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Transient Expression of a Purine-Selective Nucleoside Transporter (SPNTint) in a Human Cell Line (HeLa)

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Abstract

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 (ddl), 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 (Km = 28.1 ± 7.1 μM and 20.6 ± 5.6 μM, 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 ddl (IC50 = 46 μM) and 2CdA (IC50 =.13 μM) 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.

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Authors and Affiliations

  1. Department of Biopharmaceutical Sciences, University of California San Francisco, Box 0446, San Francisco, California, 94143

    Marci E. Schaner, Juan Wang, Karin M. Gerstin & Kathleen M. Giacomini

  2. Division of Clinical Pharmacology and Experimental Therapeutics, University of California San Francisco, San Francisco, California, 94143

    Shoshana Zevin

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  1. Marci E. Schaner
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  2. Juan Wang
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  3. Shoshana Zevin
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  4. Karin M. Gerstin
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  5. Kathleen M. Giacomini
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Correspondence to Kathleen M. Giacomini.

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Schaner, M.E., Wang, J., Zevin, S. et al. Transient Expression of a Purine-Selective Nucleoside Transporter (SPNTint) in a Human Cell Line (HeLa). Pharm Res 14, 1316–1321 (1997). https://doi.org/10.1023/A:1012148016794

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