Nucleoside Transport Inhibitor Dipyridamole Research Articles

Inhibition of nucleoside transport and synergistic potentiation of methotrexate cytotoxicity by cimicifugoside, a triterpenoid from Cimicifuga simplex

Cimicifugoside, a triterpenoid isolated from Cimicifuga simplex, which has been used as a traditional Chinese medicine due to its anti-inflammatory, analgesic or anti-pyretic action, was examined for inhibition of nucleoside transport and synergistic potentiation of methotrexate cytotoxicity. Cimicifugoside inhibited uptake of uridine, thymidine and adenosine in human leukemia U937 cells with the low nanomolar IC 50 values, but did not affect that of uracil, leucine or 2-deoxyglucose at ≤100 nM. Cimicifugoside analogs differentially inhibited uridine uptake in the order cimicifugoside > cimicifugenin (aglycon of cimicifugoside) > bugbanoside B > cimicifugenin A, O-methyl cimicifugenin and bugbanoside A. Cimicifugoside had less affinity for the binding site of nitrobenzylthioinosine (typical high-affinity inhibitor of equilibrative nucleoside transporter-1) in U937 cells, K562 cells and human erythrocyte membranes compared with the prototype nucleoside transport inhibitor dipyridamole. Cimicifugoside markedly potentiated methotrexate cytotoxicity in a culture of U937 cells and human carcinoma KB cells. Potentiation of methotrexate cytotoxicity by cimicifugoside analogs in U937 cells was in proportion to their inhibitory activity against uridine uptake. The present study demonstrates that cimicifugoside is a novel specific nucleoside transport inhibitor that displays synergistic potentiation of methotrexate cytotoxicity.

Astrocytes affect the profile of purines released from cultured cortical neurons

Adenosine (ADO) is produced by cultured neurons and astrocytes, albeit by different pathways, during in vitro stroke models (Parkinson and Xiong [2004] J. Neurochem. 88:1305-1312). Expression of ecto-5' nucleotidase (e-N), the enzyme responsible for extracellular dephosphorylation of AMP to ADO, is more abundant in astrocytes than neurons. Therefore, we tested the hypothesis that N-methyl-D-aspartate (NMDA) evokes ADO release per se from neurons, whereas dephosphorylation of extracellular adenine nucleotides contributes to NMDA-evoked ADO production in the presence of astrocytes. We used four different cell preparations-cortical rat neurons, cortical rat astrocytes, cocultures of neurons and astrocytes, and transient cocultures of neurons with astrocytes on transwell filters-to show that astrocytes contribute to NMDA-evoked increases in extracellular ADO. NMDA significantly increased ADO and inosine (INO) production from cultured cortical neurons but only increased extracellular INO production from cocultures. In neurons, the equilibrative nucleoside transport (ENT) inhibitor dipyridamole (DPR) prevented NMDA-evoked ADO and INO production, whereas the e-N inhibitor alpha,beta-methylene ADP (AOPCP) had no effect. Conversely, from both cocultures and transient cocultures DPR significantly decreased NMDA-evoked INO but not ADO generation. AOPCP inhibited NMDA-evoked production of both ADO and INO from transient cocultures. In the absence of astrocytes, NMDA evoked release of intracellular ADO and INO from cultured cortical neurons through ENT. However, in the presence of astrocytes, extracellular conversion of adenine nucleotides to ADO contributed significantly to NMDA-evoked production of this purine.

Characterization of NAD Uptake in Mammalian Cells

Recent evidence has shown that NAD(P) plays a variety of roles in cell-signaling processes. Surprisingly, the presence of NAD(P) utilizing ectoenzymes suggests that NAD(P) is present extracellularly. Indeed, nanomolar concentrations of NAD have been found in plasma and other body fluids. Although very high concentrations of NAD have been shown to enter cells, it is not known whether lower, more physiological concentrations are able to be taken up. Here we show that two mammalian cell types are able to transport low NAD concentrations effectively. Furthermore, extracellular application of NAD was able to counteract FK866-induced cell death and restore intracellular NAD(P) levels. We propose that NAD uptake may play a role in physiological NAD homeostasis.

