Nucleoside transporters in the purinome.

Neurochemistry International xxx (2014) xxx–xxx

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Neurochemistry International journal homepage: www.elsevier.com/locate/nci

Review

Nucleoside transporters in the purinome Alexandre dos Santos-Rodrigues a,1, Natalia Grañé-Boladeras b, Alex Bicket a, Imogen R. Coe a,b,⇑ a b

Department of Biology, Faculty of Science, York University, Toronto, ON, Canada Department of Chemistry and Biology, Faculty of Science, Ryerson University, Toronto, ON, Canada

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Nucleoside transporters Receptors Purinome Regulation Protein kinase C (PKC) Protein kinase A (PKA) Casein kinase II (CKII) miRNAs Phosphorylation

a b s t r a c t The purinome is a rich complex of proteins and cofactors that are involved in fundamental aspects of cellular homeostasis and cellular responses. The purinome is evolutionarily ancient and is made up of thousands of members. Our understanding of the mechanisms linking some parts of this complex network and the physiological relevance of the various connections is well advanced. However, our understanding of other parts of the purinome is less well developed. Our research focuses on the adenosine or nucleoside transporters (NTs), which are members of the membrane purinome. Nucleoside transporters are integral membrane proteins that are responsible for the flux of nucleosides, such as adenosine, and nucleoside analog drugs, used in a variety of anti-cancer, anti-viral and anti-parasite therapies, across cell membranes. Nucleoside transporters form the SLC28 and SLC29 families of solute carriers and the protein members of these families are widely distributed in human tissues including the central nervous system (CNS). NTs modulate purinergic signaling in the CNS primarily through their effects on modulating prevailing adenosine levels inside and outside the cell. By clearing the extracellular milieu of adenosine, NTs can terminate adenosine receptor-dependent signaling and this raises the possibility of regulatory feedback loops that tie together receptor signaling with transporter function. Despite the important role of NTs as modulators of purinergic signaling in the human body, very little is known about the nature or underlying mechanisms of regulation of either the SLC28 or SLC29 families, particularly within the context of the CNS purinome. Here we provide a brief overview of our current understanding of the regulation of members of the SLC29 family and highlight some interesting avenues for future research. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction 1.1. Brief overview of the purinome The purinergic signaling complex of a cell, commonly referred to as the ‘‘purinome’’, is a molecular network of purinergic ligands, receptors, enzymes, channels and transporters (Volonte and D’Ambrosi, 2009). Purinergic signaling is essential in the central nervous system (CNS) and cardiovascular system (CVS), where purinergic ligands, such as ATP and adenosine, act as autocrine and paracrine hormones. Classically, ATP is considered to be the ubiquitous carrier of chemical energy that drives many chemical reactions in the cell. Evidence of ATP as an extracellular signaling molecule was first ⇑ Corresponding author at: Department of Chemistry and Biology, Ryerson University, 350 Victoria St., Toronto, ON M5B 2K3, Canada. Tel.: +1 4169795247. E-mail address: [email protected] (I.R. Coe). 1 Current Address: Program of Neurosciences and Department of Neurobiology, Institute of Biology, Fluminense Federal University, Niterói, RJ, Brazil.

observed by Drury and Szent-Györgyi in 1929, when they demonstrated that extracellular ATP and adenosine had effects on heart rate and cardiovascular function (Drury and Szent-Györgyi, 1929). Later, experiments by Holton (1959) demonstrated that ATP release followed antidromic stimulation of sensory nerves in rabbits. The important role of ATP and adenosine in extracellular signaling was corroborated by Ginsborg and Hirst (1972) when they showed that acetylcholine release could be modulated by adenosine. The concept that ATP could act as a neurotransmitter was later inferred from these and other findings by Burnstock in 1972, and this work defined purinergic signaling and regulation as a new and exciting field of study (Burnstock, 1972). Further research has helped to elucidate the role of ATP and adenosine as players in purinergic signaling, but there is still much that remains unknown regarding the interactions between various proteins. There are thousands of distinct proteins that use purines as cofactors, implying that the purinome is diverse, ubiquitous and essential to cellular function (Haystead, 2006). The purinome is complex and dynamic, and there is a large body of information on the roles of various enzymes and various

http://dx.doi.org/10.1016/j.neuint.2014.03.014 0197-0186/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: dos Santos-Rodrigues, A., et al. Nucleoside transporters in the purinome. Neurochem. Int. (2014), http://dx.doi.org/ 10.1016/j.neuint.2014.03.014

