Transport Of Other Substrates Research Articles

Ecdysteroid Derivatives that Reverse P-Glycoprotein-Mediated Drug Resistance.

The expression of multidrug resistance P-glycoprotein (P-gp) by cancer cells represents one of the major drawbacks to successful cancer therapy. Accordingly, the development of drugs that inhibit the activity of this transporter remains a major challenge in cancer drug discovery. In this context, several new ecdysteroid derivatives have been synthesized and evaluated as P-gp inhibitors. Two of them (compounds 9 and 14) were able to resensitize CEMVbl100 and LoVoDoxo resistant cell lines to vinblastine and doxorubicin, respectively. Indeed, both compounds 9 and 14 increased the cellular accumulation of rhodamine 123 in cells expressing P-gp and stimulated basal P-glycoprotein-ATPase activity at a 1 μM concentration, demonstrating their interference with the transport of other substrates in a competitive mode. Moreover, in a medulloblastoma cell line (DAOY), compounds 9 and 14 reduced the side population representing cancer stem cells, which are characterized by a high expression of ABC drug transporters. Further, in DAOY cells, the same two compounds synergized with cisplatin and vincristine, two drugs used commonly in the therapy of medulloblastoma. Molecular docking studies on the homology-modeled structure of the human P-glycoprotein provided a rationale for the biological results, validating the binding mode within the receptor site, in accordance with lipophilicity data and observed structure–activity relationship information. Altogether, the present results endorse these derivatives as promising P-gp inhibitors, and they may serve as candidates to reverse drug resistance in cancer cells.

Impact of Q141K on the Transport of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors by ABCG2.

The ATP-binding cassette transporter ABCG2 is expressed in various organs, such as the small intestine, liver, and kidney, and influences the pharmacokinetics of drugs that are its substrates. ABCG2 is also expressed by cancer cells and mediates resistance to anticancer agents by promoting the efflux of these drugs. In the present study, we investigated the interactions between epidermal growth factor receptor tyrosine kinase inhibitors and ABCG2 by MTT assay, intracellular drug accumulation assay, and FACS. This study showed that four epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) (gefitinib, erlotinib, lapatinib, and afatinib) were transported from tumor cells as substrates of ABCG2. Q141K is a common single-nucleotide polymorphism of ABCG2 in Asians. We demonstrated that the extracellular efflux of gefitinib, erlotinib, and lapatinib was reduced by Q141K, whereas afatinib transport was not affected. In addition, all four EGFR TKIs inhibited the transport of other substrates by both wild-type and variant ABCG2 at 0.1 μM concentrations. Accordingly, epidermal growth factor receptor tyrosine kinase inhibitors may induce interactions with other drugs that are substrates of ABCG2, and single-nucleotide polymorphisms of ABCG2 may influence both the pharmacokinetics and efficacy of these anticancer agents.

Identification of Transport-critical Residues in a Folate Transporter from the Folate-Biopterin Transporter (FBT) Family

The Synechocystis Slr0642 protein and its plastidial Arabidopsis (Arabidopsis thaliana) ortholog At2g32040 belong to the folate-biopterin transporter (FBT) family within the major facilitator superfamily. Both proteins transport folates when expressed in Escherichia coli. Because the structural requirements for transport activity are not known for any FBT protein, we applied mutational analysis to identify residues that are critical to transport and interpreted the results using a comparative structural model based on E. coli lactose permease. Folate transport was assessed via the growth of an E. coli pabA abgT strain, which cannot synthesize or take up folates or p-aminobenzoylglutamate. In total, 47 residues were replaced with Cys or Ala. Mutations at 22 positions abolished folate uptake without affecting Slr0642 expression in membranes, whereas other mutations had no effect. Residues important for function mostly line the predicted central cavity and are concentrated in the core alpha-helices H1, H4, H7, and H10. The essential residue locations are consistent with a folate-binding site lying roughly equidistant from both faces of the transporter. Arabidopsis has eight FBT proteins besides At2g32040, often lacking conserved critical residues. When six of these proteins were expressed in E. coli or in Leishmania folate or pterin transporter mutants, none showed evidence of folate or pterin transport activity, and only At2g32040 was isolated by functional screening of Arabidopsis cDNA libraries in E. coli. Such negative data could reflect roles in transport of other substrates. These studies provide the first insights into the native structure and catalytic mechanism of FBT family carriers.

