with 0.2 ml of 1 N HCI, and 3 ml of Ready-Solv (Beckman Instruments, Inc., Irvine, Calif.) were added to the scintillation vial. Radioactivity was determined in a Beckman LS-230 scintillation spectrometer, 025mM DNP and counting efficiencies were determined using [3H]- or CONTROL [14C]toluene internal standards. Determination of Intracellular Water and Chloride Distri bution Ratio. Intracellular water was determined from the dif ference between the wet weight and the dry weight of a cell 50 pellet, less the [14C]inulin space as described in detail else where (2, 10). The chloride distribution ratio was measured as [MTX]e an indication of changes in membrane potential (2, 10). Chart 1. Double reciprocal plot of extracellular MTX concentration, [MTX]., versus influx at different concentrations of DNP. Influx was measured over 100 sec, and lines were plotted by the method of least squares. RESULTS Effect of DNP on MTX Influx.
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