Bacteriocins inhibit glucose PEP:PTS activity in Listeria monocytogenes by induced efflux of intracellular metabolites

Glucose transport by the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) of Listeria monocytogenes is inhibited by the bacteriocins nisin, pediocin JD and leuconocin S. To investigate the mechanism of inhibition, PTS activity assays were performed with permeabilized, bacteriocin-treated L. monocytogenes Scott A cells. In the presence of exogenous PEP, nisin stimulated the PTS while both pediocin JD and leuconocin S partially inhibited its activity. These results suggested that PTS enzymes were still active in bacteriocin-treated cells and that bacteriocin-induced PEP efflux may be a mechanism for inhibition of the PTS. To verify that PEP did efflux from bacteriocin-treated L. monocytogenes Scott A cells, intracellular and extracellular PEP were measured by HPLC. All three bacteriocins induced efflux of PEP. Nisin, pediocin JD and leuconocin S also induced efflux of AMP, ADP and ATP. These studies indicate that bacteriocin inhibition of the glucose PEP:PTS in L. monocytogenes is due to efflux of intracellular metabolites, particularly PEP.     

Glucose uptake by Listeria monocytogenes Scott A and inhibition by pediocin JD.

Glucose uptake by Listeria monocytogenes Scott A was inhibited by the bacteriocin pediocin JD and by the protonophore carbonyl cyanide m-chlorophenyhydrazone. Experiments with monensin, nigericin, chlorhexidine diacetate, dinitrophenol, and gramicidin, however, showed that glucose uptake could occur in the absence of a proton motive force. L. monocytogenes cell extracts phosphorylated glucose when phosphoenolpyruvate (PEP) was present in the assay mixture, and whole cells incubated with 2-deoxyglucose accumulated 2-deoxyglucose-6-phosphate, indicating the presence of a PEP-dependent phosphotransferase system in this organism. Glucose phosphorylation also occurred when ATP was present, suggesting that a proton motive force-mediated glucose transport system may also be present. We conclude that L. monocytogenes Scott A accumulates glucose by phosphotransferase and proton motive force-mediated systems, both of which are sensitive to pediocin JD.     

Collapse of the proton motive force in Listeria monocytogenes caused by a bacteriocin produced by Pediococcus acidilactici.

The effect of pediocin JD, a bacteriocin produced by Pediococcus acidilactici JD1-23, on the proton motive force and proton permeability of resting whole cells of Listeria monocytogenes Scott A was determined. Control cells, treated with trypsin-inactivated bacteriocin at a pH of 5.3 to 6.1, maintained a pH gradient and a membrane potential of approximately 0.65 pH unit and 75 mV, respectively. However, these gradients were rapidly dissipated in cells after exposure to pediocin JD, even though no cell lysis had occurred. The pH gradient and membrane potential of the producer cells were also unaffected by the bacteriocin. Whole cells treated with bacteriocin were twice as permeable to protons as control cells were. The results suggest that the inhibitory action of pediocin JD against L. monocytogenes is directed at the cytoplasmic membrane and that inhibition of L. monocytogenes may be caused by the collapse of one or both of the individual components of the proton motive force.     

Collapse of the proton motive force in Listeria monocytogenes caused by a bacteriocin produced by Pediococcus acidilactici.

The effect of pediocin JD, a bacteriocin produced by Pediococcus acidilactici JD1-23, on the proton motive force and proton permeability of resting whole cells of Listeria monocytogenes Scott A was determined. Control cells, treated with trypsin-inactivated bacteriocin at a pH of 5.3 to 6.1, maintained a pH gradient and a membrane potential of approximately 0.65 pH unit and 75 mV, respectively. However, these gradients were rapidly dissipated in cells after exposure to pediocin JD, even though no cell lysis had occurred. The pH gradient and membrane potential of the producer cells were also unaffected by the bacteriocin. Whole cells treated with bacteriocin were twice as permeable to protons as control cells were. The results suggest that the inhibitory action of pediocin JD against L. monocytogenes is directed at the cytoplasmic membrane and that inhibition of L. monocytogenes may be caused by the collapse of one or both of the individual components of the proton motive force.     