Structural Determinants of Substrate Specificity Differences between Human Multidrug Resistance Protein (MRP) 1 (ABCC1) and MRP3 (ABCC3)

Multidrug resistance proteins (MRPs) are members of the "C" branch of the ATP-binding cassette transporter superfamily. Human MRP1 transports a wide range of natural product drugs and structurally diverse conjugated and unconjugated organic anions. Its closest relative is MRP3. Despite their structural similarity, the homologs differ substantially in their substrate specificity. It is noteworthy that MRP1 transports glutathione (GSH) and GSH conjugates and displays GSH-stimulated transport of a number of unconjugated and conjugated compounds. In contrast, MRP3 does not transport GSH and is a poor transporter of GSH conjugates. However, both proteins transport glucuronide conjugates, such as 17beta-estradiol 17-(beta-D-glucuronide). We have constructed a series of MRP1/MRP3 hybrids and used them to identify a region of MRP1 that is critical for binding and transport of GSH conjugates such as leukotriene C(4) (LTC(4)). Substitution of this region encompassing transmembrane helices 8 and 9 and portions of cytoplasmic loops 4 and 5 of MRP1 with the equivalent region of MRP3 eliminated LTC(4) transport. Transport of other substrates was either unaffected or enhanced. We identified three residues in this region: Tyr(440), Ile(441), and Met(443), mutation of which differentially affected transport. It is noteworthy that substitution of Tyr(440) with Phe, as found in MRP3, reduced LTC(4) and GSH-stimulated estrone-3-sulfate transport without affecting transport of other substrates tested. The mutation increased the K(m) for LTC(4) 5-fold and substantially reduced photolabeling of MRP1 by both [3H]LTC(4) and the GSH derivative, azidophenacyl-[35S]GSH. These results suggest that Tyr(440) makes a major contribution to recognition of GSH and the GSH moiety of conjugates such as LTC(4).

MUTATIONAL ANALYSIS OF POLAR AMINO ACID RESIDUES WITHIN PREDICTED TRANSMEMBRANE HELICES 10 AND 16 OF MULTIDRUG RESISTANCE PROTEIN 1 (ABCC1): EFFECT ON SUBSTRATE SPECIFICITY

Human multidrug resistance protein 1 (MRP1) has a total of 17 transmembrane (TM) helices arranged in three membrane-spanning domains, MSD0, MSD1, and MSD2, with a 5 + 6 + 6 TM configuration. Photolabeling studies indicate that TMs 10 and 11 in MSD1 and 16 and 17 in MSD2 contribute to the substrate binding pocket of the protein. Previous mutational analyses of charged and polar amino acids in predicted TM helices 11, 16, and 17 support this suggestion. Mutation of Trp(553) in TM10 also affects substrate specificity. To extend this analysis, we mutated six additional polar residues within TM10 and the remaining uncharacterized polar residue in TM16, Asn(1208). Although mutation of Asn(1208) was without effect, two of six mutations in TM10, T550A and T556A, modulated the drug resistance profile of MRP1 without affecting transport of leukotriene C4, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), and glutathione. Mutation T550A increased vincristine resistance but decreased doxorubicin resistance, whereas mutation T556A decreased resistance to etoposide (VP-16) and doxorubicin. Although conservative mutation of Tyr(568) in TM10 to Phe or Trp had no apparent effect on substrate specificity, substitution with Ala decreased the affinity of MRP1 for E(2)17betaG without affecting drug resistance or the transport of other substrates tested. These analyses confirm that several amino acids in TM10 selectively alter the substrate specificity of MRP1, suggesting that they interact directly with certain substrates. The location of these and other functionally important residues in TM helices 11, 16, and 17 is discussed in the context of an energy-minimized model of the membrane-spanning domains of MRP1.

