Purine nucleobase transport in the intraerythrocytic malaria parasite

Hypoxanthine, a nucleobase, serves as the major source of the essential purine group for the intraerythrocytic malaria parasite. In this study we have measured the uptake of hypoxanthine, and that of the related purine nucleobase adenine, by mature blood-stage Plasmodium falciparum parasites isolated from their host cells by saponin-permeabilisation of the erythrocyte and parasitophorous vacuole membranes. The uptake of both [3H]hypoxanthine and [3H]adenine was comprised of at least two components; in each case there was a rapid equilibration of the radiolabel between the intra- and extracellular solutions via a low-affinity transport mechanism, and an accumulation of radiolabel (such that the estimated intracellular concentration exceeded the extracellular concentration) via a higher-affinity process. The uptake of [3H]adenine was studied in more detail. The rapid, low-affinity equilibration of [3H]adenine between the intra-and extracellular solution was independent of the energy status of the parasite whereas the higher-affinity accumulation of the radiolabel was ATP-dependent. A kinetic analysis of adenine uptake revealed that the low-affinity (equilibrative) process had a Km of approximately 1.2mM, similar to the value of 0.82 mM estimated here (using the Xenopus laevis oocyte expression system) for the Km for the transport of adenine by PfENT1, a parasite-encoded member of the 'equilibrative nucleoside/nucleobase transporter' family. The results indicate that nucleobases enter the intraerythrocytic parasite via a rapid, equilibrative process that has kinetic characteristics similar to those of PfENT1.     

Purine and pyrimidine transport and permeation in human erythrocytes

Time courses of the uptake of radiolabeled hypoxanthine, adenine and uracil were measured by rapid kinetic techniques over substrate ranges from 0.02 to 5000 microM in suspensions of human erythrocytes at 25 or 30 degrees C. At concentrations above 25 microM, the rate of intracellular phosphoribosylation of hypoxanthine and adenine was insignificant relative to their rates of entry into the cell and time courses of transmembrane equilibration of the substrates could be measured and analyzed by integrated rate analysis. Hypoxanthine and uracil are transported by simple facilitated carriers with directional symmetry, high capacity and Michaelis-Menten constants of about 0.2 and 5 mM, respectively. Adenine is probably transported by a carrier with similar properties but no saturability was detectable up to a concentration of 5 mM. Cytosine entered the cells much more slowly than the other three nucleobases, and its entry seems not to be mediated by a carrier. The hypoxanthine transporter resembles that of one group of mammalian cell lines, which does not exhibit any overlap with the nucleoside transporter and is resistant to inhibitors of nucleoside transport. Results from studies on the effects of the nucleobases on the influx and countertransport of each other were complex and did not allow unequivocal conclusions as to the number of independent carriers involved. At concentrations below 5 microM, radiolabel from adenine and hypoxanthine accumulated intracellularly to higher than equilibrium levels. Part of this accumulation reflected metabolic trapping, especially when the medium contained 50 mM phosphate. But part was due to an apparent concentrative accumulation of free adenine and hypoxanthine up to 3-fold at medium concentrations much less than 1 microM and when cells were incubated in phosphate-free medium. This concentrative accumulation could be due to the functioning of additional high-affinity, low-capacity, active transport systems for adenine and hypoxanthine, but other factors could be responsible, such as saturable binding to intracellular components.     

Purine base transport in normal and mutant erythrocytes.

The uptake of adenine and hypoxanthine in HGPRT-deficient and normal human erythrocytes was measured using a rapid filtering centrifugation technique. The transport of hypoxanthine as well as of adenine is impaired in the mutant cells. The transport of hypoxanthine into HGPRT-deficient erythrocytes differs from that into normal cells with respect to a higher accumulation capacity, to lower initial velocities and to the kinetic properties of the translocator. In addition, a higher accumulation capacity and lower initial velocities of adenine uptake could be demonstrated in mutant cells. A linkage of the purine translocator with purine phosphoribosyltransferases associated with the erythrocyte membrane is discussed.     

Initial rates of pyruvate transport in mitochondria determined by an "inhibitor-stop" technique.

