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PubMed2000

Polyamine uptake in cultured astrocytes: characterization and modulation by protein kinases.

The properties and regulation of the polyamine transport system in brain are still poorly understood. The present study shows, for the first time, the existence of a polyamine transport system in cerebellar astrocytes and suggests that polyamine uptake is mediated by a single and saturable high-affinity transport system for putrescine, spermine, and spermidine (K:(m) = 3.2, 1.2, and 1.8 microM:, respectively). Although substitution of NaCl by choline chloride produced a decrease in the putrescine, spermine, and spermidine uptake, it seems that polyamine transport in cerebellar astrocytes is not mediated by an Na(+) cotransport as in the presence of Na(+) and cholinium, polyamine uptake was much lower than when measured in a sucrose-based medium. On the other hand, ouabain, gramicidin (a Na(+) ionophore), and ionomycin (a Ca(2+) ionophore) produced a strong inhibition of polyamine uptake, suggesting that membrane potential could have an important role in the functioning of the astroglial polyamine uptake system. Moreover, protein kinase C inhibition produced an enhancement of polyamine uptake, whereas stimulation of protein kinase C with phorbol esters inhibited polyamine uptake. Alternatively, the tyrosine kinase inhibitor genistein caused a marked reduction in the uptake. No effects on polyamine uptake were observed with inhibitors and activators of cyclic AMP-dependent protein kinase or when Ca(2+)/calmodulin-dependent protein kinase II was inhibited with KN-62. These results suggest that the polyamine uptake system in cerebellar astrocytes could be modulated by protein kinase C and tyrosine kinase activities.

Animals↗
PubMed1998

Agmatine suppresses proliferation by frameshift induction of antizyme and attenuation of cellular polyamine levels.

Polyamines are required for entry and progression of the cell cycle. As such, augmentation of polyamine levels is essential for cellular transformation. Polyamines are autoregulated through induction of antizyme, which represses both the rate-limiting polyamine biosynthetic enzyme ornithine decarboxylase and cellular polyamine transport. In the present study we demonstrate that agmatine, a metabolite of arginine via arginine decarboxylase (an arginine pathway distinct from that of the classical polyamines), also serves the dual regulatory functions of suppressing polyamine biosynthesis and cellular polyamine uptake through induction of antizyme. The capacity of agmatine to induce antizyme is demonstrated by: (a) an agmatine-dependent translational frameshift of antizyme mRNA to produce a full-length protein and (b) suppression of agmatine-dependent inhibitory activity by either anti-antizyme IgG or antizyme inhibitor. Furthermore, agmatine administration depletes intracellular polyamine levels to suppress cellular proliferation in a transformed cell line. This suppression is reversible with polyamine supplementation. We propose a novel regulatory pathway in which agmatine acts as an antiproliferative molecule and potential tumor suppressor by restricting the cellular polyamine supply required to support growth.

3T3 Cells↗
PubMed2004

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity.

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.

Animals↗
PubMed1997

Oral administration of deuterium-labelled polyamines to sucking rat pups: luminal uptake, metabolic fate and effects on gastrointestinal maturation.

Non-physiological amounts of oral polyamines have been reported to induce precocious gut maturation in rat pups. The aim of the present study was to investigate organ distribution and metabolic fate of orally administered stable-isotopically labelled polyamines in rat pups. Pups received tetradeuterium-labelled putrescine (Pu-d4; 3 mumol), spermidine (Sd-d4; 5 mumol), spermine (Sp-d4; 3 mumol), or physiological saline twice daily on postnatal days 7-10 or 12-15. They were killed on days 10 and 15. We determined activities of ileal lactase (EC 3.2.1.23), maltase (EC 3.2.1.20), sucrase (EC 3.2.1.48) and diamine oxidase (EC 1.4.3.6) and established villus and crypt lengths. Polyamines and their labelling percentages in organs were determined by GC and mass fragmentography. Treatments did not affect growth rate, but caused lower weights of liver, kidneys and heart. Maltase activity increased, lactase decreased, whereas sucrase and diamine oxidase did not change. Villus and crypt lengths increased. Organ polyamine pools were labelled to different extents. Irrespective of the orally administered polyamine, all organs contained Pu-d4, SD-d4 and Sp-d4. Administered Pu-d4 and Sd-d4 were recovered mainly as Sd-d4, whereas Sp-d4 was recovered as Sp-d4 and Sd-d4. Total polyamines in a caecum, colon and erythrocytes increased, but increases were only to a minor extent with regard to labelled polyamines. Our data confirm precocious gut maturation by exogenous polyamines. Putrescine appears to be limiting factor. The exogenous polyamines were distributed among all investigated organs. They are not only used for the synthesis of higher polyamines, but also retroconverted to their precursors. Changes in erythrocyte polyamine contents suggest precocious stimulation of erythropoiesis.