Synthesis and Biological Activity of a Gemcitabine Phosphoramidate Prodrug

A gemcitabine (2',2'-difluorodeoxycytidine, dFdC) phosphoramidate prodrug designed for the intracellular delivery of gemcitabine 5'-monophosphate was synthesized. The prodrug was about an order of magnitude less active than gemcitabine against wild-type cells, and the nucleoside transport inhibitor dipyridamole reduced prodrug activity. The prodrug was more active than gemcitabine against two deoxycytidine kinase-deficient cell lines. The results suggest that the prodrug is a potent growth inhibitor that can bypass dCK deficiency at higher drug concentrations.

Glyburide Inhibits Dipyridamole‐induced Forearm Vasodilation but not Adenosine‐induced Forearm Vasodilation

The mechanism of the vasodilator response to adenosine has not been elucidated in humans. Stimulation of adenosine receptors on endothelial and vascular smooth muscle cells with subsequent endothelial release of nitric oxide and opening of adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels has been suggested. The aim of this study was to investigate the involvement of K(ATP) channels in the vasodilator response to adenosine and the nucleoside transport inhibitor dipyridamole. Methods and results In healthy male volunteers, adenosine (0.6, 1.9, 5.6, 19, 57, and 190 nmol. min(-1). dL(-1)) was infused into the brachial artery, and forearm blood flow (FBF) was measured by use of strain-gauge plethysmography. Adenosine increased the FBF ratio (FBF in experimental arm/FBF in control arm) from 1.3 +/- 0.2 to 1.2 +/- 0.2, 1.5 +/- 0.2, 2.8 +/- 0.4, 7.3 +/- 2.3, 11.1 +/- 4.1, and 12.9 +/- 3.7 for the six increasing adenosine doses, respectively. Simultaneous infusion of glyburide (INN, glibenclamide), a blocker of K(ATP) channels, did not affect this response (from 1.7 +/- 0.4 to 1.5 +/- 0.2, 2.2 +/- 0.3, 4.0 +/- 1.0, 9.3 +/- 4.0, 13.5 +/- 6.4, and 15.9 +/- 5.3 for the 6 increasing doses of adenosine, respectively; P =.439, n = 6). The increase in FBF ratio during infusion of the nucleoside transport inhibitor dipyridamole (20, 60, and 200 nmol. min(-1). dL(-1)) was significantly reduced by glyburide, as follows: from 1.2 +/- 0.1 to 1.7 +/- 0.2, 2.4 +/- 0.5, and 2.9 +/- 0.4, respectively, during saline solution and from 1.6 +/- 0.2 to 1.8 +/- 0.2, 2.1 +/- 0.3, and 2.2 +/- 0.4, respectively, during glyburide (P =.010 for effect of glyburide on response from baseline, ANOVA for repeated measures; n = 8). The vasodilator response to dipyridamole was significantly inhibited by the adenosine receptor antagonist theophylline. Opening of vascular K(ATP) channels is involved in the forearm vasodilator response to dipyridamole but not to adenosine. Differences in stimulated cell type (endothelium for adenosine versus smooth muscle cells for dipyridamole) may underlie this divergent pharmacologic profile.

Dipyridamole increases interstitial adenosine during intermittent isometric exercise but does not augment the pressor reflex