An "inhibitor-stop" technique has been developed for measuring initial rates of pyruvate transport into mitochondria. The technique uses alpha-cyano-3-hydroxycinnamate as the inhibitor and separates the mitochondria from the radioactive medium by Millipore filtration. Observed rates depend on availability of hydroxyl and other exchangeable anions within the mitochondrial matrix.     

Purine and pyridine nucleotide production in human erythrocytes

Human erythrocyte adenyl and pyridine nucleotide production has been tested in cell-free lysates and in intact cells. The main products obtained in cells incubated with adenine and nicotinic acid are adenosine triphosphate and nicotinate mononucleotide, respectively, under any experimental condition used (incubation time, base concentration). Adenine-phosphoribosyltransferase activity determined in crude lysates is about 100 times higher than nicotinate-phosphoribosyltransferase activity, while cellular adenyl nucleotide production is only three times higher than that of pyridine nucleotide. A strong intracellular regulation for the former, but not latter, synthetic process is thus suggested. Intact erythrocyte nicotinate nucleotide production is inhibited by adenine, while nicotinate-phosphoribosyltransferase activity is not. The possible regulation by adenyl nucleotides is discussed in light of the modulating action of ATP on nicotinate-phosphoribosyltransferase activity. The kinetic characteristics of both adenine- and nicotinate-phosphoribosyltransferases, determined on crude lysates, are reported.     

Expression of a novel high-affinity purine nucleobase transport function in mutant mammalian T lymphoblasts.

The single nucleoside transport function of mouse S49 lymphoblasts also transports purine bases (B. Aronow and B. Ullman, J. Biol. Chem. 261:2014-2019, 1986). This transport of purine bases by S49 cells is sensitive to inhibition by dipyridamole (DPA) and 4-nitrobenzylthioinosine, two potent inhibitors of nucleoside transport. Therefore, wild-type S49 cells cannot salvage low hypoxanthine concentrations in the presence of 10 microM DPA and 11 microM azaserine; the latter is a potent inhibitor of purine biosynthesis. Among a mutagenized wild-type population, a cell line, JPA2, was isolated which could proliferate in 50 microM hypoxanthine-11 microM azaserine-10 microM DPA. The basis for the survival of JPA2 cells under these selective conditions was expression of a unique, high-affinity purine nucleobase transport function not present in wild-type cells. JPA2 cells could transport 5 microM concentrations of hypoxanthine, guanine, and adenine 15- to 30-fold more efficiently than parental cells did. Kinetic analyses revealed that the affinity of the JPA2 transporter for all three purine bases was much greater than that of the wild-type nucleobase transport system. Moreover, nucleobase transport in JPA2 cells, unlike that in parental cells, was insensitive to inhibition by DPA, 4-nitrobenzylthioinosine, sulfhydryl reagents, and nucleosides. No alterations in nucleoside transport capability, phosphoribosylpyrophosphate levels, or purine phosphoribosyltransferase enzymes were detected in JPA2 cells. Thus, JPA2 cells express a novel nucleobase transport capability which can be distinguished from the nucleoside transport function by multiple biochemical parameters.     

cGMP and glutathione-conjugate transport in human erythrocytes.

The nature of cGMP transport in human erythrocytes, its relationship to glutathione conjugate transport, and possible mediation by multidrug resistance-associated proteins (MRPs) have been investigated. MRP1, MRP4 and MRP5 are detected in immunoblotting studies with erythrocytes. MRP1 and MRP5 are also detected in multidrug resistant COR-L23/R and MOR/R cells but at greatly reduced levels in the parent, drug sensitive COR-L23/P cells. MRP4 is detected in MOR/R but not COR-L23/R cells. Uptake of cGMP into inside-out membrane vesicles prepared by a spontaneous, one-step vesiculation process is shown to be by a low affinity system that accounts for more than 80% of the transport at all concentrations above 3 micro m. This transport is reduced by MRP inhibitors and substrates including MK-571, methotrexate, estradiol 17-beta-d-glucuronide, and S(2,4-dinitrophenyl)glutathione (DNP-SG) and also by glibenclamide and frusemide but not by the monoclonal Ig QCRL-3 that inhibits high-affinity transport of DNP-SG by MRP1. It is concluded that the cGMP exporter is distinct from MRP1 and has properties similar to those reported for MRP4. Furthermore the evidence suggests that the protein responsible for cGMP transport is the same as that mediating low-affinity DNP-SG transport in human erythrocytes.     