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A. dos Santos-Rodrigues et al. / Neurochemistry International xxx (2014) xxx–xxx

receptors in a wide variety of physiological and pathophysiological contexts (e.g. Burnstock et al., 2011; Coddou et al., 2011; Dale, 2011; Zylka, 2011; Schetinger et al., 2007; Samsel and Dzierzbicka, 2011; Ferrero, 2011; Lane et al., 2011). In contrast, relatively little is known about the members of the purinome that are responsible for the flux of purine nucleosides such as adenosine. The proteins responsible for purine nucleoside flux are the nucleoside transporters. Understanding the role of nucleoside transporters within the larger purinome is important because factors that affect the flux of adenosine (and other purine nucleosides) into and out of cells will have implications on virtually all other aspects of purinergic signaling. Additionally, purinergic signaling is a target of considerable pharmacological interest; therefore understanding the purinome will aid in drug development and drug discovery (Murray and Bussiere, 2009; Knapp et al., 2006), particularly in the CNS and the CVS. Since extracellular concentrations of purinergic ligands can be modulated by both release of ATP and uptake or release of adenosine, a rich regulatory potential for fine-tuning dynamic physiological processes exists in the regulation of nucleoside transporters, and our research focuses on seeking an enhanced understanding of these proteins. 2. Purine nucleoside transporters 2.1. General features of ENTs and CNTs Nucleoside transporter (NT) proteins are encoded by two different gene families. The SLC28 gene family encodes the concentrative nucleoside transporters, which are Na+-dependent symporters and which exist in three isoforms (CNT1–3) in humans. CNTs are predicted to possess 13 transmembrane domains with a cytoplasmatic N-terminus and an extracellular C-terminus, and include putative glycosylation sites (e.g. Kong et al., 2004) (Fig. 1a). The other major family of NTs is the SLC29 gene family, the equilibrative nucleoside transporters, which comprise four isoforms (ENT1–4) in humans and which are Na+-independent passive transporters (Rose and Coe, 2008; Kong et al., 2004; Parkinson et al., 2011). These transporters are also glycosylated, possess 11 transmembrane domains with a cytoplasmatic N-terminus and an extracellular C-terminus. They have a large extracellular loop between transmembrane domains 1 and 2, and a large intracellular loop connecting transmembrane domains 6 and 7 (Sundaram et al., 1998). They have been characterized based on their sensitivity to the nucleoside analog inhibitor, NBTI, with ENT1 being sensitive at nanomolar concentrations, while ENT2 is insensitive at these levels (although sensitive at micromolar concentrations, Parkinson et al., 2011). NBTI interacts with a high affinity binding site through noncovalent interactions on the extracellular side of the protein in the region of transmembrane domains 3–6 (Sundaram et al., 2001a) (Fig. 1b). ENTs and CNTs transport nucleosides but do not possess obvious sequence or structural similarities, nor is there any structural relationship with other transporter families. NTs are evolutionarily ancient membrane proteins (Sankar et al., 2002) and it is likely that the ENTs have evolved as part of the CNS purinome along with other components, since an ENT homolog is clearly involved in purinergic-dependent associative learning in Drosophila (Knight et al., 2010). The ENTs are typically described as being broad spectrum although ENT1 and ENT2 are major contributors to purine nucleoside transport in many tissues. In addition CNT2 and CNT3 transport purine nucleosides, while CNT1 is a pyrimidine-specific transporter. The relative contributions of the ENTs and CNTs to physiological roles of purines continue to be resolved and it is clear the role of nucleoside transporters in the purinome is complex. Many excellent reviews to date have focused on the structure and pharmacological characteristics of both ENTs and CNTs

Fig. 1. The proposed 2D topology of concentrative nucleoside transporters (a) and equilibrative nucleoside transporters (b). CNT structure is predicted to have 13 transmembrane domains as well as an intracellular N- and extracellular glycosylated C-termini. ENT structure contains 11 transmembrane domains as well as an intracellular N- and extracellular C-termini. Both proteins are glycosylated (stick structures) and possess large intracellular loops containing numerous putative phosphorylation consensus sites (not shown).

