Trophic effects of purines in neurons and glial cells

In addition to their well known roles within cells, purine nucleotides such as adenosine 5' triphosphate (ATP) and guanosine 5' triphosphate (GTP), nucleosides such as adenosine and guanosine and bases, such as adenine and guanine and their metabolic products xanthine and hypoxanthine are released into the extracellular space where they act as intercellular signaling molecules. In the nervous system they mediate both immediate effects, such as neurotransmission, and trophic effects which induce changes in cell metabolism, structure and function and therefore have a longer time course. Some trophic effects of purines are mediated via purinergic cell surface receptors, whereas others require uptake of purines by the target cells. Purine nucleosides and nucleotides, especially guanosine, ATP and GTP stimulate incorporation of [3H]thymidine into DNA of astrocytes and microglia and concomitant mitosis in vitro. High concentrations of adenosine also induce apoptosis, through both activation of cell-surface A3 receptors and through a mechanism requiring uptake into the cells. Extracellular purines also stimulate the synthesis and release of protein trophic factors by astrocytes, including bFGF (basic fibroblast growth factor), nerve growth factor (NGF), neurotrophin-3, ciliary neurotrophic factor and S-100beta protein. In vivo infusion into brain of adenosine analogs stimulates reactive gliosis. Purine nucleosides and nucleotides also stimulate the differentiation and process outgrowth from various neurons including primary cultures of hippocampal neurons and pheochromocytoma cells. A tonic release of ATP from neurons, its hydrolysis by ecto-nucleotidases and subsequent re-uptake by axons appears crucial for normal axonal growth. Guanosine and GTP, through apparently different mechanisms, are also potent stimulators of axonal growth in vitro. In vivo the extracellular concentration of purines depends on a balance between the release of purines from cells and their re-uptake and extracellular metabolism. Purine nucleosides and nucleotides are released from neurons by exocytosis and from both neurons and glia by non-exocytotic mechanisms. Nucleosides are principally released through the equilibratory nucleoside transmembrane transporters whereas nucleotides may be transported through the ATP binding cassette family of proteins, including the multidrug resistance protein. The extracellular purine nucleotides are rapidly metabolized by ectonucleotidases. Adenosine is deaminated by adenosine deaminase (ADA) and guanosine is converted to guanine and deaminated by guanase. Nucleosides are also removed from the extracellular space into neurons and glia by transporter systems. Large quantities of purines, particularly guanosine and, to a lesser extent adenosine, are released extracellularly following ischemia or trauma. Thus purines are likely to exert trophic effects in vivo following trauma. The extracellular purine nucleotide GTP enhances the tonic release of adenine nucleotides, whereas the nucleoside guanosine stimulates tonic release of adenosine and its metabolic products. The trophic effects of guanosine and GTP may depend on this process. Guanosine is likely to be an important trophic effector in vivo because high concentrations remain extracellularly for up to a week after focal brain injury. Purine derivatives are now in clinical trials in humans as memory-enhancing agents in Alzheimer's disease. Two of these, propentofylline and AIT-082, are trophic effectors in animals, increasing production of neurotrophic factors in brain and spinal cord. Likely more clinical uses for purine derivatives will be found; purines interact at the level of signal-transduction pathways with other transmitters, for example, glutamate. They can beneficially modify the actions of these other transmitters.     

Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum

The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine.     

Identification and characterization of trypanocides by functional expression of an adenosine transporter from Trypanosoma brucei in yeast

The causative agents of sleeping sickness, Trypanosoma brucei rhodesiense and T. brucei gambiense, do not synthesize purines de novo but salvage purine bases and nucleosides from their hosts. We used yeast as an expression system for functional characterization of the trypanosomal adenosine transporter TbAT1. A selection of purine analogs and flavonoids were tested for their ability to interfere with adenosine transport, with the aims of identifying (a) trypanocidal TbAT1 substrates, and (b) inhibitors of trypanosomal purine transport. Cordycepin (3'-deoxyadenosine) was a TbAT1 substrate of high activity against T. brucei rhodesiense (IC50 0.2 nM). Inhibitors of mammalian nucleoside transport were not active, while the flavonol silibinin was a potent, noncompetitive inhibitor of TbAT1-mediated adenosine transport in yeast. Silibinin also inhibited melarsen-induced lysis of bloodstream form trypanosomes. IC50 values to T. brucei rhodesiense and to human carcinoma cells were 0.6 and 140 microM, respectively, indicating a good selectivity towards the parasites. Further studies are necessary to elucidate the effects of flavonoids on trypanosomal purine transport and their potential as trypanocides.     