Interactions of mefloquine with ABC proteins, MRP1 (ABCC1) and MRP4 (ABCC4) that are present in human red cell membranes

Human erythrocyte membranes express the multidrug resistance-associated proteins, MRP1, MRP4 and 5, that collectively can efflux oxidised glutathione, glutathione conjugates and cyclic nucleotides. It is already known that the quinoline derivative, MK-571, is a potent inhibitor of MRP-mediated transport. We here examine whether the quinoline-based antimalarial drugs, amodiaquine, chloroquine, mefloquine, primaquine, quinidine and quinine, also interact with erythrocyte MRPs with consequences for their access to the intracellular parasites or for efflux of oxidised glutathione from infected cells. Using inside-out vesicles prepared from human erythrocytes we have shown that mefloquine and MK-571 inhibit transport of 3 μM [ 3H]DNP-SG known to be mediated by MRP1 (IC 50 127 and 1.1 μM, respectively) and of 3.3 μM [ 3H]cGMP thought but not proven to be mediated primarily by MRP4 (IC 50 21 and 0.41 μM). They also inhibited transport in membrane vesicles prepared from tumour cells expressing MRP1 or MRP4 and blocked calcein efflux from MRP1-overexpressing cells and BCECF efflux from MRP4-overexpressing cells. Both stimulated ATPase activity in membranes prepared from MRP1 and MRP4-overexpressing cells and inhibited activity stimulated by quercetin or PGE 1, respectively. Neither inhibited [α- 32P]8-azidoATP binding confirming that the interactions are not at the ATP binding site. These results demonstrate that mefloquine and MK-571 both inhibit transport of other substrates and stimulate ATPase activity and thus may themselves be substrates for transport. But at concentrations achieved clinically mefloquine is unlikely to affect the MRP1-mediated transport of GSSG across the erythrocyte membrane.

Phenothiazines and thioxanthenes inhibit multidrug efflux pump activity in Staphylococcus aureus.

Efflux-related multidrug resistance (MDR) is a significant means by which bacteria can evade the effects of selected antimicrobial agents. Genome sequencing data suggest that Staphylococcus aureus may possess numerous chromosomally encoded MDR efflux pumps, most of which have not been characterized. Inhibition of these pumps, which may restore clinically relevant activity of antimicrobial agents that are substrates for them, may be an effective alternative to the search for new antimicrobial agents that are not substrates. The inhibitory effects of selected phenothiazines and two geometric stereoisomers of the thioxanthene flupentixol were studied using strains of S. aureus possessing unique efflux-related MDR phenotypes. These compounds had some intrinsic antimicrobial activity and, when combined with common MDR efflux pump substrates, resulted in additive or synergistic interactions. For S. aureus SA-1199B, which overexpresses the NorA MDR efflux pump, and for two additional strains of S. aureus having non-NorA-mediated MDR phenotypes, the 50% inhibitory concentration (IC(50)) for ethidium efflux for all tested compounds was between 4 and 15% of their respective MICs. Transport of other substrates was less susceptible to inhibition; the prochlorperazine IC(50) for acriflavine and pyronin Y efflux by SA-1199B was more than 60% of its MIC. Prochlorperazine and trans(E)-flupentixol were found to reduce the proton motive force (PMF) of S. aureus by way of a reduction in the transmembrane potential. We conclude that the mechanism by which phenothiazines and thioxanthenes inhibit efflux by PMF-dependent pumps is multifactorial and, because of the unbalanced effect of these compounds on the MICs and the efflux of different substrates, may involve an interaction with the pump itself and, to a lesser extent, a reduction in the transmembrane potential.

Short- and long-term changes in proteome composition and kinetic properties in a culture of Escherichia coli during transition from glucose-excess to glucose-limited growth conditions in continuous culture and vice versa.