(Parkinson et al., 2011; Young et al., 2008; Kong et al., 2004). However, we continue to face a gap in our understanding of the role and contribution of NTs to the purinome and to purinergic signaling. In this mini-review, we highlight possible future avenues of research into the regulation of ENTs in the CNS. 3. NTs and other members of the purinome 3.1. Main sources of adenosine and crosstalk between members of the purinome Although all ENTs have been found in the brain (Anderson et al., 1999a,b; Baldwin et al., 2005; Alanko et al., 2006; Dahlin et al., 2007; for a review, see Parkinson et al., 2011), most studies focus on ENT1 and ENT2, since these were the first ENTs to be discovered. ENT3 and ENT4 were described more recently (Kong et al., 2004). Adenosine is a purine nucleoside that is usually described as a neuromodulator, rather than classical neurotransmitter since it has not been shown to be stored in synaptic vesicles. However, some reports suggest that adenosine can be released by similar dynamics to classical neurotransmitter release (Wall and Dale, 2007; Klyuch et al., 2011). Adenosine can also be released from intracellular sites via the ENTs (Wall and Dale, 2013) and possibly via CNT2 (Melani et al., 2012) suggesting that these transporters contribute to both purine nucleoside uptake and to purine release depending on the physiological context. In the CNS, under physiological conditions, ENTs may allow predominantly intracellular to extracellular flux of the purine nucleoside adenosine (for a review, see Latini and Pedata, 2001), particularly in response to glutamate stimulation and in the presence of calcium (Paes-de-Carvalho et al., 2005; Zamzow et al., 2009). In the CVS, ENTs appear to be predominantly responsible for the uptake of extracellular adenosine (i.e. facilitating an extracellular to intracellular flux) and thus



terminating adenosine receptor signaling by clearing the extracellular milieu of the purine nucleoside (Rose and Coe, 2008; Loffler et al., 2007). These findings also highlight the challenges associated with sorting out the relative contribution of the ENTs to overall purinergic signaling. Although ENTs appear to be the major NTs involved in purinergic signaling, recent evidence suggests CNT2 might also play a significant role in the modulation of adenosine levels in the brain based on studies performed in rat models. The group of Pastor-Anglada described how levels of CNT2 mRNA would be modulated by changes in concentration of extracellular adenosine in an experimental model of ictus (Medina-Pulido et al., 2013) as well as by sleep deprivation (Guillén-Gómez et al., 2004), while ENT1 mRNA levels remained unaltered. In addition, the group of Pedata reported the presence of CNT2 in plasma and vesicle membranes isolated from rat striatum supporting the relevance of CNT2 as part of the purinome (Melani et al., 2012). Extracellular adenosine interacts with metabotropic receptors, which are subdivided in four subtypes: A1, A2a, A2b and A3 (Schulte and Fredholm, 2003; Fredholm et al., 2005). A1 and A3 receptors are classically described as being coupled to Gi, which inhibits adenylyl cyclase and leads to a decrease in cAMP levels while A2a and A2b receptors are generally described as being coupled to Gs protein, which increases cAMP levels (Schulte and Fredholm, 2003; Fredholm et al., 2005). There is a long-standing interest in the nature and physiological relevance of the relationship between adenosine receptors and adenosine transporters. Studies support the concept of an interaction between CNTs and adenosine receptors (Duflot et al., 2004; Medina-Pulido et al., 2013) however the nature (if any) of the relationship between ENTs and adenosine receptors remains unknown although we anticipate a role for protein–protein interactions in the regulation of ENTs within the purinome. It is well established that the extracellular concentrations of adenosine can be modulated by manipulation or inhibition of NTs, which, in turn, can impact adenosine receptor response leading to myriad effects. Extracellular levels of adenosine can also be modulated by degradation/conversion by ecto-enzymes of adenine nucleotides found in synaptic cleft (Dunwiddie et al., 1997; Latini and Pedata, 2001). For instance, in hippocampal slices, there are variations in the source of adenosine production (Cunha et al., 1996) according to the type of electrical stimulus (high or low frequency). Under low frequency stimuli, adenosine is released to the extracellular space via nucleoside transporters, while at high frequency adenosine is generated mainly from the cleavage of released adenine nucleotides. More recently it was demonstrated that there is a similar contribution to the extracellular pool of adenosine from neurons and astrocytes in hippocampal slices (Wall and Dale, 2013), although adenosine release mediated by neurons occurs through ENTs, while adenosine from astrocytes is derived from the breakdown of adenines nucleotides. Regardless of the source of the extracellular adenosine, the activation of adenosine receptors leads to complex downstream responses and the intracellular relationship between adenosine receptor signaling and regulation of adenosine transporters still remains unclear. Intriguingly, there are also proposed intracellular actions of adenosine and other purines such as inosine and guanosine, which, for instance, can have significant roles in axon outgrowth in an uptake-dependent manner (Benowitz et al., 1998). Inhibition of purine uptake using the classic equilibrative nucleoside inhibitor, NBTI, prevents axon outgrowth in retinal ganglion cells although the underlying mechanisms responsible for this effect remain unknown (Benowitz et al., 1998). One possibility would be that such effects could be mediated by ‘‘P’’ sites, which are adenosinereactive intracellular sites associated with adenylyl cyclase (Londos and Wolff, 1977). Effects proposed to be due to ‘‘P’’ sites

