Nucleoside Transport In Cells Research Articles

Nucleoside transporters are critical to the uptake and antioxidant activity of 7,8-dihydroneopterin in monocytic cells

7,8-Dihydroneopterin protects cells intracellularly from oxidative stress-induced death, but its mode of transport across the cell membrane is unknown. Nucleosides, such as guanosine, are transported via nucleoside transporters of the equilibrative and concentrative forms. Therefore, the objective of this study was to identify which membrane transporters are responsible for 7,8-dihydroneopterin transport in cells and whether this is necessary for protection against oxidative stress. Monocytic cell lines U937, THP-1 and human monocytes were incubated with varying concentrations of 7,8-dihydroneopterin with or without nucleoside transporter inhibitors nitrobenzylthioinosine (NBMPR; ENT1), dipyridamole (DP; ENT1 and ENT2) or indomethacin (INDO; CNT). Only DP inhibited 7,8-dihydroneopterin uptake in U937 cells, while NBMPR and DP inhibited 7,8-dihydroneopterin uptake in THP-1 cells. All three inhibitors limited 7,8-dihydroneopterin uptake in human monocytes at short time points only. When the cells were incubated with 10 mM of the peroxyl radical generator 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) a 50–80% loss of cell viability was measured. 7,8-dihydroneopterin protected all cell lines against AAPH-induced cell death, which was prevented with DP in U937 cells, NBMPR in THP-1 cells and a combination of all three nucleoside inhibitors in human monocytes. These data indicate 7,8-dihydroneopterin is transported across the cell membrane of monocytic cells via equilibrative and concentrative nucleoside transporters in a cell lineage-dependent manner. The data also indicate protection from peroxyl radical-generated cell death with 7,8-dihydroneopterin is intracellular and facilitated through nucleoside transporters in monocytic cells.

Why do male mice spit soluble enzymes that hydrolyze extracellular nucleotides? Focus on “Prostatic acid phosphatase is the main acid phosphatase with 5′-ectonucleotidase activity in the male mouse saliva and regulates salivation”

