Spodoptera frugiperda (Sf9) cells express novel purine- and pyrimidine-selective nucleoside transporters

Abstract

Nucleoside fluxes in mammalian cells are mediated by a family of plasma membrane transporters that function by equilibrative or concentrative mechanisms. Members of this family can be identified on the basis of their permeant selectivities and sensitivities to various inhibitors. This study was initiated to characterize nucleoside transport processes of cultured armyworm ovary (Sf9) cells in anticipation of using Sf9 cells for the functional expression of recombinant mammalian nucleoside transporter proteins. Kinetic analysis of zero- trans influx of 3H-nucleosides in Sf9 cells revealed the presence of high ( K m; 10–40 μM) and low ( K m; ⩾0.4 mM) affinity transport processes. Influx of [ 3H]adenosine at 1 μM (high-affinity process) was inhibited only by purine nucleosides (adenosine > 3′-deoxyadenosine > formycin B > guanosine > inosine), and that of [ 3H]uridine was inhibited only by pyrimidine nucleosides (uridine > 3′-deoxyuridine > thymidine). By contrast, fluxes of [ 3H]adenosine and [ 3H]uridine at 100 μM (low-affinity process) were inhibited by both pyrimidine and purine nucleosides. These results suggested the presence of at least two high-affinity transport processes with selectivity for either purine or pyrimidine nucleosides and a low-affinity process with selectivity for both. None of the transport processes were sodium-dependent since uptake of adenosine and uridine was unaffected by elimination of the sodium gradient, and assays that employed formycin B, a non-metabolized permeant of the high-affinity purine-selecive nucleoside transport process, revealed that transport was equilibrative. Nitrobenzylthioinosine, dipyridamole, and dilazep, potent inhibitors of equilibrative nucleoside transport processes in mammalian cells, were poor inhibitors of adenosine and uridine fluxes in Sf9 cells.