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are blocked by adenosine transport inhibitors (Johnson and Shoshani, 1990; Dessauer and Gilman, 1997), but, again, the underlying mechanisms remain unclear. While mechanisms of interaction and reciprocal regulation between NTs and adenosine receptors are not clearly defined, there is evidence for a close association between ENT1 and A1 receptors in the CNS. For instance, autoradiographic studies demonstrated the presence of [3H] NBTI binding sites primarily in plexiform layers of chick retina, with a localization similar to that of A1 receptors (de Carvalho et al., 1992). Similarly, Jennings and colleagues (2001) demonstrated a co-localization between ENT1 sites and A1 receptors in different brain structures in humans suggesting that ENT1 may play an important role in regulating the neuromodulatory action of A1 receptors. This neuromodulatory role was confirmed in vitro by studies in slices of rat spinal cord (Ackley et al., 2003), where ENT1 sites were localized with A1 receptors. Moreover, inhibition of ENT1-dependent adenosine uptake contributed to an increase of adenosine extracellular levels, which led to an A1 receptor-dependent decrease in glutamatergic synaptic transmission clearly demonstrating an important role of ENT1 in the purinome of the CNS. Although there are currently no data to support a physical interaction between adenosine receptors and NTs, a complex of this kind has been described between A3 receptors and serotonin transporters (SERT) (Zhu et al., 2011), such that activation of A3 receptors can change the levels of these complexes and SERT activity (Zhu et al., 2004, 2007). Other key elements of the purinome are the enzymes, adenosine kinase (ADK) responsible for the production of AMP from adenosine, and adenosine deaminase (ADA), which generates inosine from adenosine. These enzymes are responsible for adenosine metabolism in brain (Latini and Pedata, 2001). The metabolic pathway mediated by ADA is mainly cytosolic although its expression appears to be closely associated with ENT1 sites in rat brain (Nagy et al., 1985). The ecto-enzyme form of ADA (ectoADA) appears to interact with A1 receptors in rat cortical cultured neuron (Ruiz et al., 2000), which suggests that ectoADA is involved in modulation of the A1 receptor-dependent signaling. Moreover, inhibition of ADK induces an increase in extracellular levels of adenosine in hippocampal slices which is followed by a decrease of synaptic transmission in the hippocampal CA1 area (Pak et al., 1994) confirming the role of adenosine as an important modulator of neural activity. Taken together, there is a growing body of evidence to suggest that NTs, and particularly ENT1, are closely associated, both physically and mechanistically, with the adenosine receptors, and possibly enzymes of the purinome, to modulate purinergic signaling in the CNS. 4. Mechanisms of regulation of ENTs 4.1. Regulation of ENTs at the level of the gene Current data suggest that ENTs can be regulated at various locations and through various processes within the cell (Fig. 2). ENT1 is the nucleoside transporter for which transcriptional regulation is best understood, although still at relatively simple level. Based on consensus sequences, transcriptional regulation of ENT1 could occur as the consequence of binding of a number of transcription factors including, but not limited to, CREB, GATA-1, and Sp-1 or MAZ, and Sp-1 for mENT1 and hENT1 respectively (Choi et al., 2000; Abdulla and Coe, 2007). However, the physiological significance of these putative transcriptional regulators remains unclear. More recent studies suggest that the JNK-cJun pathway negatively regulates mENT1 expression (Leisewitz et al., 2011), and that PPARa and c activation, or overexpression, results in increased hENT1-dependent transport (Montero et al., 2012), suggesting


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feedback loops that link signaling pathways, cellular homeostasis and NT gene expression. Our current understanding of the connections and the physiological consequences of altered gene expression of the NTs remains rudimentary. The exception is in our relative well-developed understanding of the relationship between adenosine signaling and ENT expression following ischemia or hypoxia in the cardiovascular system (e.g. Chaudary et al., 2004a,b; Rose et al., 2010). It is likely that similar mechanisms exist in the neurovasculature, since adenosine is critically important in cellular responses leading to both neuro- and cardioprotection. In the brain and the heart, ENT1 and ENT2 promoters are down-regulated by hypoxia inducible factor 1 (HIF-1), (Chu et al., 2013; Eltzschig et al., 2005; Morote-Garcia et al., 2009) and this regulation, which decreases the number of ENT proteins at the plasma membrane, results in increased extracellular adenosine, which promotes an adenosine receptor-dependent protective effect (Chaudary et al., 2004a,b; Eltzschig et al., 2005; Loffler et al., 2007). Loss of ENT1 leads to an inherently cardioprotected phenotype (Rose et al., 2010) clearly demonstrating the importance of ENT1 regulation in cellular homeostasis of the heart. While regulation of ENT gene expression has been clearly defined in the CVS, there is evidence that ENTs are regulated at the level of expression in other physiological situations. In the placenta, ENT expression is differentially regulated. High-glucose levels, along with subsequent increased NO production, reduce SLC29A1 promoter activity via a MAP kinase dependent pathway, while regulation of the ENT2 gene is via an NO-independent mechanism (Munoz et al., 2006; Puebla et al., 2008; Farias et al., 2010; Westermeier et al., 2011; Salomon et al., 2012). Intriguingly, we have also identified a number of splice variants for ENT2, which may potentially act in a feedback loop that regulates the presence (and thus function) of the full length, wild-type ENT2 protein (Grañé-Boladeras, 2012). Clearly, while transcriptional regulation of the NTs plays a part in their larger role in the purinome, many connections and mechanisms remain to be determined.

