Richard Marz

Chapter 16 A Rapid-Mixing Technique to Measure Transport in Suspended Animal Cells: Applications to Nucleoside Transport in Novikpff Rat Hepatoma Cells

Publisher Summary The chapter discusses techniques, which permit an operational separation of transport and metabolism. This separation can be achieved by genetic, chemical, or kinetic manipulation, or a combination thereof. The transport of various compounds across mammalian cell membranes is frequently found to occur with a rapidity which necessitates collecting data at intervals of a few seconds. By means of a dual-syringe device, suspended cells can be mixed nearly instantaneously with radioactively labeled substrate and separated from the substrate again within seconds by centrifugation into silicone oil. Depending on the cell-substrate system under investigation, initial transport velocities may be either measured directly or calculated from the time course with which equilibrium across the membrane is attained. With nonmetabolizing systems, the dual-syringe apparatus is adaptable to a variety of experimental protocols-zero-trans, equilibrium exchange, and infinite-cis—which in combination make possible a thorough kinetic characterization of a transport system.

Properties of the thymidine transport system of chinese hamster ovary cells as probed by nitrobenzylthioinosine

SummaryThe transport of thymidine into Chinese hamster ovary cells grown in suspension culture was measured under conditions in which thymidine was not metabolized, namely, when cells had been depleted of ATP. The system transporting thymidine was saturable (Kmzt=70μM), rapid (50% of transmembrane equilibrium level attained within 8 sec), and was apparently shared by other nucleosides, but not thymine or hypoxanthine. 6([4-nitrobenzyl]thio)-9-β-d-ribofuranosylpurine, “nitrobenzylthioinosine”, inhibited thymidine transport in a simple, noncompetitive fashion with an apparentKi=1.0 nM (based on total concentration of inhibitor, which significantly overestimates that of free inhibitor). The rate of expression of inhibition was slow (t1/2=17 sec) relative to the rate of association of thymidine with its transporter, and thymidine partially protected the transport system against inhibition by nitrobenzylthioinosine. The dissociation constant for the inhibitortransporter complex was estimated at about 0.1 nM, and the number of binding sites per cell at about 6×104. HeLa, P388 murine leukemia, and mouse L cells were as sensitive to nitrobenzylthioinosine inhibition of thymidine transport as Chinese hamster ovary cells; Novikoff rat hepatoma cells were much less sensitive.

Growth rate of cultured Novikoff rat hepatoma cells as a function of the rate of thymidine and hypoxanthine transport.

SummaryNovikoff rat hepatoma cells were propagated in suspension culture in the presence of 1μm methotrexate and various concentrations of hypoxanthine (or adenosine plus guanosine) and thymidine and with or without the inhibitor of nucleoside and purine transport, Persantin (dipyridamole). Methotrexate-treated cells failed to replicate and died even if the medium was supplemented with either thymidine or a purine source, but normal replication occurred when both were present. The additional presence of Persantin reduced the rate of transport of thymidine or hypoxanthine and thus their incorporation into the nucleotide pool and decreased the rate of cell replication. The growth rate of the cells was directly proportional to the rate of incorporation of thymidine (in the presence of excess hypoxanthine) or of hypoxanthine (in the presence of excess thymidine) until the normal maximum growth rate was obtained. Normal cell replication in the presence of methotrexate and Persantin occurred only when the medium was supplemented with 500 μm hypoxanthine and 30 μm thymidine. The results illustrate a dependence of the growth rate of mammalian cells on the rate of transport of essential nutrients into the cell.