In 1993, after a detailed analysis of more than 40 food items, Bardocz et al. reported that a typical human diet contributes hundreds of micromoles of polyamines per day to the gut lumen [1,2]. Subsequent studies of Sawada et al. were in accord with those observations and further indicated that ingested food is the major source of polyamines in the lumen of the upper small bowel in humans [3,4]. Shortly after a meal, polyamine concentrations in the duodenal and jejunal lumen reach almost millimolar levels, but as early as 120 min after the meal luminal polyamine content returns to the fasting level. In spite of the large amounts of polyamines arriving daily in the intestinal lumen, serum polyamine concentrations, albeit in healthy humans, are in the micromolar range . The mechanisms regulating this high supply/low utilization of luminal polyamines are at present still unclear. Disappearance of polyamines from the intestinal lumen is likely to occur due either to their rapid absorption, utilization in situ, or their rapid degradation in the gut. Regarding all described roles of luminal polyamines in normal, adaptive and neoplastic cell growth, the question of polyamine absorption/intracellular uptake in the gut and metabolic fate of luminal polyamines is of an utmost physiological and also clinical importance. The aim of this review is to summarize recent knowledge about polyamine uptake in the intestine, and to emphasize hitherto unexplored areas which deserve attention.
Disappearance of polyamines from the intestinal lumen
There is a general agreement that polyamine disappearance from the small intestinal lumen into the circulation is rapid and complete, occurring predominantly in the duodenum and proximal jejunum, and that luminal polyamines are indeed absorbed and utilized for growth processes throughout the body. For example, in the rat, luminal polyamine concentrations decreased progressively in the proximal-to-distal direction, but bore little relationship to mucosal polyamines, in either their original or metabolized form . In accord with this, radioactivity originating from luminal polyamines was detected in all growing tissues and organs, including tumour tissue, and skeletal muscle stimulated to grow by clenbuterol .
In the only study of this kind in humans reported to date , the small intestinal lumen of healthy volunteers was perfused with increasing concentrations of putrescine under steady state conditions, using a double lumen tube with proximal orifice some 30 cm distally from the ligament of Treitz. Some 60–80% of the infused putrescine disappeared, linearly with time, from the lumen. However, no putrescine per se was recovered in blood. Instead, there was a transitory increase in acetylated putrescine, and a mild but steady increase in spermidine and spermine concentration. The recovery of polyamines in blood after a ‘putrescine test meal’ was little more than 20% – which suggests that luminal putrescine when absorbed is metabolized – in the intestinal wall, in the liver, or both, and that only a small proportion of these metabolites spills over into the systemic circulation . The finding that polyamines cross the intestinal epithelial barrier by simple diffusion has been later confirmed in Caco-2 cells grown as monolayers on permeable filter supports, the model correlating well with in vivo perfusion studies . Small, charged and hydrophilic molecules like polyamines are usually absorbed via a paracellular pathway, and their absorption is facilitated by solvent drag . The results of the above studies, whilst they do not allow any firm conclusions to be drawn with respect to the bioavailability of dietary polyamines, do suggest that polyamines are absorbed from the intestinal lumen by passive diffusion, and undergo extensive metabolism before reaching the systemic circulation.
Mechanisms of polyamine uptake into intestinal epithelial cells
Although there is some controversy with regard to the kinetics of polyamine uptake in isolated cells compared with those in intact tissue , numerous in vitro studies have shown that polyamine uptake into enterocytes, across either apical or basolateral membranes, is saturable, temperature dependent, and sodium independent. In isolated rat enterocytes the Km values of uptake were about 20 μm for putrescine and 150 μm for spermidine – indicating that specific carriers are involved in the transport process. Putrescine accumulated more than 8-fold against a concentration gradient; its uptake was inhibited by KCN and various cations, not influenced by amino acids, and appeared to be shared by spermidine and spermine [12,13].