the 5′-ectonucleotidases found on the surface of most mammalian cells and in blood serum serve as a source of adenosine derived from extracellular nucleotide degradation (11). The 5′-ectonucleotidases are phosphatases whose substrate is extracellular adenosine 5′-monophosphate (AMP) generated from adenosine 5′-tri- and diphosphates (ATP and ADP) released from injured tissues to stimulate cellular responses via P2 purinergic receptors (7). A recent study by Quintero et al. (10) investigated prostatic acid phosphatase (PAP), a 5′-ectonucleotidase that plays a role in the development of adenocarcinoma of the prostate gland. These authors showed that PAP exists as two alternative splice variants, a transmembrane prostatic acid phosphatase (TMPAP) and secreted PAP (sPAP). TMPAP and sPAP are distinguished functionally from other 5′-nucleotidases through inhibition by l−(+)−tartrate, ability to cleave phosphomonoesters besides AMP, and activity over a wide pH range (i.e., 3–8). TMPAP activation inhibits endocytosis/exocytosis through interaction with snapin (10), a soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE)-associated protein (5) that negatively regulates secretory vesicle docking and fusion with plasma membranes (Fig. 1). In addition, adenosine generated by PAP is an agonist for G protein-coupled A1 adenosine receptors (A1R) that counteract the nociceptive (i.e., pain-producing) effects of ATP (4) occurring through activation of P2X receptor ATP-gated ion channels (2; Fig. 1). However, little information is available concerning physiological roles of sPAP in biological fluids. Fig. 1. Prostatic acid phosphatase (PAP) signaling in salivary glands and saliva. In this issue of American Journal of Physiology-Cell Physiology, Araujo et al. (1) attempt to understand the role of TMPAP and sPAP in a secretory organ, the submandibular gland (SMG), which secretes fluid, proteins, and other factors into saliva, thereby bathing the oral cavity with antimicrobial and digestive agents. Using PAP−/− mice, the authors evaluate effects of deletion of this 5′-ectonucleotidase on SMG function. Comparisons between PAP−/− and wild-type SMG indicate that TMPAP is primarily expressed in granular convoluted tubule (GCT) cells of the duct but not acinar cells. Remarkably, deletion of PAP increases α-adrenergic and muscarinic cholinergic receptor-mediated saliva secretion and enhances expression of genes promoting inflammation and proliferation, particularly interferon-dependent proinflammatory genes. The authors suggest that this increase in saliva secretion relates to loss of negative control by TMPAP of exocytosis (Fig. 1). PAP deletion also increases B and T cell infiltration of SMG and levels of miR-146a that have been associated with prevention of tissue damage. Together with observations of proliferating acinar cells after PAP deletion, these results strongly suggest that SMG homeostasis in PAP−/− mice is maintained by an immune response without salivary gland degeneration seen with immune cell infiltration of SMG in the autoimmune disease Sjogren's syndrome (SS; 3, 8). Another important finding is that sPAP is only present in male mouse saliva, which correlates with reduced levels of GCT cells in female versus male SMG and is consistent with sexual dimorphism in rodent salivary glands (6). The authors suggest that mouse sPAP plays a male-specific function in which licking of wounds (perhaps caused by fighting) deposits sPAP-containing saliva that induces an anti-nociceptive response in wounded tissue by participating in the generation of adenosine from released nucleotides to activate A1 receptors (Fig. 1). Since SS has a female predominance in rodents and humans (3, 8), it would be interesting to determine whether PAP in males plays a protective role in SS by promoting gland homeostasis and delay in the onset of salivary gland dysfunction and degenerative immune cell infiltration. Another question remaining is whether extracellular nucleotides are released into the oral cavity due to tissue injury of oral epithelium caused by inflammation, exposure to toxins, SS, and radiation-induced damage occurring as a side effect of cancer therapy. Once released, degradation of extracellular nucleotides by ectonucleotide diphosphohydrolases (eNTPDases), ectoATPases, and ectoADPases would generate AMP whereupon 5′-ectonucleotidases including PAP would yield adenosine that could either be taken up by nucleoside transporters in cells and salvaged or activate P1 adenosine receptors (7, 9, 11). Further study is needed to investigate whether sPAP in saliva plays a role in the regulation of purinergic signaling in the oral cavity under physiological and pathological conditions.

Differential adenosine uptake in mixed neuronal/glial or purified glial cultures of avian retinal cells: Modulation by adenosine metabolism and the ERK cascade

Adenosine is an important modulator of neuronal survival and differentiation in the CNS. Our previous work showed that nucleoside transporters (NTs) are present in cultures of chick retinal cells, but little is known about the mechanisms regulating adenosine transport in these cultures. Our aim in the present work was to study the participation of the adenosine metabolism as well as the ERK pathway on adenosine uptake in different types of retinal cultures (mixed and purified glial cultures). Kinetic analysis in both cultures revealed that the uptake reached equilibrium after 30min and presented two components. Incubation of cultures with S-(p-nitrobenzyl)-6-thioinosine (NBTI) or dipyridamole, different inhibitors of equilibrative nucleoside transporters (ENTs), produced a significant and concentration-dependent uptake reduction in both cultures. However, while dipyridamole presented similar maximal inhibitory effects in both cultures (although in different concentrations), the inhibition by NBTI was smaller in glial cultures than in mixed cultures, suggesting the presence of different transporters. Moreover, pre-incubation of [3H]-adenosine with adenosine deaminase (ADA) or adenosine kinase (ADK) inhibition with iodotubercidin promoted significant uptake inhibition in both cultures, indicating that the uptake is predominantly for adenosine and not inosine, and that taken up adenosine is preferentially directed to the synthesis of adenine nucleotides. In both cultures, the MEK inhibitors PD98059 or UO126, but not the inactive analog U0124, induced a significant and concentration-dependent uptake decrease. We have not observed any change in adenosine metabolism induced by MEK inhibitors, suggesting that this pathway is mediating a direct effect on NTs. Our results show the expression of different NTs in retinal cells in culture and that the activity of these transporters can be regulated by the ERK pathway or metabolic enzymes such as ADK which are then potential targets for regulation of Ado levels in normal or pathological conditions.

