Kinetic and Pharmacological Properties of Cloned Human Equilibrative Nucleoside Transporters, ENT1 and ENT2, Stably Expressed in Nucleoside Transporter-deficient PK15 Cells

Abstract

We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC 50 , 0.4 ؎ 0.1 nM versus 2.8 ؎ 0.3 M) and dipyridamole (IC 50 , 5.0 ؎ 0.9 nM versus 356 ؎ 13 nM), respectively. [ 3 H]NBMPR binds to ENT1 cells with a high affinity K d of 0.377 ؎ 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7.7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [ 3 H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.

Ribonucleic Acid Hydrolysis by Intestinal Explants of Neonatal Piglets

“…Guanosine also increased over the 6-hour incubation period. Taken together, these results suggests that both inosine and guanosine are poorly converted to their respective bases by intestinal explants and should be readily absorbed by specific transporters because of their low Km (30). The enterocyte intracellular catabolic enzymes seem to be responsible for the conversion of inosine and guanosine to xanthine, guanine, and uric acid in small intestine homogenates (18).…”

Nucleoside transporters in chronic lymphocytic leukaemia

“…Significant differences between the two isoforms in terms of drug recognition should be expected, since some natural nucleosides are taken up by hENT2 with a much lower affinity than hENT1. 44 Several nucleoside analogues used in the treatment of lymphoid malignancies also appear to be better substrates for hENT1 than for hENT2 (Table 2). Substrate selectivity in the CNT gene family is narrower than for ENTs.…”

Inhibition of polyadenylation reduces inflammatory gene induction

“…To investigate whether this is the case in ASM cells, we preincubated the cells with inhibitors of the adenosine transporter (nitrobenzylthioinosine, NBTI) and adenosine kinase (iodotubericidin, ITU) (Bontemps et al 1983;Newby et al 1983;Parkinson and Geiger 1996;Ward et al 2000;SenGupta et al 2002). Cells were then treated with cordycepin and TNF as previously described, RNA was isolated and subjected to RT-qPCR with normalization to GAPDH mRNA.…”