Interstitial adenosine is increased during intermittent isometric exercise (handgrip) and may be an afferent signal for the pressor reflex. The effect of the nucleoside transport inhibitor dipyridamole on interstitial adenosine concentration has never been studied in humans. The aim of the study was to determine whether dipyridamole increases interstitial adenosine during handgrip and potentiates the exercise-induced pressor reflex. In nine healthy volunteers, isometric hand-contractions (50% of maximal force) were performed with and without dipyridamole (12 mg 100 ml−1 min−1, infused into the brachial artery). Interstitial adenosine was measured in the flexor digitorum superficialis muscle using microdialysis with h.p.l.c. detection. Blood pressure was measured at 2 min intervals (Dinamap). Peroneal nerve muscle sympathetic nerve activity (MSNA) was successfully recorded in four volunteers. Intermittent handgrip increased dialysate adenosine by 0.3 ± 0.1 and 0.5 ± 0.1 nmol ml−1 in the absence and presence of dipyridamole respectively (mean ± s.e. mean; P < 0.05 for effect of dipyridamole). Dipyridamole did not augment the handgrip-induced rise in MSNA in any of the volunteers. Handgrip increased blood pressure (SBP/DBP) by 9.6 ± 2.4/4.5 ± 2.0 and 10.4 ± 2.2/7.0 ± 1.3 mmHg in the absence and presence of dipyridamole respectively (P = NS for dipyridamole effect). In none of the volunteers, dipyridamole augmented the exercise-induced rise in MSNA. Dipyridamole increases interstitial adenosine during handgrip but does not potentiate the pressor reflex. Our data do not support a role for adenosine as a trigger of the exercise-induced pressor reflex in healthy volunteers.

Differences between rat primary cortical neurons and astrocytes in purine release evoked by ischemic conditions

In the brain, the levels of adenosine increase up to 100-fold during cerebral ischernia; however, the roles of specific cell types, enzymatic pathways and membrane transport processes in regulating intra- and extracellular concentrations of adenosine are poorly characterized. Rat primary cortical neurons and astrocytes were incubated with [ 3H]adenine for 30 min to radiolabel intracellular ATP. Cells were then treated with buffer, glucose deprivation (GD), oxygen–glucose deprivation (OGD), 100 μM sodium cyanide (NaCN) or 500 μM iodoacetate (IAA) for 1 h to stimulate the metabolism of ATP and cellular release of [ 3H]purines. The nucleoside transport inhibitor dipyridamole (DPR) (10 μM), the adenosine kinase inhibitor iodotubercidin (ITU) (1 μM), the adenosine deaminase inhibitor EHNA (1 μM) and the purine nucleoside phosphorylase inhibitor BCX-34 (10 μM) were tested to investigate the contribution of specific enzymes and transporters in the metabolism and release of purines from each cell type. Our results indicate that (a) under basal conditions astrocytes released significantly more [ 3H]adenine nucleotides and [ 3H]adenosine than neurons, (b) OGD, NaCN and IAA conditions produced significant increases in [ 3H]adenosine release from neurons but not astrocytes, and (c) DPR blocked [ 3H]inosine release from both astrocytes and neurons but only blocked [ 3H]adenosine release from neurons. These data suggest that, in these experimental conditions, adenosine was formed by an intracellular pathway in neurons and then released via a nucleoside transporter. In contrast, adenine nucleotide release and extracellular metabolism to adenosine appeared to predominate in astrocytes.

Maintenance of differential methotrexate toxicity between cells expressing drug-resistant and wild-type dihydrofolate reductase activities in the presence of nucleosides through nucleoside transport inhibition

Methotrexate (MTX), a potent inhibitor of dihydrofolate reductase (DHFR), has been used widely as a chemotherapeutic agent and as a selective agent for cells expressing drug-resistant DHFR activity. MTX deprives rapidly dividing cells of reduced folates that are necessary for thymidylate synthesis and de novo purine nucleotide synthesis. However, MTX toxicity can be circumvented by salvaging thymidine (TdR) and purine nucleosides. Here we have investigated conditions under which nucleoside transport inhibition can be used to maintain differential MTX toxicity between unmodified cells and cells expressing drug-resistant DHFR activity in the presence of exogenous nucleosides. PA317 cells (a 3T3 derivative cell line) were rescued from the toxicity of 0.1 μM MTX by 1.0 μM TdR in the presence of 100 μM inosine. The nucleoside transport inhibitor dipyridamole (DP) resensitized these cells to MTX, even in the presence of exogenous nucleosides. Furthermore, PA317 cells transduced with any of three retroviruses encoding drug-resistant DHFRs remained resistant to MTX over all concentrations tested (up to 10.0 μM) in the presence of DP. Similar results were obtained in transduced HuH7 and K562 cell lines, a human hepatoma and a human leukemia cell line, respectively. We conclude that nucleoside transport inhibition increases the toxicity and selectivity of MTX in cultured cells, and therefore is an effective way to maintain differential MTX toxicity between unmodified and DHFR-modified cells. Our results support the use of nucleoside transport inhibition in in vivo selection protocols involving the liver and hematopoietic systems.

