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.