The monovalent cation "leak" transport in human erythrocytes: an electroneutral exchange process.

The mechanism of the "ground permeability" of the human erythrocyte membrane for K+ and Na+ was investigated with respect to a possible involvement of a previously unidentified specific transport pathway, because earlier studies showed that it cannot be explained on the basis of simple electrodiffusion. In particular, we analyzed and described the increase in the (ouabain+bumetanide+EGTA)-insensitive unidirectional K+ and Na+ influxes as well as effluxes (defined as "leak" fluxes) observed in erythrocytes suspended in low-ionic-strength media. Using a carrier-type model and taking into account the influence of the ionic strength on the outer surface potential according to the Gouy-Chapman theory (i.e., the ion concentration near the membrane surface), we are able to describe the altered "leak" fluxes as an electroneutral process. In addition, we can show indirectly that this electroneutral flux is due to an exchange of monovalent cations with protons. This pathway is different from the amiloride-sensitive Na+/H+ exchanger present in the human red blood cell membrane and can be characterized as a K+(Na+)/H+ exchanger.     

Effects of Ca2+-channel antagonists on nucleoside and nucleobase transport in human erythrocytes and cultured mammalian cells

Lidoflazine strongly inhibited the equilibrium exchange of uridine in human erythrocytes (Ki approximately 16 nM). Uridine zero-trans influx was similarly inhibited by lidoflazine in cultured HeLa cells (IC50 approximately to 80 nM), whereas P388 mouse leukemia and Novikoff rat hepatoma cells were three orders of magnitude more resistant (IC50 greater than 50 microM). Uridine transport was also inhibited by nifedipine, verapamil, diltiazem, prenylamine and trifluoperazine, but only at similarly high concentrations in both human erythrocytes and the cell lines. IC50 values ranged from about 10 microM for nifedipine and about 20 microM for verapamil to more than 100 microM for diltiazem, prenylamine and trifluoperazine. The concentrations required for inhibition of nucleoside transport are several orders higher than those blocking Ca2+ channels. Lidoflazine competitively inhibited the binding of nitrobenzylthioinosine to high-affinity sites in human erythrocytes, but did not inhibit the dissociation of nitrobenzylthioinosine from these sites on the transporter as is observed with dipyridamole and dilazep.     

Volume-sensitive K transport in human erythrocytes

Studies have been carried out on human erythrocytes to examine the alterations of K transport induced by swelling or shrinking the cells by osmotic and isosmotic methods. Hypotonic swelling of erythrocytes (relative cell volume, 1.20) resulted in a striking, four- to fivefold augmentation in the ouabain-resistant K influx over the value obtained at a normal cell volume. Shrinking the cells in hypertonic media resulted in a small but statistically significant reduction in K influx. Three different methods of varying cell volume gave similar results. These include the addition of sucrose and of NaCl to hypotonic media and the isosmotic (nystatin) method. The major fraction of the K influx in swollen cells is specific in its requirement for Cl or Br and is not supported by thiocyanate, iodide, nitrate, methylsulfate, or acetate. Bumetanide (0.1 mM), MK-196 (0.2 mM), and piretanide (1 mM) are poorly effective in suppressing K uptake in swollen cells, but at higher concentrations, bumetanide (1 mM) inhibits 80% of the Cl-dependent K influx in swollen cells. The bumetanide concentration required to inhibit 50% of the Cl-dependent K influx is 0.17 mM. The volume-sensitive K influx is independent of both extracellular and intracellular Na, so that the (Na + K + 2Cl) cotransport pathway is not a likely mediator of the volume-sensitive K transport. A variety of inhibitors of the Ca-activated K channel are ineffective in suppressing swelling-induced K influx. Like K uptake, the efflux of K is also enhanced by cell swelling. Swelling-activated K efflux is Cl dependent, is independent of extracellular and intracellular Na, and is observed with both hypotonic and isosmotic methods of cell swelling. The activation of K efflux by cell swelling is observed in K-free media, which suggests that the volume-sensitive K transport pathway is capable of net K efflux. The addition of external K to hypotonic media resulted in an increase in K efflux compared with the efflux in K-free media, and this increase was probably due to K/K exchange. Thus, hypotonic or isosmotic swelling of human erythrocytes results in the activation of a ouabain-resistant, Cl-dependent, Na-independent transport pathway that is capable of mediating both net K efflux and K/K exchange.     

