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  • Oxidative stress modulates nucleobase transport in microvascular endothelial cells.

Oxidative stress modulates nucleobase transport in microvascular endothelial cells.

Microvascular research (2014-07-01)
Derek B J Bone, Milica Antic, Gonzalo Vilas, James R Hammond
ABSTRACT

Purine nucleosides and nucleobases play key roles in the physiological response to vascular ischemia/reperfusion events. The intra- and extracellular concentrations of these compounds are controlled, in part, by equilibrative nucleoside transporter subtype 1 (ENT1; SLC29A1) and by equilibrative nucleobase transporter subtype 1 (ENBT1). These transporters are expressed at the membranes of numerous cell types including microvascular endothelial cells. We studied the impact of reactive oxygen species on the function of ENT1 and ENBT1 in primary (CMVEC) and immortalized (HMEC-1) human microvascular endothelial cells. Both cell types displayed similar transporter expression profiles, with the majority (>90%) of 2-chloro[(3)H]adenosine (nucleoside) uptake mediated by ENT1 and [(3)H]hypoxanthine (nucleobase) uptake mediated by ENBT1. An in vitro mineral oil-overlay model of ischemia/reperfusion had no effect on ENT1 function, but significantly reduced ENBT1 Vmax in both cell types. This decrease in transport function was mimicked by the intracellular superoxide generator menadione and could be reversed by the superoxide dismutase mimetic MnTMPyP. In contrast, neither the extracellular peroxide donor TBHP nor the extracellular peroxynitrite donor 3-morpholinosydnonimine (SIN-1) affected ENBT1-mediated [(3)H]hypoxanthine uptake. SIN-1 did, however, enhance ENT1-mediated 2-chloro[(3)H]adenosine uptake. Our data establish HMEC-1 as an appropriate model for study of purine transport in CMVEC. Additionally, these data suggest that the generation of intracellular superoxide in ischemia/reperfusion leads to the down-regulation of ENBT1 function. Modification of purine transport by oxidant stress may contribute to ischemia/reperfusion induced vascular damage and should be considered in the development of therapeutic strategies.

MATERIALS
Product Number
Brand
Product Description

Adenine, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Adenine, BioReagent, suitable for plant cell culture, ≥99%
Sigma-Aldrich
Adenine, ≥99%
Sigma-Aldrich
Hypoxanthine, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Dipyridamole, ≥98% (HPLC)
Sigma-Aldrich
Hypoxanthine, ≥99.0%
Sigma-Aldrich
Adenine, BioReagent, suitable for cell culture
Sigma-Aldrich
tert-Butyl hydroperoxide solution, 5.0-6.0 M in decane
Sigma-Aldrich
tert-Butyl hydroperoxide solution, 5.0-6.0 M in nonane
Sigma-Aldrich
tert-Butyl hydroperoxide solution, 70 wt. % in H2O
Dipyridamole, European Pharmacopoeia (EP) Reference Standard
Supelco
Adenine, Pharmaceutical Secondary Standard; Certified Reference Material
Didanosine impurity A,, European Pharmacopoeia (EP) Reference Standard
Menadione, European Pharmacopoeia (EP) Reference Standard
Supelco
Menadione (K3), analytical standard
Sigma-Aldrich
2-Chloroadenosine
Sigma-Aldrich
Menadione, crystalline
Sigma-Aldrich
Menadione, meets USP testing specifications
Dipyridamole for peak identification, European Pharmacopoeia (EP) Reference Standard
Supelco
Menadione, Pharmaceutical Secondary Standard; Certified Reference Material