Distribution of mRNA encoding a nitrobenzylthioinosine-insensitive nucleoside transporter (ENT2) in rat brain

doi:10.1016/S0169-328X(99)00164-3

Molecular Brain Research

Volume 70, Issue 2, 5 July 1999, Pages 293-297

https://doi.org/10.1016/S0169-328X(99)00164-3 Get rights and content

Abstract

Nucleoside transporters may play a role in regulating levels of extracellular adenosine and adenosine receptor activity. Two members of the equilibrative nucleoside transporter family have recently been cloned. ENT1 is potently inhibited by nitrobenzylthioinosine (NBMPR) (K_i≈1 nM) and was previously found to have a wide distribution in rat and human brain. ENT2 is insensitive to inhibition by NBMPR at low nanomolar concentrations and there is limited information describing its distribution in rat brain. The present study used RT-PCR, northern blot and in situ hybridization and detected rENT2 transcript in several brain regions including hippocampus, cortex, striatum and cerebellum. Our results indicate a wide cellular and regional distribution for ENT2 in rat brain, similar to ENT1, indicating that control of adenosine levels in brain is achieved by multiple transport processes.

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Acknowledgements

Research was supported by the Medical Research Council of Canada. F.E.P. is a scholar of the Medical Research Council of Canada. C.E.C. is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada. J.D.Y. is a Heritage Medical Scientist of the Alberta Heritage Foundation for Medical Research. C.M.A. was a trainee of the Heart and Stroke Foundation of Canada.

References (12)

C.M. Anderson et al.
Demonstration of the existence of mRNAs encoding N1/cif and N2/cit sodium/nucleoside cotransporters in rat brain
Mol. Brain Res.
(1996)
C.R. Crawford et al.
Cloning of the human equlibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line
J. Biol. Chem.
(1998)
S.Y.M. Yao et al.
Molecular cloning and functional characterization of nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative nucleoside transporter proteins (rENT1 and rENT2) from rat tissues
J. Biol. Chem.
(1997)
C.M. Anderson, W. Xiong, J.D. Geiger, J.D. Young, C.E. Cass, S.A. Baldwin, F.E. Parkinson, Distribution of...
J.D. Geiger, D.M. Fyda, Adenosine transport in nervous system tissues, in: T.L. Stone (Ed.), Adenosine in the Nervous...
M. Griffiths et al.
Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs
Nature Med.
(1997)

There are more references available in the full text version of this article.

Cited by (64)