Nucleoside transporter expression and activity is regulated during granulocytic differentiation of NB4 cells in response to all- trans-retinoic acid

NB4 cells express multiple nucleoside transporters (NTs), including: hENT1 ( es), and hENT2 ( ei), and the CNT subtype referred to as, csg; a concentrative sensitive guanosine specific transporter. csg activity is a distinguishing feature of the NB4 cell line and its presence suggests a particular requirement of these cells for guanosine salvage. Proliferation and differentiation pathways determine, in part, the number of NTs in cells and tissues. In this study, all- trans-retinoic acid (ATRA)-induced granulocytic differentiation of NB4 cells resulted in biphasic changes in guanosine transport. Transient increases in csg and es activity, the result of an increase in V max (pmol/μl s) of both transporter systems, served as early markers of differentiation while expression of a fully differentiated phenotype was accompanied by a selective loss of csg activity and the return of es activity to that of proliferating cells. Intracellular incorporation of [ 3H]-guanosine decreased as cells matured despite increased transport rates and suggested a reduced intracellular requirement of NB4-granulocytes compared to their proliferating counterparts. Whether a loss of csg activity could serve to assess clinical response to differentiation therapies is not known. Nitrobenzylthioinosine (NBMPR) binding sites within nuclear membrane (NM) preparations, suggested the presence of functional intracellular NTs. An increase in plasma membrane (PM) associated transporters coincided with the early increase in guanosine transport and a decrease in NBMPR binding to NM fractions and suggests that intracellular NTs may serve as a reserve pool for translocation to the (PM) when additional transport capacity is required. The modulation of transporters during differentiation could potentially regulate drug bioavailability and cytotoxicity and should be evaluated prior to combining differentiating agents with traditional nucleoside analogs in the treatment of APL.

Nucleoside transport in L1210 murine leukemia cells. Evidence for three transporters.

L1210 murine leukemia cells have two nucleoside transport activities that differ in their sensitivity to nitrobenzylmercaptopurine riboside (NBMPR). This study re-examines NBMPR-insensitive nucleoside transport in these cells and finds that it is mediated by two components, one Na(+)-dependent and the other Na(+)-independent. A mutant selected previously for loss of NBMPR-insensitive transport lacks only the Na(+)-independent activity. When NBMPR is used to block efflux via the NBMPR-sensitive transporter, uptake of formycin B (a nonmetabolized analog of inosine) is concentrative in both the parental and mutant cells, but the intracellular concentration of the nucleoside is 5-fold lower in the parental cells. Decreased accumulation of formycin B in the parental cells is due to efflux of the nucleoside via the NBMPR-insensitive, Na(+)-independent transporter that the mutant lacks. The Na(+)-dependent transporter appears to accept most purine, but not pyrimidine, nucleosides as substrates. Two exceptions are uridine, a good substrate, and 7-deazaadenosine, a poor substrate. In contrast, all of the nucleosides tested are substrates for the Na(+)-independent transporter. We conclude that L1210 cells have three distinct nucleoside transporters and that the specificity of the Na(+)-dependent transporter is similar to that of one of the two Na(+)-dependent nucleoside transporters seen in mouse intestinal epithelial cells.