Correlation of bioenergetic parameters with cell death in Listeria monocytogenes cells exposed to nisin.

In Listeria monocytogenes, nisin induced ATP efflux, reduced the intracellular ATP concentration within 1 min, and dissipated the proton motive force within 2 min. Efflux accounted for only 20% of the ATP depletion, suggesting that ATP hydrolysis also occurred. ATP efflux depended on nisin concentration and followed saturation kinetics. These results suggest that nisin breaches the membrane permeability barrier in a manner more consistent with pore formation than with a nonspecific detergent-like membrane destabilization.     

Role of cytoplasmic ATP in the restoration and maintenance of a membrane permeability barrier in transformed mammalian cells

Addition of ATP to medium surrounding intact, transformed 3T3 cells activates the formation of aqueous channels in the plasma membrane. This results in efflux of nucleotide pools and ions and entry into the cytosol of charged, phosphorylated species. In such permeabilized cells, glycolysis is totally dependent on the external addition of glucose, inorganic phosphate, ADP, K+, Mg2+ and NAD+ which restore lactic acid formation to levels found in untreated cells. As expected, such reconstitution of glycolytic activity is found to restore intracellular ATP levels. This is accompanied by sealing of the membrane channels so that efflux of nucleotide pools ceases. Pyruvate, a substrate for mitochondrial ATP synthesis, when provided along with ADP and inorganic phosphate also produces sealing of the membrane channels. On the other hand, reactivation of pentose phosphate shunt activity, which does not lead to ATP synthesis, does not induce restoration of the membrane permeability barrier. Furthermore, compounds which lower the internal ATP pool prevent sealing, and also render the plasma membrane more sensitive to external ATP (Rozengurt and Heppel, '79). Sealing of aqueous channels following restoration of the internal ATP pool is associated with phosphorylation of the inner membrane surface, and is unaffected by inhibitors of protein synthesis, microfilament or microtubular assembly. These results indicate the probable role of intracellular ATP in the restoration and/or maintenance of an active membrane barrier against efflux of small molecules and ions in transformed 3T3 cells.     

Growth condition-related response of Listeria monocytogenes 412 to bacteriocin inactivation

Bacteriocin inactivation of Listeria monocytogenes 412 was studied as a function of growth phase. Cells were treated with nisin (300 IU ml-1) or pediocin (320 or 2560 AU ml-1) for 20 min at 30 degrees C. Inactivation with nisin or the low concentration of pediocin was growth phase dependent, with exponentially growing cells being more susceptible than stationary cells. No effect of growth phase was observed for the high pediocin concentration. Pediocin inactivation (320 AU ml-1) of L. monocytogenes 412 exposed to osmotic (6.5% NaCl) or low-temperature (5 degrees C) stress was investigated. Pediocin failed to inactivate osmotically stressed cultures and was unable to inhibit cold-stressed cells to the same degree as unstressed cells.     

Bioenergetic mechanism for nisin resistance, induced by the acid tolerance response of Listeria monocytogenes

This study examined the bioenergetics of Listeria monocytogenes, induced to an acid tolerance response (ATR). Changes in bioenergetic parameters were consistent with the increased resistance of ATR-induced (ATR(+)) cells to the antimicrobial peptide nisin. These changes may also explain the increased resistance of L. monocytogenes to other lethal factors. ATR(+) cells had lower transmembrane pH (DeltapH) and electric potential (Deltapsi) than the control (ATR(-)) cells. The decreased proton motive force (PMF) of ATR(+) cells increased their resistance to nisin, the action of which is enhanced by energized membranes. Paradoxically, the intracellular ATP levels of the PMF-depleted ATR(+) cells were approximately 7-fold higher than those in ATR(-) cells. This suggested a role for the F(o)F(1) ATPase enzyme complex, which converts the energy of ATP hydrolysis to PMF. Inhibition of the F(o)F(1) ATPase enzyme complex by N'-N'-1,3-dicyclohexylcarbodiimide increased ATP levels in ATR(-) but not in ATR(+) cells, where ATPase activity was already low. Spectrometric analyses (surface-enhanced laser desorption ionization-time of flight mass spectrometry) suggested that in ATR(+) listeriae, the downregulation of the proton-translocating c subunit of the F(o)F(1) ATPase was responsible for the decreased ATPase activity, thereby sparing vital ATP. These data suggest that regulation of F(o)F(1) ATPase plays an important role in the acid tolerance response of L. monocytogenes and in its induced resistance to nisin.     

