Flow cytometric studies of nucleoside transport regulation in single chromaffin cells

  title={Flow cytometric studies of nucleoside transport regulation in single chromaffin cells},
  author={Raquel P. Sen and Esmerilda G Delicado and Alberto M. {\'A}lvarez and A M Brocklebank and James S. Wiley and Mar{\'i}a-Teresa Miras-Portugal},
  journal={FEBS Letters},
The present paper reveals that a fluorescent derivative of nitrobenzylthioinosine, 5‐(SAENTA‐x8)‐fluorescein, is a highly specific inhibitor of the neural NBTI‐sensitive nucleoside transporter. 5‐(SAENTA‐x8)‐fluorescein inhibited adenosine transport and [3H]NBTI binding with a K i of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated that 5‐(SAENTA‐x8)‐fluorescein specifically interacted with the NBTI‐sensitive nucleoside transporters with high affinity (K D=6 nM). Activation of… Expand
Fluorescent nucleoside derivatives as a tool for the detection of concentrative nucleoside transporter activity using confocal microscopy and flow cytometry.
Two fluorescent nucleoside analogues, uridine-furan and etheno-cytidine, were evaluated as tools to study in vivo nucleosid transporter-related functions and showed high affinity interactions with hC NT1 and hCNT3 and were shown to be substrates of both transporters. Expand
The Equilibrative Nucleoside Transporter (ENT1) can be phosphorylated at multiple sites by PKC and PKA
It is demonstrated, for the first time, that ENT1 is a phosphoprotein that can be directly phosphorylated at several sites by more than one kinase, and thus ENT1 may be subject to phosphorylation by multiple pathways. Expand
Nucleoside transporter and nucleotide vesicular transporter: Two examples of mnemonic regulation
The present work reviews studies concerning allosteric modulation of nucleoside and nucleotide vesicular transporters, as the first reported examples of mnemonic behavior in transporter proteins, presenting kinetic and allostered cooperativity, to suggest a widespread mechanism among transporter proteins. Expand
Nucleoside transporters in the purinome
A brief overview of the current understanding of the regulation of members of the SLC29 family is provided and some interesting avenues for future research are highlighted. Expand
Specific Temporal Distribution and Subcellular Localization of a Functional Vesicular Nucleotide Transporter (VNUT) in Cerebellar Granule Neurons
It is demonstrated that cerebellar granule cells express a functional VNUT that is involved in the regulation of ATP exocytosis, and the weak co-localization between VN UT and VGLUT1 suggests that the ATP and glutamate vesicle pools are segregated, as also observed in the cerebellars cortex. Expand
Adenosine transporters and receptors: key elements for retinal function and neuroprotection.
Evidence is reviewed showing that adenosine plays important functions in the mature as well as in the developing tissue, while regulation of cell survival and neurogenesis are some of the functions played byadenosine in developing retina. Expand
Quantitative autoradiography of adenosine receptors and NBTI-sensitive adenosine transporters in the brains of mice deficient in the preproenkephalin gene
There is a large body of evidence indicating important interactions between the adenosine and the opioid systems in regulating pain, opioid dependence and withdrawal. Mice lacking the proenkephalinExpand
Quantitative autoradiography of adenosine receptors and NBTI-sensitive adenosine transporters in the brains and spinal cords of mice deficient in the μ-opioid receptor gene
The results suggest there may be functional interactions between mu-receptors and A(1) adenosine receptors as well as NBTI-sensitiveAdenosine transporters in the brain but not in the spinal cord. Expand
S-(4-Nitrobenzyl)-6-thioinosine: tritium labelling at high specific activity.
  • C. N. Filer, C. Byon
  • Medicine, Chemistry
  • Journal of labelled compounds & radiopharmaceuticals
  • 2020
The one step tritiation of S-(4-nitrobenzyl)-6-thioinosine is described with characterization of the product by tritium NMR as well as mass spectrometry. The storage, stability and repurification ofExpand
Improved syntheses of 5'-S-(2-aminoethyl)-6-N-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), analogues, and fluorescent probe conjugates: analysis of cell-surface human equilibrative nucleoside transporter 1 (hENT1) levels for prediction of the antitumor efficacy of gemcitabine.
Fluorescent probes that bound at nanomolar concentrations specifically to human equilibrative nucleoside transporter 1 (hENT1) were produced in recombinant form in model expression systems and in native form in cancer cell lines. Expand


Flow cytometric quantitation of nucleoside transporter sites on human leukemic cells.
The improved properties of 5-(SAENTA-x8)-fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples. Expand
Down-regulation and recycling of the nitrobenzylthioinosine-sensitive nucleoside transporter in cultured chromaffin cells.
The dynamics of the nitrobenzylthioinosine (NBTI)-sensitive nucleoside transporter were studied in cultured chromaffin cells and a similar pattern of internalization and reappearance of nucleosid transporters was observed in cells cross-linked with non-labelled NBTI. Expand
Adenosine transporters in chromaffin cells: subcellular distribution and characterization.
Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cellsExpand
A new fluorescent probe for the equilibrative inhibitor-sensitive nucleoside transporter. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA)-chi 2-fluorescein.
SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry. Expand
Effects of phorbol esters and secretagogues on nitrobenzylthioinosine binding to nucleoside transporters and nucleoside uptake in cultured chromaffin cells.
Protein kinase C from bovine brain in the presence of ATP and effectors, decreased the number of high-affinity NBTI-binding sites in purified chromaffin cell plasma membranes, suggesting the possibility of a molecular modification at the transporter level. Expand
[3H]Nitrobenzylthioinosine Binding as a Probe for the Study of Adenosine Uptake Sites in Brain
[3H]NBI binding in brain should provide a useful probe for the study of adenosine transport in the brain, since thiopurines were very potent inhibitors of binding whereasAdenosine receptor ligands, such as cyclohexyladenosine and 2‐chloroadenosine were three to four orders of magnitude less potent. Expand
Effect of forskolin and cyclic AMP analog on adenosine transport in cultured chromaffin cells
The adenosine transport in cultured chromaffin cells was inhibited by the presence of the adenylate cyclase activator, forskolin, and a cAMP analog, suggesting that this inhibition could be mediated by a molecular modification ofAdenosine transporters, the binding with NBTI is therefore a possible parameter of this modification. Expand
Incorporation of adenosine into nucleotides of chromaffin cells maintained in primary cultures
In intact chromaffin tissue, a very low incorporation was found, but later on, and in the presence of nerve growth factor (NGF), a time dependent increase of adenosine incorporation was observed which, in 84-h old cells reached up to 54 times more than that found in intact tissue, probably in relation to granule maturation. Expand
Heterogeneity of [3H]Dipyridamole Binding to CNS Membranes: Correlation with [3H]Nitrobenzylthioinosine Binding and [3H]Uridine Influx Studies
The nitrobenzylthioinosine‐resistant [3H]dipyridamole binding sites were relatively insensitive to inhibition by all of the nucleoside transport substrates and inhibitors tested, with the exception of dipyridAMole itself. Expand
[3H]Dipyridamole Binding to Guinea Pig Brain Membranes: Possible Heterogeneity of Central Adenosine Uptake Sites
The data are consistent with [3H]DPR being a useful additional ligand for the adenosine uptake site and provide evidence that multiple uptake binding sites exist of which only about 40% are NBI‐sensitive. Expand