Biomedical subjects

G B Henderson

Publications and source records attributed to G B Henderson.

At least 19 recordsLinked to original sources

PubMed1992

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.

Adenosine Triphosphate↗

PubMed1992

Cloning, sequencing, and demonstration of polymorphism in trypanothione reductase from Crithidia fasciculata.

Trypanothione reductase (TR) is a target for drug design since it is unique to trypanosomatids, substituting for the otherwise ubiquitous enzyme, glutathione reductase. We report the cloning and sequencing of several cDNAs and genes encoding Crithidia fasciculata TR, the structure of which has recently been solved by crystallography. Single base polymorphisms are detected in cDNAs (containing 80% of the coding sequence) and two different genomic clones, including a glutamine to glutamate change in the C-terminal region of the TR coding region; other nucleotide changes are silent. Homology (from genomic clones, both of which contained signals appropriate for expression) to the Trypanosoma congolense gene was 63% at the nucleic acid level, with 68% amino acid identity; the significance of homologies to human and Escherichia coli glutathione reductase sequences is discussed. Polymorphic sites in the genomic clones included sites found in the cDNAs, indicating that differences existing in the genomic sequence are real, and propagated to RNA.

Amino Acid Sequence↗

PubMed1992

Effects of trypanothione on the biological activity of irradiated transforming DNA.

Held et al. (1984a,b) demonstrated previously that glutathione (GSH), a negatively charged thiol, is significantly less efficient in the hydrogen atom donation repair reaction with radicals induced by radiation in transforming DNA (t-DNA) than are other thiol compounds. Fahey et al. (1991a,b) postulated that the charge on thiols can influence their ability to radioprotect DNA. GSH, which is excluded from the vicinity of DNA due to its negative charge, is less protective than neutral or positively charged thiols. We have investigated this phenomenon further with trypanothione, the conjugate of glutathione and spermidine, N1,N8-bis (L-gamma-glutamyl-L-hemicystinyl-glycyl)-spermidine. Trypanothione exists in aerobic solution largely as the disulphide (T(S)2) but is maintained in the cell in the reduced form (T(SH)2) by means of an NADPH-dependent flavo-enzyme, trypanothione reductase (TR). Experimental data show that T(S)2 in the presence of TR radioprotects t-DNA in the absence of oxygen much better than GSH or spermidine alone or in combination. Little radioprotection by T(S)2 is seen when TR is not present. The results obtained with reduced trypanothione at low concentrations suggest that radioprotection of t-DNA in hypoxia occurs predominantly by H atom donation and slightly by .OH radical scavenging, and the protection is greater than that by GSH or spermidine because the polyamine moiety in trypanothione allows a greater concentration of GSH near the DNA molecule.

Bacillus subtilis↗

PubMed1991

X-ray structure of trypanothione reductase from Crithidia fasciculata at 2.4-A resolution.

Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidine conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. We report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from -2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4 degrees in the domains that form the site, with relative shifts of as much as 2-3 A in residue positions. These results provide a detailed view of the residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.

Amino Acid Sequence↗

PubMed1991

Engineering the substrate specificity of glutathione reductase toward that of trypanothione reduction.

Glutathione reductase (EC 1.6.4.2; CAS registry number 9001-48-3) and trypanothione reductase (CAS registry number 102210-35-5), which are related flavoprotein disulfide oxidoreductases, have marked specificities for glutathione and trypanothione, respectively. A combination of primary sequence alignments and molecular modeling, together with the high-resolution crystal structure of human glutathione reductase, identified certain residues as potentially being responsible for substrate discrimination. Site-directed mutagenesis of Escherichia coli glutathione reductase was used to test these predictions. The mutation of Asn-21 to Arg demonstrated that this single change was insufficient to generate the greater discrimination against trypanothione shown by human glutathione reductase compared with the E. coli enzyme. However, the mutation of Ala-18, Asn-21, and Arg-22 to the amino acid residues (Glu, Trp, and Asn, respectively) in corresponding positions in Trypanosoma congolense trypanothione reductase confirmed that this region of polypeptide chain is intimately involved in substrate recognition. It led to a mutant form of E. coli glutathione reductase that possessed essentially no activity with glutathione but that was able to catalyze trypanothione reduction with a kcat/Km value that was 10% of that measured for natural trypanothione reductases. These results should be of considerable importance in the design of trypanocidal drugs targeted at the differences between glutathione and trypanothione metabolism in trypanosomatids and their hosts.