To investigate the ability of Escherichia coli K12 MG1655 to cope with excess and limitation of a carbon and energy source, we studied the changes in kinetic properties and two-dimensional (2D) gel protein patterns of an E. coli culture. The population was transferred from glucose-excess batch to glucose-limited continuous culture (D = 0.3 h(-1)), in which it was cultivated for 500 h (217 generations) and then transferred back to glucose-excess batch culture. Two different stages to glucose-limitation were recognized: a short-term physiological adaptation characterized by a general effort in enhancing the cell's substrate scavenging ability and mutations resulting in a population exhibiting increased glucose affinity. Physiological short-term adaptation to glucose-limitation was achieved by upregulation of 12 proteins, namely MglB, MalE, ArgT, DppA, RbsB, YdcS, LivJ (precursor), UgpB (precursor), AceA, AldA, AtpA and GatY. Eight of these proteins are periplasmic binding proteins of ABC transporters. Most of them are not involved in glucose transport regulons, but rather in chemotaxis and transport of other substrates, whereas MalE and MglB have previously been shown to belong to transport systems important in glucose transport under glucose-limited conditions. Evolution under low glucose concentration led to an up to 10-fold increase in glucose affinity (from a K(s) of 366 +/- 36 microg l(-1) at the beginning to 44 +/- 7 microg l(-1)). The protein pattern of a "500-h-old" continuous culture showed a highly increased expression of MglB and MalE as well as of the regulator protein MalI. When adapted cells taken from the "500-h-old" continuous culture were transferred to batch culture, an increased expression of MalE was observed, compared with cells from un-adapted batch-grown cells. Otherwise, no significant changes were observed in the protein pattern of batch-grown populations before and after 500 h of evolution in the glucose-limited continuous culture.

Coordinate changes in drug resistance and drug-induced conformational transitions in altered-function mutants of the multidrug transporter P-glycoprotein.

The MDR1 P-glycoprotein (Pgp), responsible for a clinically important form of multidrug resistance in cancer, is an ATPase efflux pump for multiple lipophilic drugs. The G185V mutation near transmembrane domain 3 of human Pgp increases its relative ability to transport several drugs, including etoposide, but decreases the transport of other substrates. MDR1 cDNA with the G185V substitution was used in a function-based selection to identify mutations that would further increase Pgp-mediated resistance to etoposide. This selection yielded the I186N substitution, adjacent to G185V. Pgps with G185V, I186N, or both mutations were compared to the wild-type Pgp for their ability to confer resistance to different drugs in NIH 3T3 cells. In contrast to the differential effects of G185V, I186N mutation increased resistance to all the tested drugs and augmented the effect of G185V on etoposide resistance. The effects of the mutations on conformational transitions of Pgp induced by different drugs were investigated using a conformation-sensitive antibody UIC2. Ligand-binding analysis of the drug-induced increase in UIC2 reactivity was used to determine the K(m) value that reflects the apparent affinity of drugs for Pgp, and the Hill number reflecting the apparent number of drug-binding sites. Both mutations altered the magnitude of drug-induced increases in UIC2 immunoreactivity, the K(m) values, and the Hill numbers for individual drugs. Mutation-induced changes in the magnitude of UIC2 reactivity shift did not correlate with the effects of the mutations on resistance to the corresponding drugs. In contrast, an increase or a decrease in drug resistance relative to that of the wild type was accompanied by a corresponding increase or decrease in the K(m) or in both the K(m) and the Hill number. These results suggest that mutations that alter the ability of Pgp to transport individual drugs change the apparent affinity and the apparent number of drug-binding sites in Pgp.

Nutrient uptake by rat enterocytes during diabetes mellitus; evidence for an increased sodium electrochemical gradient.

1. The effect of streptozotocin-induced diabetes (7 day duration) in rats on D-glucose uptake in vivo, the unidirectional uptake of D-glucose and L-proline in vitro, the passive uptake of L-glucose in vitro and the potential difference across the brush-border membrane has been studied. 2. Diabetes resulted in an increased carrier-mediated glucose uptake both in vivo and in vitro and a stimulation of L-proline uptake at a concentration of the amino acid (0.025 mM) at which uptake was largely Na+ dependent. Diabetes was without effect on uptake using a proline concentration of 50 mM at which transport was predominantly Na+ independent. 3. A marked hyperpolarization of the brush-border membrane and an enhanced passive glucose uptake were also evident during diabetes. 4. We conclude that the stimulation of glucose uptake in vivo in diabetic intestine involves events at the brush-border membrane. The mechanisms include an increased surface area for uptake and an enhanced transmembrane electrical gradient. The latter will have a major effect on the transport of other substrates when the uptake pathway is primarily Na+ dependent.

Are there both low- and high-affinity glutamate transporters in rat cortical synaptosomes?

