Concentrations Of Purine Nucleotides Research Articles

Accelerated Extracellular Nucleotide Metabolism in Brain Microvascular Endothelial Cells in Experimental Hypercholesterolemia

Hypercholesterolemia affects the neurovascular unit, including the cerebral blood vessel endothelium. Operation of this system, especially in the context of energy metabolism, is controlled by extracellular concentration of purines, regulated by ecto-enzymes, such as e-NTPDase-1/CD39, ecto-5′-NT/CD73, and eADA. We hypothesize that hypercholesterolemia, via modulation of the activity of nucleotide metabolism-regulating ecto-enzymes, deteriorates glycolytic efficiency and energy metabolism of endothelial cells, which may potentially contribute to development of neurodegenerative processes. We aimed to determine the effect of hypercholesterolemia on the concentration of purine nucleotides, glycolytic activity, and activity of ecto-enzymes in the murine brain microvascular endothelial cells (mBMECs). We used 3-month-old male LDLR−/−/Apo E−/− double knockout mice to model hypercholesterolemia and atherosclerosis. The age-matched wild-type C57/BL6 mice were a control group. The intracellular concentration of ATP and NAD and extracellular activity of the ecto-enzymes were measured by HPLC. The glycolytic function of mBMECs was assessed by means of the extracellular acidification rate (ECAR) using the glycolysis stress test. The results showed an increased activity of ecto-5′-NT and eADA in mBMECs of the hypercholesterolemic mice, but no differences in intracellular concentration of ATP, NAD, and ECAR between the hypercholesterolemic and control groups. The changed activity of ecto-5′-NT and eADA leads to increased purine nucleotides turnover and a shift in their concentration balance towards adenosine and inosine in the extracellular space. However, no changes in the energetic metabolism of the mBMECs are reported. Our results confirm the influence of hypercholesterolemia on regulation of purine nucleotides metabolism, which may impair the function of the cerebral vascular endothelium.Graphical abstractThe effect of hypercholesterolemia on the murine brain microvascular endothelial cells (mBMECs). An increased activity of ecto-5′-NT and eADA in mBMECs of the LDLR−/−/Apo E−/− mice leads to a shift in the concentration balance towards adenosine and inosine in the extracellular space with no differences in intracellular concentration of ATP. Figure was created with Biorender.com.

Molecular determinants of inhibition of UCP1-mediated respiratory uncoupling

Brown adipose tissue expresses uncoupling protein 1 (UCP1), which dissipates energy as heat, making it a target for treating metabolic disorders. Here, we investigate how purine nucleotides inhibit respiration uncoupling by UCP1. Our molecular simulations predict that GDP and GTP bind UCP1 in the common substrate binding site in an upright orientation, where the base moiety interacts with conserved residues R92 and E191. We identify a triplet of uncharged residues, F88/I187/W281, forming hydrophobic contacts with nucleotides. In yeast spheroplast respiration assays, both I187A and W281A mutants increase the fatty acid-induced uncoupling activity of UCP1 and partially suppress the inhibition of UCP1 activity by nucleotides. The F88A/I187A/W281A triple mutant is overactivated by fatty acids even at high concentrations of purine nucleotides. In simulations, E191 and W281 interact with purine but not pyrimidine bases. These results provide a molecular understanding of the selective inhibition of UCP1 by purine nucleotides.

Inosine pranobex enhances human NK cell cytotoxicity by inducing metabolic activation and NKG2D ligand expression.

Inosine pranobex (IP) is a synthetic immunomodulating compound, indicated for use in the treatment of human papillomavirus‐associated warts and subacute sclerosing panencephalitis. Previous studies demonstrate that the immunomodulatory activity of IP is characterized by enhanced lymphocyte proliferation, cytokine production, and NK cell cytotoxicity. The activation of NKG2D signaling on NK cells, CD8+ T cells, and γδ T cells also produces these outcomes. We hypothesized that IP alters cellular immunity through the induction of NKG2D ligand expression on target cells, thereby enhancing immune cell activation through the NKG2D receptor. We tested this hypothesis and show that exposure of target cells to IP leads to increased expression of multiple NKG2D ligands. Using both targeted metabolic interventions and unbiased metabolomic studies, we found that IP causes an increase in intracellular concentration of purine nucleotides and tricarboxylic acid (TCA) cycle intermediates and NKG2D ligand induction. The degree of NKG2D ligand induction was functionally significant, leading to increased NKG2D‐dependent target cell immunogenicity. These findings demonstrate that the immunomodulatory properties of IP are due to metabolic activation with NKG2D ligand induction.

