Influence of 4-pyridone-3-carboxamide-1Β-D-ribonucleoside (4PYR) on activities of extracellular enzymes in endothelial human cells

ABSTRACT

Previous studies demonstrated that human endothelial cells were capable to phosphorylate 4-pyridone-3-carboxamide-1β-D-ribonucleoside (4PYR) to monophosphate (4PYMP) and formed another metabolite—an analog of NAD (4PYRAD). Elevated levels of 4PYMP and 4PYRAD had an adverse effect on energy balance—depressed adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) concentration in human endothelial cells. Ecto-enzymes such as ecto-nucleoside triphosphate diphosphohydrolase (eNTPD); ecto-5′-nucleotidase (e5’NT); and ecto-adenosine deaminase (eADA) are involved in controlling of inflammation and platelet aggregation. This study aimed to evaluate influence of 4PYR and its metabolites on activities of extracellular enzymes in human endothelial cells. Endothelial cells (endothelial cell line HMEC-1) were treated with 100 uM 4PYR for 0, 24, 48, or 72 hours. After incubation, intact HMEC-1 cells were incubated with suitable substrate. Simultaneously, in another path of experiments intracellular concentration of 4PYMP and 4PYRAD had been analyzed. Conversion of extracellular nucleotides into their products and intracellular concentration of 4PYMP and 4PYRAD were measured by high performance liquid chromatography (HPLC). We demonstrated that eNTPD and e5’NT activities increase after 72 hours of cell treatment with 4PYR as compared to control (0.40 ± 0.02 versus 0.29 ± 0.02 nmol/min/mg protein; 13.3 ± 0.6 versus 8.30 ± 0.34 nmol/min/mg protein, respectively, mean ± SEM). eADA activity decreases after 24 hours of cells treatment with 4PYR as compared to control (1.55 ± 0.06 versus 1.92 ± 0.13 nmol/min/mg protein, respectively, mean ± SEM). 4PYR and its derivatives have positive effect on ecto-enzymes related with ATP degradation pathway. We conclude that these increases in extracellular enzyme activities are an adaptive response to decreased intracellular ATP and NAD arising from 4PYR uptake. These changes may protect the cells from the inflammatory result of external ATP degradation.     

4-Pyridone-3-Carboxamide-1β-D-Ribonucleoside Metabolism in Endothelial Cells and Its Impact on Cellular Energetic Balance

4-Pyridone-3-carboxamide-1β-D-ribonucleoside (4PYR) is a naturally occurring compound related to nicotinamide that could be metabolized to mono-, di-, and triphosphates of 4PYR (4PYMP, 4PYDP, 4PYTP) and nicotinamide adenine dinucleotide (NAD) analogue (4PYRAD) in all types of cells. Previous studies demonstrated that formation of 4PYMP and 4PYTP was dependent on adenosine kinase activity. Pathway of 4PYRAD production is not yet identified, but most likely this process involves production of 4PYMP. This study aimed to evaluate influence of 4PYR on metabolism of endothelial cells and to test effect of nucleoside transport inhibitors. 4PYR was obtained by chemical synthesis. Endothelial cell line (HMEC-1) was incubated for 24 or 48 hours with 100 μM 4PYR. After incubation, cells were separated from medium and analyzed for concentrations of ATP, NAD, and 4PYR metabolites by using reversed-phase high performance liquid chromatography. We demonstrated progressive accumulation of 4PYR metabolites in endothelium that reached 33.2 ± 0.8 nmol/mg protein for 4PYMP and 5.25 ± 0.17 nmol/mg protein for 4PYRAD after 48-hour incubation with 4PYR. Dipyridamole protected from accumulation of 4PYR metabolites in endothelial cells. We conclude that endothelium is capable to convert 4PYR into intracellular metabolites and this causes disruption of cell energy balance. Nucleoside transport inhibition with dipyridamole could protect endothelium from this effect. This finding could be of clinical relevance in conditions associated with accumulation of 4PYR such as chronic renal disease.     