Uptake across the apical membrane of the enterocyte, in particular, has received much attention. In isolated brush border-membrane vesicle studies about 30% of polyamines apparently ‘taken up’ was non-specifically membrane bound. Uptake across the apical membrane appeared to be temperature dependent, pH dependent (the highest uptake being at physiological pH when polyamines are fully charged), and saturable. Transmembrane uptake also was Na+ independent, and inhibited by structurally and electrically related compounds. Km values were similar in all these studies (not higher than 20–30 μm) [14–20]. Thus it appears that polyamines are initially rapidly bound to the outer surface of the membrane, and subsequently, and relatively slowly, transported across the lipid bilayer via specific carriers.
Transport across the contraluminal pole of the enterocyte is also carrier mediated. In isolated basolateral membrane vesicles oriented predominantly right side out, the uptake of polyamines was associated with carriers of high affinity (Km not exceeding 12–13 μm) . Such transporters may well be responsible for the uptake of the minute amounts of polyamines normally present in blood, across the basolateral membrane of rapidly proliferating enterocytes. The extrusion process of polyamines across the basolateral membrane, in contrast, uses a low-affinity carrier, is more dependent on the electrochemical properties of the membrane and more sensitive to inhibition by cations, than the apical transporter. Inhibition of the extrusion process by structurally related substances was stronger when cations were present in the extravesicular medium .
In the basal state whether uptake is apical or basolateral may be unimportant. In proliferating cells, however, import across the basolateral membrane may become dominant. Indeed it has been shown that basolateral rather than apical uptake of polyamines is of great importance in rapidly proliferating cells of the small intestinal mucosa [22,23].
Mitogen induced stimulation and regulation of polyamine uptake in intestinal epithelial cells
The intestinal epithelium is one of most rapidly proliferating tissues in the body, and therefore, inherently, has a high demand for polyamines. During the last decade many studies showed that enhanced polyamine uptake in intestinal epithelial cells is as reliable a marker for cell proliferation as ornithine decarboxylase (ODC) activity. Enhanced uptake of putrescine by the small intestine has been associated with the lectin increased proliferation of the rat small intestine  and as an adaptive response of the gut to fasting . Uptake effects may be specific for particular polyamines: re-feeding of fasted rats had little effect on the already elevated putrescine uptake but increased spermidine uptake . This adaptive up-regulation of polyamine uptake exists also at the level of isolated membrane vesicles, in rats exposed to starvation by tumour , and in fasted rats . The factors determining the selectivity of intestinal polyamine uptake remain to be elucidated but it has recently been determined that the intestinal brush border contains at least three selective polyamine binding sites . Regulation of these diverse carrier systems may be dictated by particular phases of the cell cycle which in turn may vary with particular growth-inducing stimuli.
Thus polyamine uptake is highest in the proliferative stage of intestinal and colonic epithelial cells, as well as in colon cancer cells [26,27] and even weak growth stimuli, such as a change in cell culture medium  or deoxycholic acid , will induce significant up-regulation of polyamine uptake.
Epidermal growth factor (EGF), a potent growth stimulus present in both blood and intestinal lumen under in vivo conditions, evoked a 4-fold increase in putrescine uptake in proliferating Caco-2 cells and its rapid conversion to spermidine and spermine . This EGF stimulated putrescine uptake is thought to occur through recruitment of the polyamine carrier proteins from the cytosol surrounding the apical membrane, without apparent morphological changes of the putative polyamine carrier itself [30,31]. Other growth stimuli in the gut, such as insulin, may act in a similar way in enhancing polyamine uptake in the proliferating intestine .
The most abundant polyamine in the small intestinal and colonic lumen, putrescine, is not only rapidly taken up but also rapidly converted to metabolically active spermidine and spermine, and, in the cell, directed to the nucleus, where it interacts with oligopeptides and exerts its growth related actions . Treatment with EGF enhances not only putrescine synthesis and uptake, but also its conversion to spermidine and spermine; this occurs by up-regulation of SAMDC activity. Colon cancer cells treated with EGF rapidly convert both synthesized and absorbed putrescine, and therefore utilize the entire available polyamine pool .
