Intracellular Polyamine Content Research Articles

Amifostine: a tonic or toxin to myeloid progenitors

It should come as no surprise, that the aminothiol prodrug, Amifostine (EthyolTM), whose actions as a hematopoietic promoter were recently realized, should now be considered to exert opposing biologic effects in leukemic cells [1]. A product of the United States Defense Department research emanating from the cold war, the relative selective cytoprotection afforded by amifostine to normal cells compared to tumor cells seemed at first glance somewhat difficult to accept. Indeed, relative differences in alkaline phosphatase distribution and pH within the tumor milieu appeared plausible [2–4], but insufficient to explain the agent’s ability to potentiate antineoplastic cytotoxicity in selected malignancies while conferring no protection to leukemic cells and protecting normal tissues [5–8]. The answer to this apparent dichotomy lies in amifostine’s chemical derivation and structural resemblance to endogenous polyamines, abundant but highly regulated organic cations involved in the homeostatic balance of cell death and survival. The dephosphorylated form of amifostine, WR-1065, and its symmetrical disulfide oxidation product, WR33278, display striking homology to spermidine and spermine, respectively [9,10]. In the oxidized extracellular environment, WR-33278 is believed to predominate, gaining entry into cells as a substrate for the polyamine facilitative transporter [11,12]. The activity of these transporters are strictly regulated by the feedback inhibitor, antizyme, which serves as a gate-keeper to limit intracellular polyamine content [13,14]. Within the cell, WR-33278 can be reduced to WR-1065. These novel aminothiols can then modulate cell behavior in two ways. They can compete with the naturally occurring polyamines for binding to DNA [15] and as substrates for amine oxidases [16]. Like endogenous polyamines, they induce the activity of spermine/spermidine acetyl transferase (SSAT), the first enzyme in polyamine catabolism, which is transcriptionally activated by the erythroid-specific transcription factor GATA-1 as an early event in erythroid differentiation [16,17]. In addition, these molecules can activate unique patterns of gene expression via polyamine-independent mechanisms, such as the redox sensitive tumor suppressor gene, p53 [18]. Like the polyamines, the cationic charge of the aminothiols facilitates their avid DNA binding, where they exert their many biologic activities including activation of early response genes, thymidine kinase, NFk-B, and other genes involved in the maintenance of genomic integrity [19–21]. Polyamines and polyamine analogues, like classical thiol antioxidants, afford protection against nuclear oxidation by serving as scavengers of reactive oxygen intermediates [22–24]. How then can we expect divergent effects on cell survival in normal, myelodysplastic and leukemic progenitors? Proliferating tumor cells maintain a high cellular polyamine content via ornithine decarboxylase-controlled biosynthesis; thereby blocking cell import by the induction of antizyme. Significant changes in polyamine homeostasis in tumor cells can be catastrophic, resulting in growth arrest and/or programmed cell death. Although active import of WR33278 is inhibited [25], passive inward diffusion of WR-1065 accelerates polyamine degradation by induction of SSAT, resulting in lowered apoptotic threshold. Sensing exceedingly high intracellular polyamine content, antizyme binds to and destabilizes ornithine decarboxylase, triggering its rapid proteosome degradation [26,27]. In normal cells where polyamine generation is not upregulated, the disulfide is actively imported into the cell, affording added defense against oxidative stress and resistance to apoptosis induction. The latter is indeed the case for normal hematopoietic progenitors,

Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets.

This study was conducted to determine a role for cortisol in regulating intestinal ornithine decarboxylase (ODC) activity and to identify the metabolic sources of ornithine for intestinal polyamine synthesis in suckling pigs. Thirty-two 21-day-old suckling pigs were randomly assigned to one of four groups with eight animals each and received daily intramuscular injections of vehicle solution (sesame oil; control), hydrocortisone 21-acetate (HYD; 25 mg/kg body wt), RU-486 (10 mg/kg body wt, a potent blocker of glucocorticoid receptors), or HYD plus RU-486 for two consecutive days. At 29 days of age, pigs were killed for preparation of jejunal enterocytes. The cytosolic fraction was prepared for determining ODC activity. For metabolic studies, enterocytes were incubated for 45 min at 37 degrees C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 1 mM [U-(14)C]arginine, 1 mM [U-(14)C]ornithine, 1 mM [U-(14)C]glutamine, or 1 mM [U-(14)C]proline plus 1 mM glutamine. Cortisol administration increased intestinal ODC activity by 230%, polyamine (putrescine, spermidine, and spermine) synthesis from ornithine and proline by 75-180%, and intracellular polyamine concentrations by 45-83%. Polyamine synthesis from arginine was not detected in enterocytes of control pigs but was induced in cells of cortisol-treated pigs. There was no detectable synthesis of polyamines from glutamine in enterocytes of all groups of pigs. The stimulating effects of cortisol on intestinal ODC activity and polyamine synthesis were abolished by coadministration of RU-486. Our data indicate that an increase in plasma cortisol concentrations stimulates intestinal polyamine synthesis via a glucocorticoid receptor-mediated mechanism and that proline (an abundant amino acid in milk) is a major source of ornithine for intestinal polyamine synthesis in suckling neonates.