Deletion of equilibrative nucleoside transporter 2 disturbs energy metabolism and exacerbates disease progression in an experimental model of Huntington's disease
2023, Neurobiology of Disease
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease, characterized by motor dysfunction and abnormal energy metabolism. Equilibrative nucleoside transporter 1 (ENT1) and ENT2 are the major nucleoside transporters in cellular plasma membrane of the brain. Yet, unlike ENT1 whose function has been better investigated in HD, the role of ENT2 in HD remains unclear. The present study aimed to investigate the impacts of ENT2 deletion on HD using a well-characterized mouse model (R6/2). Microarray analysis, quantitative real-time polymerase chain reaction, and immunostaining of ENT2 in postmortem human brain tissues were conducted. R6/2 mice with or without genetic deletion of ENT2 were generated. Motor functions, including rotarod performance and limb-clasping test, were examined at the age of 7 to 12 weeks. Biochemical changes were evaluated by immunofluorescence staining and immunoblotting at the age of 12 to 13 weeks. In regard to energy metabolism, levels of striatal metabolites were determined by liquid chromatography coupled with the fluorescence detector or quadrupole time-of-flight mass spectrometer. Mitochondrial bioenergetics was assessed by the Seahorse assay. The results showed that ENT2 protein was detected in the neurons and astrocytes of human brains and the levels in the postmortem brain tended to be higher in patients with HD. In mice, ENT2 deletion did not alter the phenotype of the non-HD controls. Yet, ENT2 deletion deteriorated motor function and increased the number of aggregated mutant huntingtin in the striatum of R6/2 mice. Notably, disturbed energy metabolism with decreased ATP level and increased AMP/ ATP ratio was observed in R6/2-Ent2^−/− mice, compared with R6/2-Ent2^+/+ mice, resulting in the activation of AMPK in the late disease stage. Furthermore, ENT2 deletion reduced the NAD⁺/NADH ratio and impaired mitochondrial respiration in the striatum of R6/2 mice. Taken together, these findings indicate the crucial role of ENT2 in energy homeostasis, in which ENT2 deletion further impairs mitochondrial bioenergetics and deteriorates motor function in R6/2 mice.
Of adenosine and the blues: The adenosinergic system in the pathophysiology and treatment of major depressive disorder
2021, Pharmacological Research
Citation Excerpt :
However, it must be noted that studies using in situ hybridization have found evidence for marked ENT1 mRNA expression in both the hippocampus and the cerebellum of rats [83]. ENT2 expression extensively overlaps that of ENT1 [84], with mRNA expression being observed in cortical, striatal, thalamic, hippocampal, and cerebellar neurons [85]. Unlike ENTs 1 and 2, ENT3 is primarily located in the intracellular space, having an especially relevant role in lysosomal functioning [86].
Major depressive disorder (MDD) is the foremost cause of global disability, being responsible for enormous personal, societal, and economical costs. Importantly, existing pharmacological treatments for MDD are partially or totally ineffective in a large segment of patients. As such, the search for novel antidepressant drug targets, anchored on a clear understanding of the etiological and pathophysiological mechanisms underpinning MDD, becomes of the utmost importance.
The adenosinergic system, a highly conserved neuromodulatory system, appears as a promising novel target, given both its regulatory actions over many MDD-affected systems and processes. With this goal in mind, we herein review the evidence concerning the role of adenosine as a potential player in pathophysiology and treatment of MDD, combining data from both human and animal studies.
Altogether, evidence supports the assertions that the adenosinergic system is altered in both MDD patients and animal models, and that drugs targeting this system have considerable potential as putative antidepressants. Furthermore, evidence also suggests that modifications in adenosine signaling may have a key role in the effects of several pharmacological and non-pharmacological antidepressant treatments with demonstrated efficacy, such as electroconvulsive shock, sleep deprivation, and deep brain stimulation. Lastly, it becomes clear from the available literature that there is yet much to study regarding the role of the adenosinergic system in the pathophysiology and treatment of MDD, and we suggest several avenues of research that are likely to prove fruitful.
Nucleoside transporters in the purinome
2014, Neurochemistry International
Citation Excerpt :
In this mini-review, we highlight possible future avenues of research into the regulation of ENTs in the CNS. 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).
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.
Functional expression of drug transporters in glial cells: Potential role on drug delivery to the CNS
2014, Advances in Pharmacology
Drug permeability in the central nervous system (CNS) across blood–brain and blood–cerebrospinal fluid barriers is an important determinant of neurological disorders therapeutic efficacy and is highly regulated by the expression of membrane-associated transporters belonging to the ATP-binding cassette (ABC) and solute carrier (SLC) superfamilies. Functional expression of ABC efflux transporters exists not only at the brain barriers (primary biochemical barrier) but also in astrocytes, microglia, neurons, and oligodendrocytes can significantly restrict drug penetration into these cells, thus creating a secondary biochemical barrier to permeability in brain parenchyma. In contrast, SLC members primarily contribute to the uptake of endogenous substrates (i.e., hormones, neurotransmitters) and pharmacological agents and can play a critical role in maintaining CNS homeostasis and drug response. In this chapter, we review the functional expression and localization of drug transporters in the brain, their role in CNS drug delivery, and their regulation in neuropathological conditions.
Differential adenosine uptake in mixed neuronal/glial or purified glial cultures of avian retinal cells: Modulation by adenosine metabolism and the ERK cascade
2011, Biochemical and Biophysical Research Communications
Citation Excerpt :
Three subtypes of concentrative transporters promote nucleoside influx in a sodium-dependent manner [7]. On the other hand, there are four described subtypes of equilibrative transporters, which promote sodium-independent nucleoside transport according to the concentration gradient [7], and ENT1 and ENT2 are the predominant isoforms expressed in the CNS [8,9]. Concerning the enzymes, adenosine kinase (ADK) and ADA are the main players in adenosine inactivation.
Adenosine is an important modulator of neuronal survival and differentiation in the CNS. Our previous work showed that nucleoside transporters (NTs) are present in cultures of chick retinal cells, but little is known about the mechanisms regulating adenosine transport in these cultures. Our aim in the present work was to study the participation of the adenosine metabolism as well as the ERK pathway on adenosine uptake in different types of retinal cultures (mixed and purified glial cultures). Kinetic analysis in both cultures revealed that the uptake reached equilibrium after 30 min and presented two components. Incubation of cultures with S-(p-nitrobenzyl)-6-thioinosine (NBTI) or dipyridamole, different inhibitors of equilibrative nucleoside transporters (ENTs), produced a significant and concentration-dependent uptake reduction in both cultures. However, while dipyridamole presented similar maximal inhibitory effects in both cultures (although in different concentrations), the inhibition by NBTI was smaller in glial cultures than in mixed cultures, suggesting the presence of different transporters. Moreover, pre-incubation of [³H]-adenosine with adenosine deaminase (ADA) or adenosine kinase (ADK) inhibition with iodotubercidin promoted significant uptake inhibition in both cultures, indicating that the uptake is predominantly for adenosine and not inosine, and that taken up adenosine is preferentially directed to the synthesis of adenine nucleotides. In both cultures, the MEK inhibitors PD98059 or UO126, but not the inactive analog U0124, induced a significant and concentration-dependent uptake decrease. We have not observed any change in adenosine metabolism induced by MEK inhibitors, suggesting that this pathway is mediating a direct effect on NTs. Our results show the expression of different NTs in retinal cells in culture and that the activity of these transporters can be regulated by the ERK pathway or metabolic enzymes such as ADK which are then potential targets for regulation of Ado levels in normal or pathological conditions.
Behavioral effects of elevated expression of human equilibrative nucleoside transporter 1 in mice
2011, Behavioural Brain Research
Citation Excerpt :
Adenosine can be formed intracellularly or extracellularly and is transported through cell membranes via nucleoside transporters [3,4]. Nucleoside transport in neurons and glia is predominantly mediated by equilibrative nucleoside transporters 1 and 2 (ENT1, ENT2) [5,6]. To examine the role of ENTs in regulating adenosine levels and adenosine receptor activity, transgenic mice that express human ENT1 (hENT1) under the control of a neuron specific enolase promoter have been developed [7].
Adenosine concentrations are regulated by purinergic enzymes and nucleoside transporters. Transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1) have been generated (Parkinson et al., 2009 [7]). The present study tested the hypothesis that mice homozygous and heterozygous for the transgene exhibit differences in hENT1 mRNA and protein expression, and in behavioral responses to caffeine and ethanol, two drugs with adenosine-dependent actions. Real time polymerase chain reaction (PCR) was used to identify mice heterozygous and homozygous for the transgene. Gene expression, determined by real time PCR of cDNA reverse transcribed from cerebral cortex RNA, was 3.8-fold greater in homozygous mice. Protein abundance, determined by radioligand binding assays using 0.14 nM [³H]S-(4-nitrobenzyl)-6-thioinosine ([³H]NBTI), was up to 84% greater in cortex synaptosome membranes from homozygous than from heterozygous mice. In western blots with an antibody specific for hENT1, a protein of approximately 40 kDa was strongly labelled in cortex samples from homozygous mice, weakly labelled in samples from heterozygous mice and absent from samples from wild type mice. In behavioral assays, transgenic mice showed a greater response to ethanol and a reduced response to caffeine than wild type littermates; however, no significant differences between heterozygous and homozygous mice were detected. These data indicate that the difference in ENT1 function between wild type and heterozygous mice was greater than that between heterozygous and homozygous mice. Therefore, either heterozygous or homozygous hENT1 transgenic mice can be used in studies of ENT1 regulation of adenosine levels and adenosine dependent behaviors.