The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system in streptococci.

The protein, HPr, a necessary component of the phosphoenolpyruvate phosphotransferase system (PTS) in bacteria, was purified from Streptococcus salivarius by column chromatography. The purified preparation gave only one band when analyzed by sodium dodecylsulfate gel electrophoresis or by isoelectric focusing in polyacrylamide gel (pI = 4.85). However, electrophoresis in Tris-containing buffers under non-denaturing conditions revealed 2 bands that could be phosphorylated by PEP in the presence of enzyme I of the PTS or by ATP with the HPr kinase. Homogeneous preparations of these 2 forms could be obtained by preparative electrophoresis. Each preparation exhibited only 1 band when analyzed by electrophoresis under non-denaturing conditions, indicating that the doublet observed before preparative electrophoresis was not an electrophoretic artefact. The electrophoretic mobility of each protein was not modified following heat-treatment at 100 degrees C for 20 min or storage at -40 degrees C for several months. Both HPr proteins catalyzed in vitro the PEP-dependent phosphorylation of glucose, but at a rate slightly lower than that observed with a preparation of HPr containing both forms of the protein. Both forms were also able to transfer the phosphate group from PEP to the other specific PTS proteins known in S salivarius. Rabbit polyclonal antibodies directed against each form reacted with both proteins. The presence of the 2 forms of HPr was detected in fresh cellular extracts of S salivarius; however, their intracellular ratio varied according to growth conditions. A doublet was also found in many other streptococcal species tested (S mutans, S sobrinus, S sanguis, S thermophilus, S bovis, S rattus) and also in L lactis.(ABSTRACT TRUNCATED AT 250 WORDS)     

乙酸菌糖磷酸化作用的研究

对7种乙酸菌(Acetitomaculum ruminis, Acetobacterium woodii, Eubacterium limosum和分离株A2、A4、A10、H3HH)的葡萄糖和2脱氧葡萄糖磷酸化作用进行了研究。尽管所有机体都存在磷酸化反应,但它们在依赖PEP和ATP的比例上有实质性区别。分离菌株A10具有最高的依赖PEP的葡萄糖磷酸化活力(1162nmol·L-1·mg-1·min-1),A10、H3HH和E.limosum都具有葡萄糖磷酸转移酶系统(phosphotransferase systemPTS)。相反,Ａ.ruminis、A.woodii、A2和A4则不具有PTS活力。这七株菌的葡萄糖依赖ATP的磷酸化活力都高于依赖PEP的磷酸化活力,但其程度有所不同。A10和H3HH的葡萄糖PTS可通过胞外葡萄糖诱导,并且其比活在对数期随培养时间延长而增加。此外,还检测到A10和H3HH对麦芽糖和果糖的依赖ATP和PEP的磷酸化活力。     

Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of eugenol against L. monocytogenes and Lactobacillus sakei