Amino Acid Sequence↗

PubMed1991

Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease.

Hemoglobin is an important nutrient source for intraerythrocytic malaria organisms. Its catabolism occurs in an acidic digestive vacuole. Our previous studies suggested that an aspartic protease plays a key role in the degradative process. We have now isolated this enzyme and defined its role in the hemoglobinolytic pathway. Laser desorption mass spectrometry was used to analyze the proteolytic action of the purified protease. The enzyme has a remarkably stringent specificity towards native hemoglobin, making a single cleavage between alpha 33Phe and 34Leu. This scission is in the hemoglobin hinge region, unraveling the molecule and exposing other sites for proteolysis. The protease is inhibited by pepstatin and has NH2-terminal homology to mammalian aspartic proteases. Isolated digestive vacuoles make a pepstatin-inhibitable cleavage identical to that of the purified enzyme. The pivotal role of this aspartic hemoglobinase in initiating hemoglobin degradation in the malaria parasite digestive vacuoles is demonstrated.

Amino Acid Sequence↗

PubMed1991

Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei.

Dihydroorotate dehydrogenase (DHODase) has been purified 400-fold from the rodent malaria parasite Plasmodium berghei to apparent homogeneity by Triton X-100 solubilization followed by anion-exchange, Cibacron Blue F3GA-agarose affinity, and gel filtration chromatography. The purified enzyme has a molecular mass of 52 +/- 2 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of 55 +/- 6 kDa by gel filtration chromatography, and it has a pI of 8.2. It is active in monomeric form, contains 2.022 mol of iron and 1.602 acid-labile sulfurs per mole of enzyme, and does not contain a flavin cofactor. The purified DHODase exhibits optimal activity at pH 8.0 in the presence of the ubiquinone coenzyme CoQ6, CoQ7, CoQ9, or CoQ10. The Km values for L-DHO and CoQ6 are 7.9 +/- 2.5 microM and 21.6 +/- 5.5 microM, respectively. The kcat values for both substrates are 11.44 min-1 and 11.70 min-1, respectively. The reaction product orotate and an orotate analogue, 5-fluoroorotate, are competitive inhibitors of the enzyme-catalyzed reaction with Ki values of 30.5 microM and 34.9 microM, respectively. The requirement of the long-chain ubiquinones for activity supports the hypothesis of the linkage of pyrimidine biosynthesis to the electron transport system and oxygen utilization in malaria by DHODase via ubiquinones [Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].

Animals↗

PubMed1991

Evidence for cAMP and cholate extrusion in C6 rat glioma cells by a common anion efflux pump.

C6 rat glioma cells were investigated for a shared unidirectional efflux system for cAMP and cholate. [3H]Cholate was accumulated (at pH 7.3) by scraped C6 cell monolayers via a process which was rapid initially and then slowed to a steady state after 10 min at 37 degrees C. Release of the accumulated label was also rapid (t1/2 = 2 min), was essentially complete within 15 min, and exhibited energy dependence since it could be blocked by antimycin A. Half-maximal inhibition by antimycin A occurred at 0.87 microM, and maximal inhibition exceeded 90%. Various other compounds also inhibited [3H]cholate efflux. The most effective was prostaglandin A1, which reduced efflux half-maximally at a concentration of 0.14 microM. Other inhibitors, prostaglandin B1, verapamil, probenecid, and bromosulfophathalein, produced half-maximal inhibition at 5.3, 42, 78, and 110 microM, respectively. Cholate efflux was also blocked by 40 microM vincristine. Initial influx of [3H]cholate was not affected by antimycin A, prostaglandin A1, or vincristine and hence was attributed to a process separate from efflux. C6 rat glioma cells also have the ability to produce high intracellular levels of cAMP in response to isoproterenol and to release cAMP into the medium via a carrier-mediated efflux system. When measured under the same conditions employed for cholate efflux, the efflux of cAMP was found to be sensitive to each of the inhibitors of cholate efflux. Moreover, plots of cAMP efflux versus varying concentrations of prostaglandin A1, antimycin A, prostaglandin B1, verapamil, and probenecid showed similar response curves and comparable values for half-maximal These results indicate that C6 rat glioma cells contain a unidirectional efflux pump for cholate and that this same system also appears to mediate the unidirectional efflux of cAMP. These findings support the hypothesis that various cells contain efflux pumps which exhibit a broad specificity for large organic anions of diverse structure and that the function of these efflux pumps resides primarily in cellular anion detoxification. Analogous efflux pumps for hydrophobic drugs are overproduced in tumor cells exhibiting multidrug resistance.