4.2. Regulation of ENTs at the protein level The first equilibrative nucleoside transporter to be cloned and characterized was hENT1 (Griffiths et al., 1997). Before cloning, the transport activity was ascribed to an es transporter (equilibrative, sensitive), based on sensitivity to inhibition by nitrobenzylthioinosine (NBTI). Nagy and colleagues (1990) demonstrated that an acute exposure of cultured cells (including those from the CNS) to ethanol led to decreased adenosine uptake and that this effect was primarily due to effects on the es transporter. Moreover, this ethanol sensitivity was PKA- and PKC-dependent suggesting that es (subsequently confirmed to be ENT1) was subject to post-translational regulation by kinases (Nagy et al., 1991; Coe et al., 1996a,b). Intriguingly, other studies (Delicado et al., 1991) showed that PKC activation induced a decrease of es transporters in cultured chromaffin cells, which led to a reduction of adenosine uptake. Similar effects on adenosine uptake by PKA and PKC were also observed in single chromaffin and neuroblastoma cells (Sen et al., 1998, 1999). PKC activation can also increase hENT1dependent nucleoside flux (Coe et al., 2002) in certain cells and this may occur via activation of adenosine receptors and the MAP kinase pathway (Grden et al., 2008). In hippocampal nerve terminals, A2a receptor activation induced an increase of adenosine uptake, in a PKC-dependent manner (Pinto-Duarte et al., 2005), which is interesting since this is a non-classical A2a pathway. Other studies have demonstrated that inhibition of adenosine transport by glucose is dependent on PKC, nitric oxide and ERKs in human fetal endothelial cells (Montecinos et al., 2000) and in B-lymphocytes (Sakowicz et al., 2005). In chick retina, inhibition of ERKs was also able to decrease adenosine uptake (dos SantosRodrigues et al., 2011) although the underlying mechanism is not clear. TGF-b1 is also able to negatively modulate ENT1 protein levels in a nitric oxide-dependent pathway (Vega et al., 2009). Taking all the current data to date together, three kinases seem to play a broad role in ENT1 regulation: CKII, PKA and PKC. Putative

Fig. 2. Cartoon representing putative regulatory mechanisms, interactions and pathways for NTs in the CNS. Regulation of NTs in the CNS may occur via crosstalk and PPI between NTs, receptors and other proteins, via transcriptional regulation (through promoter activity or miRNA interactions), via post-translational regulation (through de/ phosphorylation, glycosylation) and via regulated localization (mediated endocytosis, recycling).


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CKII phosphorylation sites have been predicted in both ENT1 and ENT2 sequences (Kiss et al., 2000; Stolk et al., 2005; Robillard et al., 2008; Hammond et al., 2004) and a mENT1 splice variant (mENT1b) is predicted to have a potential CKII phosphorylation site at Ser254, within the large intracellular loop located between TM 6 and 7 (Kiss et al., 2000; Handa et al., 2001). Further studies correlated CKII phosphorylation with an increased presence of the ENTs at the membrane plus an increase in substrate translocation and altered inhibitor sensitivity (Bone et al., 2007). However, the underlying mechanism of the CKII regulation remains unclear. While our understanding of the regulatory pathways and physiological relevance of kinase-dependent regulation of ENTs continue to be obscure, we now know that ENT1 can be directly phosphorylated, in vitro, within large intracellular loop between transmembrane domains 6 and 7 by both PKC and PKA, showing that this is a potential mechanism of regulation of ENTs by kinase-dependent pathways (Reyes et al., 2011). Less is known about the post-translational regulation of the other ENTs or CNTs, although mENT2 possesses potential PKC phosphorylation sites in the first and third intracellular loops (Kiss et al., 2000). Based on analysis by NetPhosK (Blom et al., 2004), human ENT2 has putative PKC, PKA and CKII phosphorylation sites, some of which are highly conserved among species, suggesting that direct phosphorylation may be a mechanism of regulation of this isoform. Lu and colleagues (2010), showed that adenosine accumulation, due to chronic morphine treatment, was attributable to the alteration of adenosine uptake via ENT2, and changes in PKC activity were correlated with the attenuation of ENT2 function resulting in an accumulation of extracellular adenosine (Lu et al., 2010) thus strengthening a mechanistic link between PKC signaling and ENT2 function. In addition to phosphorylation, glycosylation of ENTs appears to regulate their function. ENT1 and ENT2 possess N-linked glycosylation (confirmed by mutational assays) and glycosylation appears to be involved in nucleoside transport, structure and/or inhibitor binding affinity, for ENT1 structure (Vickers et al., 1999; Sundaram et al., 2001b; Ward et al., 2003), while absence of glycosylation of ENT2 led to disrupted plasma membrane localization, suggesting that glycosylation is required for proper targeting and/or trafficking (Ward et al., 2003). The extent to which these post-translational modifications play a role in the function of ENTs in the CNS remains to be determined.