Metabolic pathway of 4-pyridone-3-carboxamide-1β-d-ribonucleoside and its effects on cellular energetics

4-pirydone-3-carboxamide-1β-d-ribonucleoside (4PYR) is an endogenous nucleoside that could be converted to triphosphates, diphosphates, monophosphates and an analogue of NAD − 4PYRAD. Elevated level of these compounds have been reported in chronic renal failure, cancer and active HIV infection. However, little is known about the effect on cell functionality and the metabolic pathways. This study tested effects of 4PYR in different cell types on nucleotide, energy metabolism and clarified enzymes that are involved in conversions of 4PYR.We have found that human neuroblastoma cells, human malignant melanoma cells, human adipose-derived stem cells, human bone marrow-derived stem cells, human dermal microvascular endothelial cells and human embryonic kidney cells, were capable to convert 4PYR into its derivatives. This was associated with deterioration of cellular energetics. Incubation with 4PYR did not affect mitochondrial function, but decreased glycolytic rate (as measured by extracellular acidification) in endothelial cells. Silencing of adenosine kinase, cytosolic 5′-nucleotidase II and nicotinamide nucleotide adenylyltransferase 3, blocked metabolism of 4PYR. Incubation of endothelial cells with 4PYR decreased AMP deaminase activity by 40%.The main finding of this paper is that human cells (including cancer type) are capable of metabolizing 4PYR that lead to deterioration of energy metabolism, possibly as the consequence of inhibition of glycolysis. This study, it was also found that several enzymes of nucleotide metabolism could also contribute to the 4PYRconversions     

Metabolism of 4-pyridone-3-carboxamide-1β-d-ribonucleoside (4PYR) in primary murine brain microvascular endothelial cells (mBMECs).

Abstract

4-pyridone-3-carboxamide-1β-D-ribonucleoside (4PYR) is a derivative of nicotinamide found physiologically in human body fluids that can be metabolized to mono-, di- or triphosphate derivatives (4PYMP, 4PYDP and 4PYTP respectively) and an analogue of NAD - the 1-β-D-ribonucleoside-4-pyridone-3-carboxamide adenine dinucleotide (4PYRAD) in human cells. The European Uremic Toxin Work Group (EUTox) has classified 4PYR as a uremic toxin that adversely affects endothelium.

This study aimed to investigate the metabolism of 4PYR in murine brain microvascular endothelial cells (mBMECs). Incubation of mBMECs with 4PYR was carried out for 0, 24, 48 or 72?h. After incubation, a medium was removed and cellular concentrations of ATP, ADP, NAD, 4PYMP and 4PYRAD were analyzed using reversed-phase HPLC.

4PYR was metabolized by mBMECs to 4PYMP and 4PYRAD that reached concentrations of 2?±?0.7 and 0.6?±?0.2?nmol/mg protein (mean?±?SEM), respectively, after 72?h incubation. However, unlike with endothelial cells studied so far this process has no effect on energy balance in the cell as indicated by maintained ATP/ADP ratio and adenine and nicotinamide intracellular pools. Further studies are required to explain whether the difference in 4PYR metabolism is related to differences between species or organs.     

Pharmacological inhibition of AMP-deaminase in rat cardiac myocytes

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC(50) = 0.5 micro M. AMPDI was much less effective with isolated cardiomyocytes (IC(50) = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies.     

Effects of 4-Pyridone-3-carboxamide-1β-D-ribonucleoside on adenine nucleotide catabolism in the aortic wall; Implications for atherosclerosis in ApoE-/-LDLR-/- mice

ABSTRACT

4-Pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) is an endogenously produced nucleoside that had been identified as a substrate for intracellular phosphorylation to form intracellular nucleotides. Previous studies demonstrated that 4PYR adversely affects metabolism of endothelial cells that is known risk factor for atherosclerosis. The purpose of this study was to evaluate effects of 4PYR on the progression of atherosclerosis and changes in extracellular nucleotides degradation on the surface of the vessel wall in the murine model. Methods. Two month old ApoE-/-LDLR-/- mice were subcutaneously injected with 4PYR (4P) twice per day for one month or with saline in controls (C). Then, at the age of eight month hydrolysis rates of ATP, AMP and adenosine were evaluated in the intact aorta sections by HPLC based assays. Oil Red O (ORO) staining that indicates lipid deposition was quantified spectrophotometrically after extraction from the vessel. Serum amyloid A (SAA) content was analyzed with ELISA. Results. Adenosine deamination rate (activity of eADA) increased from 8.7±1.4 nmol/min/cm² in C to 16.0±2.6 nmol/min/cm² in 4P (p<0.05). AMP dephosphorylation rate (activity of e5NT) and ATP hydrolysis rate (activity of eNTPD) were not different between C and 4P. ORO staining in the aorta of 4P mice increased by 75% as compared to C (p<0.01) while SAA content was similar in both groups. Conclusions. This data demonstrated that prolonged exposure to 4PYR of ApoE-/-LDLR-/- mice results in sustained elevation of vascular eADA activity and increased ORO staining indicating endothelial impairment and accelerated atherosclerosis.     

