PURPOSE: 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.
PURPOSE: 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.
Authors: B A Larder; A Kohli; S Bloor; S D Kemp; P R Harrigan; R T Schooley; J M Lange; K N Pennington; M H St Clair Journal: J Virol Date: 1996-09 Impact factor: 5.103