4.3. Future perspectives in the regulation of ENTs: regulation by microRNAs and PPI While transcriptional regulation via promoter of ENTs continues to be an area of interest, we have recently identified the microRNAs (miRNAs) as potential key regulators of NTs. miRNAs are a class of non-coding RNA consisting of 21–24 nucleotides, which work through mediation of post-transcriptional gene silencing by controlling the translation of mRNA into proteins. There are over one thousand miRNAs already described (Kozomara and Griffiths-Jones, 2011). Mature miRNAs, when present in the cytoplasm, interact with Argonaute to form miRNA-induced silencing complexes (miRISCs) which target mRNAs in the 30 -UTR for two types of regulation: mRNA degradation or inhibition of mRNA translation (Pasquinelli, 2012). Bioinformatic tools, such as TargetScan, PicTar, miRanda, predict miRNA targets, which can then direct in vitro experimentation to confirm real interactions between putative miRNA targets (such as an NT) and a specific miRNA. There are a number of reports describing the regulation of membrane transporters by miRNAs, including GLUT3 (Fei et al., 2012), GLUT4 (Lu et al., 2010) and GLT-1 (Morel et al., 2013), the latter of which occurs within the CNS.

There are currently limited data to suggest miRNA regulation of purinergic systems although the importance of their potential role was recently noted (see Volonte et al., 2012). Our preliminary analyses suggest that NTs are indeed targets of miRNA regulation (dos Santos-Rodrigues et al., 2013). Table 1 shows a list of potential miRNAs that could be interacting with human and mouse ENT1 and ENT2 based on in silico analyses using the database TargetScan (http://www.targetscan.org/vert_61/). We report findings from this database because it discriminates between conserved and poorly conserved families of miRNAs. A combined search of a number of databases is possible using other analysis tools (e.g. miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/ predictedmirnagene.html) although the level of rigor in terms of conservation appears to vary considerably between analysis tools. The data we present in Table 1 were confirmed in at least 4 out of 5 databases (Diana-mT, miRanda, PITA, miRWalk and TargetScan), except for mouse ENT2, which is not indexed in the miRWalk database and the output represents sites that are identified as being conserved for miRNA families broadly conserved among vertebrates. Interestingly, based on our TargetScan analysis, potential miRNA target sites are not completely conserved between species although, intriguingly, there is conservation of six specific miRNAs for the ENT2 homologs. Moreover, there are many more potential miRNAs identified for ENT2 than ENT1, possibly suggesting that ENT2 is highly regulated by miRNAs. Confirmation of miRNAs involved in regulating NTs and the physiological relevance of this regulation remains completely unknown. This is an area of research that is currently almost completely unexplored. Regulation via protein–protein interactions (PPIs) is another form of regulation that we are just beginning to understand. It is likely that NTs form complexes with a variety of proteins to regulate location or function although the identity of partners and nature of the interaction remains unclear for most NTs. Precedent for PPIs between transporters and receptors (in the form of heterodimers) in the CNS has been reported (Genedani et al., 2005) and we can speculate that complex formation between members of the purinome (such as receptors and transporters, or transporters and metabolic enzymes) is likely. Thus, post-translational modifications may be a regulatory mechanism that controls PPIs to modify function or location. A major goal of future research should be aimed at connecting purinergic signaling with intracellular pathways that regulate or modulate NT function – thus building a deeper understanding of the network of receptors, kinases and transporters that comprise the purinome.