Amine Oxidase (Copper-Containing)↗
PubMed1997

Fasting and postprandial polyamine concentrations in the human digestive lumen.

Polyamines are essential to cellular proliferation and differentiation. The gastrointestinal tract could represent a major source of polyamines in the body; however, there is little information regarding the presence of polyamines in the human intestinal chyme, and the source of these intraluminal polyamines remains unclear. The aims of our study were to determine the concentrations and flow rates of polyamines in the human intestinal lumen and to estimate the contribution from food to these concentrations. Polyamine concentrations and flow rates were determined after 12 h of fasting in jejunal (n = 25) and ileal (n = 9) effluents collected by the slow-marker perfusion technique. Kinetic studies were performed after water ingestion (no polyamines) in the jejunum (n = 6) and ileum (n = 5) and in the jejunum after a yogurt test meal (polyamine content: 2.8 mumol putrescine, 2.1 mumol cadaverine, 2.1 mumol spermidine, and 1.9 mumol spermine; n = 9). There were significant polyamine concentrations in the lumen of the human gut during the fasting state, suggesting endogenous secretion. Higher polyamine concentrations were observed in the jejunum than in the ileum (P < 0.05), suggesting proximal absorption. Kinetic studies showed a 25% transitory increase in the jejunal putrescine flow rate after ingestion of the yogurt test meal, suggesting that dietary polyamines are fully absorbed.

Adult↗
PubMed2000

Dietary soy protein is associated with reduced intestinal mucosal polyamine concentration in male Wistar rats.

Quantitation of polyamine levels has been correlated with biomarkers of proliferation in the colon mucosa where dysregulated epithelial hyperproliferation is associated with colorectal cancer risk. This study was performed to assess the response of polyamine measurements to dietary factors in an animal model. Male Wistar rats were fed purified diet or diets substituted by 20% lard fat, 20% beet fiber and 20% soy protein. After 2 wk, mucosal polyamines were measured along intestinal tracts by HPLC. In rats fed the control diet (n = 10), mucosal polyamines were found at high levels in the duodenum, jejunum and ileum but at low levels in the cecum, colon and rectum. Compared with rats fed the control diet, those fed the 20% lard diet showed greater polyamine levels in the large intestine (P < 0.05, n = 10), but those fed the 20% fiber diet exhibited lower polyamine levels in the small intestine (P < 0.05, n = 9). However, rats fed the 20% soy protein diet had lower polyamine levels in both small and large intestines (P < 0.05, n = 15). Significant linear correlations were observed between rectal polyamine levels and the dietary energy intakes in these four diet groups (r = 0.972-0.991, P < 0.001). Supplementation of 0.1% soy isoflavones to the basal diet or 0.3% DL-methionine to the 20% soy protein diet for 4 wk did not affect polyamine levels. The results indicate that soy protein reduced mucosal polyamine levels, at least in part, through reduction of energy intakes. Further studies are warranted to verify that polyamine levels in intestinal mucosa are useful as an intermediate endpoint of the dietary risk factors.

Animals↗
PubMed1993

Effects of novel polyamines on cell-free polypeptide synthesis catalyzed by Thermus thermophilus HB8 extract.

