Uridine transport in novikoff rat hepatoma cells and other cell lines and its relationship to uridine phosphorylation and phosphorolysis

Abstract

AbstractTime courses of [3H]uridine uptake as a function of uridine concentration were determined at 25° in untreated and ATP‐depleted wild‐type and uridine kinase‐deficient Novikoff cells and in mouse L and P388 cells, Chinese hamster ovary cells and human HeLa cells. Short term uptake was measured by a rapid sampling technique which allows sampling of cell suspensions in intervals as short as one and one‐half seconds. The initial segments of the time courses were the same in untreated, wild‐type cells in which uridine is rapidly phosphorylated and in cells in which uridine phosphorylation was prevented due to lack of ATP or uridine kinase. The initial rates of uptake, therefore, reflected the rate of uridine transport. Uridine uptake, however, was approximately linear for only five to ten seconds at uridine concentrations from 20–160 μM and somewhat longer at higher concentrations. In phosphorylating cells the rate of uridine uptake (at 80 μM) then decreased to about 20–30% of the initial rate and this rate was largely determined by the rate of phosphorylation rather than transport. At uridine concentrations below 1 μM, however, the rate of intracellular phosphorylation in Novikoff cells approached the transport rate. The apparent substrate saturation of phosphorylation suggests the presence of a low Km uridine phosphorylation system in these cells. The “zero‐trans” (zt) Km for the facilitated transport of uridine as estimated from initial uptake rates fell between 50 and 240 μM for all cell lines examined. The zero‐trans Vmax values were also similar for all the lines (4–15 pmoles/μ1 cell H2O.sec). The time courses of uridine uptake by CHO cells and the kinetic constants for transport were about the same whether the cells were propagated (and analyzed for uridine uptake) in suspension or monolayer culture. When Novikoff cells were preloaded with 10 μM uridine the apparent Km and Vmax values (infinite‐trans) were two to three times higher than the corresponding zero‐trans values. Uridine transport was inhibited in a simple competitive manner by several other ribo‐ and deoxyribonucleosides. All nucleosides seem to be transported by the same system, but with different efficiencies. Uridine transport was also inhibited by hypoxanthine, adenine, thymine, Persantin, papaverin, and o‐nitrobenzylthioinosine, and by pretreatment of the cells with p‐chloromercuri‐benzoate, but not by high concentrations of cytosine, D‐ribose or acronycin. The inhibition of uridine transport by Persantin involved changes in both V and K. Because of the rapidity of transport, some loss of intracellular uridine occurred when cells were rinsed in buffer solution to remove extracellular substrate, even at 0°. This loss was prevented by the presence of a transport inhibitor, Persantin, in the rinse fluid or by separating suspended cells from the medium by centrifugation through oil. Metabolic conversion of intracellular uridine were also found to continue during the rinse period. The extent of artifacts due to efflux and metabolism during rinsing increased with duration of the rinse.