Degradation of brown adipocyte purine nucleotides regulates uncoupling protein 1 activity

ObjectiveNon-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation. MethodsWith mass spectrometry, purine nucleotide metabolites were quantified in cellular homogenates and supernatants of cultured primary brown adipocytes. We also determined oxygen consumption in response to a β-adrenergic agonist. ResultsUpon adrenergic activation, brown adipocytes decreased the intracellular concentration of inhibitory nucleotides (ATP, ADP, GTP and GDP) and released the respective degradation products. At the same time, an increase in cellular calcium occurred. None of these phenomena occurred in white adipocytes or myotubes. The brown adipocyte expression of enzymes implicated in purine metabolic remodeling is altered upon cold exposure. Pharmacological and genetic interference of purine metabolism altered uncoupling protein 1 mediated uncoupled respiration. ConclusionAdrenergic stimulation of brown adipocytes lowers the intracellular concentration of purine nucleotides, thereby contributing to uncoupling protein 1 activation.

Comparison of human erythrocyte purine nucleotide metabolism and blood purine and pyrimidine degradation product concentrations before and after acute exercise in trained and sedentary subjects

This study aimed at evaluating the concentration of erythrocyte purine nucleotides (ATP, ADP, AMP, IMP) in trained and sedentary subjects before and after maximal physical exercise together with measuring the activity of purine metabolism enzymes as well as the concentration of purine (hypoxanthine, xanthine, uric acid) and pyrimidine (uridine) degradation products in blood. The study included 15 male elite rowers [mean age 24.3 ± 2.56 years; maximal oxygen uptake (VO2max) 52.8 ± 4.54 mL/kg/min; endurance and strength training 8.2 ± 0.33 h per week for 6.4 ± 2.52 years] and 15 sedentary control subjects (mean age 23.1 ± 3.41 years; VO2max 43.2 ± 5.20 mL/kg/min). Progressive incremental exercise testing until refusal to continue exercising was conducted on a bicycle ergometer. The concentrations of ATP, ADP, AMP, IMP and the activities of adenine phosphoribosyltransferase (APRT), hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and phosphoribosyl pyrophosphate synthetase (PRPP-S) were determined in erythrocytes. The concentrations of hypoxanthine, xanthine, uric acid and uridine were determined in the whole blood before exercise, after exercise, and 30 min after exercise testing. The study demonstrated a significantly higher concentration of ATP in the erythrocytes of trained subjects which, in part, may be explained by higher metabolic activity on the purine re-synthesis pathway (significantly higher PRPP-S, APRT and HGPRT activities). The ATP concentration, just as the ATP/ADP ratio, as well as an exercise-induced increase in this ratio, correlates with the VO2max level in these subjects which allows them to be considered as the important factors characterising physical capacity and exercise tolerance. Maximal physical exercise in the group of trained subjects results not only in a lower post-exercise increase in the concentration of hypoxanthine, xanthine and uric acid but also in that of uridine. This indicates the possibility of performing high-intensity work with a lower loss of not only purine but also pyrimidine.

Effects of purine nucleotide administration on purine nucleotide metabolism in brains of heroin-dependent rats

Heroin is known to enhance catabolism and inhibit anabolism of purine nucleotides, leading to purine nucleotide deficiencies in rat brains. Here, we determined the effect of exogenous purine nucleotide administration on purine nucleotide metabolism in the brains of heroin-dependent rats. Heroin was administrated in increasing doses for 9 consecutive days to induce addiction, and the biochemical changes associated with heroin and purine nucleotide administration were compared among the treated groups. HPLC was performed to detect the absolute concentrations of purine nucleotides in the rat brain cortices. The enzymatic activities of adenosine deaminase (ADA) and xanthine oxidase (XO) in the treated rat cortices were analyzed, and qRT-PCR was performed to determine the relative expression of ADA, XO, adenine phosphoribosyl transferase (APRT), hypoxanthine-guaninephosphoribosyl transferase (HGPRT), and adenosine kinase (AK). Heroin increased the enzymatic activity of ADA and XO, and up-regulated the transcription of ADA and XO. Alternatively, heroin decreased the transcription of AK, APRT, and HGPRT in the rat cortices. Furthermore, purine nucleotide administration alleviated the effect of heroin on purine nucleotide content, activity of essential purine nucleotide metabolic enzymes, and transcript levels of these genes. Our findings therefore represent a novel, putative approach to the treatment of heroin addiction.

Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation

The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3+T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2–200µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP and GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5′-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.

Effect of acute exercise on cardiovascular hemodynamic and red blood cell concentrations of purine nucleotides in hypertensive compared with normotensives rats

The mechanisms of exercise-induced health benefits are complex and not fully understood. This study investigated the effects of exercise and hypertension on cardiovascular hemodynamic responses and red blood cell (RBC) concentrations of purine nucleotides using normotensive and hypertensive rat models in vivo. Sprague Dawley rats (SDRs) and spontaneously hypertensive rats (SHRs) were exercised on a treadmill for 15 min at a speed of 10 m/min and 5% grade. Blood samples were obtained from each rat before, during, and after exercise for measurement of adenosine 5'-triphosphate (ATP) and guanosine 5'-triphosphate (GTP) concentrations in RBCs by a validated high-performance liquid chromatography assay. They were returned to a restrainer after exercise, and hemodynamic recording collected continuously up to 6 h. Two separate groups (SDRs and SHRs) without exercise were used as controls. Biomarker data were compared between SDRs and SHRs using analysis of variance and t test and difference considered significant at p < 0.05. The study has demonstrated for the first time a difference in the postexercise effect between SDRs and SHRs. The 15 min of exercise significantly increased systolic blood pressure (SBP) (129 ± 16 to 162 ± 26 mmHg) and heart rate (HR) (416 ± 29 to 491 ± 26 bpm) in SDRs (p < 0.05), but not in SHRs. The postexercise hemodynamic effects were more profound in SHRs. SBP and diastolic blood pressure (DBP) also fell significantly in the control group of SHRs (SBP 184 ± 14 to 152 ± 29 mmHg and DBP 149 ± 9 to 120 ± 14 mmHg, p < 0.05 for both) towards the end of the experiment but not in the SDR group. The RBC concentrations of ATP and GTP increased after exercise in both SDRs and SHRs which were significantly correlated with the postexercise hemodynamic effect (p < 0.05). SHRs were more tolerant to increases in HR and SBP induced by exercise, and have more profound postexercise hemodynamic effects than SDRs. The hemodynamic effects were linked closely with RBC concentrations of ATP and GTP in both SDRs and SHRs.

GMP reductase modulates purine nucleotide concentrations and uncoupling protein 1 (UCP1) activity

GMP reductase modulates purine nucleotide concentrations and uncoupling protein 1 (UCP1) activity

Simultaneous Determination of Purine and Pyrimidine Metabolites in Hprt-Deficient Cell Lines

Genetic mutations in the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT), are known to cause Lesch–Nyhan syndrome and Kelley–Seegmiller syndrome. In patients, purine metabolism is different from that of normal persons. We have previously developed a method for simultaneously determining the concentration of purine and pyrimidine nucleosides and nucleotides. This system was applied to determine the concentrations of nucleosides and nucleotides in HPRT-deficient cell lines. The amount of inosine 5′-monophosphate (IMP) was different in Lesch–Nyhan syndrome, Kelley–Seegmiller syndrome, and control cell lines. The difference in the amount of IMP confirmed the mutation of the enzyme.

Anti-Ischemia Drugs have no Effect on the In Vivo Metabolism of ATP by RBC in Normotensive Restrained Rats#

The objective is to determine the effect of anti-ishemia agents on metabolism of adenosine-5'-triphosphate (ATP) in red blood cell (RBC) in a normotensive rat model. Male Sprague Dawley (SD) rats weighing between 300 - 400 g were used. Each rat received either saline (control), or 5 mg/kg of diltiazem (DTZ), losartan, amlodipine or dipyrida- mole by subcutaneous injection (sc) twice daily for 5 doses. Blood samples were collected using a Stopping Solution from each rat at time 0 (before the last dose), and sequentially after over 6 hours following the last dose via an indwelling carotid artery catheter. In addition, hemodynamic recordings were collected throughout the experiment. Concentrations of ATP and other purine nucleotides in the RBC were determined by a validated HPLC. Data between groups were analyzed by ANOVA and paired t-test, and differences between groups considered significant when p < 0.05. The results showed that the concentrations of ATP and the other purine nucleotides in RBC of the rats treated with the anti-ischemia drugs were not different from the control rats (Table 1). The concentrations of ATP and guanosine-5'-triphosphate (GTP) were higher towards the end of the experiment, but the increase was significant only after amlodipine and losartan (p < 0.05). The increase of ATP and AMP concentrations correlated with decrease of diastolic blood pressure (DBP) in the rats. The study concluded that the anti-ischemia drugs tested in the current study have no effect on RBC concentrations of ATP in the restraining rat model.

