Evaluation of cellular purine transport and metabolism in the Caco-2 cell using comprehensive high-performance liquid chromatography method for analysis of purines

ABSTRACT

Using Caco-2 cells and our previously developed high-performance liquid chromatography method for quantification of purine bases, nucleosides, and nucleotides, we evaluated cellular purine transport and uptake. The analytes were separated using YMC-Triart C18 column with gradient elution. We used Caco-2 cells as intestinal model cells and monitored purine transport across a monolayer for 2 h. The degree of change of purine concentrations in the permeate was very slight; however, it was possible to simultaneously determine these parameters for all purines because of our method's high sensitivity. In the present study, the purine bases (adenine, guanine, hypoxanthine, and xanthine) showed a relatively high permeability as compared with the nucleosides (adenosine, guanosine, inosine, and xanthosine). Increased concentration of metabolites in the permeate was also observed following the addition of purines. In a cell uptake assay, both the cell culture medium (extracellular) and the cells extracted from Caco-2 with acetonitrile:water (7:3) (intracellular) were measured. The additional nucleoside did not increase significantly within the cells. On the other hand, we observed that nucleotide, such as ATP, increased in the cell in a time-dependent manner following the addition of nucleoside. The additional nucleosides were considered to be rather recycled via the salvage pathway than metabolized to purine bases and/or uric acid in the cell. Such differences might have affected the increase in the serum uric acid levels depending on purine form.     

Simultaneous determination of nucleosides and nucleotides in dietary foods and beverages using ion-pairing liquid chromatography–electrospray ionization-mass spectrometry

A method using ion-pairing liquid chromatography–electrospray ionization (ESI)-mass spectrometry (MS) was developed for the simultaneous determination of 23 types of purine or pyrimidine nucleosides and nucleotides in dietary foods and beverages. Dihexylammonium acetate (DHAA) was used as an ion-pairing agent and an ultra performance liquid chromatography (UPLC™) system with a reversed-phase column and a gradient program was employed for the separation of nucleosides and nucleotides. Positive-ion ESI-MS was applied for the detection of nucleosides, and negative-ion ESI-MS was used for nucleotides. Lower limits of quantitation ranged from 0.02 μmol/L (UMP and AMP) to 1.3 μmol/L (CDP). The present method was validated, and sufficient reproducibility and accuracy was obtained for the quantitative measurement of the 23 types of nucleosides and nucleotides. The method was subsequently applied to their determination in a range of Japanese foods and beverages that are considered to contain significant amounts of umami flavor compounds. Because dietary purine nucleosides and nucleotides are known to be related to hyperuricemia and gout, the determination of their concentrations in dietary foods is useful for both evaluating umami flavor and assessing the effects of dietary food on purine metabolism.     

Development of a FIA system with immobilized enzymes for specific post-column detection of purine bases and their nucleosides separated by HPLC column.

A sensitive and highly selective method for the simultaneous determination of purine bases and their nucleosides is proposed. An amperometric flow-injection system with the two immobilized enzyme reactors (guanase immobilized reactor and purine nucleoside phosphorylase/xanthine oxidase co-immobilized reactor) is used as the specific post-column detection system of HPLC, to convert compounds separated by a reversed-phase. HPLC column to electroactive species (hydrogen peroxide and uric acid) which can be detected at a flow-through platinum electrode. The proposed detection system is specific for a group of purine bases and purine nucleosides and does not respond for purine nucleotides and pyrimidine bases. The linear determination ranges are from 10 pmol to 5 nmol for four purine bases (hypoxanthine, xanthine, guanine, and adenine) and four purine nucleosides (inosine, xanthosine, guanosine, and adenosine). The detection limits are 1.2-5.5 pmol.     

Docking simulation with a purine nucleoside specific homology model of deoxycytidine kinase, a target enzyme for anticancer and antiviral therapy

