Mechanism of Spermidine Uptake in Cultured Mammalian Cells and Its Inhibition by Some Polyamine Analogues

Abstract

Transport pathways for spermidine (Spd) were characterized in mammalian cells in culture of different origin, i.e. L 1210, P 388, C 6, U 251, Balb/c 3T3 normal and transformed by virus SV40 (SV40/3T3). The kinetic constants (K_m and V_max) for ¹⁴C-Spd uptake were found to be different in these cells. Spd uptake was inhibited by spermine and putrescine in all cells. Preloading of these cells with system A and other amino acids, including ornithine, usually did not affect Spd uptake, except in L 1210 and C 6 cells, where Spd uptake was accelerated by 2-aminoisobutyric acid, demonstrating that in these two cell lines the polyamines share the system A pathway. Iso-osmotic replacement of Na⁺ by choline chloride in the assay medium resulted in a decrease in Spd uptake which suggests that Spd uptake is Na⁺ activated. In all cells, Spd uptake was inhibited by gramicidin and the Ca²⁺ ionophore A 23187. The degree of inhibition varied among the cells. Valinomycin (K⁺ ionophore) inhibited Spd uptake by C 6, P 388, Balb/c 3T3 and SV40/3T3 but not by L 1210 and U 251 cells. Treatment with N-ethylmaleimide or p-chloromercuribenzene sulfonate abolished the Spd uptake by U 251 and P 388 cells, while the same treatment to L 1210, C 6, Balb/c 3T3 and SV40/3T3 cells did not affect appreciably the uptake process. Some newly synthesized polyamine analogues inhibited the Spd uptake of all cells.

Identification and Characterization of a Polyamine Permease from the Protozoan Parasite Leishmania major

“…Ca 2ϩ is also known to affect polyamine transport into eukaryotes, although the effects are disparate. Ca 2ϩ stimulates polyamine uptake in human fibroblasts (48) and breast cancer cells (47), whereas the divalent cation inhibits polyamine transport into embryonic mesenchymal cells (49) and Neurospora crassa (50). Because the calcium ionophore A23687 inhibited putrescine uptake into TUB::HD309-LmPOT1 T. brucei, it is possible that putrescine transport via LmPOT1 is Ca 2ϩ -dependent.…”

Effects of a series of homologous α,ω‐dimethylaminoalkanes on cell proliferation: binding and uptake of putrescine by a human glioblastoma cell line (U251) in culture

“…Since the putrescine acceptor sites were supposed to be involved in putrescine uptake (4), and could be considered as a potential target for anticancer drug design, our experiments have been carried out in order to characterize their specificity. We observed that methylation of the two primary amine functions of putrescine did not prevent recognition by the putrescine binding site.…”

Unusual aspects of the polyamine transport system affect the design of strategies for use of polyamine analogues in chemotherapy

“…Values for this uptake velocity from a number of different mammalian cell lines suggest that 2-5 nmol/10 6 cells per h is a common range [7,[17][18][19]23,24]. At this velocity the spermidine or spermine level of a cell could be doubled in 1 h!…”

“…Although there may be some cell-type variation, kinetic studies of mammalian polyamine uptake generally suggest an apparent K m of spermine for the transporter at 1 µM or less [7,[17][18][19][20]. The triamine spermidine demonstrates almost the same affinity as spermine, while putrescine is found to be a poorer substrate with an apparent K m in the 5-10 µM range.…”