Animals↗

PubMed1991

An iron-carboxylate bond links the heme units of malaria pigment.

The intraerythrocytic malaria parasite uses hemoglobin as a major nutrient source. Digestion of hemoglobin releases heme, which the parasite converts into an insoluble microcrystalline material called hemozoin or malaria pigment. We have purified hemozoin from the human malaria organism Plasmodium falciparum and have used infrared spectroscopy, x-ray absorption spectroscopy, and chemical synthesis to determine its structure. The molecule consists of an unusual polymer of hemes linked between the central ferric ion of one heme and a carboxylate side-group oxygen of another. The hemes are sequestered via this linkage into an insoluble product, providing a unique way for the malaria parasite to avoid the toxicity associated with soluble heme.

Animals↗

PubMed1990

Preliminary crystallographic analysis of trypanothione reductase from Crithidia fasciculata.

Trypanothione reductase, a flavoprotein disulfide reductase specific to trypanosomatid parasites, has been crystallized by vapor diffusion of a protein solution (10 mg/ml) against 22% polyethylene glycol (average Mr 8000) containing 100 mM-ammonium sulfate. Crystals of a size suitable for structure determination by X-ray diffraction have been obtained by seeding protein solutions with smaller crystals. The space-group is P21 (a = 60.9 A, b = 161.8 A, c = 58.4 A, beta = 99.1 degrees). The molecular mass and volume of the unit cell suggest that there is a dimer of the enzyme in the asymmetric unit, and this is confirmed by self-rotation functions calculated using data to 4.5 A resolution. The crystals diffract to beyond 3 A resolution. Crystals of another P21 form (a = 91.3 A, b = 114.4 A, c = 92.0 A, beta = 141.3 degrees) are observed to grow under similar conditions.

Animals↗

PubMed1990

Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata.

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate (L-CA) to L-5,6-dihydroorotate (L-DHO), which is the third enzyme in de novo pyrimidine biosynthesis. The enzyme was purified from two parasitic protozoa, Crithidia fasciculata (about 16,000-fold) and Plasmodium berghei (about 790-fold). The C. fasciculata enzyme had a native molecular weight (Mr) of 42,000 +/- 5000, determined by gel filtration chromatography, and showed a single detectable protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Mr 44,000 +/- 3000. The DHOase from P. berghei had a native molecular weight of 40,000 +/- 4000 and a subunit molecular weight on SDS-PAGE of 38,000 +/- 3000. The DHOase from both parasites, in contrast to the mammalian enzyme which resides on a trifunctional protein of the first two enzymes of the pathway, carbamoyl-phosphate synthase and aspartate transcarbamylase, is monomeric and has no oligomeric structure as studied by chemical cross-linking with dimethyl suberimidate. The rate of cyclization of L-CA by the C. fasciculata enzyme was relatively high at acidic pH, decreasing to a very low rate at alkaline pH. In contrast, the rate of ring cleavage of L-DHO was very low at acidic pH and increased to a higher rate at alkaline pH. These pH-activity profiles gave an intersection at pH 6.6. The Km and kcat for L-CA were 0.846 +/- 0.017 mM and 39.2 +/- 6.4 min-1, respectively; for L-DHO, they were 25.85 +/- 2.67 microM and 258.6 +/- 28.5 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Growth inhibition by homofolate in tumor cells utilizing a high-affinity folate binding protein as a means for folate internalization.