Adenosine down-regulates the surface expression of dipeptidyl peptidase IV on HT-29 human colorectal carcinoma cells: implications for cancer cell behavior

Dipeptidyl peptidase IV (DPPIV) is a multifunctional cell-surface protein that, as well as having dipeptidase activity, is the major binding protein for adenosine deaminase (ADA) and also binds extracellular matrix proteins such as fibronectin and collagen. It typically reduces the activity of chemokines and other peptide mediators as a result of its enzymatic activity. DPPIV is aberrantly expressed in many cancers, and decreased expression has been linked to increases in invasion and metastasis. We asked whether adenosine, a purine nucleoside that is present at increased levels in the hypoxic tumor microenvironment, might affect the expression of DPPIV at the cell surface. Treatment with a single dose of adenosine produced an initial transient (1 to 4 hours) modest (approximately 10%) increase in DPPIV, followed by a more profound (approximately 40%) depression of DPPIV protein expression at the surface of HT-29 human colon carcinoma cells, with a maximal decline being reached after 48 hours, and persisting for at least a week with daily exposure to adenosine. This down-regulation ofDPPIV occurred at adenosine concentrations comparable to those present within the extracellular fluid of colorectal tumors growing in vivo, and was not elicited by inosine or guanosine. Neither cellular uptake of adenosine nor its phosphorylation was necessary for the down-regulation of DPPIV. The decrease in DPPIV protein at the cell surface was paralleled by decreases in DPPIV enzyme activity, binding of ADA, and the ability of the cells to bind to and migrate on cellular fibronectin. Adenosine, at concentrations that exist within solid tumors, therefore acts at the surface of colorectal carcinoma cells to decrease levels and activities of DPPIV. This down-regulation of DPPIV may increase the sensitivity of cancer cells to the tumor-promoting effects of adenosine and their response to chemokines and the extracellular matrix, facilitating their expansion and metastasis.     

The extracellular versus intracellular mechanisms of inhibition of TCR-triggered activation in thymocytes by adenosine under conditions of inhibited adenosine deaminase

The absence or low levels of adenosine deaminase (ADA) in humans result in severe combined immunodeficiency (SCID), which is characterized by hypoplastic thymus, T lymphocyte depletion and autoimmunity. Deficiency of ADA causes increased levels of both intracellular and extracellular adenosine, although only the intracellular lymphotoxicity of accumulated adenosine is considered in the pathogenesis of ADA SCID. It is shown that extracellular but not intracellular adenosine selectively inhibits TCR-triggered up-regulation of activation markers and apoptotic events in thymocytes under conditions of ADA deficiency. The effects of intracellular adenosine are dissociated from effects of extracellular adenosine in experiments using an adenosine transporter blocker. We found that prevention of toxicity of intracellular adenosine led to survival of TCR-cross-linked thymocytes in long-term (4 days) assays, but it was not sufficient for normal T cell differentiation under conditions of inhibited ADA. Surviving TCR-cross-linked thymocytes had a non-activated phenotype due to extracellular adenosine-mediated, TCR-antagonizing signaling. Taken together the data suggest that both intracellular toxicity and signaling by extracellular adenosine may contribute to pathogenesis of ADA SCID. Accordingly, extracellular adenosine may act on thymocytes, which survived intracellular toxicity of adenosine during ADA deficiency by counteracting TCR signaling. This, in turn, could lead to failure of positive and negative selection of thymocytes, and to additional elimination of thymocytes or autoimmunity of surviving T cells.     

The liver as a major site of immunological elimination of murine trypanosome infection, demonstrated with the liver perfusion model.