Effects of novel, naturally occurring polyamines on protein synthesis catalyzed by Thermus thermophilus cell-free extract were investigated. The results revealed the physiological importance of a branched quaternary polyamine, tetrakis(3-aminopropyl) ammonium, in thermophile protein biosynthesis. Longer polyamines than triamine supported the polypeptide synthesis at high temperature, though both the activity and the optimum temperature varied depending on polyamines added. The highest activity was found when tetrakis(3-aminopropyl)ammonium and a tetraamine were simultaneously present. The optimum temperature of the reaction supported by the combination of the branched polyamine and spermine was the highest and in accord with the optimum temperature of the bacterial growth. These results suggested an essential role of the quaternary amine in protein synthesis in vivo. This amine effectively stabilized the ternary complex between ribosomes, the messenger, and phenylalanyl-tRNA, and this stabilization may account, at least in part, for its action on the present reaction. In contrast, another branched polyamine, tris(3-aminopropyl)amine supported the activity only moderately even in the presence of another polyamine, though the tris amine stabilized the ternary complex as effectively as the quaternary amine. This result suggests the presence of another essential site for polyamine action in the thermophile polypeptide synthesis, in addition to the stabilization of the ternary complex. The effects of polyamines on MS2 RNA directed reaction resembled those on poly(U) directed polypeptide synthesis, indicating that polyamines are essential in protein biosynthesis directed by natural messengers in vivo. The quaternary amine inhibited the aminoacylation of tRNA(Phe), and the inhibition was canceled by the addition of another polyamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed2004

Polyamine synthesis from proline in the developing porcine placenta.

Polyamines (putrescine, spermidine, and spermine) are essential for placental growth and angiogenesis. However, little is known about polyamine synthesis in the porcine placenta during conceptus development. The present study was conducted to test the hypothesis that arginine and proline are the major sources of ornithine for placental polyamine production in pigs. Placentae, amniotic fluid, and allantoic fluid were obtained from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, and 110 of the 114-day gestation (n = 6 per day). Placentae as well as amniotic and allantoic fluids were analyzed for arginase, proline oxidase, ornithine aminotransferase (OAT), ornithine decarboxylase (ODC), proline transport, concentrations of amino acids and polyamines, and polyamine synthesis using established radiochemical and chromatographic methods. Neither arginase activity nor conversion of arginine into polyamines was detected in the porcine placenta. In contrast, both proline and ornithine were converted into putrescine, spermidine, and spermine in placental tissue throughout pregnancy. The activities of proline oxidase, OAT, and ODC as well as proline transport, polyamine synthesis from proline, and polyamine concentrations increased markedly between Days 20 and 40 of gestation, declined between Days 40 and 90 of gestation, and remained at the reduced level through Day 110 of gestation. Proline oxidase and OAT, but not arginase, were present in allantoic and amniotic fluids for the production of ornithine (the immediate substrate for polyamine synthesis). The activities of these two enzymes as well as the concentrations of ornithine and total polyamines in fetal fluids were highest at Day 40 but lowest at Days 20, 90, and 110 of gestation. These results indicate that proline is the major amino acid for polyamine synthesis in the porcine placenta and that the activity of this synthetic pathway is maximal during early pregnancy, when placental growth is most rapid. Our novel findings provide a new base of information for future studies to define the role of proline in fetoplacental growth and development.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1988

Polyamine levels and gastrin receptors in colon cancers.

Polyamines and gastrin receptors (GR) were studied in samples of colon cancer and mucosa from 40 patients and in control mucosa from 11 patients without cancer. Polyamines (i.e., putrescine, spermidine, spermine) are essential for growth and differentiation. The concentration of polyamines is elevated in rapidly proliferating normal tissues and in some cancers. The presence of GR in human colon cancers has been previously reported. The purpose of the present study was twofold: (1) to determine whether polyamine levels are elevated in colon cancers and in adjacent normal colon mucosa compared to colon mucosa from patients without cancer; and (2) to examine the relationship between polyamine levels and GR in colon cancers. Polyamine levels in colon cancers were significantly higher than in the normal colon mucosa from the same patients. The polyamines, spermidine and spermine, were significantly higher in colon mucosa from patients with cancer compared to patients without cancer. Spermidine and the spermidine:spermine ratio, an index of cell proliferation, were increased in colon cancers with GR compared to cancers without GR. There were no significant correlations between polyamine levels and the following: patient age, CEA level, site of cancer, stage, or differentiation. Because polyamine levels are increased in colon mucosa from patients with cancer, measurement of polyamines may detect patients at risk for subsequent development of colon cancer. Increased levels of polyamines in colon cancers with GR is evidence that gastrin may play a trophic role in human colon cancers.

Carcinoma↗
PubMed1997

Fasting gastric fluid and fecal polyamine concentrations in premature infants.