The spice oil components eugenol and cinnamaldehyde possess activity against both gram-positive and gram-negative bacteria, but the mechanisms of action remain obscure. In broth media at 20 degrees C, 5 mM eugenol or 30 mM cinnamaldehyde was bactericidal (>1-log reduction in the number of CFU per milliliter in 1 h) to Listeria monocytogenes. At a concentration of 6 mM eugenol was bactericidal to Lactobacillus sakei, but treatment with 0.5 M cinnamaldehyde had no significant effect. To investigate the role of interference with energy generation in the mechanism of action, the cellular and extracellular ATP levels of cells in HEPES buffer at 20 degrees C were measured. Treatment of nonenergized L. monocytogenes with 5 mM eugenol, 40 mM cinnamaldehyde, or 10 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 5 min prevented an increase in the cellular ATP concentration upon addition of glucose. Treatment of energized L. monocytogenes with 40 mM cinnamaldehyde or 10 microM CCCP caused a rapid decline in cellular ATP levels, but 5 mM eugenol had no effect on cellular ATP. Treatment of L. sakei with 10 mM eugenol prevented ATP generation by nonenergized cells and had no effect on the cellular ATP of energized cells. CCCP at a concentration of 100 microM had no significant effect on the cellular ATP of L. sakei. No significant changes in extracellular ATP were observed. Due to their rapidity, effects on energy generation clearly play a major role in the activity of eugenol and cinnamaldehyde at bactericidal concentrations. The possible mechanisms of inhibition of energy generation are inhibition of glucose uptake or utilization of glucose and effects on membrane permeability.     

Technical Note: Bioluminescence Measurements of the Antilisterial Activity of Nisin: Comparison with Ampicillin and Streptomycin

The influence of nisin on intracellular ATP and cell numbers of Listeria monocytogenes strain Scott A was determined and compared with the effect of ampicillin and streptomycin under similar conditions. In the presence of nisin (3–12 μg/ml), intracellular ATP and cell numbers decreased rapidly during the first hour at 35°C and extracellular ATP increased. Cell numbers and intracellular ATP increased after 3 h of incubation. No effect was observed in cells treated with ampicillin (3–12 μg/ml) and streptomycin (15–60 μg/ml) during the first hour. However, concentrations of ≥3 μg/ml ampicillin and ≥30 μg/ml streptomycin were listeriostatic after 3 h of incubation. Progressive loss of viability and reduction of intracellular ATP were observed in resting cells in PBS (pH 7.2) containing increasing concentrations of the antimicrobials. Rapid accumulation of extracellular ATP, observed immediately after treatment with nisin but not with the antibiotics, supports the reported collapse of proton motive force in L. monocytogenes by nisin.     

Use of ATP bioluminescence to determine the bacterial sensitivity threshold to a bacteriocin

A new ATP bioluminescence‐based method was developed to determine the effectiveness of nisin on a sensitive strain of Lactococcus cremoris. The principle of the method is to quantify the release of adenylic‐nucleotides (AN) by a sensitive strain under the action of the bacteriocin, with the complex luciferin–luciferase. Nisin‐induced leakage of AN included ATP from a sensitive L. cremoris to the external medium immediately after the contact with the bacteria. The growth of L. cremoris was correlated with the extracellular AN content. The extracellular ATP and AN concentration exhibited a linear correlation to the logarithm of the nisin concentration. For the determination of the effectiveness threshold, the concentration of AN was more sensitive and more reliable than the direct quanti?cation of ATP. The effectiveness threshold, corresponding to a 100% inhibition of L. cremoris growth, was obtained for a null concentration of intracellular nucleotides, i.e. for a AN_tot:AN_ext ratio = 1. For an initial concentration of 1.4 × 10⁷ bacteria/mL, the nisin effectiveness threshold is 3.4 ± 0.01 mg nisin/L. It is possible to detect effectiveness threshold concentration by taking into account the physiological state of the cells. Copyright © 2003 John Wiley & Sons, Ltd.     