A novel nucleotide found in human erythrocytes, 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside triphosphate

We report the identification of a hitherto unknown nucleotide that is present in micromolar concentrations in the erythrocytes of healthy subjects and accumulates at levels comparable with the ATP concentration in erythrocytes of patients with chronic renal failure. The unknown nucleotide was isolated and identified by liquid chromatography with UV and tandem mass detection, (1)H nuclear magnetic resonance and infrared spectroscopy as 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside triphosphate (4PYTP), a structure indicating association with metabolism of the oxidized nicotinamide compounds. Subsequently, we demonstrated formation of 4PYTP in intact human erythrocytes during incubation with the chemically synthesized nucleoside precursor 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR). We noted preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP as well as a decrease in erythrocyte ATP concentration during incubation with 4PYR. Both the 4PYR phosphorylation and ATP depletion were blocked by an inhibitor of adenosine kinase. Plasma concentration of 4PYR was detectable but very low (0.013 +/- 0.006 microm) in contrast with the high daily urine excretion of this compound (26.7 +/- 18.2 micromol/24 h) in healthy subjects, indicating much greater renal clearance than other nicotinamide metabolites, nucleosides, or creatinine. We also noted a 40-fold increase in 4PYR plasma concentration in patients with chronic renal failure (0.563 +/- 0.321 microm). We suggest that 4PYTP formation in the erythrocytes is a hitherto unknown process aimed at sequestering potentially toxic 4PYR in a form that could be safely transported and subsequently released and excreted during passage of erythrocytes through the kidney.     

Modulation of AMP deaminase in rat hearts subjected to ischemia and reperfusion by purine riboside

Changes in AMP deaminase (AMPD) activity influence heart function and progression of heart disease, but the underlying mechanism is unknown. We evaluated the effect of purine riboside (Purr) on the activity of AMPD in perfused rat hearts and in isolated rat cardiomyocytes. Brief perfusion of the pre-ischemic heart with 200 micro M Purr resulted in activation of AMPD, more pronounced degradation of the adenine nucleotides, and reduced recovery of the adenine nucleotide pool during reperfusion. Brief incubation of rat cardiomyocytes with 200 micro M Purr also activated AMPD, while prolonged exposure resulted in enzyme inhibition. We conclude that Purr activates AMPD, whereas metabolites of this compound may inhibit the enzyme.     

Effect of 4-Pyridone-3-Carboxamide Ribonucleoside (4PYR)-Potential Cardiovascular Toxin in Perfused Rat Heart

We recently described a new nicotinamide derivative: 4-pyridone-3-carboxamide ribonucleoside (4PYR) and its conversion to intracellular metabolites (4PYR monophosphate: 4PYMP and 4PYR adenylate diphosphate: 4PYRAD). The aim of this study was to clarify the metabolism and physiological effects of brief exposure to 4PYR in perfused rat heart. Rat hearts were perfused in Langendorff mode. After 15 min equilibration, 100 μM 4PYR (or solvent in controls) was infused into coronary circulation for 5 min. Coronary flow was recorded with electromagnetic flow meter and left ventricular mechanical function was assessed with intraventricular baloon by constructing pressure–volume relations. After perfusion hearts were freeze-clamped and analyzed using HPLC for phosphocreatine, creatine, ATP with metabolites as well as 4PYR metabolites. 4PYR infused into the coronary circulation was rapidly converted in the heart into 4PYMP and 4PYRAD with concentrations reaching 85.6 ± 46.9 and 43.9 ± 6.4 nmol/g dry weight, respectively, while control concentrations were below 20 nmol/g. 4PYR had no effect on baseline coronary flow (11.9 ± 2.3 ml/min versus 11.0 ± 2.7 ml/min in control) or stimulated by shear stress (23.2 ± 4.5 ml/min versus 23.1 ± 5.2 ml/min in control). Both systolic and diastolic left ventricular mechanical function were not affected by 4PYR. No difference was noted for heart rate. Myocardial concentrations of ATP or phosphocreatine were also not affected by 4PYR. We conclude that 4PYR has no immediate effect on coronary endothelium or cardiomyocyte functions such as coronary flow, rhythm, diastolic properties, or contractility despite rapid incorporation into intracellular metabolites. This study also indicates the lack of effect on purinergic receptors.     

Pharmacological Inhibition of AMP-Deaminase in Rat Cardiac Myocytes

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC ₅₀ = 0.5 μ M. AMPDI was much less effective with isolated cardiomyocytes (IC ₅₀ = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies.     

Activities of purine converting enzymes in heart,liver and kidney mice LDLR−/− and Apo E−/−

Nucleotide metabolism plays a major role in a number of vital cellular processes such as energetics. This, in turn, is important in pathologies such as atherosclerosis.