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Molecular Brain Research

Abstract

Section snippets

Acknowledgements

References (12)

Demonstration of the existence of mRNAs encoding N1/cif and N2/cit sodium/nucleoside cotransporters in rat brain

Mol. Brain Res.

Cloning of the human equlibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line

J. Biol. Chem.

Molecular cloning and functional characterization of nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative nucleoside transporter proteins (rENT1 and rENT2) from rat tissues

J. Biol. Chem.

Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs

Nature Med.

Cited by (64)

Deletion of equilibrative nucleoside transporter 2 disturbs energy metabolism and exacerbates disease progression in an experimental model of Huntington's disease

Of adenosine and the blues: The adenosinergic system in the pathophysiology and treatment of major depressive disorder

Nucleoside transporters in the purinome

Functional expression of drug transporters in glial cells: Potential role on drug delivery to the CNS

Differential adenosine uptake in mixed neuronal/glial or purified glial cultures of avian retinal cells: Modulation by adenosine metabolism and the ERK cascade

Behavioral effects of elevated expression of human equilibrative nucleoside transporter 1 in mice

Short communicationDistribution of mRNA encoding a nitrobenzylthioinosine-insensitive nucleoside transporter (ENT2) in rat brain

Abstract

Section snippets

Acknowledgements

Mol. Brain Res.

J. Biol. Chem.

J. Biol. Chem.

Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs

Nature Med.

Short communication
Distribution of mRNA encoding a nitrobenzylthioinosine-insensitive nucleoside transporter (ENT2) in rat brain