Facilitated transport of inosine and uridine in cultured mammalian cells is independent of nucleoside phosphorylases

Abstract

The zero- trans uptake of uniformly and base-labeled inosine and uridine was measured at 25°C in suspensions of Novikoff rat hepatoma cells, Chinese hamster ovary cells, mouse L cells, mouse S49 lymphoma cells and a purine-nucleoside phosphorylase-deficient subline thereof (NSU-1), and in monolayer culture of mouse 3T3 and L cells. The initial velocities of uptake of both nucleosides were about the same in all cell lines investigated, regardless of the position of the label or of the substrate concentration between 3 and 300 μM or whether or not the cells possessed uridine or purine-nucleoside phosphorylase activity. The kinetic parameters for the facilitated transport of uridine and inosine were also similar in phosphorylase positive and negative cell lines ( K = 120–260 μM and V = 6–40 pmol/μl cell water per s) and the transport activities of the cells exceeded their total phosphorylase activities by at least 10-fold for uridine and 1–2-fold for inosine. Chromatographic fractionation of the intracellular contents and of the culture fluid showed that the free nucleosides appeared intracellularly prior to and more rapidly than their phosphorolysis products. During the initial 20–60 s of uptake of U- 14C-labeled nucleosides the rates of intracellular appearance of ribose-1- P and base were about the same. After several minutes of incubation, on the other hand, the main intracellular component was ribose-1- P whereas the base attained a low intracellular steady-state concentration and accumulated in the medium due to exit transport. Other nucleosides, dipyridamole and nitrobenzylthionosine, specifically inhibited the transport of uridine and inosine, and depressed the intracellular accumulation of ribose-1- P and the formation of base commensurate with that inhibition. The data indicate that the metabolism of inosine and uridine by the various cell lines can be entirely accounted for by the facilitated transport