The isolated liver perfusion model has been used to investigate immunological elimination of bacteria and yeasts but not for analysis of mechanisms of immunological destruction of extracellular parasitic protozoa. Extracellular trypanosomes are eliminated primarily through antibody (and complement?)-promoted hepatic (Kupffer cell) uptake and destruction. We studied the suitability of the isolated liver model system for analyzing the mechanism of immune elimination of mouse-specific Trypanosoma musculi and identified several factors which can complicate such analyses: (i) mechanical trapping of trypanosomes that are quite large (for example, reproducing forms or epimastigotes) or are nonviable and, therefore, nondeformable; (ii) variable species and concentrations of cytadhesive molecules; and (iii) the integrity and composition of the trypanosomal surface coat. There was a substantial difference between hepatic retention of infused T. musculi organisms coated with a specific antibody and those devoid of antibody when both were suspended in normal mouse serum. The difference appeared sufficient to allow accurate quantitative studies of immune destruction in the liver. Studies of whole mice indicated that quantitative investigations of immunological elimination of trypanosomes from the bloodstream are likely to be complicated by problems such as cytadherence of parasites to host endothelial cells and mechanical trapping. Uptake by the liver and spleen appeared more reliable. Thus, the isolated liver perfusion model should significantly benefit studies to elucidate the mechanisms of immune elimination of extracellular trypanosomes.     

Identification of antibody classes and Fc receptors responsible for phagocytosis of Trypanosoma musculi by mouse macrophages

The phagocytosis of Trypanosoma musculi by macrophages in the presence of specific antibodies was investigated. In 14-day-infected mice, opsonic antibodies were detected in serum, and phagocytosis of parasites by peritoneal macrophages was observed. The mechanism of T. musculi phagocytosis was analyzed. The binding of trypanosomes to peritoneal macrophages and J774 cells was observed in the presence of serum from hyperimmune mice and from mice infected 14 or 28 days earlier, but not in the presence of control mouse serum or sera from 7-day-infected mice. Binding was partially inhibited by mouse monoclonal immunoglobulins G1 (IgG1) or IgG2a and almost completely inhibited by a mixture of both. Binding was also partially inhibited by the anti-Fc gamma 1/gamma 2b receptor monoclonal antibody 2.4G2. Binding of T. musculi was also induced by fractions of serum from 28-day-infected mice obtained by protein A-Sepharose chromatography. Only the IgG1-rich fraction eluted at pH 6.0 and the IgG2a-rich fraction eluted at pH 4.5 promoted binding which could be almost completely inhibited by monoclonal IgG1 and IgG2a. These data indicate that IgG1 and IgG2a anti-T. musculi antibodies are responsible for the phagocytosis of T. musculi by mouse macrophages and both Fc gamma 2a and Fc gamma 1/gamma 2b receptors are involved. Such a mechanism is likely to account for the elimination of parasites in T. musculi-infected mice.     

Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase.

Toxoplasma gondii lacks the capacity to synthesize purines de novo, and adenosine kinase (AK)-mediated phosphorylation of salvaged adenosine provides the major route of purine acquisition by this parasite. T. gondii AK thus represents a promising target for rational design of antiparasitic compounds. In order to further our understanding of this therapeutically relevant enzyme, an AK cDNA from T. gondii was overexpressed in E. coli using the pBAce expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques. Kinetic analysis of TgAK revealed Km values of 1.9 microM for adenosine and 54.4 microM for ATP, with a k(cat) of 26.1 min(-1). Other naturally occurring purine nucleosides, nucleobases, and ribose did not significantly inhibit adenosine phosphorylation, but inhibition was observed using certain purine nucleoside analogs. Adenine arabinoside (AraA), 4-nitrobenzylthioinosine (NBMPR), and 7-deazaadenosine (tubercidin) were all shown to be substrates of T. gondii AK. Transgenic AK knock-out parasites were resistant to these compounds in cell culture assays, consistent with their proposed action as subversive substrates in vivo.     

Interaction between murine natural killer cells and trypanosomes of different species.

