Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells

Abstract

The uptake of intracellular putrescine and spermidine was examined in B16 melanoma cells. It was found that difluoromethylornithine preferentially induced putrescine transport (28-fold) compared to that for spermidine (3.5-fold). Putrescine uptake was partially Na' dependent, whereas spermidine uptake was not. Inhibition studies with the two polyamines showed that putrescine was a poor competitive inhibitor of spermidine uptake, exhibiting a Ki of 69-7.5 pM, whereas the estimated K, for putrescine uptake was only 5.36 pM. By contrast, spermidine inhibition of putrescine transport produced a non-linear Eadie-Scatchard plot suggesting that putrescine was taken up by a spermidine-sensitive and a spermidine-insensitive process. The estimated spermidine Ki for inhibition of the spermidine-sensitive process was 0.12.5 pM. Using a series of polypyridinium quaternary salts to inhibit transport, no correlation between inhibition of putrescine uptake and inhibition of spermidine uptake was seen. Finally, the photoaffinity label, 1 ,12-di(N5-azido-2-nitrobenzoy1)spermine selectively inactivated the putrescine transporter(s) without affecting spermidine uptake. From these observations, it was concluded that multiple polyamine transporters are present on B16 melanoma cells and that separate, distinct transporter(s) account for the uptake of putrescine and spermidine in this cell-line following induction with difluoromethylornithine. The present of different transporters for the two polyamines indicates that expression of uptake activity for putrescine and spermidine may be under separate cellular control.The uptake of extracellular polyamines has been shown to occur in many different cell types [I -81. The system responsible for uptake is highly inducible by a number of different stimuli [l, 2, 5, 9, 101 and may also be regulated by the extent of cell differentiation [ll]. Several recent studies have shown that the uptake of polyamines into mammalian cells can not be explained by a single transport system [8,12, 131. One of these studies has used various inhibitors of putrescine and spermidine uptake to show the existence of a non-homogeneous uptake system [12] while another has demonstrated non-Michaelis-Menten uptake behaviour that suggests the presence of multiple transporters [8]. Further support for multiple transporters has come from studies with cells cloned for the human polyamine transporter [14]. In this work, clones with different uptake characteristics were isolated and identified. However, it is still unclear whether different transporters exist for each of the polyamines, whether the activities of these transporters are under common cellular control, and to what extent polyamine uptake by the different systems contributes to overall intracellular polyamine homeostasis.We have previously shown [1.5] that the uptake of putrescine into B16 melanoma cells is highly inducible by the ornithine decarboxylase inhibitor difluoromethylornithine (F,MeOrn). This increase in uptake is a common phenomenon Correspondence to ...

Transport of polyamines in Drosophila S2 cells: kinetics, pharmacology and dependence on the plasma membrane proton gradient

“…In contrast, neither putrescence nor cadaverine inhibited transport with high affinity. These data indicate that, unlike some other cultured cell lines [32], the spermine/spermidine transport activity in S2 cells is relatively insensitive to putrescine. We note that this does Table 1 Endogenous polyamine transport activity of Drosophila S2 cells Cell suspensions were assayed for transport activity at both 1 and 5 min.…”

Characterization of Spermidine Transport System in a Cyanobacterium,Synechocystis sp. PCC 6803

“…In some mammalian cell lines, such as rat pneumocytes [13,37], polyamine transport has been described to share a highly specific transport system by the different polyamines. However, many different polyamine transport systems have been described in hamster ovary [17] and in Escherichia coli [9]. The present study revealed that putrescine and spermine were able to significantly inhibit the uptake of spermidine in Synechocystis cells by about 40 % and 50 %, respectively ( Table 1).…”

Upregulation of Polyamine Transport in Human Colorectal Cancer Cells

“…While the difference in K m between putrescine and spermidine can exist in a single transporter, the differences in normal/induced uptake indicate different characteristics of the carrier transporting each of the polyamines, suggesting different carriers. The existence of at least two transporters has been proposed by other groups as well [19][20][21] There is a debate whether the inward and outward transport of polyamines are mediated by similar or different carriers. Wallace et al suggest the existence of separate transporters for uptake and export of polyamines after showing effects on the former but not on the later using competitive polyamine-uptake inhibitors on human cancer cells [22].…”