BACKGROUND: The role of milk polyamines in the development of the gastrointestinal tract of human infants is presently unknown. Polyamine concentrations are higher in human milk than in infant formulas. The aim of the present study was to gather data on luminal polyamines by measuring gastric fluid and fecal polyamine concentrations in premature infants during the postnatal period. We further compared gastric fluid polyamine concentrations with those reported for milk and looked for possible relationships between luminal polyamine concentrations, age, and growth rate. METHODS: High-performance liquid chromatography was used for the measurement of polyamine concentrations in both fecal and gastric fluid samples. RESULTS: Ninetieth centiles for gastric polyamines during the first week were 62, 28, 82, and 14 microM for putrescine, spermidine, spermine, and cadaverine, respectively. These values are higher than those reported for human milk and infant formulas. Polyamine concentrations were unrelated to either age or growth rate. Ninetieth centiles for fecal polyamines during the first week were 7668, 5176, 53, and 75 microM for cadaverine, putrescine, spermidine, and spermine, respectively. CONCLUSIONS: Fasting gastric fluid polyamine concentrations in premature infants are higher than those reported for either human milk or infant formulas. The high fecal cadaverine and putrescine concentrations are probably of bacterial origin.

Aging↗
PubMed2003

NF-kappaB-mediated IAP expression induces resistance of intestinal epithelial cells to apoptosis after polyamine depletion.

Apoptosis plays a crucial role in maintenance of intestinal epithelial integrity and is highly regulated by numerous factors, including cellular polyamines. We recently showed that polyamines regulate nuclear factor (NF)-kappaB activity in normal intestinal epithelial (IEC-6) cells and that polyamine depletion activates NF-kappaB and promotes resistance to apoptosis. The current study went further to determine whether the inhibitors of apoptosis (IAP) family of proteins, c-IAP2 and XIAP, are downstream targets of activated NF-kappaB and play a role in antiapoptotic activity of polyamine depletion in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine not only activated NF-kappaB activity but also increased expression of c-IAP2 and XIAP. Specific inhibition of NF-kappaB by the recombinant adenoviral vector containing IkappaBalpha superrepressor (AdIkappaBSR) prevented the induction of c-IAP2 and XIAP in polyamine-deficient cells. Decreased levels of c-IAP2 and XIAP proteins by inactivation of NF-kappaB through AdIkappaBSR infection or treatment with the specific inhibitor Smac also overcame the resistance of polyamine-depleted cells to apoptosis induced by the combination of tumor necrosis factor (TNF)-alpha and cycloheximide (CHX). Although polyamine depletion did not alter levels of procaspase-3 protein, it inhibited formation of the active caspase-3. Decreased levels of c-IAP2 and XIAP by Smac prevented the inhibitory effect of polyamine depletion on the cleavage of procaspase-3 to the active caspase-3. These results indicate that polyamine depletion increases expression of c-IAP2 and XIAP by activating NF-kappaB in intestinal epithelial cells. Increased c-IAP2 and XIAP after polyamine depletion induce the resistance to TNF-alpha/CHX-induced apoptosis, at least partially, through inhibition of the caspase-3 activity.

Animals↗
PubMed1993

Role of polyamines in the regulation of proliferation and hormone production by insulin-secreting cells.

This paper focuses on the mechanisms regulating proliferation and insulin production by normal and tumoral pancreatic beta-cells. In particular, the evidence for involvement of polyamines is reviewed. Pancreatic islet cells contain high levels of polyamines, and based on findings obtained using enzyme-directed inhibitors, it appears that putrescine and spermidine are necessary for proinsulin biosynthesis, whereas spermine may exert a stimulatory or permissive role in RNA transcription-stabilization and long-term insulin release. Islet polyamine content is not altered by short-term secretory stimulation, nor is the acute secretory response impeded by polyamine synthesis inhibitors, making it unlikely that these amines play any major role in short-term insulin release. Various mitogens increase islet polyamine contents and DNA synthesis, but increases in cytosolic polyamines do not seem to mediate their mitogenicity. Nuclear polyamine content is not altered by the inhibitors, suggesting that maintenance of polyamines within this organelle may be sufficient to sustain elevated DNA synthesis. In tumoral RINm5F cells, polyamine depletion results in decreased proliferation and increased cellular content of insulin and insulin secretory granules without affecting insulin mRNA levels or translation. Moreover, polyamine-depleted RINm5F cells display improved substrate metabolism and sensitivity of the stimulus-secretion coupling. Possible levels of polyamine interaction with Ca2+ metabolism are discussed.