Selective potentiation of lometrexol growth inhibition by dipyridamole through cell-specific inhibition of hypoxanthine salvage.

The novel antifolate lometrexol (5,10-dideazatetrahydrofolate) inhibits de novo purine biosynthesis, and co-incubation with hypoxanthine abolishes its cytotoxicity. The prevention of hypoxanthine rescue from an antipurine antifolate by the nucleoside transport inhibitor dipyridamole was investigated for the first time in nine human and rodent cell lines from seven different tissues of origin. In A549, HeLa and CHO cells, dipyridamole prevented hypoxanthine rescue and so growth was inhibited by the combination of lometrexol, dipyridamole and hypoxanthine, but in HT29, HCT116, KK47, MDA231, CCRF CEM and L1210 cells dipyridamole had no effect and the combination did not inhibit growth. Dipyridamole inhibited hypoxanthine uptake in A549 but not in CCRF CEM cells. Dipyridamole prevented the hypoxanthine-induced repletion of dGTP pools, depleted by lometrexol, in A549 but not in CCRF CEM cells. Thus, the selective growth-inhibitory effect of the combination of lometrexol, dipyridamole and hypoxanthine is apparently due to the dipyridamole sensitivity (ds) or insensitivity (di) of hypoxanthine transport. Both the human and murine leukaemic cells are of the di phenotype. If this reflects the transport phenotype of normal bone marrow it would suggest that the combination of lometrexol, dipyridamole and hypoxanthine might be selectively toxic to certain tumour types and have reduced toxicity to the bone marrow.

Extracellular ATP and adenosine cause apoptosis of pulmonary artery endothelial cells.

ATP acts as an intracellular energy source and an extracellular signaling molecule. We report that extracellular ATP causes apoptosis in pulmonary artery endothelial cells, as assessed by morphological changes and internucleosomal DNA degradation. We investigated the mechanism of this effect using release of tritiated soluble DNA as a marker for apoptosis. We conclude that the metabolite adenosine is responsible for the apoptotic effect of ATP, since nucleotides that can be degraded to adenosine, as well as adenosine itself, cause DNA damage, whereas nonmetabolizable ATP analogs and uridine 5'-triphosphate are inactive. Furthermore, the ecto-5'-nucleotidase inhibitor alpha, beta-methylene-ADP blocks ATP-induced DNA fragmentation. The adenosine receptor agonist 5'-N-ethylcarboxamide adenosine does not cause DNA fragmentation, and adenosine receptor antagonists do not block adenosine-induced apoptosis. However, the nucleoside transport inhibitor dipyridamole prevents extracellular ATP-induced DNA cleavage. These findings indicate that ATP- and adenosine-mediated apoptosis are mediated via intracellular events rather than through cell surface receptor(s). The adenosine metabolites inosine, hypoxanthine, and xanthine do not cause apoptosis. The adenosine analogs 3-deazaadenosine and MDL-28842, which are not metabolized and are S-adenosylhomocysteine hydrolase inhibitors, also cause DNA fragmentation. Therefore, we speculate that extracellular ATP and adenosine cause apoptosis of pulmonary artery endothelial cells by altering methylation reactions that require S-adenosylmethionine as the methyl donor. We speculate that ATP released from cells undergoing cytolysis or degranulation may cause endothelial cell death. Endothelial cell apoptosis may be important in acute vascular injury or in limiting angiogenesis.

Characterization of inhibitor-sensitive and -resistant adenosine transporters in cultured human fetal astrocytes.

