Hypoxanthine uptake and release by equilibrative nucleoside transporter 2 (ENT2) of rat microvascular endothelial cells.
The cardioprotective actions of adenosine are terminated by its uptake into endothelial cells with subsequent metabolism through hypoxanthine to uric acid. This process involves xanthine oxidase-mediated generation of reactive oxygen species (ROS), which have been implicated in the vascular dysfunction observed in ischemia-reperfusion injury. The equilibrative nucleoside transporter, ENT2, mediates the transfer of hypoxanthine into cells. We hypothesize that ENT2 also mediates the cellular release of hypoxanthine, which would limit the amount of intracellular hypoxanthine available for xanthine oxidase-mediated ROS production. Rat microvascular endothelial cells (MVECs) were isolated from skeletal muscle by lectin-affinity purification. The transport of [(3)H]hypoxanthine was assessed using an oil-stop method, and hypoxanthine metabolites were identified by thin-layer chromatography. MVECs accumulated hypoxanthine with a K(m) of 300 microM and a V(max) of 2.8 pmol microl(-1) s(-1). ATP-depleted cells loaded with [(3)H]hypoxanthine released the radiolabel with kinetics similar to that obtained for [(3)H]hypoxanthine influx. The uptake and release of [(3)H]hypoxanthine were both blocked by ENT2 inhibitors with similar order of potency. Thus, ENT2 mediates both the influx and efflux of hypoxanthine. Inhibition of ENT2 in MVECs might be expected to increase the amount of intracellular hypoxanthine available for metabolism by xanthine oxidase and enhance the intracellular production of ROS.
Department of Physiology and Pharmacology, M216 Medical Sciences Building, University of Western Ontario London, ON, Canada N6A 5C1.
Kevin R
KR
Robillard
Derek B J
DB
Bone
James R
JR
Hammond
D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D001692:Biological Transport; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D003847:Deoxyglucose / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism*; D033721:Equilibrative Nucleoside Transporter 1 / Q000378:metabolism; D033722:Equilibrative-Nucleoside Transporter 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019271:Hypoxanthine / Q000032:analysis / Q000378:metabolism*; D008297:Male; D008833:Microcirculation / Q000166:cytology / Q000378:metabolism; D018482:Muscle, Skeletal / Q000098:blood supply*; D051381:Rats; D017208:Rats, Wistar; D012402:Rotenone / Q000494:pharmacology; D014316:Tritium; D014475:Uncoupling Agents / Q000494:pharmacology
D000963:Antimetabolites; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; C438652:Slc29a1 protein, rat; C438655:Slc29a2 protein, rat; D014475:Uncoupling Agents; D012402:Rotenone; D014316:Tritium; D019271:Hypoxanthine; D000493:Allopurinol; D003847:Deoxyglucose