Membrane transport of nucleobases: interaction with inhibitors.

1. The kinetic properties and the mechanism of nucleobase transport and transport inhibition are briefly reviewed. 2. Many purine derivatives even when bearing large substituents on N9 and C6 are inhibitors of nucleobase transport, some are also substrates. 3. Papaverine and other benzyl-isoquinolines are efficient inhibitors of facilitated transport of nucleobases. 4. Papaverine is a noncompetitive inhibitor of nucleobase transport in human erythrocytes. 5. Reduction of the aromatic isoquinoline to the tetrahydro form causes loss of inhibitory activity whereas replacement of methoxy groups by ethoxy groups leads to increased activity. 6. Papaverine also inhibits sodium dependent active nucleobase transport in pig kidney cells. 7. The nucleoside transport inhibitors dipyridamole and dilazep have no effect on facilitated diffusion transport of nucleobases, but inhibit in micromolar concentrations active sodium dependent nucleobase transport in pig kidney cells.

Nucleobase and nucleoside transport in mammalian cells.

Membrane transport of nucleobases and nucleosides has been an actively pursued research field for the past 25 years. Not only are these substances of physiological interest; derivatives are in clinical use or under investigation for their pharmacological activity against viral and neoplastic disease. An understanding of the molecular pharmacology of these substances includes a detailed knowledge of how they reach their intracellular targets. Membrane transport systems which have so far been found in all cells examined play an important role in this process. Since the transporters are minor membrane components, little is known about them on a molecular basis. This review discusses methodological approaches used to measure initial rates of membrane transport and summarizes current knowledge of the various transport systems which have been characterized with these kinetic methods.

Inhibition of purine nucleobase transport in human erythrocytes and cell lines by papaverine. Investigation of structure-activity relationship.

Papaverine was found to be an effective inhibitor of hypoxanthine transport not only in human erythrocytes, but also in the human cell lines HL60 (myeloic) and U937 (monocytic). IC50 values for inhibition of hypoxanthine influx ranged from 6 to 20 microM. In erythrocytes papaverine was found to be a non-competitive inhibitor of hypoxanthine equilibrium-exchange transport with a Ki value of approximately 13 microM, which is in close agreement with the respective IC50 values estimated for zero-trans influx of hypoxanthine. In addition papaverine also had a slight inhibitory effect on unmediated nucleobase transport, most likely due to a perturbation of the membrane lipid environment. Several papaverine analogs were tested for their inhibitory effect on nucleobase transport. Only ethaverine was as effective as papaverine. Drotaverine and berberine were moderately inhibitory while laudanosine had no inhibitory effect at all. Isoquinoline acted as a very weak inhibitor.

Allopurinol transport in human erythrocytes.

The mechanism of allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] transport into human erythrocytes was investigated with an inhibitor stop assay. Allopurinol transport could be resolved into two components: (1) a saturable system and (2) a non-saturable process, which most likely represents non-facilitated diffusion. Allopurinol transport had a Km of 268 mumol/L and a Vmax of 28 pmol/microL intracellular volume/sec; the non-saturable component was 0.0195/sec. Mutual inhibition studies showed that the competitive Ki values of hypoxanthine and adenine on allopurinol transport were 120 and 3 mumol/L, respectively. These Ki values as well as the IC50 values of 100-150 mumol/L for hypoxanthine and 3-10 mumol/L for adenine were similar to the corresponding transport Km values of these bases, which are 128 and 8 mumol/L, respectively. The Ki of allopurinol on hypoxanthine transport was 274 mumol/L and thus nearly identical to its Km. Thus in erythrocytes the uricostatic agent allopurinol is an alternative substrate for the purine transport system, but lacks the exceptional high affinity it has for xanthine oxidase. This could explain the paradoxical clinical side effect of allopurinol, namely that it can provoke an attack of gout. Theophylline, a methylated purine, inhibited allopurinol transport with an IC50 of 200-400 mumol/L. Oxypurinol [4,6-dihydroxypyrazolo(3,4-d)pyrimidine], the main metabolite of allopurinol, also inhibited allopurinol transport with an IC50 of 20-40 mumol/L. This is noteworthy, since allopurinol and oxypurinol do not share the same transport system in the kidney.