Table 1 Potential regulatory miRNAs of h/mENT1 and h/mENT2 based on analysis of 30 UTRs using the target prediction database TargetScan (http://www.targetscan.org/). miRNA miR-17 miR-20a miR-20b miR-34a miR-34b-5p miR-34c miR-93 miR-106a miR-106b miR-124 miR-141 miR-200a miR-449a miR-449b miR-449c miR-506 miR-519d

hENT1

hENT2

mENT1

U U U

mENT2 U U U

U U U U U U U

U U U U

U U U U U U U


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5. ENTs in disease and dysfunction A number of studies have demonstrated that ENTs are present in, or correlated with, disease and dysfunction, in a variety of tissues including in the CNS. While there are no known human diseases due to a mutation in hENT1 or hENT2, mutations in hENT3, result in a spectrum of disorders including H syndrome (HuberRuano et al., 2012; Molho-Pessach et al., 2008), PHID (Pigmented Hypertrichosis with Insulin dependent Diabetes mellitus) syndrome (Cliffe et al., 2009) and syndromic forms of histiocytosis (Faisalabad histiocytosis and familial RDD/SHML (fRDD)) (Morgan et al., 2010). Also, ENT3 null mice present a type of progressive macrophage-dominated histiocytosis (Hsu et al., 2012). The lack of any apparent disease or syndrome related to mutations in ENT1 and ENT2 might suggest that these two NTs are embryonic lethal if mutated or absent. Surprisingly, this is not the case and mice in which ENT1 has been knocked out are viable (Choi et al., 2004). However, subsequent work has confirmed that ENT1 null mice exhibit significant cognitive dysfunctions such as anxiety-like behavior, decreased ethanol intoxication and increased alcohol drinking (Choi et al., 2004; Chen et al., 2007; Ruby et al., 2011). These effects are likely to be due to disruptions in the purinergic signaling given the critical role adenosine plays in the brain particularly in response to ethanol. Many studies over more than 20 years show a strong link between ENT1 (or es) and ethanol. The loss of ENT1 results in alterations in A1 adenosine receptordependent signaling, which has been shown to be involved in both ethanol effects and anxiety behaviors. Thus, in wild-type mice, ethanol inhibition of adenosine uptake via ENT1 leads to activation of A1 receptors, which contributes to the intoxicating and addictive effects of ethanol (Choi et al., 2004). Some of the effects related with increased consumption of alcohol seem to be mediated by glutamatergic signaling (via NMDA receptors) in the nucleus accumbens (NAc), which can be regulated by adenosine (Chen et al., 2010; Nam et al., 2011). More recently, it was also shown that these behaviors are regulated by decreased adenosine mediated A2a receptor signaling in the dorsomedial striatum (Nam et al., 2013). Additionally, ENT1 overexpressing mice are more sensitive to ethanol than wild type (Kost et al., 2011). While these studies are almost exclusively murine, it is likely that similar mechanisms exist in humans, since polymorphisms in ENT1 are associated with alcohol abuse phenotype in women (Kim et al., 2011) and alcoholism with a history of withdrawal seizures (Gass et al., 2010). There is a substantial body of data to suggest a role of ENT1 in the effective functioning of the purinome of the brain but interestingly, it also appears to be important in biomineralization since ENT1 knockout mice develop features related to diffuse idiopathic skeletal hyperostosis (DISH) (Warraich et al., 2013). DISH is a pathology characterized by ectopic calcification of spinal tissues which can lead to pain and stiffness in the spine and which affects middle-aged and elderly people. This novel finding implicating ENT1 in biomineralization opens a new avenue for research in the role of the NTs in the purinome. The absence of ENT1 in null mice raises extracellular circulating adenosine that can disrupt purinergic signaling in a number of physiological contexts. In the heart, this effect promotes responses that are cardio-protective (Rose et al., 2010) and explains the basis for the long-standing clinical use of drugs that interact and block NTs to enhance beneficial purinergic responses (Szentmiklosi et al., 2011). Inhibition of ENT1 and ENT2 in the CVS by drugs such as dipyridamole (Persantin™), results in increased levels of extracellular adenosine, potentiating adenosine-receptor signaling pathways that are cardioprotective (Rose and Coe, 2008; Loffler et al., 2007). However, since individual patient NT profiles in the