The phosphoenolpyruvate:glucose phosphotransferase system of Salmonella typhimurium. The phosphorylated form of IIIGlc

Enzyme IIIGlc of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) of Salmonella typhimurium can occur in two forms: phosphorylated and nonphosphorylated. Phosphorylated IIIGlc (P-IIIGlc) has a slightly lower mobility during sodium dodecyl sulphate/polyacrylamide gel electrophoresis than IIIGlc. In bacterial extracts both phosphoenolpyruvate (the physiological phosphoryl donor of the PTS) as well as ATP can phosphorylate IIIGlc. The ATP-catalyzed reaction is dependent on phosphoenolpyruvate synthase, however, and is due to prior conversion of ATP to phosphoenolpyruvate. The phosphoryl group of phosphorylated IIIGlc is hydrolysed after boiling in sodium dodecyl sulfate but phosphorylated IIIGlc can be discriminated from IIIGlc if treated with this detergent at room temperature. We have used the different mobilities of IIIGlc and P-IIIGlc to estimate the proportion of these two forms in intact cells. Wild-type cells contain predominantly P-IIIGlc in the absence of PTS sugars. In an S. typhimurium mutant containing a leaky ptsI17 mutation (0.1% enzyme I activity remaining) both forms of IIIGlc occur in approximately equal amounts. Addition of PTS sugars such as glucose results, both in wild-type and mutant, in a dephosphorylation of P-IIIGlc. This correlates well with the observed inhibition of non-PTS uptake systems by PTS sugars via nonphosphorylated IIIGlc.     

Transport of trehalose in Salmonella typhimurium.

We have studied trehalose uptake in Salmonella typhimurium and the possible involvement of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in this process. Two transport systems could recognize and transport trehalose, the mannose PTS and the galactose permease. Uptake of trehalose via the latter system required that it be expressed constitutively (due to a galR or galC mutation). Introduction of a ptsM mutation, resulting in a defective IIMan/IIIMan system, in S. typhimurium strains that grew on trehalose abolished growth on trehalose. A ptsG mutation, eliminating IIGlc of the glucose PTS, had no effect. In contrast, a crr mutation that resulted in the absence of IIIGlc of the glucose PTS prevented growth on trehalose. The inability of crr and also cya mutants to grow on trehalose was due to lowered intracellular cyclic AMP synthesis, since addition of extracellular cyclic AMP restored growth. Subsequent trehalose metabolism could be via a trehalose phosphate hydrolase, if trehalose phosphate was formed via the PTS, or trehalase. Trehalose-grown cells contained trehalase activity, but we could not detect phosphoenolpyruvate-dependent phosphorylation of trehalose in toluenized cells.     

Protective Effects of Pyridoxal Phosphate Against Glucose Deprivation-Induced Damage in Cultured Hippocampal Neurons

Abstract: When hippocampal cultures were deprived of glucose, massive release of lactate dehydrogenase (LDH), an indicator of neuronal death, occurred via NMDA receptor activation. Addition of pyridoxal phosphate (PLP; 1 and 10 µ M ) inhibited this LDH release in a concentration-dependent manner. Prior exposure to PLP evoked more potent inhibitory effects on LDH release compared with those treated at the onset of glucose deprivation. Furthermore, PLP inhibited the reduction of intracellular content of pyruvate induced by glucose deprivation, which was accompanied by the reversal of intracellular ATP depletion. A noteworthy elevation of extracellular glutamate in response to glucose deprivation was completely reversed by addition of PLP. Aminooxyacetic acid, a potent inhibitor of PLP-dependent enzymes, antagonized the effects of PLP on LDH release, pyruvate production, and ATP formation. These results suggest that PLP protects neurons from glucose deprivation-induced damage by enhancing the formation of energy-yielding products and relieving extracellular load of glutamate. The observed phenomena further indicate that PLP might be used prophylactically against neuronal death induced by metabolic disorders.     

Genetic and biochemical characterization of the phosphoenolpyruvate:glucose/mannose phosphotransferase system of Streptococcus thermophilus

In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIAB(Man), two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIAB(Man), IIC(Man), IID(Man), and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIAB(Man) as well as membrane fragments containing IIC(Man) and IID(Man). These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIAB(Man).     