5'-Phosphorylation, catalyzed by human deoxycytidine kinase (dCK), is a crucial step in the metabolic activation of anticancer and antiviral nucleoside antimetabolites, such as cytarabine (AraC), gemcitabine, cladribine (CdA), and lamivudine. Recently, crystal structures of dCK (dCKc) with various pyrimidine nucleosides as substrates have been reported. However, there is no crystal structure of dCK with a bound purine nucleoside, although purines are good substrates for dCK. We have developed a model of dCK (dCKm) specific for purine nucleosides based on the crystal structure of purine nucleoside bound deoxyguanosine kinase (dGKc) as the template. dCKm is essential for computer aided molecular design (CAMD) of novel anticancer and antiviral drugs that are based on purine nucleosides since these did not bind to dCKc in our docking experiments. The active site of dCKm was larger than that of dCKc and the amino acid (aa) residues of dCKm and dCKc, in particular Y86, Q97, D133, R104, R128, and E197, were not in identical positions. Comparative docking simulations of deoxycytidine (dC), cytidine (Cyd), AraC, CdA, deoxyadenosine (dA), and deoxyguanosine (dG) with dCKm and dCKc were carried out using the FlexX docking program. Only dC (pyrimidine nucleoside) docked into the active site of dCKc but not the purine nucleosides dG and dA. As expected, the active site of dCKm appeared to be more adapted to bind purine nucleosides than the pyrimidine nucleosides. While water molecules were essential for docking experiments using dCKc, the absence of water molecules in dCKm did not affect the ability to correctly dock various purine nucleosides.     

Isolation of the hexameric form of purine nucleoside phosphorylase from E. coli. Comparative study of trimeric and hexameric forms of the enzyme

The presence of two forms (high and low molecular weight ones) of purine nucleoside phosphorylase II (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was demonstrated. The high molecular weight form of the enzyme was purified, and the properties of both forms were compared. The enzyme forms were shown to differ in their quaternary structure (trimeric and hexameric), molecular weight of the native enzyme and its subunits (85,000 and 28,000 for the trimer, 150,000 and 25,000 for the hexamer, respectively) as well as substrate specificity (the trimer is specific for all major purine nucleosides, while the hexamer does not cleave adenine nucleosides). Adenosine is a competitive inhibitor of the hexameric form with respect to deoxyguanosine (Ki = 1.16 X 10(-3) M); the Km value for deoxyguanosine is 9.85 X 10(-5) M. The isoelectric point for the both forms of the enzyme in the presence of 9 M urea is about 5.5. Both forms have a pH optimum of phosphorolytic activity between 6.5 and 7.0.     

Brain nucleoside recycling

The rate limiting reactions of nucleotide synthesis are modulated by intracellular fluctuations of nucleoside triphosphate concentrations. This topic has been mostly studied at the level of the de novo nucleotide synthesis from simple precursors. However, there are districts, such as brain, which rely more heavily on the salvage of preformed purine and pyrimidine rings, mainly in the form of nucleosides. This raises the following question: how do these districts maintain the right balance between the purine and pyrimidine pools? We believe that it is now safe to state that a cross talk exists between the extra- and intracellular metabolism of purine and pyrimidine nucleosides in the brain. The extracellular space is the major site of nucleoside generation through successive dephosphorylations of released triphosphates, whereas brain cytosol is the major site of multiple phosphorylations of uptaken nucleosides at their 5′-position. Modulation of both extracellular nucleoside generation by membrane bound ectonucleotidases, and intracellular nucleoside phosphorylation by cytosolic kinases might contribute to maintain the right extra- and intracellular purine and pyrimidine nucleotide balance in the brain.     

Purine metabolism in cultured aortic and coronary endothelial cells

Purine salvage pathways in cultured endothelial cells of macrovascular (pig aorta) and microvascular (guinea pig coronary system) origin were investigated by measuring the incorporation of radioactive purine bases (adenine or hypoxanthine) or nucleosides (adenosine or inosine) into purine nucleotides. These precursors were used at initial extracellular concentrations of 0.1, 5, and 500 microM. In both types of endothelial cells, purine nucleotide synthesis occurred with all four substrates. Aortic endothelial cells salvaged adenine best among purines and nucleosides when applied at 0.1 microM. At 5 and 500 microM, adenosine was the best precursor. In contrast, microvascular endothelial cells from the coronary system used adenosine most efficiently at all concentrations studied. The synthetic capacity of salvage pathways was greater than that of the de novo pathway. As measured using radioactive formate or glycine, de novo synthesis of purine nucleotides was barely detectable in aortic endothelial cells, whereas it readily occurred in coronary endothelial cells. Purine de novo synthesis in coronary endothelial cells was inhibited by physiological concentrations of purine bases and nucleosides, and by ribose or isoproterenol. The isoproterenol-induced inhibition was prevented by the beta-adrenergic receptor antagonist propranolol. The end product of purine catabolism in aortic endothelial cells was found to be hypoxanthine, whereas coronary endothelial cells degraded hypoxanthine further to xanthine and uric acid, a reaction catalyzed by the enzyme xanthine dehydrogenase.     