The involvement of natural killer (NK) cells in the immunological resistance of mice to murine-specific Trypanosoma musculi was evaluated. Murine NK cells were found to be unable to kill or inhibit T. musculi or to protect recipients from infection. In addition, the ability of spleen cells from normal mice and from mice on day 3 of T. musculi infection, at the time of maximum NK augmentation, to kill Trypanosoma cruzi and Trypanosoma lewisi was evaluated. Spleen cells from normal mice displayed significant killing of both T. cruzi and T. lewisi. Furthermore, augmented spleen cells from T. musculi-infected mice were considerably more effective than normal spleen cells in killing both T. cruzi and T. lewisi. The activity of NK cells toward YAC-1 tumor target cells was inhibited in a dose-dependent fashion by either live T. musculi or extracts of T. musculi, but not by extracts of rat-specific T. lewisi. The results suggest that well-adapted protozoan parasites may be nonsusceptible to the natural cell-mediated resistance mechanisms of their hosts. Their nonsusceptibility could result from the ability to elaborate substances that either inactivate NK cells or block NK cell interaction with complementary sites on the parasite surface.     

Enzymatic and extraenzymatic role of ecto-adenosine deaminase in lymphocytes

Summary: Adenosine deaminase (ADA, EC 3.5.4.4) is an enzyme of the purine metabolism which has been the object of considerable interest mainly because the congenital defect causes severe combined immunodeficiency (SCID). In the last 10 years, ADA, which was considered to be cytosolic, has been found on the cell surface of many ceils and, therefore, it can be considered an ecto-enzyme. There is recent evidence about a specific role of ecto-ADA, which is different from bat of intracellular ADA. Apart from degrading extracellular adenosine (Ado) or 2'-deoxyadenosLne (dAdo), which are toxic for lymphocytes, ecto-ADA has an extraenzymatic function via its interaction with GD26. ADA/CD26 interaction results in co-stimulatory signals in T cells. This co-stimulation is blocked by HIV-1, thus evidencing a role for ecto-ADA in the pathophysiology of AIDS. The fact that, besides CD26, ADA can interact with different cell-surface proteins opens new perspectives in the research for a role of ecto-ADA in the function of the immune system and in the Interactions that take place between different cells in the development of the immune system. The most interesting aspect is the possible participation of the ecto-enzyme in cell-to-cell contacts during ontogenesis and maturation of immunocompetent cells.     

Enzymatic and extraenzymatic role of adenosine deaminase 1 in T-cell-dendritic cell contacts and in alterations of the immune function

Adenosine deaminase 1 (ADA1) is an enzyme of the purine metabolism whose congenital defect leads to severe combined immunodeficiency (SCID). Although classically considered a cytosolic enzyme, early evidence from work in brain synaptosomes suggested that the enzyme could be an ectoenzyme. In lymphoid cells, ectoenzymatic activity of ADA1 was also found. The obvious role of this enzyme located on the cell surface of lymphocytes and monocytes was to deaminate adenosine, making it less available for uptaking and metabolism, and also for adenosine-receptor activation. Quite unexpectedly, ADA1 was shown to act extraenzymatically. In addition, cell surface ADA1-binding proteins have been identified. Interestingly, the interaction of ADA1 with these anchoring proteins leads to costimulation of T-cell activation. Recent studies performed with professional antigen-presenting cells and T lymphocytes have shown that ADA1 can bridge the two cell types together by a cross-linking established between different anchoring molecules in each cell. Some aspects of ADA action are similar to that of growth factors. In fact, ADA1 is a member of the adenosine deaminase growth factor (ADGF) family. Some molecular mechanisms that occur in ADA-related SCID and the role ADA1 may play in acquired immunodeficiency are also reviewed here.     

Adenosine deaminase and purine nucleoside phosphorylase activity in spleen cells of aged mice

The activities of two enzymes of purine metabolism, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), were determined in spleen lymphocytes from mice of various ages. We found that in the older animals, which have depressed responses to concanavalin A and phytohemagglutinin, there is a decrease in the activity of PNP but normal activity of ADA. The decline of PNP activity was seen at 7.5 months of age and appears to be concurrent with a decline in T-cell function. These results suggest that a decrease in PNP activity may be a contributing factor in the immunodeficient state of the aged.     

Immunohistochemical localization of adenosine deaminase in primary afferent neurons of the rat

Adenosine deaminase (ADA) was detected immunohistochemically in neuronal cell bodies of dorsal root ganglia (DRG) of the rat. ADA-immunoreactivity was confined exclusively to small type B ganglion neurons in cervical, thoracic and lumbar sensory ganglia; large type A neurons in sensory ganglia were devoid of immunostaining for ADA. It was consistently found that only a small proportion of type B neurons in DRG contain immunohistochemically detectable ADA. It is suggested that the expression of high ADA levels is a distinguishing feature of a subpopulation of type B DRG neurons and, further, that ADA in these neurons may reflect their utilization of purines (adenosine or adenine nucleotides) as transmitters or cotransmitters.     