A cortisol surge mediates the enhanced polyamine synthesis in porcine enterocytes during weaning.

This study was conducted to determine whether a cortisol surge mediates the enhanced expression of intestinal ornithine decarboxylase (ODC) in weanling pigs. Piglets were nursed by sows until 21 days of age, when 40 pigs were randomly assigned into one of four groups (10 animals/group). Group 1 continued to be fed by sows, whereas groups 2-4 were weaned to a corn and soybean meal-based diet. Weanling pigs received intramuscular injections of vehicle solvent (sesame oil), RU-486 (a potent blocker of glucocorticoid receptors; 10 mg/kg body wt), and metyrapone (an inhibitor of adrenal cortisol synthesis; 5 mg/kg body wt), respectively, 5 min before weaning and 24 and 72 h later. At 29 days of age, pigs were used to prepare jejunal enterocytes for ODC assay and metabolic studies. To determine polyamine (putrescine, spermidine, and spermine) synthesis, enterocytes were incubated for 45 min at 37 degrees C in 2 ml Krebs-bicarbonate buffer containing 1 mM [U-(14)C]arginine, 1 mM [U-(14)C]ornithine, 1 mM [U-(14)C]glutamine, or 1 mM [U-(14)C]proline plus 1 mM glutamine. Weaning increased intestinal ODC activity by 230% and polyamine synthesis from ornithine, arginine, and proline by 72-157%. Arginine was a quantitatively more important substrate than proline for intestinal polyamine synthesis in weaned pigs. Administration of RU-486 or metyrapone to weanling pigs prevented the increases in intestinal ODC activity and polyamine synthesis, reduced intracellular polyamine concentrations, and decreased villus heights and intestinal growth. Our results demonstrate an essential role for a cortisol surge in enhancing intestinal polyamine synthesis during weaning, which may be of physiological importance for intestinal adaptation and remodeling.

Identification and characterization of testis specific ornithine decarboxylase antizyme (OAZ-t) gene: expression in haploid germ cells and polyamine-induced frameshifting.

Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. However, in the testis, where polyamines such as spermidine and spermine exist in high concentrations, their roles still remains to be elucidated. We have cloned a testis-specific gene encoding an ornithine decarboxylase antizyme known to control intracellular concentrations of polyamines in a feedback manner. The mRNA encoding the protein named ornithine decarboxylase antizyme in testis (OAZ-t) was specifically expressed in haploid germ cells. In contrast, the mRNA level of somatic ornithine decarboxylase antizyme 1 (OAZ1) decreased markedly at the late stages of haploid germ cell differentiation. OAZ-t mRNA was first observed in 23-day-old mice, whereas the OAZ-t protein was detected much later, at 35 days after birth. Further experiments on OAZ-t revealed that polyamines were capable of inducing a frameshifting at the frameshift sequence of OAZ-t mRNA, resulting in the translation of OAZ-t, as was the case with the somatic OAZ1. Transfection of OAZ-t cDNA inactivated the ornithine decarboxylase activity in the HEK293 cells. Results indicate that the expression of OAZ-t is controlled at both transcriptional and translational levels, and that OAZ-t likely plays a key role in spermatogenesis by regulating the intracellular concentration of polyamines in haploid germ cells.

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.