The kinetic characteristics of [3H]adenosine uptake, the extent to which accumulated [3H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis-Menten kinetic values of KT and Vmax, and the sensitivities with which nucleoside transport inhibitors blocked [3H] adenosine accumulations were determined in cultured human fetal astrocytes. KT and Vmax values for accumulations of [3H]-labeled purines using 15-s incubations in the absence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and the adenosine kinase inhibitor 5'-iodotubercidin (ITU) were 6.2 microM and 0.15 nmol/min/mg of protein for the high-affinity and 2.6 mM and 21 nmol/min/mg of protein for the low-affinity components, respectively. In the presence of EHNA and ITU, where < 4% of accumulated [3H] adenosine was metabolized, transport per se was measured, and kinetic values for KT and Vmax were 179 microM and 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [3H]adenosine was rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of "concentrative" uptake in that the intracellular levels of [3H]-labeled purines (adenosine plus its metabolites) were 1.4-fold higher than in the medium. No apparent concentrative accumulations of [3H]adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [3H]-adenosine transport; for the inhibitor-sensitive components the IC50 values were 0.7 nM for NBI, 1.3 nM for DPR, and 3.3 nM for dilazep, and for the inhibitor-resistant component the IC50 values were 2.5 microM for NBI, 5.1 microM for dilazep, and 39.0 microM for DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor-sensitive and -resistant adenosine transporters in nontransformed human cells.

Influence of both salvage and DNA damage response pathways on resistance to chemotherapeutic antimetabolites

The resistance of 3 human embryo fibroblast cell lines to the antimetabolites methotrexate (MTX), N-phosphonacetyl- l-aspartate (PALA) and 5-fluorouracil (5-FU) has been studied. The cell lines were of common genetic origin, all originating from the normal KMS parental cell line, which was irradiated with 60Co to produce the immortalised derivative KMST which, in turn, was transfected with an activated N- ras oncogene to produce the tumourigenic KN-NM cell line. Previous work from this group, using dialysed versus nondialysed serum, has provided evidence for the involvement of salvage pathways of purine and pyrimidine biosynthesis in the increased resistance to antimetabolites of those cell lines (KMST and KN-NM) tending towards increased tumourigenicity. The present study has extended this work by using the nucleoside and nucleobase transport inhibitor dipyridamole, to further assess the contribution of the salvage pathways to the increased cellular resistance to the three antimetabolites. The salvage pathways were found to contribute to the resistance of cell lines to PALA and MTX, but had no effect on the resistance to 5-FU. The addition of excess uridine in the case of PALA, and hypoxanthine plus thymidine in the case of MTX, could be used to “rescue” cells from the effects of dipyridamole-induced salvage pathway inhibition. The data will be discussed in relation to 1. the effect of limited substrate availability, 2. the induction of DNA damage and DNA damage-response pathways, and 3. DNA-damage protection by the salvage pathways of purine and pyrimidine biosynthesis.

Failure of orally administered dipyridamole to enhance the antineoplastic activity of fluorouracil in combination with leucovorin in patients with advanced colorectal cancer: a prospective randomized trial.

A randomized trial was performed to investigate the ability of the nucleoside transport inhibitor dipyridamole (DP) to enhance the antitumor activity of fluorouracil (5-FU)/leucovorin (folinic acid [FA]). One hundred eighty-one untreated patients with advanced colorectal cancer were randomized to receive 5-FU 600 mg/m2 plus FA 300 mg/m2 on days 2 to 4 with or without DP 75 mg orally three times daily on days 1 to 5. Cycles were repeated every 3 weeks. Only patients with documented tumor progression before therapy were eligible. 5-FU pharmacokinetics using high-performance liquid chromatography (HPLC) were assessed in 11 nonrandomized patients receiving paired cycles with or without DP. One hundred seventy-four patients were assessable for toxicity and response. There was no significant difference in toxicity, except DP-related headache in 24% of patients. An objective response rate of 15% (one complete response [CR] and 13 partial responses [PRs]) for 5-FU/FA and 13% (two CRs and nine PRs) for 5-FU/FA/DP was observed. The dose-intensity of 5-FU delivered was significantly higher (1.09- to 1.16-fold) for the DP-containing arm. Pharmacokinetic parameters of 5-FU did not differ significantly, except for a prolonged half-life (t1/2) induced by DP. The median time to progression (P = .8) and the median survival time (11.6 months for 5-FU/FA v 9.3 months for 5-FU/FA/DP; P = .14, log-rank test) were not different between treatment arms. Orally administered DP did not improve the antineoplastic activity of 5-FU/FA in patients with advanced colorectal cancer when used at this dose and schedule. The observed increase in 5-FU dose-intensity for FU/FA/DP was not clinically relevant.