Three month old atherosclerotic mice with knock outs for LDLR and apolipoprotein E (ApoE) were used for the experiments. Activities of AMP-deaminase (AMPD), ecto5′-nucleotidase (e5NT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) were measured in heart, liver and kidney cortex and medulla by analysing conversion of substrates into products using HPLC.

The activity of ecto5′-nucleotidase differ in hearts of LDLR^?/? and ApoE^?/? mice with no differences in ADA and AMPD activity. We noticed highest activity of e5NT in kidney medulla of the models.

This model of atherosclerosis characterize with an inhibition of enzyme responsible for production of protective adenosine in heart but not in other organs and different metabolism of nucleotides in kidney medulla.     

Metabolism of 4-Pyridone-3-Carboxamide-1-ß-D- Ribonucleoside Triphosphate and Its Nucleoside Precursor in the Erythrocytes

We recently discovered new nucleotides (4-pyridone-3-carboxamide-1-β -D-ribonucleoside phosphates) in human erythrocytes. To establish the precursor compound and pathways of nucleotide derivative formation and breakdown, human erythrocytes were incubated for 3 hours with 0.3 mM 4-pyridone-3-carboxamide-1-β -D-ribonucleoside (4PYR) and erythrocyte concentrations of 4PYR and adenine nucleotides were followed. 4PYR triphosphate increased from 16.1 ± 0.6 μ M to 74.9 ± 9.17 and 4PYR monophosphate increased from 5 μ M to 254.7 ± 13.9 μ M. Conversely, incubation with 0.3 mM 4-pyridone-3-carboxamide (4PY) did not lead to additional 4PYR nucleotide formation. 4PYR nucleotides were catabolized to 4PYR. We conclude that 4PYR nucleotides are formed in erythrocytes by nucleoside kinase-mediated 4PYR phosphorylation and catabolized by 5′nucleotidase-mediated dephosphorylation.     

Modulation of AMP Deaminase in Rat Hearts Subjected to Ischemia and Reperfusion by Purine Riboside

Changes in AMP deaminase (AMPD) activity influence heart function and progression of heart disease, but the underlying mechanism is unknown. We evaluated the effect of purine riboside (Purr) on the activity of AMPD in perfused rat hearts and in isolated rat cardiomyocytes. Brief perfusion of the pre-ischemic heart with 200 μ M Purr resulted in activation of AMPD, more pronounced degradation of the adenine nucleotides, and reduced recovery of the adenine nucleotide pool during reperfusion. Brief incubation of rat cardiomyocytes with 200 μ M Purr also activated AMPD, while prolonged exposure resulted in enzyme inhibition. We conclude that Purr activates AMPD, whereas metabolites of this compound may inhibit the enzyme.     

Effect of AMP-Deaminase 3 Knock-Out in Mice on Enzyme Activity in Heart and Other Organs

Recent findings suggest that inhibition of AMP-deaminase (AMPD) could be effective therapeutic strategy in heart disease associated with cardiac ischemia. To establish experimental model to study protective mechanisms of AMPD inhibition we developed conditional, cardiac specific knock-outs in Cre recombinase system. AMPD3 floxed mice were crossed with Mer-Cre-Mer mice. Tamoxifen was injected to induce Cre recombinase. After two weeks, hearts, skeletal muscle, liver, kidney, and blood were collected and activities of AMPD and related enzymes were analyzed using HPLC-based procedure. We demonstrate loss of more than 90% of cardiac AMPD activity in the heart of AMPD3 -/- mice while other enzymes of nucleotide metabolism such as adenosine deaminase, purine nucleoside phosphorylase were not affected. Surprisingly, activity of AMPD was also reduced in the erythrocytes and in the kidney by 20%–30%. No change of AMPD activity was observed in the skeletal muscle and the liver.     

Enzymes of the purine interconversion system in chronic lymphatic leukemia: decreased purine nucleoside phosphorylase and adenosine deaminase activity.

Activities of adenosine deaminase (ADA), adenosine kinase (AK), adenine phosphoribosyltransferase (APRT), hypoxanthine guanine phosphoribosyltransferase (HGPRT), and purine nucleoside phosphorylase (PNP), all enzymes of the purine interconversion system, were determined in lymphocytes of 25 patients with chronic lymphatic leukemia (CLL) and in 23 controls. A statistically significant decrease of PNP activities and a reduction of ADA activities at borderline levels were found in the patients, whereas for the other enzymes assayed no deviation from normal values was observed.     