Exercise improves hemodynamic profiles and increases red blood cell concentrations of purine nucleotides in a rodent model

To study the effect of exercise on hemodynamic profiles and red blood cell (RBC) concentrations of adenosine-5'-triphosphate (ATP). Male Sprague-Dawley (SD) rats (n = 9) were exercised on a treadmill for 15 min at a speed of 10 m/min with a 5% gradient after an hour settling down in a restrainer. Blood samples were collected via an indwelling carotid artery catheter using a 'Stopping Solution' from each rat before, during and after exercise. Hemodynamic recordings were collected continuously throughout the experiment. Concentrations of ATP and other purine nucleotides in the RBCs were determined by a validated high-performance liquid chromatography (HPLC) assay. A control group (n = 12) was treated the same way except without the exercise. Data between groups were analyzed by analysis of variance (ANOVA) and Student's t-test, and differences were considered significant when p < 0.05. Exercise increased systolic blood pressure (SBP; 141±23 vs. 132±17 mmHg) and heart rate (HR; 420±33 vs. 397±41 bpm), but decreased diastolic blood pressure (DBP; 105±18 vs. 110±14 mmHg). This was followed by a postexercise condition when SBP, DBP, and HR were decreased for the remainder of the experiment. RBC concentrations of ATP and guanosine-5'-triphosphate (GTP) increased significantly during exercise and continued to increase for 5 hours postexercise (1.5±0.75 vs. 0.96 vs. 0.36 mM for ATP; and 0.14±0.061 vs. 0.058±0.030 mM) (p < 0.05). Exercise increased RBC ATP concentrations in a rodent model, which was correlated with the decrease in BP and HR postexercise.

Adenine, guanine and pyridine nucleotides in blood during physical exercise and restitution in healthy subjects

Maximal physical exertion is accompanied by increased degradation of purine nucleotides in muscles with the products of purine catabolism accumulating in the plasma. Thanks to membrane transporters, these products remain in an equilibrium between the plasma and red blood cells where they may serve as substrates in salvage reactions, contributing to an increase in the concentrations of purine nucleotides. In this study, we measured the concentrations of adenine nucleotides (ATP, ADP, AMP), inosine nucleotides (IMP), guanine nucleotides (GTP, GDP, GMP), and also pyridine nucleotides (NAD, NADP) in red blood cells immediately after standardized physical effort with increasing intensity, and at the 30th min of rest. We also examined the effect of muscular exercise on adenylate (guanylate) energy charge—AEC (GEC), and on the concentration of nucleosides (guanosine, inosine, adenosine) and hypoxanthine. We have shown in this study that a standardized physical exercise with increasing intensity leads to an increase in IMP concentration in red blood cells immediately after the exercise, which with a significant increase in Hyp concentration in the blood suggests that Hyp was included in the IMP pool. Restitution is accompanied by an increase in the ATP/ADP and ADP/AMP ratios, which indicates an increase in the phosphorylation of AMP and ADP to ATP. Physical effort applied in this study did not lead to changes in the concentrations of guanine and pyridine nucleotides in red blood cells.

Cladribine inhibits a diltiazem-induced increase in red blood cell purine nucleotide concentrations in a zebrafish model

Minimizing drug interactions is paramount to improving the efficacy and tolerability of cancer therapy. The zebrafish represents an innovative cancer model due to highly conserved genetics and inherent capacity for high-throughput chemical screening. This pilot study extends the utility of the zebrafish to a preclinical model for pharmacodynamics by examining the interaction of the nucleoside analogue, cladribine with the calcium channel blocker, diltiazem. Cladribine (0.7–3.5 mM) and/or diltiazem (2.4 mM), was injected intraperitoneally into adult zebrafish and red blood cell (RBC) lysates were assayed by HPLC for levels of purine nucleotides (e.g. ATP), potential biomarkers of cardiovascular health. Diltiazem increased RBC ATP concentrations, which were inhibited by co-injection of cladribine. These results suggest a novel drug interaction and highlight the feasibility of the zebrafish as an in vivo model for pharmacodynamic studies.