Adenine and hypoxanthine transport in human erythrocytes: distinct substrate effects on carrier mobility.

Transport of adenine and hypoxanthine in human erythrocytes proceeds via two mechanisms: (1) a common carrier for both nucleobases and (2) unsaturable permeation 4-5-fold faster for adenine for hypoxanthine. The latter process was resistant to inactivation by diazotized sulfanilic acid. Carrier mediated transport of both substrates was investigated using zero-trans and equilibrium exchange protocols. Adenine displayed a much higher affinity for the carrier (Km approximately 5-8 microM) than hypoxanthine (Km approximately 90-120 microM) but maximum fluxes at 25 degrees C were generally 5-10-fold lower for adenine (Vmax approximately 0.6-1.4 pmol/microliters per s) than for hypoxanthine (Vmax approximately 9-11 pmol/microliters per s). The carrier behaved symmetrically with respect to influx and efflux for both substrates. Adenine, but not hypoxanthine reduced carrier mobility more than 10-fold. The mobility of the unloaded carrier, calculated from the kinetic data of either hypoxanthine or adenine transport, was the same thus providing further evidence that these substrates share a common transporter and that their membrane transport is adequately described by the alternating conformation model of carrier-mediated transport.

Concordant changes of pyrimidine metabolism in blasts of two cases of acute myeloid leukemia after repeated treatment with ara-C in vivo.

Though data from cell lines are abundant, the reason for the development of resistance to 1-beta-D arabinofuranosylcytosine (ara-C) in vivo remains unresolved. A broad interpatient variation of metabolic parameters has further complicated interpretation of the results. The present study compares ara-C metabolism in leukemic blasts of two patients with newly diagnosed disease, before and after repeated treatment with ara-C containing chemotherapy regimens in vivo. Membrane transport of ara-C was unchanged after treatment. In addition, cell-free extracts of blasts obtained after treatment failure showed an unchanged cytidine deaminase activity. Though deoxycytidine kinase activity in cell extracts was unaltered or increased after treatment failure, the activity in situ, measured as the rate of 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) formation, was decreased. This could be shown to be due to an expansion of the deoxycytidine triphosphate (dCTP) pool. The severalfold increase in dCTP pool was accompanied by a decrease in thymidine triphosphate (dTTP) pool and correlated with a decrease in deoxycytidylate deaminase (dCMP-deaminase) activity in cell free extracts. Low dCMP-deaminase activity had been shown to confer an ara-C resistant phenotype to cell lines in vitro. Data presented in this paper show that a selection for leukemic blasts with low dCMP-deaminase activity can also be favored by ara-C containing treatment regimens in vivo. Our data suggest that this mechanism might contribute to treatment failure.

Influence of cell cycle on glutathione-S-transferase, selenium-dependent glutathione peroxidase, superoxide dismutase and glutathione levels in human myeloid leukaemia cell lines.

An important biological function of glutathione (GSH) resides in the detoxication reactions mediated by enzymes such as glutathione-S-transferase (GSTs) and glutathione peroxidase (GPX). An increasing body of evidence implies that GSH and these enzymes play important roles in determining the sensitivity of tumours against cytotoxic drugs like quinone antibiotics, in particular adriamycin (Adr). In the present study, we have analysed the effects of cell-cycle on GSH and GSH-dependent enzymes in an attempt to explain cell-cycle specificity of these antileukaemic drugs which were shown to be involved in free-radical-type reactions. Determination of GSH, GST, GPX and superoxide dismutase in cell-cycle-enriched fractions of five different human myeloid leukaemia cell lines (KG1, K562, U937, ML-1 and ML-2) yielded results identical to those obtained in random cultures, which implies that neither GSH nor GSH-related enzymes are cell-cycle regulated. These findings argue against the presumption that cell-cycle specificity of cytotoxic drugs like Adr could be due to the glutathione-dependent metabolism in myeloid leukaemia cell lines.