Purine nucleoside phosphorylase from human erythrocytes: a kinetic study of the fully separated isoenzymes.

Human red cell lysates contain at least seven electrophoretically distinct isoenzymes of purine-nucleoside phosphorylase (PNPase); the proportion of more anodal bands increases as the erythrocyte ages, suggesting that the native enzyme is subjected to progressive post-translational modifications. The age dependent electrophoretic changes observed in the hemolysate are associated with the downward curvature of the Lineweaver-Burk double reciprocal plot at high inosine-substrate concentrations unlike the single-banded PNPase from tissue cultures of rapidly dividing cells. Thanks to the high resolution power of the ion-exchange HPLC technique utilized we have been able to fully separate all the seven isoenzymes and correlate structural to functional modifications in PNPase from human erythrocytes. Our results indicate that the downward curvature of Lineweaver-Burk plot is not due to a mixture of isoforms with low and high Km for inosine but that the allosteric activation by the inosine substrate is the direct consequence of structural modification(s) on the "primary" form of the enzyme.     

Rubidium transport in X-irradiated human erythrocytes

Outflow of 86Rb, a radioactive analogue of potassium, from human erythrocytes X-irradiated in vitro was studied with the following results. (1) The 86Rb level in the supernatants of irradiated and control cell suspensions reflected mainly 86Rb outflow and much less its active re-uptake. (2) The effect of irradiation on 86Rb outflow was more pronounced at a low temperature (4 degrees C) than at 37 degrees C; the lowest dose of X-radiation exhibiting a significant effect on 86Rb outflow at 4 degrees C was 2.5 Gy. (3) K/Rb exchange did not seem to play an appreciable role in radiation-induced 86Rb outflow. (4) Calcium and its accumulation in irradiated cells was not found to be the cause of the effect of radiation on 86Rb outflow. (5) The effect of radiation on 86Rb outflow was higher in low Na medium but it was not inhibited by bumetanide. Rb/Na counter- or co-transport do not therefore seem to be involved in radiation-induced Rb+ outflow.     

Purine base transport in Neurospora crassa.

Observations presented in this paper point to the presence of dual transport mechanisms for the base adenine in Neurospora crassa. Competition for transport, as well as growth inhibition studies using an ad-1 auxotroph, show that the purine bases adenine, guanine, and hypoxanthine share at least one transport mechanism which is insensitive to adenosine, cytosine, and a variety of other purine base analogues. On the other hand, uptake of adenine by an ad-8 mutant strain unable to transport [8-14C]hypoxanthine at any concentration was not inhibited by guanine or hypoxanthine. This observation demonstrates the existence of an adenine-specific transport system which was also found to be insensitive to inhibition by other purine base analogues, adenosine or cytosine. Recombination analysis of ad-8 by wild-type crosses showed that the inability to transport [8-14C]hypoxanthine was a consequence of the ad-8 lesion or a closely linked mutation. Saturation plots of each system gave intermediary plateaus and nonlinear reciprocal plots which, based on comparison with pure enzyme kinetic analysis, suggest that either each system consists of two or more uptake systems, at least one of which exhibits cooperativity, or that each system is a single uptake mechanism which possesses more than two binding sites where the relative affinity for the purine base first decreases and then increases as the sites are filled.     

Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter

Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V(max) of 3.4 +/- 1 pmol.microl(-1).s(-1), with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs (K(m) = 96 +/- 37 microM; V(max) = 1.6 +/- 0.3 pmol.microl(-1).s(-1)) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine (K(i) = 19 +/- 7 microM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na(+) or H(+). PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.