Differences in the activity of adenosine deaminase and of purine nucleoside phosphorylase and in the sensitivity to deoxypurine nucleosides between subpopulations of mouse thymocytes

The activities of adenosine deaminase (ADA) and of purine nucleoside phosphorylase (PNP) were measured in thymocyte subpopulations separated by peanut agglutinin (PNA), in unseparated thymocytes, in lymph node and in spleen cells. The PNA+ thymocyte subpopulation exhibited the highest ADA activity of all cells studied. The lowest PNP activity was found in the PNA- subpopulation of thymocytes. PNA+ cells, moreover, exhibited a more intensive DNA synthesis than the PNA- cells, and a greater sensitivity to deoxyadenosine toxicity in the presence of erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA). The two thymocyte subpopulations exhibited a similar sensitivity to deoxyguanosine (dG) toxicity.     

Activities of purine metabolising enzymes in lymphocytes of neonates and young children: correlates with immune function

Depressed activities of the following purine enzymes have been shown to result in immunodeficiencies: adenosine deaminase (ADA), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and purine nucleoside phosphorylase (PNP). These enzymes and adenosine kinase (AK) were measured in cord blood lymphocytes of premature and small-for-gestational age infants since they have partial immunodeficiencies of unknown biochemical etiology which can persist for many years. We also measured these enzymes in 3 infants with various immunodeficiencies. Activities were compared with appropriate matched control groups. The results indicated normal ADA and PNP but significantly depressed AK (P less than 0.05) and HGPRT (P less than 0.001) activities in 10 premature/SGA infants when compared to 35 full-term normal infants. In the 3 immunodeficient children the results were as follows: Child 1 had a 2- to 3-fold decrease in ADA with normal PNP and AK activities; Child 2 had a 2- to 3-fold decrease in AK, 4-fold decrease in HGPRT with normal PNP and ADA activities; Child 3 had confirmed AIDS and a 4-fold decrease in ADA, 6-fold decrease in HGPRT with normal PNP activity. The possible role of these depressed purine enzyme activities found in lymphocytes is discussed in relation to the imparied immunity seen in these infants.     

Simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography with fluorescence derivatization

A sensitive method was developed for the simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography. The adenyl purines (adenine, adenosine, AMP, ADP, ATP and cyclic AMP) were derivatized using 2-chloroacetaldehyde for fluorescence detection, and the reaction and separation conditions were reinvestigated to improve sensitivity for small volume sample analysis. Each derivatized purine was separated on a Capcell Pack SG120A column with mobile phase consisting of 0.05 M citric acid-0.1 M dipotassium hydrogen phosphate (pH 4.0)-methanol (97+3). The detection limits were 100-1000 fmol/ml by fluorescence detection, some 500 times better than previous reports. The proposed method was applied to determine adenyl purines in human plasma. The purine levels were as follows: ATP (9.2-22.2 pmol/ml), ADP (5.5-22.2 pmol/ml), AMP (0.8-3.2 pmol/ml). Other purines, adenine, adenosine, cAMP were lower than 0.1 pmol/ml.     

Deoxycoformycin (pentostatin): clinical pharmacology, role in the chemotherapy of cancer, and use in other diseases