Red blood cells as a delivery system for AZT

3′-Azido-3′-deoxythymidine 5′-phosphate (AZT-MP) was synthesized with the aim of checking whether it represents a prodrug of AZT. AZT-MP was then encapsulated in human erythrocytes by a procedure of hypo-tonic hemolysis and isotonic resealing and found to be dephosphorylated to the corresponding nucleoside (AZT), which was subsequently released outside the red cells. Encapsulated AZT-MP did not interfere with the major metabolic properties of erythrocytes. The dephosphorylation reaction had an apparent Km of 1.6 mM, a pH optimum of 7.4, and was inhibited by 10-5m Pb2+. ATP, TMP, and UMP had no effect on AZT-MP dephosphorylation. AZT permeated the erythrocyte membrane predominantly by nonfacilitated diffusion, but a slow release of AZT-MP was also observed. AZT-MP in human plasma was then dephosphorylated at a rate of 170 nmol/h/ml. Influx of AZT-MP in human erythrocytes was almost undetectable at concentrations below 1 mm. At 2 mri extraerythrocytic AZT-MP influx took place at a rate of 10 nmol/min/ml cells and was not inhibited by the nucleoside transport inhibitors dipyridamole and nitrobenzyl 6-thioinosine. Thus, AZTMP-loaded erythrocytes can perform as a slow delivery system for AZT, potentially avoiding the peaks of drug concentration commonly found after the oral or intravenous administration of nucleoside analogues.

Effect of the carbocyclic nucleoside analogue MDL 201,112 on inhibition of interferon-γ-induced priming of Lewis (LEW/N) rat macrophages for enhanced respiratory burst and MHC class II Ia+ antigen expression

The effects of the carbocyclic nucleoside MDL 201,112 and the purine nucleoside adenosine on the interferon-gamma (IFN-gamma)-induced priming of macrophages (m phi s) for the respiratory burst and major histocompatibility class II (MHC class II) Ia+ antigen expression were compared. Priming of purified, peritoneal m phi s from Lewis (LEW/N) rats for 18 h with recombinant rat IFN-gamma (rRaIFN-gamma) in the presence of either adenosine (100 microM) or MDL 201,112 (10 microM) resulted in a fourfold decrease in superoxide anion (O2-) production after stimulation with opsonized zymosan. Both agents were effective even when added 2 or 4 h after rRaIFN-gamma treatment. Peritoneal m phi s from LEW/N rats stimulated with LPS/rRaIFN-gamma were observed to secrete immunoreactive and bioactive TNF-alpha over 18 h in vitro and this cytokine could be dose-dependently inhibited by MDL 201,112. MDL 201,112 did not bind to classical A1 or A2 receptors on rat brain homogenates. Physiological levels of adenosine deaminase, or treatment with the nucleoside transport inhibitor dipyridamole, reversed the effects of adenosine; however, these agents at physiological concentrations had little or no effect on the inhibition of O2- release mediated by MDL 201,112. Furthermore, incubation of LEW/N m phi s for 18 h in vitro with rRaIFN-gamma resulted in significant enhancement of MHC class II Ia+ antigen expression, and these levels could be blocked by nearly 50% by either MDL 201,112 (10 microM) or adenosine (100 microM). MDL 201,112 and adenosine were also effective in decreasing m phi opsonized zymosan-stimulated O2- levels and MHC class II Ia+ antigen expression in vivo. The effects of MDL 201,112 on the down-regulation of heat-killed M. tuberculosis-activated LEW/N m phi MHC class II Ia+ antigen expression in vitro appear to be mediated by a novel pathway, because there was no rank order of potency of ADO A1 or A2 agonist/antagonists (CCPA, NECA, XAC, or CPT) in our in vitro system. In summary, our data provide compelling evidence that immunoregulatory carbocyclic nucleoside analogues such as MDL 201,112 or adenosine appear to regulate LEW/N rat m phi activation through novel molecular mechanisms and may have important therapeutic implications for acute and chronic inflammatory diseases.