A subline (JT-1) of L1210 mouse leukemia cells that contains elevated levels of a high-affinity folate binding protein is sensitive to growth inhibition by homofolate. Inhibition was observed at nanomolar concentrations of folate or 5-formyltetrahydrofolate where the high-affinity binding protein is the predominant uptake route for folate compounds. At 1.0 nM folate, inhibition of growth by 50% occurred at 0.7 nM homofolate, and maximal inhibition exceeded 90% at homofolate concentrations above 10 nM. Homofolate also inhibited the uptake of 1.0 nM [3H]folate by L1210/JT-1 cells in 72-hr cultures, and the extent of uptake inhibition by 1.0 and 20 nM homofolate was comparable to the inhibition of cell growth by the same concentrations of homofolate. At a growth-limiting concentration of 5-formyltetrahydrofolate (0.5 nM), half-maximal inhibition of L1210/JT-1 cell growth occurred at 1.0 nM homofolate. When excess concentrations of folate (5 microM) or 5-formyltetrahydrofolate (0.5 microM) were added to the medium, no growth inhibition was observed for homofolate at concentrations up to 100 microM. Parental cells lacking the folate binding protein did not respond to homofolate either at growth-limiting (0.5 microM) or excess (5.0 microM) levels of folate. Binding measurements showed that homofolate has a high affinity for the folate-binding protein (Ki = 0.03 nM) but interacts poorly with the reduced-folate transport system (Ki = 203 microM). These results indicate that homofolate inhibits the growth of L1210 cells when intracellular folates are acquired via the high-affinity folate binding protein. The basis for this inhibition appears to be competition by homofolate for substrate binding and internalization.

Animals↗

PubMed1990

Biosynthesis of the trypanosomatid metabolite trypanothione: purification and characterization of trypanothione synthetase from Crithidia fasciculata.

Trypanothione synthetase from Crithidia fasciculata has been purified ca. 14,500-fold to homogeneity in an overall yield of 40%. The pure enzyme catalyzed the synthesis of N1- and N8-glutathionylspermidine and N1,N8-bis(glutathionyl)spermidine (trypanothione) from ATP/magnesium, glutathione (GSH), and spermidine, N1- and N8-glutathionylspermidines being intermediates of trypanothione synthesis. The enzyme showed a sharp pH optimum of 7.5-7.75 for the synthesis of both mono- and diglutathionylspermidine conjugates. It was highly specific for its physiological substrates ATP/Mg2+, GSH, spermidine, and N1- and N8-glutathionylspermidine with Km values of 400 microM, 914 microM, 1.07 mM, 20 microM, and 7 microM, respectively. Trypanothione synthetase was active in the monomeric form with Mr = 87,000 and absorption maxima lambda max = 225 and 280 nm (A280/A260 = 1.85). Trypanothione synthetase is a new member of the ATP-dependent class of ligases which form amide linkage with concomitant production of ADP and orthophosphate.

Amide Synthases↗

PubMed1990

Characteristics of a novel transport system for folate compounds in wild-type and methotrexate-resistant L1210 cells.

The growth requirements and transport characteristics of folate compounds in a methotrexate-resistant L1210/R81 cell line were compared with parental cells. Concentrations for half-maximal growth of the resistant cells with folate (350 nM) and 5-formyltetrahydrofolate (20 nM) were found to be higher by 2.7-fold and 20-fold, respectively, relative to the parent, suggesting that changes had occurred in the transport of these folate compounds. Transport measurements revealed that the resistant cells have lost the capacity to transport methotrexate and other folate compounds via the reduced-folate transport system but that a second previously undescribed transport system is present. Uptake of folate via this second route is energy dependent, exhibits saturation kinetics, can be inhibited by substrate analogues, and is activated by a reduction in pH. The pH effect is substantial since uptake at 5.0 microM folate can be increased 10-fold by decreasing the pH from 7.4 to 6.2. The observed Kt for half-maximal influx of folate at pH 6.8 was 5.2 microM, and the Vmax was 0.55 pmol/min/mg of protein. Ki values for methotrexate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate were 10.2, 3.2, and 2.7 microM, respectively. Transport activity was not affected by depleting internal stores of folate by growth in folate-free medium, and this same transport system was also present in comparable amounts in wild-type cells. In the latter case, interfering uptake via the reduced-folate transport system was blocked by bromosulfophthalein and thiamine pyrophosphate. Methotrexate and 5-formyltetrahydrofolate were also transported via this alternative, pH-dependent route, whereas little or no uptake of 5-methyltetrahydrofolate could be detected. The results indicate that antifolate resistance in L1210/R81 cells was induced by inactivating the reduced-folate transport system and by utilizating a secondary route which has a low capacity for transporting methotrexate but whose activity is sufficient to permit cell growth in medium supplemented with folate or 5-formyltetrahydrofolate.

Animals↗

PubMed1990

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.

Animals↗

PubMed1990

Hemoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle.