Animals↗
PubMed1998

Differences in transglutaminase mRNA after polyamine depletion in two cell lines.

Polyamines serve as natural substrates for the transglutaminase that catalyzes covalent cross-linking of proteins and is involved in cellular adhesion and proliferation. This study tests the hypothesis that intracellular polyamines play a role in the regulation of transglutaminase expression in rat small intestinal crypt cells (IEC-6 cell line) and human colon carcinoma cells (Caco-2 cell line). Treatment with alpha-difluoromethylornithine (DFMO; a specific inhibitor of polyamine synthesis) significantly depleted the cellular polyamines putrescine, spermidine, and spermine in both cell lines. In IEC-6 cells, polyamine depletion was associated with a decrease in the levels of transglutaminase mRNA. In Caco-2 cells, however, polyamine depletion significantly increased the levels of transglutaminase mRNA and enzyme activity. In both cell lines, ornithine decarboxylase mRNA levels increased and protooncogene c-myc mRNA decreased in the presence of DFMO. Addition of polyamines to cells treated with DFMO reversed the effect of DFMO on the levels of mRNA for these genes in both lines. There was no significant change in the stability of transglutaminase mRNA between control and DFMO-treated IEC-6 cells. In contrast, the half-life of mRNA for transglutaminase in Caco-2 cells was dramatically increased after polyamine depletion. Spermidine, when given together with DFMO, completely prevented increased half-life of transglutaminase mRNA in Caco-2 cells. These results indicate that 1) expression of transglutaminase requires polyamines in IEC-6 cells but is inhibited by these agents in Caco-2 cells, 2) polyamines modulate transglutaminase expression at the level of mRNA through different pathways in these two cell lines, and 3) posttranscriptional regulation plays a major role in the induction of transglutaminase mRNA in polyamine-deficient Caco-2 cells.

Animals↗
PubMed2005

Polyamines are necessary for synthesis and stability of occludin protein in intestinal epithelial cells.

Occludin is an integral membrane protein that forms the sealing element of tight junctions and is critical for epithelial barrier function. Polyamines are implicated in multiple signaling pathways driving different biological functions of intestinal epithelial cells (IEC). The present study determined whether polyamines are involved in expression of occludin and play a role in intestinal epithelial barrier function. Studies were conducted in stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) associated with a highly differentiated phenotype. Polyamine depletion by alpha-difluoromethylornithine (DFMO) decreased levels of occludin protein but failed to affect expression of its mRNA. Other tight junction proteins, zonula occludens (ZO)-1, ZO-2, claudin-2, and claudin-3, were also decreased in polyamine-deficient cells. Decreased levels of tight junction proteins in DFMO-treated cells were associated with dysfunction of the epithelial barrier, which was overcome by exogenous polyamine spermidine. Decreased levels of occludin in polyamine-deficient cells was not due to the reduction of intracellular-free Ca(2+) concentration ([Ca(2+)](cyt)), because either increased or decreased [Ca(2+)](cyt) did not alter levels of occludin in the presence or absence of polyamines. The level of newly synthesized occludin protein was decreased by approximately 70% following polyamine depletion, whereas its protein half-life was reduced from approximately 120 min in control cells to approximately 75 min in polyamine-deficient cells. These findings indicate that polyamines are necessary for the synthesis and stability of occludin protein and that polyamine depletion disrupts the epithelial barrier function, at least partially, by decreasing occludin.

CDX2 Transcription Factor↗
PubMed2001

NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells.

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.

Animals↗
PubMed2000

EGF-stimulated polyamine accumulation in the colon carcinoma cell line, Caco-2.