Propentofylline and other adenosine transport inhibitors increase the efflux of adenosine following electrical or metabolic stimulation of rat hippocampal slices.

Propentofylline is a novel neuroprotective agent that has been shown to act as an adenosine transport inhibitor as well as an adenosine receptor antagonist. In the present series of experiments we have compared the effects of propentofylline with those of known adenosine transport inhibitors and receptor antagonists on the formation of adenosine in rat hippocampal slices. The ATP stores were labeled by incubating the slices with [3H]adenine. The total 3H overflow and the overflow of endogenous and 3H-labeled adenosine, inosine, and hypoxanthine were measured. Adenosine release, secondary to ATP breakdown, was induced both by hypoxia/hypoglycemia and by electrical field stimulation. Propentofylline (20-500 microM) increased the release of endogenous and radiolabeled adenosine, without increasing the total release of purines. Thus, the drug altered the pattern of released purines, i.e., increasing adenosine and decreasing inosine and hypoxanthine. This pattern, which was observed when purine release was induced both by electrical field stimulation and by hypoxia/hypoglycemia, was shared by the nucleoside transport inhibitor dipyridamole (1 microM) and by mioflazine (1 microM) and nitrobenzylthioinosine (1 microM). By contrast, other xanthines, including theophylline (100 microM) and 8-cyclopentyltheophylline (10 microM), enprofylline (100 microM), or torbafylline (300 microM), if anything, increased the total release of purines without alterations of the pattern of release. These results indicate that nucleoside transport inhibitors can decrease the release of purines from cells and at the same time increase the concentration of extracellular adenosine, possibly by preventing its uptake and subsequent metabolism. This change in purine metabolism may be beneficial with regard to cell damage after ischemia. The results also indicate that propentofylline behaves in such a potentially beneficial manner.

Organ culture as a model for investigating the effects of antimetabolites and nucleoside transport inhibitors on rodent colonic mucosa

The in-vitro effects of hydroxyurea 5-FU and 5-FUdR have been extensively studied in experimental systems employing cell-line techniques. In this study we investigated the effects of these drugs on the levels of incorporation of labeled nucleosides into DNA in explants of intact rat colonic mucosa maintained in organ culture. The effects of the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR) and dipyridamole--which are modulators of antimetabolite cytotoxicity--on the incorporation of tritiated thymidine ([3H]TdR) into DNA were also studied. The incorporation of tritiated TdR into DNA was reduced by hydroxyurea but was not altered by either 5-FU or 5-FUdR. The levels of tritiated deoxyuridine were reduced by 5-FU and 5-FUdR in separate experiments; this is in keeping with thymidylate synthase inhibition. NBMPR and dipyridamole also reduced 3H-TdR incorporation into DNA. These results can be explained in terms of the known mechanisms of action of these drugs. This experimental model is therefore useful in assessing the effects of antimetabolites and nucleoside transport inhibitors in intact colonic mucosa.

Tissue-specific expansion of uridine pools in mice: Effects of benzylacyclouridine, dipyridamole and exogenous uridine