The malaria parasite Plasmodium falciparum uses host erythrocyte hemoglobin as a major nutrient source. We report the purification of P. falciparum digestive vacuoles and characterization of the degradative process therein. Vacuoles were isolated by a combination of differential centrifugation and density gradient separation. The pure vacuoles were capable of degrading hemoglobin to small fragments with a pH optimum of 5-5.5. Proteolysis in the vacuoles appears to be an ordered process, requiring an aspartic protease to clip intact hemoglobin before other proteolytic activities can function efficiently. The vacuoles do not contain other hydrolases commonly found in lysosomes and therefore appear to be unique proteolytic organelles designed specifically to degrade hemoglobin.

Animals↗

PubMed1990

Folate-binding proteins.

Folate-binding proteins of three major classes have been observed in various bodily fluids and in the plasma membrane and cytoplasm of normal and neoplastic cells. A major class, the high-affinity folate-binding proteins, show a preferential and tight binding of folic acid relative to reduced folates and methotrexate and consist of water-soluble and membrane-associated forms. Soluble forms of the high-affinity binders are present in serum and milk and in the growth medium of certain cultured cell lines, whereas membrane-associated forms are observed on the surface of various cells and tissues. The binders in serum have no clearly defined function, whereas the milk binders serve to accumulate and stabilize reduced-folate compounds in milk and they may also facilitate the absorption of folates by the intestinal mucosa of neonates. Membrane-bound forms of high-affinity folate-binding proteins mediate the transport of folate compounds across plasma membranes and appear to utilize endocytosis as the transport mechanism. Membrane-associated high-affinity binding proteins contain covalently bound phospholipids and hydrophobic C-terminal amino acid sequences that are absent in the soluble forms. The remaining protein portions of these binders show considerable sequence homology. The second class is composed of folate-binding proteins that reside solely in the plasma membrane and are structurally and mechanistically distinct from the high-affinity binders. These proteins function in transport, exhibit varied substrate specificities that accommodate reduced-folate compounds with equal or higher affinity than folate, and do not utilize endocytosis as the mechanism for substrate internalization. The third class of folate-binding proteins consists of enzymes that reside in the cytoplasm of cells.

Animals↗

PubMed1989

Mediation of cellular anion detoxification in leukemic cells by unidirectional efflux pumps.

Methotrexate influx in L1210 cells is mediated almost entirely by a single system, whereas efflux occurs via three routes, the influx carrier functioning in reverse and two additional systems which are operationally unidirectional. The two unidirectional routes show considerable similarities in energy-dependence and inhibitor sensitivities but can be separated by their differential inhibition by bromosulfophthalein (BSP), probenecid, and vincristine. The predominant route is inhibited by BSP and vincristine, whereas the other route is sensitive to probenecid. A search for additional anion substrates for the unidirectional efflux systems for methotrexate led to the finding that cholate exits L1210 cells via the same two unidirectional efflux systems as methotrexate. Cholate efflux is carrier-mediated, energy-dependent, and unidirectional, and it can be inhibited by several compounds which inhibit the efflux of methotrexate. Moreover, the same concentration of each compound which produced a half-maximal inhibition of methotrexate efflux also inhibited the efflux of cholate by 50%. Cholate efflux occurred predominantly (85%) via the BSP-sensitive route. The observation that methotrexate and cholate share the same efflux systems in L1210 cells suggest that perhaps other large anions are also accommodated by these systems since methotrexate and cholate differ in net negative charge and have few common structural features. The hypothesis advanced from these studies has been that the function of the unidirectional efflux systems is to extrude various organic anion catabolites which might otherwise become toxic if allowed to accumulate within cells. Possible intracellular anions in this latter category include sulfonated or carboxylated steroids, bilirubin, and products of vitamin D and prostaglandin catabolism. Unidirectional efflux pumps have been identified for other anionic compounds including cyclic AMP and oxidized glutathione, and in both cases similarities were apparent with the efflux of methotrexate and cholate. The most compelling comparison was with prostaglandin A1 which inhibited the efflux of cyclic AMP, methotrexate, and cholate half-maximally at the same low concentration of 0.1 microM. Energy-dependent unidirectional efflux pumps for large neutral or cationic drugs have also been identified in cells with acquired multidrug resistance. The latter efflux activity is thought to be mediated by a membrane glycoprotein (p170) which is also sensitive to several of the various inhibitors (reserpine, verapamil, and quinidine) which reduce the efflux of methotrexate and cholate.

Animals↗