Evidence for separate carriers for purine nucleosides and for pyrimidine nucleosides in the renal brush border membrane.

The aim of the present study was to test if the transport of all nucleosides in rat renal brush border membranes occurs via a common carrier or if specific carriers exist for various groups of nucleosides. We measured the inward transport of radiolabeled nucleosides into brush border vesicles. The effect of unlabeled nucleosides present inside of the vesicles (trans-stimulation) or outside of the vesicles (cis-inhibition) was studied. Uphill influx of a nucleoside into the vesicles could be driven by the efflux of another nucleoside (trans-stimulation) if they were both purines or both pyrimidines but not if one nucleoside was a purine and the other one a pyrimidine. Thus, there exist a carrier that transports various purine nucleosides, and a carrier that transports various pyrimidine nucleosides, but the tested purine nucleosides and the tested pyrimidine nucleosides do not appear to be transported by the same carrier. Uridine and thymidine were similarly potent for the inhibition of cytidine transport whereas uridine was much more potent than thymidine for the inhibition of adenosine transport. This suggests that cytidine and adenosine can use different carriers. Preincubation of the vesicles with N-ethylmaleimide resulted in a marked decrease of the rate of transport of purine nucleosides but it had little effect on the transport of pyrimidine nucleosides. These data are best explained by the presence in the renal brush border membrane of two carriers, one for purine nucleosides, the other one for pyrimidine nucleosides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deoxynucleic guanidine; synthesis and incorporation of purine nucleosides into positively charged DNG oligonucleotides

The synthesis of purine nucleosides capable of making the guanidinium linkage is described for the first time starting from the corresponding 2'-deoxynucleosides. The positively charged mixed base DNG oligomer containing guanine was synthesized on solid-phase using CPG as support from 3' to 5' direction using the precursor building block nucleosides.     

An enzymatic transglycosylation of purine bases

An enzymatic transglycosylation of purine heterocyclic bases employing readily available natural nucleosides or sugar-modified nucleosides as donors of the pentofuranose fragment and recombinant nucleoside phosphorylases as biocatalysts has been investigated. An efficient enzymatic method is suggested for the synthesis of purine nucleosides containing diverse substituents at the C6 and C2 carbon atoms. The glycosylation of N(6)-benzoyladenine and N(2)-acetylguanine and its O(6)-derivatives is not accompanied by deacylation of bases.     

Structures of human purine nucleoside phosphorylase complexed with inosine and ddI

Human purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effect on B-cell function. PNP is highly specific for 6-oxopurine nucleosides and exhibits negligible activity for 6-aminopurine nucleosides. The catalytic efficiency for inosine is 350,000-fold greater than for adenosine. Adenine nucleosides and nucleotides are deaminated by adenosine deaminase and AMP deaminase to their corresponding inosine derivatives which, in turn, may be further degraded. Here we report the crystal structures of human PNP in complex with inosine and 2('),3(')-dideoxyinosine, refined to 2.8A resolution using synchrotron radiation. The present structures provide explanation for ligand binding, refine the purine-binding site, and can be used for future inhibitor design.     

Interconversion and uptake of nucleotides, nucleosides, and purine bases by the marine bacterium MB22.

Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases. Nucleosides and purine bases formed were taken up by distinct transport systems. We found a high-affinity common transport system for adenine, guanine, and hypoxanthine, with a Km of 40 nM. This system was inhibited noncompetitively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 microM and another one for inosine and adenosine with a Km of 1.4 microM. The nucleoside transport systems exhibited both mixed and noncompetitive inhibition by different nucleosides other than those translocated; purine and pyrimidine bases had no effect. The transport of nucleosides and purine bases was inhibited by dinitrophenol or azide, thus suggesting that transport is energy dependent. Inside the cell all of the substrates were converted mainly into guanosine, xanthine, and uric acid, but also anabolic products, such as nucleotides and nucleic acids, could be found.     

Promotion or retardation of the growth of adenine auxotrophs ofCandida albicans by purines,pyrimidines and nucleosides

A study has been made of the growth responses to purine and pyrimidine metabolites shown by sixteen ultraviolet-induced adenine requiring mutants ofCandida albicans blocked at early stages in purine biosynthesis. The salient findings establish that, inC. albicans, (1) the pathway for the conversion of adenine to guanine is not reversible, (2) exogenous nucleotides are not utilized, and the purine and pyrimidine components of exogenous nucleosides must be converted to the free base form before utilization and (3) cytosine and guanine competitively inhibit different steps in the utilization of exogenous adenine.     

