Distinguishing between membrane transport and intracellular metabolism of fluorodeoxyuridine in Ehrlich ascites tumor cells by application of kinetic and high performance liquid chromatographic techniques.

Abstract

Bidirectional fluxes, net transport, and cellular metabolic profiles following incubation of Ehrlich ascites tumor cells for short intervals with [3H]fluorodeoxyuridine were examined to characterize the relationship between transport and metabolism of this nucleoside. This approach indicates that the uptake sequence of 3H is characterized by (i) a very rapid initial uptake phase that is linear with time, extrapolates through the point of origin, and represents the initial rate of fluorodeoxyuridine transport into the cell. This is followed by (ii) a fall in the net rate of 3H uptake due to the contribution of an efflux component for fluorodeoxyuridine and, to some extent, fluorouracil as the nucleoside and base accumulate within the cell. As these exit components increase, cellular fluorodeoxyuridine reaches a steady state within 15 s. (iii) At this point, net uptake of 3H has declined to a velocity which remains constant for at least the next 100 s and reflects almost entirely the rate of cellular accumulation and retention of fluorodeoxyuridine monophosphate. Over this interval of observation and under these experimental conditions, only fluorodeoxyuridine, fluorouracil, and fluorodeoxyuridine monophosphate are detected within the cell. Low concentrations of thymidine (10 pM) block the late linear uptake phase for 3H, but have only a small effect on the initial uptake velocity. The effect of thymidine on the late linear phase is due primarily to a marked inhibition of the metabolism of fluorodeoxyuridine to fluorodeoxyuridine monophosphate rather than an inhibition of fluorodeoxyuridine transport. When cells are first loaded with high levels of nonlabeled fluorodeoxyuridine (10 mM) and then pulsed with [3H]fluorodeoxyuridine, there is a trans-stimulation of [3H]fluorodeoxyuridine influx and a transient increase in the steady state fluorodeoxyuridine level within the cell (countertransport), both characteristics of a carrier-mediated process. Efflux studies confirm that this enhanced uptake of [3H]fluorodeoxyuridine is related to a rapidly exchangeable component which appears to be free in the intracellular water. These observations (i) point to the critical role of experimental design and data analysis in the study of membrane transport properties of nucleosides or other compounds that are rapidly metabolized within cells, and (ii) demonstrate the evanescent nature of trans-