Characterization of the inducible polyamine transporter in bovine lymphocytes

Abstract

The polyamine uptake system in bovine lymphocytes was activated by concanavalin A. The system was common to putrescine, spermidine and spermine. The K, values for uptake activities of putrescine, spermidine and spermine were 3.7 pM, 0.38 pM and 0.23 pM in that order. The uptake activity was inhibited by carbonyl cyanide m-chlorophenylhydrazone, gramicidin D or valinomycin in the presence of 20 mM IS+ suggesting that polyamine uptake depends on the membrane potential. The uptake activity appeared 10 h after addition of concanavalin A, and the maximum was reached at 28 h indicating that induction of the polyamine transporter precedes the initiation of DNA synthesis. Addition of polyamine antimetabolites, such as cr-difluoromethylornithine and ethylglyoxal bis(guanylhydrazone), to the medium enhanced at least eightfold the induction of the polyamine transporter. The induction was repressed by addition of 50 pM spermidine or spermine, but not putrescine. We propose here that the induction of the membrane-potential-dependent polyamine transporter is regulated by the intracellular level of spermidine and spermine.Polyamines (putrescine, spermidine and spermine) are organic cations widely distributed in nature. The significance of polyamines in cell growth and metabolic processes has been well recognized (for reviews, see [l-31). Increases in polyamine contents are observed in various cells stimulated to proliferate.While cellular polyamines are mainly produced via de nova synthesis, the specific polyamine transport system has been demonstrated in both prokaryotes [4, 51 and eukaryotes [6 -91. Pohjanpelto (61 reported that putrescine uptake is greatly increased in human fibroblasts stimulated to proliferate. In mouse mammary explants [7] it has been shown that polyamine transport can be stimulated by insulin and prolactin, hormones that stimulate growth of explants. In human skin fibroblasts [8], epidermal growth factor stimulated both ornithine decarboxylase and polyamine transport. Thus, the physiological significance of polyamine uptake as well as polyamine biosynthesis is considered to increase intracellular polyamine levels during cellular responses to proliferative stimuli.The polyamine biosynthetic pathway is very highly regulated by the polyamines themselves [3, 101. An increase in the intracellular content of polyamines reduces the activity of ornithine decarboxylase, the first enzyme in polyamine biosynthesis, and this reduction by polyamines is controlled mainly at the translational level [ll]. However, the molecular basis of regulation of the polyamine transport system is still not known.In the present study we examined the iiiducibility of the polyamine uptake system in bovine lymphocytes, and found that induction of the polyamine transporter is regulated by the intracellular level of spermidine and spermine.Correspondence to K. Igarashi, Faculty of Pharmaceutical Sciences, Inohana Campus, Chiba University, 1-8-1 Inohana, Chiba, Japan 280Abbreviations. ConA, concanavalin A; DFMO, cr-difluoromethylornithine...

Differential post‐transcriptional control of ornithine decarboxylase and spermidine‐spermine N1‐acetyltransferase by polyamines

“…The results illustrate how polyamine catabolism as regulated by SSAT and polyamine biosynthesis as regulated by ODC can be rapidly and simultaneously controlled by intracellular pools. These responses, together with that of polyamine transport which is also known to be regulated by polyamines [7][8][9], function in a coordinated manner to sensitively maintain intracellular polyamine pools at a relatively constant level [3]. Such a homeostatic system would ensure an adequate supply of polyamines for cell growth while at the same time preventing cellular toxicity due to polyamine excess.…”

“…These effectors are short-lived and differentially regulated by intracellular polyamine pools [3]. Exposure to exogenous polyamines causes polyamine biosynthesis (reviewed in [5,6]) and uptake [7][8][9] to decrease and polyamine catabolism to increase *Corresponding author. Fax: (1) (716) 845-8857.…”

Regulation of spermidine/spermine N1‐acetyltransferase by intracellular polyamine pools

“…These data indicate that, under basal conditions, steady-state levels of SSAT expression are probably maintained in a partially up-regulated state by basal polyamine pools since a decline in these pools results in a decrease in SSAT activity and mRNA. Polyamine transport and the polyamine biosynthetic enzymes, ODC and SAMDC, seem to be similarly maintained under basal conditions but via an inverse relationship since a decline in pools causes an increase in their expression [1,26] and vice versa [ 1,2].…”

“…The former was apparent from experiments with exogenous polyamines and the latter by pool depletion with specific enzyme inhibitors. The biosynthetic enzymes, ODC and SAMDC [l], and the polyamine transport system [2,31,32,35], are also sensitively upregulated and down-regulated by changes in intracellular polyamine pools. Unlike SSAT, however, the regulatory relationships for these systems are negative in nature -as pools decrease, enzyme and transport activities increase and vice versa.…”

Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells

“…Several investigators have shown that cells fed with spermidine or spermine will rapidly incorporate these until intracellular polyamine levels are slightly above normal (1-2 h), at which time uptake is abruptly terminated [14][15][16][17]. We [18] and others [19] have demonstrated that such attenuation of polyamine uptake could be prevented by the simultaneous inhibition of protein synthesis with cycloheximide.…”