Identification of Adenosine ENT1 Transporters in Astrocytes and
their Modulation by Adenosine A1 Receptors
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1
University of Coimbra, Center for Neurosciences of Coimbra, Institute of Biochemistry, Faculty of Medicine, Portugal
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2
Institute of Biology, Federal Fluminense University, Laboratory of Cellular Neurobiology, Department of Neurobiology, Program of Neurosciences, Brazil
Adenosine is an important neuromodulator in the CNS. Adenosine mainly controls neuronal activity through a synaptic action on inhibitory A1 receptors and facilitatory A2A receptors, although it has also been proposed to control glial functions (Fredholm et al., 2005, Int Rev Neurobiol 63:191-270). The extracellular levels of adenosine are tightly controlled: adenosine builds-up extracellularly as a function of neuronal activity and adenosine transporters play a key role in the rapid removal of this extracellular adenosine; this mainly occur through equilibrative nucleoside transporters (ENT1 and ENT2), which are located both in neurons and astrocytes. Using primary cultures of brain astrocytes, we now aimed at defining the relative contribution of ENT1 and ENT2 in the clearance of extracellular adenosine. Astrocytic cultures were prepared from cerebral cortices of 4-5-day postnatal Wistar rats and maintained in cultures for 3 weeks. 3H-Adenosine uptake by astrocytes was linear from 15 to 300 seconds. Since NBTI (nitrobenzylthioinosine) is a selective antagonist of ENT1, we used this compound to define the relative contributions of ENT1 and ENT2 for 3H-adenosine uptake. We observed that ENT1 was responsible for 68.75±8.04% (n=3) of the total 3H-adenosine uptake through equilibrative nucleoside transporters. Western blot analysis confirmed the presence of ENT1 in cultured astrocytes and 3H-NBTI displayed a saturable binding with a KD of 2.4±0.6 nM (95% confidence interval of 1.3-3.6 nM, n=3) and a Bmax of 18.2±1.3 fmol/mg protein (n=3). Since we have previously shown that adenosine receptors control adenosine uptake (Pinto-Duarte et al., 2005, J Neurochem 93: 595-604), we next began investigating if the activation of adenosine receptors modifies the uptake of adenosine by astrocytes. Activation of A1 receptors with CPA (100 nM) decreased 3H-adenosine uptake by 22.5±6.9% (n=3, p<0.05) and increased the KD of 3H-NBTI binding by 75-107%, without evident modification of Bmax (n=2). These data identify ENT1 as the main adenosine transporter in astrocytes and show that the activation of adenosine A1 receptors might control the rate adenosine clearance by astrocytes.
Supported by CNPq and FCT
Conference:
11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009.
Presentation Type:
Poster Presentation
Citation:
Rodrigues
AS,
Matos
MA,
De-Carvalho
PR,
Agostinho
P and
Cunha
RA
(2009). Identification of Adenosine ENT1 Transporters in Astrocytes and
their Modulation by Adenosine A1 Receptors.
Front. Neurosci.
Conference Abstract:
11th Meeting of the Portuguese Society for Neuroscience.
doi: 10.3389/conf.neuro.01.2009.11.117
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Received:
11 Aug 2009;
Published Online:
11 Aug 2009.
*
Correspondence:
Paula Agostinho, University of Coimbra, Center for Neurosciences of Coimbra, Institute of Biochemistry, Faculty of Medicine, Alicante, Portugal, pagost@cnc.cj.uc.pt