Synthesis of D-Fructofuranosylpurine Nucleosides

Abstract

The Mitsunobu reaction has been applied to the formation of purine nucleosides of D-fructofuranose. The use of O-benzyl protection results in a predominance of the β-configuration in these novel compounds and both α- and β-D-fructofuranosyladenine are obtained in stereochemically pure form.     

Purine nucleoside-mediated protection of chemical hypoxia-induced neuronal injuries involves p42/44 MAPK activation

Hypoxia in brain may lead to cell death by apoptosis and necrosis. Concomitant is the formation of purine nucleosides, e.g. adenosine, a powerful endogenous neuroprotectant. Despite vigorous studies, many aspects of the mechanisms involved in purine-based protection are still unclear. In this study, we wanted to investigate the effect of purine nucleosides on cellular responses to chemical hypoxia. O(2)-sensitive neuronal pheochromocytoma (PC12)-cells, which are widely used as a model system for sympathetic ganglion-like neurons, were subjected to chemical hypoxia induced with rotenone, an inhibitor of mitochondrial complex I. Adenosine and its relatives guanosine and inosine were tested for their neuroprotective capability to improve neurite outgrowth and viability. In addition, cell lysates were analyzed for mitogen-activated-protein-kinases (MAPK) activation by anti-active and anti-total MAPKinase immunoblotting. Adenosine, guanosine and inosine significantly inhibited the loss of viability after hypoxic insult. In combination with NGF, purine nucleosides also partially rescued neurite outgrowth. The MEK-1/-2 inhibitor PD098059 inhibited purine nucleoside-mediated protection up to 85.23% and also markedly decreased neurite formation induced by NGF and purine nucleosides in hypoxic cells. Immunoblot analysis revealed a strong activation of MAPKinase upon incubation of cells with adenosine, guanosine or inosine. In combination with NGF an additive effect was observed. Results suggested that activation of the MAPKinase pathway plays a vital role in purine nucleoside-mediated protection of neuronal cells following hypoxic insult.     

A preparative method for the enzymatic 5'-monophosphorylation of nucleosides

A simple procedure to prepare a stable preparation of bovine liver adenosine kinase was developed. The adenosine kinase was used to phosphorylate a variety of purine nucleosides using ATP or [gamma-32P]ATP as the phosphoryl donor. A convenient scheme to purify the nucleotides was also developed.     

Effect of Adenosine and Deoxyadenosine on the Incorporation and Breakdown of Thymidine in Escherichia coli K-12

Deoxyadenosine (AdR) and adenosine (AR) enhance the incorporation of thymidine (TdR) into bacterial deoxyribonucleic acid (DNA) by the inhibition of TdR phosphorolysis in vivo. Neither of the purine nucleosides has an effect on the reaction catalyzed by TdR phosphorylase in vitro. AdR induces TdR phosphorylase and both purine nucleosides induce purine nucleoside phosphorylase. AR can stimulate uptake of more TdR into bacterial DNA than AdR.     

Purine metabolism in microplasmodia of Physarum polycephalum.

The uptake and utilization of purine nucleosides and purines in microplasmodia of Physarum polycephalum were investigated. The results revealed a unique pattern, namely that exogenous purine nucleosides are readily taken up and metabolised, while free purine bases are hardly taken up. The pathways of incorporation have been elucidated in studies with whole cells and with cell-free extracts. The ribonucleosides (adenosine, inosine and guanosine) can be converted into ribonucleotides in two ways; either directly catalysed by a kinase or by a phosphorolytic cleavage to the free base (adenine, hypoxanthine and guanine respectively) which can then be activated by a purine phosphoribosyltransferase. Apparently the purine phosphoribosyltransferases do not react with exogenous purine bases. The deoxyribonucleosides (deoxyadenosine, deoxyinosine and deoxyguanosine) are also phosphorolysed by purine nucleoside phosphorylase to adenine, hypoxanthine and guanine respectively. A portion of deoxyadenosine is directly phosphorylated to dAMP. It appears that only a minor part of the soluble nucleotide pool can be synthesised from exogenous supplied nucleosides and that none of the deoxyribonucleosides specifically label DNA. There is no catabolism of the purine moiety. In agreement with the above findings, we have found that analoguees of purine nucleosides are more toxic than their corresponding purine base analogues.     

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.