Cladribine inhibits a diltiazem-induced increase in red blood cell purine nucleotide concentrations in a zebrafish model

Cladribine inhibits a diltiazem-induced increase in red blood cell purine nucleotide concentrations in a zebrafish model

Electrophysiological Characterization of Mitochondrial Uncoupling Protein 1

Uncoupling proteins (UCP1- UCP5) are integral transport proteins of the inner mitochondrial membrane (IMM). They mediate transmembrane ion leak, thus dissipating the electrochemical proton gradient across the IMM and uncoupling mitochondrial respiration and ATP synthesis. UCPs are involved in thermogenesis, reducing fat deposition, and attenuating reactive oxygen species production by mitochondria to protect the cell against oxidative damage and ageing. The mechanism of ion conductance of UCPs has long remained elusive due to the lack of direct methods for measuring ion currents produced by them. To resolve this problem, we applied the patch-clamp technique to the whole inner membrane of mitochondria from the brown adipose tissue to identify and characterize currents produced by the family's founding member, UCP1. In our experiments, both the cytoplasmic and matrix faces of the IMM were exposed to solutions that did not contain any ions normally permeable through ion channels or transporters, except for H+ and OH-. Under these conditions, we identified a current that showed all signature properties of UCP1: it was strongly potentiated by micromolar concentrations of unsaturated fatty acids, inhibited by removing endogenous membrane fatty acids with bovine serum albumin, and blocked by micromolar concentrations of purine nucleotides. Moreover, the current was absent in the IMM of the kidney COS-7 cells, in agreement with the fact that UCP1 is specifically expressed in the brown adipose tissue. The transport molecule mediating this current was activated by membrane depolarization and showed robust tail currents upon return to the negative potentials. In a symmetrical pH 5.0 solution the current amplitude was negligible as compared to a symmetrical pH 8.0 solution, suggesting that the current was carried by OH- but not H+. Our results indicate that UCP1 is a low-conductance OH- channel weakly activated by membrane depolarization.

Regulation of Acanthamoeba castellanii alternative oxidase activity by mutual exclusion of purine nucleotides; ATP's inhibitory effect

The effects of different adenine and guanine nucleotides on the cyanide-resistant respiration (i.e. alternative oxidase (AcAOX) activity) of mitochondria from the amoeba A. castellanii mitochondria were studied. We found that guanine nucleotides activate AcAOX to a greater degree than adenine nucleotides, and that nucleoside monophosphates were more efficient activators than nucleoside di- or triphosphates. The extent of the nucleotides' influence on AcAOX was dependent on the medium's pH and was more pronounced at pH 6.8, which is optimal for AcAOX activity. In contrast to other purine nucleosides, we demonstrate, for the first time, that ATP has an inhibitory effect on AcAOX activity. Since we also observed the inhibition by ATP in the mitochondria of another protozoon, such as Dictyostelium discoideum, and the yeast, Candida maltosa, it may be a regulatory feature common to all purine nucleotide-modulated non-plant AOXs. The physiological importance of this discovery is discussed. Kinetic data show that the binding of GMP (a positive allosteric effector) and the binding of ATP (a negative allosteric effector) to AcAOX are mutually exclusive. ATP's inhibition of the enzyme can be overcome by sufficiently high concentrations of GMP, and conversely, GMP's stimulation can be overcome by sufficiently high concentrations of ATP. However, an approximately three times lower concentration of GMP compared to ATP gives a half maximal effect on AcAOX activity. This is indicative of a higher binding affinity for the positive effector at the same or, at least overlapping, nucleotide-binding sites on AcAOX. These results suggest that AcAOX activity in A. castellanii mitochondria might be controlled by the relative intracellular concentrations of purine nucleotides.