Evaluation of radioimmunoassays: comparison of dose interpolation calculations by four parameter logistic and spline functions.

Two computerized methods for dose interpolation calculation were compared. Generated data sets with a known coefficient of variation as well as laboratory RIA data were analysed. The four parameter logistic method, which is based on an approximation of the mass action law, performed better than the Spline method, a procedure which makes no a priori assumptions about the data. Correct weighting of the data was important for obtaining satisfactory fits. The determination of the response error relationship proved to be the most satisfactory approach in obtaining suitable weighting factors.

Inhibition of nucleoside and nucleobase transport and nitrobenzylthioinosine binding by dilazep and hexobendine.

The transport of 500 microM uridine by human erythrocytes and S49, P388 and L1210 mouse leukemia cells, Chinese hamster ovary (CHO) cells and Novikoff rat hepatoma cells was inhibited strongly by dilazep and hexobendine with similar concentration dependence, but the sensitivity of transport in the various cell types varied greatly; IC50 values ranged from 5-30 nM for human erythrocytes and S49 and P388 cells to greater than 1 microM for CHO and Novikoff cells. The binding of nitrobenzylthioinosine (NBTI) to high-affinity sites on these cells (Kd approximately equal to 0.5 nM) was inhibited by hexobendine and dilazep in a similar pattern. Furthermore, these drugs, just as dipyridamole and papaverine, inhibited the dissociation of NBTI from high-affinity binding sites but only at concentrations 10-100 times higher than those inhibiting uridine transport. In contrast, high uridine concentrations (greater than 2 mM) accelerated the dissociation of NBTI. Dilazep also inhibited the transport of hypoxanthine, but only in those cell lines whose transporter is sensitive to inhibition by uridine and dipyridamole. Adenine transport was not inhibited significantly by dilazep in any of the cell lines tested, except for a slight inhibition in Novikoff cells. [14C]Hexobendine equilibrated across the plasma membrane in human erythrocytes within 2 sec of incubation at 25 degrees, but accumulated to 6-10 times the extracellular concentration in cells of the various cultured lines. Uptake was not affected by high concentrations of uridine, NBTI or dipyridamole. Hexobendine inhibited the growth of various cell lines to a lesser extent (IC50 = greater than or equal to 100 microM) than dipyridamole (IC50 = 15-40 microM). At 40 microM, however, it completely inhibited the growth of S49 cells that had been made nucleoside dependent by treatment with methotrexate or pyrazofurin.

Adenosine uptake, transport, and metabolism in human erythrocytes.

Using rapid kinetic techniques, we have determined the kinetics of zero-trans influx and equilibrium exchange of adenosine, and its uptake and in situ phosphorylation at 25 degrees C in human erythrocytes which were pretreated with 2'-deoxycoformycin to inhibit deamination of adenosine. Both the Km and Vmax for adenosine transport were about 300 times higher than those for the in situ phosphorylation of adenosine (Km about 0.2 microM), so that the first order rate constants for both processes were about the same. In contrast, the first order rate constant for adenosine deamination by untreated, intact cells was about 20% of that of adenosine transport or phosphorylation. These kinetic properties of the various steps, in combination with substrate inhibition of adenosine phosphorylation above 1 microM adenosine, assure that, at extracellular concentrations of physiological relevance (less than 1 microM), adenosine is very rapidly and efficiently salvaged by the erythrocytes and converted to ATP, whereas at extracellular concentrations of 10 microM or higher, practically all adenosine transported into the cells is deaminated. When the concentration of adenosine was 0.1 microM, a 10% (v/v) suspension of erythrocytes depleted the extracellular fluid of adenosine within 1 min of incubation at 25 degrees C.