It has been also postulated that polyamine uptake in intestinal epithelial cells could be regulated by amino acid supply, via a mechanism involving dysregulation of the protein regulating polyamine transport, the ODC antizyme. Exposure of Caco-2 cells to a medium deprived from l-methionine or l-leucine activated spermidine uptake, and decreased the intracellular level of ODC antizyme. However, exposure to limited amounts of amino acid increased transport without altering the ODC antizyme complex level. Therefore, it may be possible that antizyme independent mechanisms, sensitive to intracellular amino acid content, also may regulate polyamine uptake in intestinal epithelial cells [34,35].
As to in vivo conditions, ODC is paradoxically expressed along the crypt–villus axis, its activity being the highest not in rapidly proliferating crypt cells, but in fully differentiated cells of the villus tip . This may imply that luminal nutrients in some way induce ODC activity in villus cells. A recent study, investigating expression of ODC antizyme mRNA in small intestinal cells isolated from the villus tip, mid-villus, and the crypts including the crypt base, may provide some additional light on this problem . In this study, antizyme mRNA was found to be expressed in exactly the opposite order as is the case with ODC: it was the highest in the crypt, and almost non-detectable in the villus tip. In a subsequent study done in crypt-like IEC-6 cells, it was shown that antizyme mRNA falls to non-detectable levels after ODC activity was inhibited by α-difluoromethyl ornithine (DFMO), but that it started rising again some 4 h after the addition of exogenous putrescine. It was concluded that the inhibitory effect of ODC antizyme on both polyamine synthesis and uptake may be dependent on polyamine induced regulation of antizyme-gene transcription or mRNA stability, and that polyamines involved in this regulation originate exclusively from the extracellular sources. The inhibition of both synthesis and uptake is thought to be dependent on polyamine induced regulation of antizyme-gene transcription, and the polyamines involved in this regulation are believed to originate exclusively from extracellular sources .
Polyamines arrive in the intestinal lumen in almost millimolar concentrations, disappear from the lumen rapidly and completely, but their content in systemic circulation still hardly exceeds 10–20 μm. In spite of this apparent paradox of high supply/low utilization of luminal polyamines, it has been repeatedly shown that luminal polyamines are indeed absorbed in sufficient amounts and utilized for growth support throughout the body. In addition, they are involved in normal, adaptive and neoplastic epithelial cell proliferation in the gut. Finally, there is an interesting hypothesis that, after degradation to succinate, putrescine is utilized as a non-specific and instant energy source in the gut .
The mechanisms of polyamine absorption and their uptake into epithelial cells of the gut, resulting from the studies outlined in this article, are summarized in Figure 1. Because of the importance of polyamines in neoplastic cell growth, understanding the mechanisms of polyamine uptake is of utmost clinical relevance. The amount of polyamines available for neoplastic cell growth can be depleted by simultaneously reducing the dietary intake of polyamines and the bacterial generation of polyamines; by inhibiting cellular synthesis of polyamines and their uptake from the extracellular space; and by reducing the absorption of polyamines from the gut lumen.
To apply the knowledge on the mechanisms of the uptake and bioavailability of exogenous polyamines to clinical oncology may be the most important goal of polyamine research in the next decade.
The author is indebted to Professor Wolfgang F. Caspary for his lasting support; and to Susan Bardocz, Patrick Brachet, Alex R. Khomutov, John L. Mitchell, Jürgen Stein, Lyudmila Turchanowa, Edward Seidel and Jian-Ying Wang for inspiring discussions. The author’s own studies summarized in this article were done with generous support from the Alexander von Humboldt Foundation (Bonn, Germany) and the Special Trustees of Guy’s Hospital (London, UK).
• Of special interest
•• Of outstanding interest
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