Distribution of Equilibrative, Nitrobenzylthioinosine‐Sensitive Nucleoside Transporters (ENT1) in Brain

Abstract

Nucleoside transport processes may play a role in regulating endogenous levels of the inhibitory neuromodulator adenosine in brain. The cDNAs encoding species homologues of one member of the equilibrative nucleoside transporter (ENT) gene family have recently been isolated from rat (rENT1) and human (hENT1) tissues. The current study used RT-PCR, northern blot, in situ hybridization, and [ 3 H]nitrobenzylthioinosine autoradiography to determine the distribution of mRNA and protein for ENT1 in rat and human brain. Northern blot analysis indicated that hENT1 mRNA is widely distributed in adult human brain. 35 Slabeled sense and antisense riboprobes, transcribed from a 153-bp segment of rENT1, were hybridized to fresh frozen coronal sections from adult rat brain and revealed widespread rENT1 mRNA in pyramidal neurons of the hippocampus, granule neurons of the dentate gyrus, Purkinje and granule neurons of the cerebellum, and cortical and striatal neurons. Regional localization in rat brain was confirmed by RT-PCR. Thus, ENT1 mRNA has a wide cellular and regional distribution in brain, indicating that this nucleoside transporter subtype may be important in regulating intra-and extracellular levels of adenosine in brain. Key Words: Adenosine-Adenosine transport-Nucleoside transport-ENT1-es nucleoside transport-In situ hybridization-RT-PCR-Nitrobenzylthioinosine. J. Neurochem. 73, 867-873 (1999).Several CNS effects, including decreased alertness following a prolonged wakeful state (Porkka-Heiskanen et al., 1997) and neuroprotection following stroke or seizure (von Lubitz et al., 1997), have been described for the purine nucleoside adenosine. These effects are mediated by adenosine receptors, of which four subtypes have been identified and are termed A 1 , A 2A , A 2B , and A 3 . Stimulation of adenosine A 1 receptors stabilizes membrane potentials and decreases excitatory neurotransmitter release. Through these mechanisms, adenosine can limit toxic accumulation of Ca 2ϩ during ischemia and prevent cellular damage (Rudolphi et al., 1992). Adenosine A 1 receptors may also mediate ethanolinduced motor incoordination in cerebellum (Dar, 1997), contribute to synaptic plasticity in hippocampus (de Mendonça and Ribeiro, 1997), and produce antinociception in spinal cord (Sollevi, 1997;Sawynok, 1998). Stimulation of adenosine A 2A receptors has direct vasodilatory effects on cerebral vessels (Winn et al., 1981) and inhibits neutrophil and platelet activities (Miller and Hsu, 1992), leading to improved blood flow during times of reduced ATP synthesis (von Lubitz et al., 1997). Excitatory actions have also been described for A 2A receptors in brain (Sebastião and Ribeiro, 1996), as have antagonistic interactions with dopamine D 2 receptors in the basal ganglia (Ferré et al., 1997).Nucleoside transporters salvage extracellular nucleosides for intracellular synthesis of nucleotides. Nucleoside transport processes may be of further importance as regulators of endogenous adenosine levels and adenosine receptor stimulation....

Homo‐ and heteroexchange of adenine nucleotides and nucleosides in rat hippocampal slices by the nucleoside transport system

“…Therefore, ENT1 equilibrative nucleoside transporter could be involved in this effect, which is in line with the observation that the effect of ATP was temperature dependent. This finding corroborates with the distribution of this transporter in the central nervous system, including the hippocampus, and its role in the transmembrane carriage of extracellular nucleosides (Glass et al, 1996;Anderson et al, 1999). The observation that adenosine was more efficient than uridine in eliciting [ H]purine efflux in the hippocampus.…”

“…Yet another possibility is that ATP is secreted by CD39 protein, recently recognized as an ATP-transporting protein (Bodas et al, 2000). As the ENT1 nucleoside transporters are distributed on nerve terminals and also on astrocytes and endothelial cells (Anderson et al, 1999), the cellular source of ATP-evoked purine efflux could be either nerve terminals, glial cells or endothelial cells. Since no similar ATP-or ADP-induced purine accumulation was observed in isolated hippocampal nerve terminals (Cunha, 2001b), it seems likely that nonneuronal elements expressing the ENT1 transporter provide a major contribution to this effect.…”

Ecto-Nucleotidases and Nucleoside Transporters Mediate Activation of Adenosine Receptors on Hippocampal Mossy Fibers by P2X7Receptor Agonist 2′-3′-O-(4-Benzoylbenzoyl)-ATP

“…Cellular release and uptake of adenosine in the brain are mediated by several types of membranous nucleoside transporters (Hyde et al, 2001;Lee et al, 2001;Baldwin et al, 2004). Recent work has shown that NTrans are expressed and functional in rodent hippocampus (Anderson et al, 1999;Sperlagh et al, 2003). Indeed, challenging NTrans with inosine (Yao et al, 1997;Li et al, 2001) reversed the outward current induced by BzATP (Fig.…”

Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity

“…First, microarray data from FACSpurified neurons show that both developing (P7 and P16) and adult (older than P20) neurons express ENT1 and ENT2 (43,44). Second, in situ hybridization studies in rat brain tissue demonstrate that cortical tissue and the hippocampal CA1 region express ENT1 (45,46). We used this information to manipulate adenosine release from CA1 neurons by administering inosine intracellularly as a competitive substrate to adenosine.…”