SPODOPTERA-FRUGIPERDA (SF9) CELLS EXPRESS NOVEL PURINE-SELECTIVE AND PYRIMIDINE-SELECTIVE NUCLEOSIDE TRANSPORTERS

Nucleoside fluxes in mammalian cells are mediated by a family of plasm a membrane transporters that function by equilibrative or concentrativ e 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 process es of cultured armyworm ovary (Sf9) cells in anticipation of using Sf9 cells for the functional expression of recombinant mammalian nucleosi de transporter proteins. Kinetic analysis of zero-trans influx of H-3- nucleosides in Sf9 cells revealed the presence of high (K-m;10-40 mu M ) and low (K-m;greater than or equal to 0.4 mM) affinity transport pro cesses. Influx of [H-3] adenosine at 1 mu M (high-affinity process was inhibited only by purine nucleosides (adenosine > 3'-deoxyadenosine > formycin B > guanosine > inosine), and that of [H-3]uridine was inhib ited only by pyrimidine nucleosides (uridine > 3'-deoxyuridine > thymi dine). By contrast, fluxes of [H-3]adenosine and [H-3]uridine at 100 m u M (low-affinity process) were inhibited by both pyrimidine and purin e nucleosides. These results suggested the presence of at least two hi gh-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 upt ake of adenosine and uridine was unaffected by elimination of the sodi um gradient, and assays that employed formycin B, a non-metabolized pe rmeant of the high-affinity purine-selective nucleoside transport proc ess, revealed that transport was equilibrative. Nitrobenzylthioinosine , dipyridamole, and dilazep, potent inhibitors of equilibrative nucleo side transport processes in mammalian cells, were poor inhibitors of a denosine and uridine fluxes in Sf9 cells.