BACKGROUND: Polyamines (putrescine, spermidine and spermine) are ubiquitous molecules indispensable for cell proliferation. In the intestinal lumen they are present in high amounts. Polyamine accumulation in proliferating cells of the intestinal mucosa is high, and it occurs both by enhanced synthesis and by increased uptake from the lumen. AIMS: To study mitogen-induced polyamine accumulation in the gut, we treated proliferating Caco-2 cells with epidermal growth factor (EGF) and measured the activity of ornithine decarboxylase (ODC) and putrescine uptake. Furthermore, we investigated whether EGF-induced changes in the apical membrane could be responsible for the effect of EGF on polyamine uptake in Caco-2 cells. METHODS: Putrescine uptake, ODC activity and intracellular polyamine content were evaluated in the presence of 100 ng/ml EGF. To study the mechanisms of EGF-stimulated polyamine uptake, apical membrane vesicles were isolated, and putrescine uptake into the vesicles measured. Possible enrichment in brush border membrane cytoskeleton proteins (ezrin and villin) was assessed by Western blot. RESULTS: Treatment with EGF induced an increase in ODC activity, which occurred within the first minutes of treatment and reached peak values after 3 h. In contrast, an increase in putrescine uptake was more sustained, with peak levels at 12 h. Both synthesis and uptake contributed to an over 60% increase in intracellular putrescine and spermidine after EGF treatment. There were no detectable changes in apical membrane cytoskeleton (as concluded by the absence of ezrin and villin enrichment in EGF-treated Caco-2 cells). However, in apical membrane vesicles isolated from EGF-pretreated cells, putrescine uptake was enhanced twofold. CONCLUSIONS: EGF stimulates both synthesis and uptake of polyamines in Caco-2 cells. Enhanced synthesis seems to ensure rapid supply with polyamines in the earliest stages of growth, while the uptake is responsible for the maintenance of high polyamine intracellular levels during late growth phases. EGF-stimulated polyamine uptake is apparently not a consequence of structural changes in the apical membrane, but is likely to occur by a distinct EGF-induced alteration of the polyamine transporter itself.

Biological Transport↗
PubMed1990

Polyamine uptake by human colon carcinoma cell line CaCo-2.

The intracellular concentrations of the polyamines are highly regulated and high polyamine concentrations are associated with rapidly proliferating cells. Hormones, nutrients and growth factors that stimulate the proliferation of the intestinal epithelium, increase the intracellular polyamine concentration mainly by activating ODC expression. Other cell types stimulated to proliferate satisfy their requirement for polyamines by increasing polyamine uptake. In the present study, we investigated polyamine uptake by a human colon carcinoma cell line, CaCo-2. Uptake of putrescine, spermidine and spermine by CaCo-2 cells was saturable and temperature dependent and all polyamines appear to share a common carrier. The carrier of differentiated cells had an apparently higher affinity and lower activity than the carrier of replicating cells. Culture of CaCo-2 cells on porous filters showed that polyamine accumulation occurred mainly through the basolateral membrane in replicating cells, while an increase in the rate of apical uptake was observed after differentiation. A significant increase in polyamine uptake and in ODC expression resulted from fresh medium replacement, a well-known stimulus to proliferation; no change in uptake occurred after ODC inhibition by DFMO. We conclude that CaCo-2 cells are able to increase their polyamine concentration by both enhanced synthesis and increased polyamine uptake.

Biogenic Polyamines↗
PubMed1990

Polyamine metabolism and uptake during Phaseolus vulgaris lectin, PHA-induced growth of rat small intestine.

Kidney bean lectin, PHA, stimulated the hyperplastic and hypertrophic growth of rat small intestine. This growth was preceded by a rapid accumulation of polyamines in the small intestine. However, since the lectin had little effect on in situ polyamine biosynthesis, most of the polyamines must have been of extracellular origin. To investigate the source of polyamines, both the luminal uptake and basolateral transport of polyamines by the rat small intestine were measured in vivo. Luminal polyamine uptake was apparently by passive diffusion, non-saturable, linearly dependent on concentration and its extent was not stimulated by PHA. In contrast, 14C-polyamines injected intraperitoneally were probably taken up by a transport system(s). Moreover, basolateral polyamine transport was stimulated in a time-dependent manner when small intestinal growth was stimulated by PHA. However, in keeping with the finding of polyamine accumulation prior to demonstrable growth of the tissue, stimulation by PHA of the polyamine transport system also preceded small intestinal growth. Stimulation of polyamine transport by luminal factors is possibly a general mechanism involved in intestinal adaptation.

Adaptation, Physiological↗