Nucleoside/nucleobase transport and metabolism by microvascular endothelial cells isolated from ENT1^−/− mice

Abstract

Bone DB, Choi DS, Coe IR, Hammond JR. Nucleoside/nucleobase transport and metabolism by microvascular endothelial cells isolated from ENT1 Ϫ/Ϫ mice. Am J Physiol Heart Circ Physiol 299: H847-H856, 2010. First published June 11, 2010; doi:10.1152/ajpheart.00018.2010.-Nucleoside and nucleobase uptake is integral to mammalian cell function, and its disruption has significant effects on the cardiovasculature. The predominant transporters in this regard are the equilibrative nucleoside transporter subtypes 1 (ENT1) and 2 (ENT2). To examine the role of ENT1 in more detail, we have assessed the mechanisms by which microvascular endothelial cells (MVECs) from ENT1 Ϫ/Ϫ mice transport and metabolize nucleosides and nucleobases. Wild-type murine MVECs express mainly the ENT1 subtype with only trace levels of ENT2. These cells also have a Na ϩ -independent equilibrative nucleobase transport mechanism for hypoxanthine (ENBT1). In the ENT1Ϫ/Ϫ cells, there is no change in ENT2 or ENBT1, resulting in a very low level of nucleoside uptake in these cells, but a high capacity for nucleobase accumulation. Whereas there were no significant changes in nucleoside transporter subtype expression, there was a dramatic increase in adenosine deaminase and adenosine A 2a receptors (both transcript and protein) in the ENT1 Ϫ/Ϫ tissues compared with WT. These changes in adenosine deaminase and A 2a receptors likely reflect adaptive cellular mechanisms in response to reduced adenosine flux across the membranes of ENT1 Ϫ/Ϫ cells. Our study also revealed that mouse MVECs have a nucleoside/nucleobase transport profile that is more similar to human MVECs than to rat MVECs. Thus mouse MVECs from transgenic animals may prove to be a useful preclinical model for studies of the effects of purine metabolite modifiers on vascular function. adenosine; microvasculature; heart; transgenic; equilibrative nucleoside transporter ALL MAMMALIAN CELLS express at least one form of equilibrative transporter responsible for the cellular uptake and release of endogenous nucleosides such as adenosine. The predominant transporters are equilibrative nucleoside transporter subtypes 1 (ENT1; SLC29A1) and 2 (ENT2; SLC29A2) (3). The inhibition of ENTs has been shown to enhance the cardioprotective (21) and neuroprotective (13) actions of adenosine. These transporters are also responsible for the cellular uptake of cytotoxic nucleoside analogs used to treat cancer and viral infections (25).An ENT1-null (ENT1 Ϫ/Ϫ ) mouse model has been generated by Choi and colleagues (10). The ENT1 Ϫ/Ϫ mice are phenotypically normal with the only difference from wild-type (WT) littermates being a slight (Ͻ10%) decrease in body weight and some differences in their behavioral characteristics. The ENT1-null mice are less sensitive to the intoxicating effects of ethanol compared with their WT littermates (10), and they also display less anxiety-like behavior (9). It has been noted that there is less tonic A 1 adenosine receptor signaling in the brains of ENT1 Ϫ/Ϫ mice (10), reflecting either re...

Loss of equilibrative nucleoside transporter 1 in mice leads to progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis in humans

“…Genotyping was performed as described. (33) Given the increased reported prevalence of DISH in males (25% of men versus 15% of women over 50 years of age), (34) male mice were used for all experiments. Mice were euthanized at the following ages: 1 month (4-4.5 weeks), 2 months (8-11 weeks), 4 months (16-18 weeks), 6 months (26-30 weeks), and 12þ months (12-17 months).…”

“…Moreover, there is no significant difference in spontaneous mortality rates up to 6 months of age. To date, there have been no reports of phenotypic changes in skeletal tissues related to mineralization (32,33,41,42) ; however, all reported studies were conducted on mice less than 4 months of age.…”

Molecular Basis of Nucleobase Transport Systems in Mammals

“…Hypoxanthine uptake is neither inhibited by dipyridamole in mouse primary microvascular endothelial cells, which exhibit considerable transport activity of 2-chloroadenosine by Ent1, nor significantly altered in cells from Ent1-deficient mice. 34) However, one study reported the involvement of ENT1 in hypoxanthine transport in a murine T-lymphoma cell line, S49, which was mostly inhibited by 10 nM of NBMPR. 35) 4.2.…”

“…However, because the term "ENBT1" was previously used to denote a putative transporter and a system that mediates dipyridamole-insensitive hypoxanthine transport in cells such as primary human cardiac microvascular endothelial cells, 34,46,47) the use of "ENBT1" as an alias for SLC43A3 would cause confusion. The present review advocates the use of either SLC43A3 or the same alongside ENBT1.…”

Tubuloglomerular feedback and renal function in mice with targeted deletion of the type 1 equilibrative nucleoside transporter

“…Nevertheless, the effect of prolonged activation of A1AR by CHA or adenosine on TGF responsiveness has not been examined. Finally, accelerated degradation of adenosine may affect TGF responses since an upregulation of adenosine deaminase has been found in microvascular endothelial cells of ENT1-deficient mice (2). This possibility cannot be excluded although we did not find an increased expression of adenosine deaminase mRNA in kidney tissue.…”