Cell Model for Studying Nucleoside Transporters, a Key Component of the Blood‐Testis Barrier

Abstract

Sertoli cells in the testis represent the principle element of the Blood‐Testis Barrier (BTB). Equilibrative nucleoside transporters (ENTs) are responsible for the transport of nucleosides across rodent BTB and immunohistochemistry suggests that hENT1 is located on the basal membrane and hENT2 is located on the apicolateral membrane of Sertoli cells (Klein et al., 2013). Therefore, the ENTs are of particular interest in studying the disposition of nucleoside reverse transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in chemical structure to nucleosides. This study characterized the transport of a specific nucleoside, uridine, to determine the relative roles of ENT1 and ENT2 in nucleoside transport in HeLa S3 cells, a potential model system for the study of ENT selectivity, using the ENT specific inhibitor S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR). Uptake of 20 nM [3H]uridine was linear for 7 minutes and 100 nM NBMPR blocked 77% of this uptake, with the remaining uptake blocked by 100 uM NBMPR (to the same level produced by 5 mM unlabeled uridine). ENT1 and ENT2 are known to have different sensitivities for NBMPR and, consistent with this difference, inhibition of [3H]uridine uptake produced by increasing concentration of NBMPR proved to be biphasic, with IC50s of 11.1 nM (ENT1) and 7.2 uM (ENT2). Consequently, uridine uptake measured in the presence of 100 nM NBMPR was taken to represent ENT2‐mediated transport; subtracting that from total uptake represented ENT1‐mediated transport. The kinetics of ENT1‐ and ENT2‐mediated [3H]uridine uptake into HeLa S3 cells showed no difference in Jmax (16.1 and 12.3 fmol cm−2 min−1) and a three‐fold difference in Km (13.3 and 35.2 uM). The resulting 3.5‐fold difference in intrinsic clearance for uridine uptake (i.e., Jmax/Km) for hENT1 and hENT2 mediated transport (1.21 mL cm−2 min−1 and 0.35 mL cm−2 min−1, respectively), accounted for the observed inhibition of uridine transport produced by 100 nM NBMPR, indicating that at low concentrations, hENT1 is primarily responsible for uridine uptake in these cells. Together, these data suggest that HeLa S3 cells are an adequate model for studying the characteristics of nucleoside transporters present in the BTB.Support or Funding InformationSupported by R01GM123643This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.