HPLC assay with UV detection for determination of RBC purine nucleotide concentrations and application for biomarker study in vivo

ATP and other purine nucleotides are important biomarkers for ischemia and may have considerable potential as targets for management of ischemic heart disease and stroke. The main objective of the study is to develop a rapid HPLC assay, which has adequate sensitivity and specificity for measuring concentrations of ATP, ADP, AMP, GTP, GDP and GMP in erythrocytes (RBC). The assays used ion-pair chromatography coupled with ultraviolet detection at 254 nm to separate and detect the purine nucleotides. Using 50–100 μL of RBC lysate as blank biologic matrix, the assay was linear from 100 to 2000 μg/mL for ATP and ADP, and 20–400 μg/mL for AMP, GTP, and GDP with coefficients of determination ( r 2) >0.99. GDP and GMP were not measurable in the study because of low concentrations and interference from endogenous materials, respectively. The intra-assay and inter-assay variations over a period of 1 year were less than 10% and 20%, respectively for most of the nucleotides. The assay was successfully applied to two pilot biomarker studies to measure RBC concentrations of the purine nucleotides in rats under restraining and exercise conditions. Preliminary results showed that the RBC concentrations of ATP and GTP were higher in the spontaneously hypertensive rats (SHR) compared to the Sprague–Dawley (SD) rats, and that exercise increased RBC concentrations of ATP in rats treated with the calcium channel blocker diltiazem.

IMP dehydrogenase basal activity in MOLT‐4 human leukaemia cells is altered by mycophenolic acid and 6‐thioguanosine

Objective. Depletion of guanine and deoxyguanine nucleotides by inhibition of inosine 5'‐monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) or introduction of 6‐thioguanine nucleotide antimetabolites are two principles of retarding cell proliferation by interference with the cellular purine nucleotide pool. IMPDH activity may be a promising pharmacodynamic biomarker during immunosuppressive and anticancer pharmacotherapy. The aim of the study was to investigate the impact of mycophenolic acid (MPA) and 6‐thioguanosine (tGuO) on IMPDH basal activity. Material and methods. We studied the IMPDH basal activity (i.e. the enzyme activity following inhibitor exposure, but measured in absence of the inhibitor) in response to increasing concentrations of the IMPDH inhibitor MPA and the antimetabolite tGuO in MOLT‐4 human leukaemia cells. In parallel, IMPDH gene expression and cellular purine nucleotide concentrations were examined. Results. A biphasic concentration‐dependent influence of MPA on the IMPDH basal activity was observed. At concentrations⩽IC50, MPA increased the IMPDH basal activity. The increase was associated with elevated expression of IMPDH2. Despite increased expression, the basal enzyme activity decreased following exposure to high MPA concentrations. The IMPDH2 expression increased modestly in response to tGuO exposure. However, the IMPDH basal activity decreased when the cells were exposed to a proliferation‐blocking tGuO concentration. Conclusions. These findings demonstrate that IMPDH basal activity is influenced by MPA and tGuO, and suggest that reduced IMPDH basal activity is related to the proliferation‐blocking effects of these agents.

Nucleotides and inorganic phosphates as potential antioxidants

Highly reactive OH radicals, formed in an iron-ion catalyzed Fenton reaction, are implicated in many pathological conditions. The quest for Fenton reaction inhibitors, either radical scavenger or metal-ion chelator antioxidants, spans the previous decades. Purine nucleotides were previously studied as natural modulators of the Fenton reaction; however, the modulatory role of purine nucleotides remained in dispute. Here, we have resolved this long-standing dispute and demonstrated a concentration-dependent biphasic modulation of the Fenton reaction by nucleotides. By electron spin resonance measurements with 0.1 mM Fe(II), we observed an increase of *OH production at low purine nucleotide concentrations (up to 0.15 mM), while at higher nucleotide concentrations, an exponential decay of *OH concentration was observed. We found that the phosphate moiety, not the nucleoside, determines the pro/antioxidant properties of a nucleotide, suggesting a chelation-based modulation. Furthermore, the biphasic modulation mode is probably due to diverse nucleotide-Fe(II) complexes formed in a concentration-dependent manner. At ATP concentrations much greater than Fe(II) concentrations, multiligand chelates are formed which inhibit the Fenton reaction owing to a full Fe(II) coordination sphere. In addition to natural nucleotides, we investigated a series of base- or phosphate-modified nucleotides, dinucleotides, and inorganic phosphates, as potential biocompatible antioxidants. Ap5A, inorganic thiophosphate and ATP-gamma-S proved highly potent antioxidants with IC50 values of 40, 30, and 10 microM, respectively. ATP-gamma-S proved 100 and 20 times more active than ATP and the potent antioxidant Trolox, respectively. In the presence of 30 microM ATP-gamma-S no *OH was detected after 5 min in the Fenton reaction mixture. The most potent antioxidants identified inhibit the Fenton reaction by forming full coordination sphere chelates.