Adenine nucleotide metabolism and nucleoside transport in human erythrocytes under ATP depletion conditions.

The adenine nucleotides of human red cells were labeled by incubation of the cells with [3H]adenosine. Then, the cells were incubated in Tris-saline with various supplements that cause the loss of cellular ATP, and the degradation products were quantitated as a function of time of incubation at 37 degrees C. Incubation of the cells with 2.5 or 5 mM iodoacetate, iodoacetamide or 1 mM HCHO in combination with 5 mM KF and 50 mM deoxyglucose, 50 mM D-glucose or 10 mM inosine was most efficient in depleting the cells of ATP (100% in 0.5-1 h) without causing cell lysis. In iodoacetate- and iodoacetamide-treated cells practically all catabolism of ATP occurred via ADP----AMP----IMP----inosine----hypoxanthine with hypoxanthine accumulating in the medium. In HCHO-treated cells and in cells incubated in Tris-saline or in Tris-saline with deoxyglucose with and without KF, a substantial proportion of ATP (up to 50%) was catabolized via ADP----AMP----adenosine----inosine----hypoxanthine. Under all conditions, AMP deamination and IMP and AMP hydrolysis were rate-limiting reactions. IMP degradation was more rapid in iodoacetamide- and HCHO-treated than in iodoacetate-treated red cells. It was also more rapid in fresh than in outdated red cells, and it was inhibited by Pi. Treatment with iodoacetamide and HCHO under ATP-depletion conditions resulted in a 60-80% inhibition of uridine transport by the cells. Treatment with iodoacetate or deoxyglucose plus KF had only minor effects on nucleoside transport; thus, cells treated in this manner might be useful for studying the transport of adenosine and deoxyadenosine under conditions were their phosphorylation is prevented.

Enzymological aspects of disorders in purine metabolism.

Congenital enzyme defects of purine synthesis de novo and the salvage pathway are responsible for excessive uric acid production and are often associated with hyperuricemia and gout. On the other hand, defects of enzymes essential for the purine nucleotide cycles are the biochemical basis of dysfunction of the immune system. The influence of several congenital enzyme deficiencies on the regulation of biosynthesis de novo, on the regulation of purine nucleotide concentrations, and on adenosine concentration, as well as the effect on purine transport through cell membranes are discussed. The determination of enzymes involved in purine metabolism in noncongenital diseases seems to be of diagnostic importance. As examples, enzyme activities in lymphocytes of leukemic patients, and the determination of serum guanase activity in patients with liver dysfunction are described.

Regulation of purine uptake in normal and neoplastic cells.

Purine bases and purine nucleosides pass the cell membrane by facilitated diffusion. For purine bases two different carrier proteins seem to exist. Purine bases are trapped intracellularly immediately after passage of the cell membrane by the action of purine phosphoribosyltransferases (PRTs). Comparison of kinetic data of transport and intracellular enzyme reactions shows that intracellular metabolism is rate limiting for the whole uptake process. Since phosphate stimulates the uptake of bases, limited availability of phosphoribosylpyrophosphate (PRPP) might play a regulatory role. Purine nucleosides apparently enter cells via a common carrier. Of the nucleosides under investigation, only adenosine was taken up in significant amounts. Uptake of adenosine is mainly determined by the ratio of adenosine deaminase (ADA) and adenosine kinase (AK) activities. For uptake of purine nucleotides sequential action of ecto-5'-nucleotidase (ecto-5'-NT), nucleoside carrier and intracellular metabolism is necessary. Cells without ecto-5'-NT activity did not accumulate radioactivity from nucleotides. Proliferating neoplastic cells (K 562 and HL 60 cells) showed enhanced uptake of purine bases and nucleosides, when compared to quiescent cells (erythrocytes and granulocytes). From initial rates of uptake and intracellular enzyme activities it could be concluded that this enhanced uptake was due to alterations of enzyme pattern in the neoplastic cells.

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.