Kinetics of sodium dependent glutamic acid transport have been studied in rat cortical synaptosomes at sufficiently high glutamic acid concentrations ([G]) to delineate the "low affinity" transporter. Computer optimization techniques were used to fit the data to models which account for the sodium and substrate dependence of uptake. The data fit about equally well models consisting of two carriers (Model 1) or one carrier plus a linear component (Model 2). However, the results of further studies were inconsistent with Model 1, but totally consistent with Model 2. Thus the results are incompatible with the presence of both high- and low-affinity carriers. The carrier model found in previous studies of high affinity glutamate transport predicts the effects of high [G] and [Na] observed in the present study. The biphasic effect of [Na] on velocity of uptake is the logical consequence of the operation of this model. The rate equation for this model has been utilized to define and compute kinetic parameters which characterize the transporter. These kinetic functions are remarkably similar in shape and magnitude to previous estimates from the studies of the high affinity transport (low [G]). The results of other studies by the author which corroborate and expand the predictions of the kinetic model are discussed. These have been combined with the present results to formulate a rather comprehensive model of glutamate function. This model can be used to describe function in terms of mathematical equations and to make predictions from these equations. These equations relate velocity of uptake and the kinetic parameters to sodium and substrate concentration, velocity to membrane potential, distribution ratio to the electrochemical potential, and release to time, compartment sizes, and exchange constants. Such processes as concentration in the presynaptic terminal, depolarization induced release, re-uptake following stimulus induced release, and postsynaptic depolarization are all possible consequences of the operation of this model. The wide applicability of the model to the transport of other substrates in addition to glutamate is discussed.

Chronic hypoglycemia increases brain glucose transport.

Glucose transport into the brain is depressed in chronically hyperglycemic (diabetic) rats. To determine whether hypoglycemia has the opposite effect, brain transport of hexoses and other substrates was examined in chronically and acutely hypoglycemic rats. We produced chronic hypoglycemia by implanting insulin-secreting tumors or insulin-releasing osmotic mini-pumps or by repeated injection of protamine zinc insulin (PZI) and acute hypoglycemia by intravascular injection of regular insulin. Blood-brain barrier (BBB) transport was measured using the brain uptake index (BUI) method. In the three models of chronic hypoglycemia, brain glucose extraction was increased compared with controls. The extraction of deoxyglucose and several other hexoses was also increased by chronic hypoglycemia. Acute hypoglycemia had no effect on brain transport. The transport of other substrates was either not affected or depressed, suggesting increased brain hexose transport is specific. Studies of freeze-blown brain in insulinoma-engrafted rats showed that brain glucose levels were depressed while creatine phosphate, ATP, and glucose 6-phosphate were maintained. Tumor removal led to a reversion of brain glucose transport to control rates but only after 5-25 days. These findings support the view that glucose transport across the BBB is modulated by chronic alterations in the ambient glucose concentration. They also may explain why some patients with chronic hypoglycemia tolerate low blood glucose concentrations.

The trophic role of glycolic acid in coastal seawater. I. Heterotrophic metabolism in seawater and bacterial cultures

Glycolic acid, a known algal excretory product, represents a potentially important energy source for heterotrophic bacteria in marine waters. Measurements of heterotrophic uptake and mineralization by the natural microbes in the plankton indicate quantitative use of glycolic acid comparable to other common microbial substrates. This activity shows vertical and horizontal variations that correlate in a general way with primary productivity. Glycolic acid appears to be present at higher concentrations than other low molecular weight substrates, and it also shows a lower rate of turnover. It is mineralized (respired) to a greater extent than other recorded substrates, an average of 70% of total uptake. Two-thirds of a total of 141 colonies of bacteria cultured from seawater on marine agar proved capable oftaking up and respiring glycolic acid. These same bacteria, however, were unable to utilize glycolic acid for growth when it was the sole carbon source. Glycolate may therefore occur at higher concentrations and show erratic fluctuations in natural waters because microbial use is not tied to glycolate production but to the presence of other substrates. The fact that so many bacteria are able to metabolize it indicates a possibly important trophic role as an energy source. It is suggested that glycolic acid may be a major source of energy for active transport of other substrates by marine bacteria.