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

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

Nucleoside transport in acute leukaemia and lymphoma: close relation to proliferative rate

The proliferation of mammalian cells requires nucleosides which are provided either by de novo synthesis or by influx of nucleosides via membrane transporters with subsequent metabolic trapping. In this study the density of nucleoside transporters in freshly-isolated blast cells from patients with leukaemias and lymphomas was quantitated by equilibrium binding of 3H-nitrobenzylmercaptopurine riboside (NBMPR). In acute myeloid leukaemia (AML) the density of NBMPR binding sites on blast cells ranged from 3800 to 24,200 sites/cell and this value correlated with the 3H-thymidine labelling index (1-20%) which was used to measure proliferative rate (r = 0.80, P less than 0.001). Cells from patients with Burkitt's lymphoma, other B-cell lymphomas, T-lymphoblastic lymphoma and large cell lymphoma gave a 20-fold range of NBMPR site densities (from 3700 to 75,300 sites/cell) and site numbers correlated closely with the labelling index (r = 0.87, P less than 0.001). Non-proliferating cells from patients with chronic lymphocytic leukaemia expressed the lowest density of NBMPR binding sites (850-2900 sites/cell). Comparison of bone marrow and peripheral blood blasts confirmed the positive correlation between NBMPR binding sites and labelling index for four individual patients. In contrast, the density of NBMPR binding sites on lymphoblasts from non-T acute lymphoblastic leukaemia (ALL) was low (2300-7400 sites/cell) and showed little dependence on proliferation over a wide range of labelling indices (1-20%). No correlation was observed between NBMPR site density and cell size measured by the intracellular water space. Thus an increased proliferative rate of AML or lymphoma is associated with higher numbers of nucleoside transporters in the cell membrane.

Adenosine transport by cultured glial cells from chick embryo brain

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [ 3H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with K m = 370 μM and V max = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na + from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells ( K m = 12 μM; V max = 0.34 nmol/ min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N 6-benzyladenosine. In decreasing order of potency, N 6-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.

Nucleoside transport in cells and membrane vesicles from Escherichia coli K12.

Osmotic shock treatment of cells of Escherichia coli K12 caused a reduction in the transport of nucleosides into the cells. The strains used carried mutations in the nucleoside catabolizing enzymes. This indicated that the decrease in transport capacity was not due to loss of these enzymes during the shock treatment. Membrane vesicles, prepared from the same strains, showed a limited transport of cytidine, deoxycytidine, and uridine. Transport of purine nucleosides and of thymidine was very low in vesicles lacking the appropriate nucleoside phosphorylases and no significant stimulation was observed if the nucleoside phosphorylases were present in the membrane vesicles. These results all indicate that components outside the cytoplasmic membrane are important for nucleoside transport. Selection for resistance to fluorodeoxycytidine yielded mutants which were unable to transport any nucleoside, even when the nucleoside phosphorylases were present in high amounts. This finding is consistent with a requirement for a specific transport process prior to the initial enzymatic attack on the incoming nucleoside.

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. IV. Nucleoside transport in cells depleted of nucleotides by treatment with KCN.

AbstractIncubation of Novikoff rat hepatoma cells in glucose‐free basal medium containing 2 mM KCN results in a rapid and almost complete loss of uracil and adenine nucleotides. By following the fate of radioactivity from 3H‐nucleoside pulse‐labeled cells during incubation with KCN it was shown that the nucleotides are degraded to nucleosides and bases which are released into the culture fluid. Depletion of the cells of nucleotides by incubation with KCN allows a direct analysis of the kinetics of uridine transport into the cell, since KCN‐treated cells fail to phosphorylate uridine. Uridine uptake follows normal Michaelis‐Menten kinetics with an apparent Kn of about 50 μm at 18°C. Uptake is by facilitated diffusion since it does not require energy and uridine is not transported against a concentration gradient. The effects of KCN are largely prevented by the presence of 10 mM glucose in the medium. They are also rapidly reversed by resuspending the cells in fresh medium without KCN. Upon removal of KCN, the cells rapidly regenerate their nucleotide pools and resume growth at the normal rate.