Pentostatin (2'-deoxycoformycin, dCF) is a purine nucleoside analog and a product of the fermentation of Streptomyces antibioticus. It is a tight-binding inhibitor of adenosine deaminase (ADA), an enzyme essential in the cellular metabolism of purines. Children with congenital absence of ADA suffer from atrophy of lymphoid tissues and severe combined immune deficiency (SCID) syndrome. It was hypothesized that pentostatin would be lymphocytotoxic and this proved to be true; this finding prompted its investigation in lymphoid neoplasms. It was anticipated that pentostatin would be most active in neoplasms with high intracellular concentrations of ADA, e.g. acute lymphocytic leukemia (ALL), particularly of the T-cell variety. Although pentostatin proved to be active in ALL, large doses were required and major toxic effects outweighed therapeutic benefits. By contrast, pentostatin proved to be exceptionally active in hairy cell leukemia (HCL), a B-cell neoplasm with low intracellular concentrations of ADA. Pentostatin has since been shown to possess activity in chronic lymphocytic leukemia, prolymphocytic leukemia, cutaneous T-cell lymphomas, adult T-cell lymphoma-leukemia, and low grade non-Hodgkin's lymphomas. It potentiates the activity of vidarabine against viruses and against the cells of acute myeloid leukemia. Pentostatin is inactive in melanoma and renal carcinoma, but has not been adequately evaluated in other solid tumors. The toxic effects of pentostatin include renal failure, central nervous system (CNS) depression, immunosuppresion, keratoconjunctivitis, and opportunistic infections. In the absence of pre-existing bone marrow compromise, pentostatin produces only mild myelosuppression. Aside from its use as an antineoplastic agent, pentostatin has potential applications as an immunosuppressive drug, as an antiviral agent, as an antimalarial compound, and in the protection of cells of the CNS from damage induced by ischemia and anoxia. Clinical studies with pentostatin are ongoing, and its roles in the management of neoplastic and non-neoplastic diseases have yet to be fully defined.     

Immune and nonimmune regulation of the population of Trypanosoma musculi in infected host mice.

A clear understanding of the population dynamics of trypanosome infections is lacking. In the case of murine Trypanosoma musculi infections, there are no answers to questions concerning (i) the nature of the prolonged plateau phase during which the number of parasites present in the host remains nearly constant (is it a static or dynamic steady state?); (ii) the origin of new parasites, if the plateau is a dynamic steady state, given the relatively early disappearance of generative forms from the bloodstream; and (iii) the role, if any, of a putative ablastin (reproduction-restricting antibody) in regulating the population dynamics of T. musculi infections. We describe here the results of studies of the number and distribution of mature and reproductive forms (RF) in the blood and peritoneal space of both immunocompetent and cyclophosphamide-treated mice throughout the course of infection. While the RF disappeared from the blood within a few days after parasite inoculation, a high fraction (20 to 30%) of the parasites in the peritoneal space were RF throughout the course of infection and, thus, represented a source of new parasites. If an ablastin is responsible for inhibiting RF in the blood, it appeared to have no effect on RF in the peritoneal space. The results of this investigation support the conclusion that the control of the dynamics of T. musculi infections is largely nonimmunological (until cure of the infection) and probably is exercised by the supply of nutrients and reproduction-inhibiting (nonimmunological) and maturation-promoting factors that affect the generative fraction of the population.     

Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter

Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [³H]adenosine 1.8-fold within 30 s, approx. half of the [³H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [¹⁴C]Inosine, [¹⁴C]hypoxanthine and [³H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC₅₀ approx. 0.7 μM), but not nitrobenzylthioinosine (15 μM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 μM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [³H]adenosine and [¹⁴C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. gondii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purimes may also be present.     

Formycin B, Purine Nucleoside Phosphorylase and Lymphocyte Function

A genetic deficiency of purine nucleoside phosphorylase (NP) is associated with immune dysfunction that specifically affects T cells. An absence of adenosine deaminase (ADA), the preceding enzyme in the pathway of purine recycling, is associated with failure of both T and B lymphocyte function.

Formycin B, an analogue of inosine and an inhibitor of purine nucleosidase phosphorylase, was used in cell culture systems to stimulate deficiency of this enzyme. The cells used were normal circulating lymphocytes and lymphoblastoid cell lines (LCL) with T and B cell characteristics. Formycin B inhibited the growth of these cells, but its primary effect was found to be due to a mechanism other than purine nucleoside phosphorylase inhibition.

The possibility that formycin B was inhibiting adenosine deaminase, for which it is a product analogue, was studied by the anaylsis of reaction progress curves using the integrated rate equation. The molecular structure of formycin B, whilst preventing its phosphorylytic cleavage by purine nucleoside phosphorylase, could enable this compound to function as a substrate for adenosine kinase. The resultant formation of formycin B nucleotide probably causes the observed inhibition of growth in cultured cells.