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The regulation of putrescine transport in difluoromethylornithine-treated B16 melanoma cells by extracellular Ca2+ has been investigated. It was found that physiological concentrations of Ca2+ were essential for optimum uptake of putrescine and spermidine. Mg2+, albeit at higher concentrations, also could potentiate polyamine transport. The maximum rate of putrescine uptake increased from 1698 +/-: 67 pmol/min/mg DNA in the absence of Ca2+ to 3100 +/- 98 pmol/min/mg DNA in the presence of 0.5 mM Ca2+. There was no change in K-m. While Ca2+ enhanced transport of both putrescine and spermidine it did not affect the uptake of deoxyglucose, thymidine or leucine. Putrescine did not alter Ca2+ fluxes suggesting that the two cations do not share a common transport system. The effects of Ca2+ on putrescine uptake appeared to be mediated extracellularly firstly because Ca2+ did not potentiate putrescine uptake in the presence of A23187 and secondly, because the effects of Ca2+ were completely inhibited by the lanthanide Tb3+, which binds to calcium-dependent proteins and does not readily cross biological membranes. Ca2+ did not affect putrescine transport in the absence of extracellular Na+. Moreover, the rate of putrescine uptake in the absence of Ca2+ was similar to that in the absence of extracellular Na+. The results from this study indicate that polyamine transport is stimulated by extracellular Ca2+ and suggest that Ca2+ is required for activity of the Na+-dependent transporter only. This transporter appears to possess a regulatory binding site for divalent cations. (C) 1997 Elsevier Science Ltd.

Previous studies in which investigators have induced the rate of polyamine uptake in vitro have used either inhibitors of polyamine biosynthesis or growth factors that induce cell proliferation. Recently, however, we have described the induction of putrescine uptake in cultured adult mouse hepatocytes and have shown that uptake is independent of both intracellular polyamine levels and proliferation. Although proliferation was not apparent in those studies, data suggested that, after isolation, the cells entered G of the cell cycle. In this study, we have examined whether the induction of putrescine uptake is a function of entry into the cell cycle and whether uptake activity is essential for optimal progression into the S phase. Using ribonuclease reductase subunit M1 as a marker of entry into the cell cycle, we have shown that hepatocytes enter G during the first 4 hr of culture. Both putrescine uptake and ornithine decarboxylase activity increased as the cells entered G. Treatment of the cells with retinoic acid (10 to 33 μmol/L) prevented them from entering G and also inhibited the induction of the putrescine transporter by up to 90%. In contrast, initiation of G to S phase transition markedly down‐regulated the activity of the transporter. Thus induction of the putrescine transporter in isolated hepatocytes appears to be a G‐specific event. Culturing the hepatocytes in the presence of 1,1′‐bis[3‐(1′‐methyl‐[4,4′‐bipyridinium]‐1‐yl)‐propyl]‐4,4′‐bipyridinium, a potent competitive inhibitor of putrescine uptake, resulted in a 47% decrease in intracellular putrescine. Measurement of the distribution of tracer H polyamines showed a loss of intracellular polyamines and an accumulation of extracellular polyamines when cells were treated with 1,1′‐bis[3‐(1′‐methyl‐[4,4′‐bipyridinium]‐1‐yl)‐propyl]‐4,4′‐bipyridinium, indicating that the re‐uptake of effluxed polyamines contributes to intracellular polyamine homeostasis in cultured hepatocytes. DNA synthesis was significantly inhibited in 1,1′‐bis[3‐(1′‐methyl‐[4,4′‐bipyridinium]‐1‐yl)‐propyl]‐4,4′‐bipyridinium–treated cells, and this effect was completely reversed by the addition of 200 μmol/L extracellular putrescine. We concluded that putrescine uptake is important for maintaining high intracellular putrescine levels required for optimal G to S phase transition in isolated mouse hepatocytes. (HEPATOLOGY 1991;14:1243–1250.)

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 69-75 microM, whereas the estimated Km for putrescine uptake was only 5.36 microM. 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 0.125 microM. 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-nitrobenzoyl)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.