The concentration of uridine (Urd) in murine tissues appears to be controlled by Urd catabolism, concentrative Urd transport, and the non-concentrative, facilitated diffusion of Urd. Previous reports document the tissue-specific disruption of these processes, and subsequently intracellular pools of free Urd in mice, by the administration of exogenous Urd (250 mg/kg) or the Urd phosphorylase (EC 2.4.2.3; uracil:ribose-1-phosphate phosphotransferase) inhibitor 5-benzylacyclouridine (BAU) (240 mg/kg). We now report the effect of combinations of BAU (120 mg/kg, p.o.), the nucleoside transport inhibitor dipyridamole (DP) (25 mg/kg, i.p.), and exogenous Urd (250 mg/kg, i.v.) on Urd pools in mice. This dose of BAU increased Urd pools 2- to 6-fold, in a tissue-specific manner, for up to 5 hr. DP increased Urd pools 3-fold in spleen, over a 4-hr period, but did not affect other tissues. Administration of BAU 1 hr prior to exogenous Urd resulted in a 50- to 100-fold expansion of tissue Urd pools which returned to normal within 4hr, except in spleen, where the Urd concentration was normal after 6 hr. Administration of DP 1 hr prior to exogenous Urd caused a tissue-specific 40- to 100- fold increase in Urd pools which, except in spleen, returned to normal within 2 hr. The marked additive effects of these combinations were in contrast to those obtained following the administration of BAU 1 hr prior to DP. This regimen increased Urd pools from 4- to 9-fold, in a tissue-specific manner. In addition, Urd pools remained elevated for up to 9 hr, except in spleen where the Urd concentration was elevated for up to 15 hr. Analysis of enzyme activities indicated that DP does not enhance the inhibitory effect of BAU against murine liver Urd phosphorylase. However, DP did inhibit the plasma clearance of BAU, and this effect may partially explain the apparent synergistic effect of this combination. In spite of the prolonged and dramatic expansion of tissue Urd pools produced by BAU + DP, the total Ura nucleotide content in spleen, gut and colon tumor 38 (CT38) increased by less than 70% over a 12- hr period following administration of this combination. These findings are discussed in light of their biochemical and therapeutic implications.

Effect of herpes simplex virus type 1 infection on nucleoside transport in HeLa S3 cells

The initial velocity of thymidine uptake was measured in HeLa S3 cells infected with herpes simplex virus type 1 (HSV-1). The rate of nucleoside influx into the cells was shown to increase from as early as 1 h post-infection (p.i.) up to 8 h p.i. This increased uptake was shown to be attributable to a progressively increasing contribution from passive diffusion superimposed upon normal transport. Thus, the specific nucleoside transport system was still operating with unaltered kinetic parameters 8 h after infection. Despite the inhibition of host cell protein synthesis and its replacement by the synthesis of virus-specified proteins, the numbers and affinity of the nucleoside transporters in cells 8 h after infection were virtually unchanged. The increased transport of thymidine in infected cultures was resistant to the nucleoside transport inhibitor dipyridamole, and was correlated with entry of a normally impermeant solute (sucrose) into infected cells. These data suggest that the system for the carrier-mediated facilitated diffusion of nucleosides remains intact in HSV-infected cells, but that progressively increasing passive diffusion takes place. Passive diffusion is the major process operating late after virus infection.

Role of hypoxanthine and thymidine in determining methotrexate plus dipyridamole cytotoxicity

The nucleoside transport inhibitor dipyridamole can potentiate the cytotoxicity of methotrexate by a mechanism that was thought to be related to the inhibition of thymidine salvage. In human ovarian carcinoma cells thymidine only partly reversed the in vitro cytotoxicity of methotrexate plus dipyridamole at sub-millimolar concentrations, above which the cytotoxicity of thymidine itself became evident. Hypoxathine with thymidine, or hypoxanthine alone at a higher concentration, completely reversed methotrexate and methotrexate plus dipyridamole cytotoxicity. The effects of dipyridamole on cellular cyclic adenosine monophosphate (cAMP) levels and on 3H-methotrexate efflux in 2008 cells were examined. At 10 μmol/l, dipyridamole did not alter cAMP content or methotrexate influx in ovarian carcinoma cells, but reduced the rate of efflux of 3H-methotrexate by 25%. In Chinese hamster ovary cells and their folypolyglutamyl synthase-deficient variant AUX B1, the reduced methotrexate efflux by dipyridamole was not due to increased polyglutamation, since increased retention was observed in both cell lines. The data support the hypothesis that dipyridamole potentiated the activity of methotrexate by inhibiting the salvage of hypoxanthine, and to a lesser extent, that of thymidine. The ability of dipyridamole to increase the cellular retention of methotrexate was probably a non-specific action of dipyridamole on the cell membrane, and may have a role in the observed synergy.