title: Oxidative stress modulates nucleobase transport in microvascular endothelial cells.
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.
authors: Department of Physiology and Pharmacology, Western University, London, ON, Canada. Electronic address: derek.bone@nih.gov.
authors: Derek B J
authors: DB
authors: Bone
authors: Department of Physiology and Pharmacology, Western University, London, ON, Canada.
authors: Milica
authors: M
authors: Antic
authors: Department of Pharmacology, University of Alberta, Edmonton, AB, Canada. Electronic address: gvilas@ualberta.ca.
authors: Gonzalo
authors: G
authors: Vilas
authors: Department of Physiology and Pharmacology, Western University, London, ON, Canada; Department of Pharmacology, University of Alberta, Edmonton, AB, Canada. Electronic address: james.hammond@ualberta.ca.
authors: James R
authors: JR
authors: Hammond
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology; D001692:Biological Transport; D015687:Cell Hypoxia; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D033721:Equilibrative Nucleoside Transporter 1 / Q000187:drug effects / Q000378:metabolism*; D033722:Equilibrative-Nucleoside Transporter 2 / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D011687:Purines / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors
chemical_list: D000975:Antioxidants; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D016877:Oxidants; D011687:Purines; C438649:SLC29A1 protein, human; C438654:SLC29A2 protein, human; D013481:Superoxides; D019271:Hypoxanthine; D000241:Adenosine
keywords: Adenosine; Endothelium; Hypoxanthine; Reactive oxygen species; Solute transporters; Vascular