Uptake of polyamines by cultured cells has been shown to be influenced by growth rate and/or differentiation. In this study, we have investigated whether the fully differentiated, non-proliferating adult mouse hepatocyte is capable of accumulating extracellular putrescine. When hepatocytes were cultured from 4 to 48 h, uptake of putrescine was found to increase substantially with time spent in culture. The Vmax for putrescine uptake increased 22-fold during this period with no change in apparent Km. Treatment of the cells with cycloheximide or actinomycin D at concentrations that did not affect cell viability inhibited the induction of putrescine uptake. Endogenous putrescine levels increased from 19.7 nmol/mg DNA after 4 h in culture to over 500 nmol/mg DNA after 48 h in culture. This increase was accompanied by a loss of over 90% of ornithine decarboxylase activity. Spermidine levels did not change over this time period, whereas spermine levels decreased by 35%. Difluoromethylornithine prevented the observed increase in intracellular putrescine but did not affect putrescine uptake. The increase in putrescine transport was not inhibited by culturing the hepatocytes in a high concentration of putrescine, spermidine or spermine. Moreover, the induction process was not stimulated by foetal calf serum but was selectively inhibited by the differentiating agents dimethylsulfoxide and retinoic acid. The results from those studies show that cultured mouse hepatocytes express a putrescine transport system that is poorly regulated by extracellular polyamines. The expression of the transporter requires the synthesis of mRNA and protein, and appears to be related to a time-dependent change in hepatocyte phenotype.

Serine dehydratase activity is absent from the rat foetal liver and normally appears in the immediate postnatal period. In foetal hepatocytes cultured from livers of various gestational ages, enzyme activity can be induced only in the simultaneous presence of dexamethasone and dibutyryl cyclic AMP in the culture medium. Adrenalin and glucagon can replace dibutyryl cyclic AMP. Actinomycin D and cordycepin both repress the response, a result that suggests the induction of enzyme synthesis involves the initial transcription of the enzyme gene(s). Inducibility is assessed in cultures prepared from foetuses aged between 15 and 19 days of gestation after 48 h of culture. No induction is obtained in cells from 15 day foetuses, only a marginal induction from 16 day foetuses, and a substantial induction from older foetuses. In cultures from older foetuses, 6–18 fold inductions are already demonstrable after 24 h of culture. While hepatocytes from more mature foetuses are able to acquire inductibility during culture, cells taken from 15 day foetuses do not develop in the same manner in spite of being maintained under identical conditions. These results suggest that a differentiation event occurs in vivo at about day 16 of foetal development which renders the hepatocyte inducible when cultured. Cells taken prior to this stage do not appear to acquire inducibility. This system represents a case of enzymic differentiation and requires the simultaneous presence of two inducer molecules. The mechanism of induction may represent a unique system in cellular differentiation.

Several bipyridinium, tetrapyridinium and hexapyridinium quaternary salts have been found to be potent inhibitors of putrescine uptake into B16 melanoma cells which had previously been treated with difluoromethylornithine. In general, the potency of inhibitors increased as the number of quaternary centres increased. A relationship between the distance apart of the positively charged nitrogen atoms and the potency of the salts as inhibitors of uptake has been established by comparison with a number of diaminoalkanes. It was found that an inter-nitrogen distance of 0.6-0.7 nm or 1.0-1.1 nm was optimal for high activity. This finding is significant in determining structural features of the polyamine transport system.

We have examined the effect of difluoromethylornithine on the ability of B16 melanoma cells to take up putrescine and the 4,4'-dipyridyl herbicide paraquat. Pretreatment with difluoromethylornithine for 24 hr enhanced putrescine uptake by inducing the maximum capacity of the transport system without affecting the Km for the substrate. Paraquat uptake was minor compared with that of putrescine and was not affected by difluoromethylornithine. Neither putrescine, spermidine or spermine at concentrations up to 100 microM inhibited the accumulation of paraquat. However, paraquat competitively inhibited putrescine transport (Ki = 54 +/- 10 microM). Exposure of the B16 melanoma cells for 24 hr to increasing concentrations of paraquat produced a dose-dependent inhibition of DNA synthesis. Difluoromethylornithine pretreatment did not affect paraquat toxicity. These data show that paraquat is not taken up into B16 melanoma cells by the uptake system responsible for transporting putrescine. Moreover, it is likely that the difluoromethylornithine inducible polyamine transport system in B16 melanoma cells is characteristically different to that previously described in normal mammalian lung since the latter is reportedly capable of transporting both putrescine and paraquat.