Increased ATPase activity is responsible for acid sensitivity of nisin-resistant Listeria monocytogenes ATCC 700302

The growth of the foodborne pathogen Listeria monocytogenes can be controlled by nisin, an antimicrobial peptide. A spontaneous mutant of L. monocytogenes shows both resistance to nisin and increased acid sensitivity compared to the wild type. Changes in the cell membrane correlated with nisin resistance, but the mechanism for acid sensitivity appears unrelated. When hydrochloric or lactic acid is added to cultures, intracellular ATP levels drop significantly in the mutant (P < 0.01) compared to the results seen with the wild type. Characterization of the F(0)F(1) ATPase, which hydrolyzes ATP to pump protons from the cell cytoplasm, shows that the enzyme is more active in the mutant than in the wild type. These data support a model in which the increased activity of the mutant ATPase upon acid addition depletes the cells' supply of ATP, resulting in cell death.     

ATP-dependent fructose uptake system in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

The bacterial phosphoenolpyruvate (PEP)-dependent group translocation system (PTS) requires the presence of both membrane-bound and cytoplasmic components to phosphorylate and translocate sugar. Deinococcus radiodurans has a functional fruA gene coding for the membrane-bound components of the fructose-specific PTS. However, fruB gene coding for the fructose-specific cytosolic components of PTS is a pseudogene. Yet, this bacterium metabolized fructose readily. In vitro studies showed that both cell membranes and cytoplasmic fractions of the cells were needed for fructose phosphorylation. Further studies showed that fructose phosphorylation required ATP, not PEP, as the phosphate donor. Unlike most PEP-dependent PTS systems, fructose phosphorylation is sensitive to sodium fluoride, a kinase inhibitor. Fructose phosphorylation was also inhibited in the presence of antiserum against a kinase phosphorylation site. Rhodobacter capsulatus has a functional fruA–fruB system. Complementation assays by reconstituting the membrane fraction of D. radiodurans to the cytoplasmic fraction of R. capsulatus resulted in a PEP-dependent fructose phosphorylation, whereas mixing the membranes of R. capsulatus and the deinococcal cytosol in vitro resulted in an ATP-dependent fructose phosphorylation.     

Methotrexate efflux in L1210 cells. Kinetic and specificity properties of the efflux system sensitive to bromosulfophthalein and its possible identity with a system which mediates the efflux of 3',5'-cyclic AMP

Methotrexate exits L1210 mouse leukemia cells via multiple routes that include a unidirectional efflux component which is sensitive to bromosulfophthalein. This efflux component has been characterized in the present study after eliminating the contribution from the other efflux routes by treatment of the cells with an active ester of methotrexate and by reducing the assay pH to 6.2. The remaining efflux at pH 6.2 was greater than 90% sensitive to bromosulfophthalein. This route was also inhibited by probenecid, prostaglandin A1, diamide, 1-methyl-3-isobutylxanthine, various metabolic inhibitors, and by transfer of the cells to a buffer containing high concentrations of KCl. The inhibition by prostaglandin A1 was exceptionally potent and reached 50% at a concentration of 0.5 microM. An enhancement in efflux occurred upon the addition of glucose or by transfer of the cells to a non-saline buffer. When parameters relating to cellular energetics were measured, a reduction in ATP level was associated with the inhibition of efflux by probenecid, carbonylcyanide m-chlorophenylhydrazone, valinomycin, and antimycin A, whereas the increase in efflux by glucose was accompanied by an increase in intracellular ATP. Changes in ATP, however, were not associated with the inhibition by various other compounds or additions or with the enhancement in efflux by the non-anionic buffer. When the relative sensitivity of methotrexate efflux to bromosulfophthalein, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and lactic anhydride was compared with other anion transport systems, differences in specificity indicated that methotrexate was not exiting the cells via the bicarbonate/chloride exchange carrier, the lactate/H+ co-transport system, or a system which mediates the efflux of phthalate. However, a correlation was apparent between the sensitivity of methotrexate efflux to inhibition by prostaglandin A1, probenecid, and certain metabolic inhibitors and the ability of these same compounds to inhibit the unidirectional efflux of 3',5'-cyclic AMP in other cell lines, suggesting that methotrexate may share a common efflux route with cyclic nucleotides.