CVS are highly variable (Marvi et al., 2010), the consequence of unique individual profiles on drug responses remains unclear. There are a number of pathophysiological situations where changes in ENT expression or protein levels appear to be correlated with a disease state but where the mechanisms and significance are unknown. For instance, intestinal tissue taken from patients with inflammatory bowel disease (IBD) shows hENT expression levels that are significantly higher in IBD patients than in healthy tissue, which implies a dysregulation of hENT expression levels due to disease (Wojtal et al., 2009). In Alzheimer Disease (AD), ENT1 protein levels are reduced to less than 25% of normal in red blood cell membranes from Alzheimer patients (Mohanty et al., 2010), possibly indicating a compensatory response to oxidative stress in these individuals, since hypoxia leads to decreased expression of ENT1, which results in amplified adenosinergic signaling to promote cellular survival (Eltzschig et al., 2005). The role of ENTs in these diseases remains to be clarified. Recently, a new drug (T1–11) was developed which acts at two targets: as A2a adenosine receptors agonist and as an inhibitor of ENT1. This compound is being studied for its efficacy in the treatment of Huntington’s disease in animal models (Huang et al., 2011) and once again, shows the role of the ENTs as contributors to the purinome. Although there is a body of data on NTs describing their roles in determining efficacy of a range of drugs in the treatment of a variety of diseases (e.g. Cano-Soldado and Pastor-Anglada, 2012; Molina-Arcas and Pastor-Anglada, 2010), there remain many unknown aspects of the basic cell biology of the NTs, which limit our ability to maximize their roles and potential as drug portals or drugs targets. Concluding remarks The purine transporters of the purinome are still not well understood despite their critically important roles as routes of efflux and uptake of purinergic ligands. Purinergic signaling is physiologically essential in many cellular settings including particularly the cardiovasculature and brain, and understanding aspects of the basic cell biology of the NTs in purinergic signaling is therefore clinically relevant. Future research is aimed at significantly enhancing our understanding of these important members of the purinome. Acknowledgements ASR was a recipient of a postdoctoral fellowship from CAPES, Brazil. Support for work described in this review by IRC has been provided over the years by the Natural Science and Engineering Council, Canada (NSERC) and the Canadian Institutes of Health Research (CIHR). References Abdulla, P., Coe, I.R., 2007. Characterization and functional analysis of the promoter for the human equilibrative nucleoside transporter gene, hENT1. Nucleosides, Nucleotides Nucleic Acids 26, 99–110. Ackley, M.A., Governo, R.J., Cass, C.E., Young, J.D., Baldwin, S.A., King, A.E., 2003. Control of glutamatergic neurotransmission in the rat spinal dorsal horn by the nucleoside transporter ENT1. J. Physiol. 548, 507–517. Alanko, L., Porkka-Heiskanen, T., Soinila, S., 2006. Localization of equilibrative nucleoside transporters in the rat brain. J. Chem. Neuroanat. 31, 162–168. Anderson, C.M., Baldwin, S.A., Young, J.D., Cass, C.E., Parkinson, F.E., 1999a. Distribution of mRNA encoding a nitrobenzylthioinosine-insensitive nucleoside transporter (ENT2) in rat brain. Brain Res. Mol. Brain Res. 70, 293– 297. Anderson, C.M., Xiong, W., Geiger, J.D., Young, J.D., Cass, C.E., Baldwin, S.A., Parkinson, F.E., 1999b. Distribution of equilibrative, nitrobenzylthioinosinesensitive nucleoside transporters (ENT1) in brain. J. Neurochem. 73, 867–873.


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Nucleoside transporters in human placenta.

Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters.

A Novel and Fast Purification Method for Nucleoside Transporters.

Effects of phorbol esters and secretagogues on nitrobenzylthioinosine binding to nucleoside transporters and nucleoside uptake in cultured chromaffin cells.

The Role of Flexible Loops in Folding, Trafficking and Activity of Equilibrative Nucleoside Transporters.

Dilazep analogues for the study of equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2).

Nucleoside transporters and human organic cation transporter 1 determine the cellular handling of DNA-methyltransferase inhibitors.

Erlotinib, gefitinib, and vandetanib inhibit human nucleoside transporters and protect cancer cells from gemcitabine cytotoxicity.

Clearance of rapid adenosine release is regulated by nucleoside transporters and metabolism.

Equilibrative Nucleoside Transporters 1 and 4: Which One Is a Better Target for Cardioprotection Against Ischemia-Reperfusion Injury?

Glucose and nucleoside transporters of human erythrocytes: effects of detergents on immunoadsorption of a membrane protein to its monoclonal antibody.

TGF-β-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3.

The evolution of silicon transporters in diatoms.

Alpha-carboxy nucleoside phosphonates as universal nucleoside triphosphate mimics.

Multicomponent reactions in nucleoside chemistry.

Treatment of HIV infection: the antiretroviral nucleoside analogues. Nucleoside analogues: monotherapy.

Transporters in plant sulfur metabolism.

Ion transporters in brain tumors.

Treatment of HIV infection: the antiretroviral nucleoside analogues. Nucleoside analogues: combination therapy.

Multidrug resistance in MCF-7 human breast cancer cells is associated with increased expression of nucleoside transporters and altered uptake of adenosine.

How do glutamate transporters function as transporters and ion channels?

The nucleoside uridine isolated in the gas phase.

Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters.

Sodium-dependent bile salt transporters of the SLC10A transporter family: more than solute transporters.