Metabolism of 4-pyridone-3-carboxamide-1-??-d-ribonucleoside (4PYR) in rodent tissues and in vivo

Our previous studies identified 4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) phosphates in human erythrocytes. We demonstrated formation of these nucleotides by phosphorylation of 4PYR and potential toxicity due to disruption of erythrocyte energy balance. This study aimed to evaluate the ability of the other cell types to phosphorylate 4PYR to characterize function and toxicity of these compounds. Homogenates of rat heart, kidneys, and liver were used to study the rate of 4PYR phosphorylation in the presence of ATP. In another experiment, 4PYR was administered into mouse as repeated subcutaneous injections and into rats as intraperitoneal infusion. After 7 days, heart, liver, kidney, lungs, and skeletal muscle were collected, and the concentration of 4PYR nucleotides was evaluated. HPLC was used to measure 4PYR and 4PYR nucleotides in homogenate and specimens from in vivo experiments. 4PYR was rapidly phosphorylated by the liver homogenate (390 ± 27 nmol/min/g wet wt). Significant rates were reported in the heart and kidneys' homogenates: 34.3 ± 4.3 nmol/min/g and 33.2 ± 9.2 nmol/min/g, respectively. Phosphorylation of 4PYR was almost completely inhibited by adenosine kinase inhibitor 5'-iodotubercidin. Administration of 4PYR in vivo resulted in accumulation of 4PYR monophosphate in the liver, heart, skeletal muscle, and lung (20-220 nmol/g dry wt) except kidney (<1 nmol/g). In contrast to erythrocytes, no 4PYR triphosphate formation (<1 nmol/g) was observed in any of the organs studied. We conclude that not only the erythrocytes but also other cell types are capable of phosphorylating 4PYR to form 4PYR monophosphate. Potential toxicity or physiological role of 4PYR in peripheral organs could be considered, but mechanisms will be different from that in erythrocytes.     

Metabolism of 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside triphosphate and its nucleoside precursor in the erythrocytes

We recently discovered new nucleotides (4-pyridone-3-carboxamide-1-beta -D-ribonucleoside phosphates) in human erythrocytes. To establish the precursor compound and pathways of nucleotide derivative formation and breakdown, human erythrocytes were incubated for 3 hours with 0.3 mM 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) and erythrocyte concentrations of 4PYR and adenine nucleotides were followed. 4PYR triphosphate increased from 16.1 +/- 0.6 micro M to 74.9 +/- 9.17 and 4PYR monophosphate increased from 5 micro M to 254.7 +/- 13.9 micro M. Conversely, incubation with 0.3 mM 4-pyridone-3-carboxamide (4PY) did not lead to additional 4PYR nucleotide formation. 4PYR nucleotides were catabolized to 4PYR. We conclude that 4PYR nucleotides are formed in erythrocytes by nucleoside kinase-mediated 4PYR phosphorylation and catabolized by 5'nucleotidase-mediated dephosphorylation.     

Polymorphism of S-adenosylhomocysteine hydrolase in Italy

S-adenosylhomocysteine hydrolase (SAHH) polymorphism has been investigated in the Italian population. Three common alleles, SAHH*1, SAHH*2 and SAHH*3, have been observed and the estimated gene frequencies are 0.968, 0.023 and 0.009, respectively. SAHH activity has been assayed in 50 healthy individuals and the mean activity was 0.043 +/- 0.017 mumol uric acid/min/g Hb at 37 degrees C. Five heterozygotes for adenosine deaminase deficiency and three heterozygotes for purine nucleoside phosphorylase deficiency showed SAHH within the range of the normal distribution. The effects of some thiol reagents on red blood cell SAHH electrophoretic pattern have been investigated.     

Adenine phosphoribosyltransferase: A simple spectrophotometric assay and the incidence of mutation in the normal population

The significance of partial deficiency of erythrocyte adenine phosphoribosyltransferase (APRT), reported in a number of subjects with gout, has been investigated by studying its incidence in 700 normal blood donors. Three clearly deficient subjects were found, an incidence not significantly different from that in patients with abnormalities of urate metabolism. A new assay method for APRT is described in which an erythrocyte lysate is incubated with adenine and phosphoribosylpyrophosphate (PRPP) for a given time; both hemoglobin and adenine nucleotide (AMP) are then precipitated with lanthanum phosphate; the change in absorbance of adenine at 260 nm reflects the extent of its conversion to AMP by APRT.This work was supported by the National Health and Medical Research Council of Australia.     

Erythrocytic adenosine monophosphate as an alternative purine source in Plasmodium falciparum

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.