Nucleoside transporter expression and function in cultured mouse astrocytes

Abstract

Uptake of purine and pyrimidine nucleosides in astrocytes is important for several reasons: (1) uptake of nucleosides contributes to nucleic acid synthesis; (2) astrocytes synthesize AMP, ADP, and ATP from adenosine and GTP from guanosine; and (3) adenosine and guanosine function as neuromodulators, whose effects are partly terminated by cellular uptake. It has previously been shown that adenosine is rapidly accumulated by active uptake in astrocytes (Hertz and Matz, Neurochem Res 14:755-760, 1989), but the ratio between active uptake and metabolism-driven uptake of adenosine is unknown, as are uptake characteristics for guanosine. The present study therefore aims at providing detailed information of nucleoside transport and transporters in primary cultures of mouse astrocytes. Reverse transcription-polymerase chain reaction identified the two equilibrative nucleoside transporters, ENT1 and ENT2, together with the concentrative nucleoside transporter CNT2, whereas CNT3 was absent, and CNT1 expression could not be investigated. Uptake studies of tritiated thymidine, formycin B, guanosine, and adenosine (3-s uptakes at 1-4 degrees C to study diffusional uptake and 1-60-min uptakes at 37 degrees C to study concentrative uptake) demonstrated a fast diffusional uptake of all four nucleosides, a small, Na(+)-independent and probably metabolism-driven uptake of thymidine (consistent with DNA synthesis), larger metabolism-driven uptakes of guanosine (consistent with synthesis of DNA, RNA, and GTP) and especially of adenosine (consistent with rapid nucleotide synthesis), and Na(+)-dependent uptakes of adenosine (consistent with its concentrative uptake) and guanosine, rendering neuromodulator uptake independent of nucleoside metabolism. Astrocytes are accordingly well suited for both intense nucleoside metabolism and metabolism-independent uptake to terminate neuromodulator effects of adenosine and guanosine.

Mouse Equilibrative Nucleoside Transporter 2 (mENT2) Transports Nucleosides and Purine Nucleobases Differing from Human and Rat ENT2

“…21) Therefore, it is difficult to explain the high-affinity uptake via mENT2 observed in the present study by species difference. On the other hand, ENTs were recently reported to be metabolism-driven transporters, 15) and the salvage pathways for nucleotide synthesis are not necessary for NT-deficient cells (Xenopus oocytes and NT-deficient PK15 cells), suggesting that the sufficient activity of intracellular metabolism in transfectants may lead to the high-affinity transport mediated by ENT2.…”

“…15) Furthermore, tumor cells-bearing mouse has been used for the developments of improved chemotherapeutic strategies. 16) Therefore, the functional characterization of mENT2 using a variety of nucleosides and nucleobases is physiologically and pharmacologically important.…”

Purinergic mechanisms in neuroinflammation: An update from molecules to behavior

“…Adenosine is principally metabolized in the CNS by adenosine kinase, an enzyme expressed in highest concentrations in astrocytes (Studer et al, 2006). Extracellular concentrations of adenosine in the CNS are regulated by three nucleoside transporters expressed on astrocytes (Peng et al, 2005), with adenosine release, uptake and metabolism forming an 'adenosine-cycle', described in detail by Boison (2008). Following metabolic stress and cell damage, for example, under conditions of high neuronal activity or brain injury, the extracellular concentration of adenosine is dramatically elevated where it acts, in concert with ATP, as an alarm molecule, promoting or inhibiting neuroinflammation, depending on a host of complex factors, which are only beginning to be understood.…”

Energy Metabolism in Astrocytes: High Rate of Oxidative Metabolism and Spatiotemporal Dependence on Glycolysis/Glycogenolysis

“…Stimulation of astrocytes by neuronally released glutamate generates an increase in free cytosolic Ca 2 + concentration ([Ca 2 + ] i ) in the astrocytes, which either can remain locally and lead to exocytotic glutamate release from astrocytes, or travel through an astrocytic syncytium as a Ca 2 + wave, mediated by transport of IP 3 through gap junctions and/or by ATP release and subsequent stimulation of purinergic receptors (Cornell-Bell et al, 2004;Haas et al, 2006). Exocytotic glutamate release and propagation of Ca 2 + waves (including reestablishment of a low resting [Ca 2 + ] i ) require energy, and release of ATP as a transmitter represents utilization of an energy-rich molecule, which subsequently has to be re-constituted by active uptake of adenosine and the sequential re-synthesis of adenosine monophosphate (AMP), adenosine diphosphate (ADP), and ATP (Peng et al, 2005). No details or quantitative estimates of energy demand for these functions and filopodial motility are available.…”