Transient expression of a purine-selective nucleoside transporter (SPNTint) in a human cell line (HeLa).
OBJECTIVE
The goal of this study was to develop a mammalian expression system for the cloned rat intestinal, Na(+)-dependent, purine-selective nucleoside transporter (SPNTint) and to study the interactions of nucleosides and nucleoside analogs with this transporter.


METHODS
Lipofection was used to transfect HeLa cells with a mammalian expression vector (pcDNA3) containing the cDNA insert encoding SPNTint. Nucleoside transport activity was measured using [3H]inosine, [3H]uridine, [3H]-dideoxyinosine (ddI), and [3H]-2-chloro-2'-deoxyadenosine (2CdA) as model substrates.


RESULTS
Expression of SPNTint was observed between 36 and 90 h post-transfection, with maximal expression at 66 h. At 66 h, Na(+)-stimulated uptake of [3H]inosine in cells transiently transfected with SPNTint was approximately threefold greater than that in cells transfected with empty vector (p < 0.05). The Na(+)-stimulated uptake of both inosine and uridine was saturable (K(m) = 28.1 +/- 7.1 microM and 20.6 +/- 5.6 microM, respectively) in the transfected cells and was significantly inhibited by the naturally occurring nucleosides (1 mM) inosine and uridine and to a lesser extent by thymidine. The nucleoside analogs ddI (IC50 = 46 microM) and 2CdA (IC50 = 13 microM) also significantly inhibited the Na(+)-stimulated uptake of [3H]inosine. A Na(+)-stimulated uptake of [3H]2CdA was observed suggesting that 2CdA is also a permeant of SPNTint.


CONCLUSIONS
HeLa cells transiently transfected with SPNTint represent a useful tool to study the kinetics and interactions of drugs with SPNTint.
Schaner
M E
ME
Department of Biopharmaceutical Sciences, University of California San Francisco 94143, USA.
Wang
J
J
Zevin
S
S
Gerstin
K M
KM
Giacomini
K M
KM
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017338:Cladribine / Q000378:metabolism; D016049:Didanosine / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007288:Inosine / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins; D011684:Purine Nucleosides / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D014162:Transfection; D014529:Uridine / Q000037:antagonists & inhibitors / Q000378:metabolism
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D011684:Purine Nucleosides; C065171:cif nucleoside transporter; D017338:Cladribine; D007288:Inosine; D012964:Sodium; D016049:Didanosine; D014529:Uridine