Human Erythrocyte Nucleoside Transporter ENT1 Functions at Ice-cold Temperatures

Abstract

The functionality of human erythrocyte nucleoside transporter ENT1 was examined at ice-cold temperatures (ICT; measured temperature, 0.5-0.7 degrees C) using rightside-out membrane vesicles (ROVs). The uptake of uridine, an ENT1 substrate, showed saturation kinetics and was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), a specific ENT1 inhibitor, at both 23 degrees C and ICT. [3H]Uridine uptake was markedly trans-stimulated by preloading ROVs with unlabeled uridine or ribavirin, another ENT1 substrate, and the overshoot phenomenon was observed at ICT. Similarly, [3H]ribavirin uptake was markedly trans-stimulated by unlabeled ribavirin or uridine at ICT. The trans-stimulated uptake of [3H]uridine at ICT was inhibited by ENT1 inhibitors/substrates such as NBMPR, dipyridamole, adenosine, and ribavirin in a concentration-dependent manner. The inhibition of [3H]uridine uptake by NBMPR and dipyridamole at ICT was also observed in intact red blood cells. Like uridine uptake, [3H]D-glucose uptake by ROVs, which is mediated by facilitative glucose transporter GLUT1, was trans-stimulated by unlabeled D-glucose at ICT, and the overshoot phenomenon was observed. In contrast, the ability of ATP-dependent transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein via multidrug resistance-associated protein 5 in inside-out membrane vesicles disappeared at ICT. These results clearly indicate that human erythrocyte transporters such as ENT1 function even at very low temperatures near 0 degrees C. The significance of these findings in transporter research is discussed.

Transport Characteristics of Ribavirin in Human Erythrocyte Membrane Vesicles

“…ROVs were prepared from human erythrocytes by the method described previously 7,8) . Briefly, 5 ml of fresh blood was washed with 25 ml of buffer B (137 mM NaCl, 2.7 mM KCl, 10.6 mM Na2HPO4, 8.5 mM KH2PO4, 1 mM MgCl2, 100 M EGTA, pH 7.4) by centrifugation.…”

“…IOVs were prepared from human erythrocytes by the method described previously 7,9) . Briefly, erythrocytes from 5 ml of fresh blood were washed with 40 ml of 0.9% NaCl by centrifugation.…”

“…The uptake of ribavirin by ROVs was measured by a rapid filtration technique, as described previously 7,10) . …”

“…In other words, ENT1 is functioning at ice-cold temperatures near 0 . Surprisingly, even overshoot uptake (uphill transport) of uridine was observed at ice-cold temperatures under certain conditions 7) . These studies have given us important information in order to estimate erythrocyte ENT1 function properly; addition of an ENT1 inhibitor such as NBMPR in the stop solution (the solution to stop the uptake reaction and to wash the membrane vesicles) is essential.…”

“…These studies have given us important information in order to estimate erythrocyte ENT1 function properly; addition of an ENT1 inhibitor such as NBMPR in the stop solution (the solution to stop the uptake reaction and to wash the membrane vesicles) is essential. In fact, about 90% of uridine taken up by erythrocyte membrane vesicles was effluxed during washing process using ice-cold stop solution without NBMPR, and therefore it was impossible to evaluate transport characteristics of uridine at the membrane level 7) . We suppose that this may be one of the reasons why no study on ribavirin transport in erythrocyte membrane vesicles has been reported so far.…”