Characterization of the photoreceptor(s) responsible for the regulation of the intracellular polyamine level and the putative participation of heterotrimeric G-proteins in the signal transduction chain

In previous publications we have demonstrated a photoregulation of the intracellular polyamine content during chloroplast development. In the present paper action spectra of the changes in the intracellular polyamine levels of putrescine (Put), spermidine (Spd) and spermine (Spm) are presented, giving first evidence for the existence of three photoreceptor systems: a protochlorophyllide photoreceptor is possibly responsible for the inhibition of Put and Spd formation during chloroplast development; a blue-light photoreceptor that probably mediates in general the formation of polyamines, possibly in the semiquinone form in the case of Spm; and, finally, a red-light (possibly the PSII reaction centre) photoreceptor that could be responsible for the induction of the polyamine increase. The function and the physiological role of these three photoreceptor systems are discussed. Although chloroplast photodevelopment and the inhibition of polyamine biosynthesis have the same photoreceptor, it seems that this inhibition is not directly linked to the signal transduction chain of chlorophyll biosynthesis. However, there are hints that a separate transduction chain, in which heterotrimeric G-proteins are involved, leads to the inhibition of polyamine biosynthesis. Dark-grown cultures, supplemented with a non-hydrolysable GTP analogue, GTP-γ-S, as well as cells treated with cholera (CTX) and pertussis (PTX) toxins, simulate polyamine changes similar to those of cells grown under light qualities that do not significantly influence chlorophyll biosynthesis. A possible hypothesis for the mode of interaction of polyamine regulation and chloroplast development is presented.

The c-myc gene regulates the polyamine pathway in DMSO-induced apoptosis.

It is accepted that apoptosis is a gene-controlled process of cellular self-destruction. It occurs during physiological regulation and in pathological situations in the life of a cell. In the immune system, several different intracellular and extracellular factors have been associated with the induction of apoptosis, and the final responses depend on the cell system and the acquired signals. In lymphoid cells, dexamethasone-induced apoptosis is associated with c-myc downregulation in cells that remain in G0-G1 until the point of death. Ornithine decarboxylase (ODC), a key enzyme involved in polyamine biosynthesis, is regulated by c-myc, which is a transcriptional activator implicated not only in the control of cell proliferation and differentiation but also in programmed cell death. As dimethylsulphoxide (DMSO) induces apoptosis in the RPMI-8402 human pre-T cell line, the present study analysed the involvement of the c-myc proto-oncogene and polyamine pathway as mediators of apoptosis. Cell growth, programmed cell death, c-myc expression, ODC activity and intracellular polyamine content were detected after DMSO and difluoromethylornithine (DFMO) treatment. DMSO-treated cells exhibit a decrease in ODC activity and polyamine levels associated with cell growth arrest and programmed cell death induction. The expression of c-myc proto-oncogene, as its mRNA or protein, is specifically down-regulated. DFMO, a well defined polyamine biosynthesis inhibitor, completely blocks ODC activity, resulting in growth inhibition but not apoptosis. Moreover, in these samples no evidence of changes of c-myc expression were found. The results obtained suggest that, in RPMI-8402 cells, DMSO provokes a c-myc-dependent decrease of ODC activity followed by a depletion of intracellular polyamine levels, associated with programmed cell death and cell growth arrest.

Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells.

We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells.

Agmatine modulates polyamine content in hepatocytes by inducing spermidine/spermine acetyltransferase.

Agmatine has been proposed as the physiological ligand for the imidazoline receptors. It is not known whether it is also involved in the homoeostasis of intracellular polyamine content. To show whether this is the case, we have studied the effect of agmatine on rat liver cells, under both periportal and perivenous conditions. It is shown that agmatine modulates intracellular polyamine content through its effect on the synthesis of the limiting enzyme of the interconversion pathway, spermidine/spermine acetyltransferase (SSAT). Increased SSAT activity is accompanied by depletion of spermidine and spermine, and accumulation of putrescine and N1-acetylspermidine. Immunoblotting with a specific polyclonal antiserum confirms the induction. At the same time S-adenosylmethionine decarboxylase activity is significantly increased, while ornithine decarboxylase (ODC) activity and the rate of spermidine uptake are reduced. This is not due to an effect on ODC antizyme, which is not significantly changed. All these modifications are observed in HTC cells also, where they are accompanied by a decrease in proliferation rate. SSAT is also induced by low oxygen tension which mimics perivenous conditions. The effect is synergic with that promoted by agmatine.

Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation.

Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation Cell proliferation and transformation induced by growth factor stimulation or by carcinogens, viruses, or oncogenes are characterized by an associated increase in polyamine levels, which is mediated by increased polyamine biosynthesis and enhanced uptake of polyamines. Polyamine biosynthesis is catalyzed particularly, in the level of ornithine decarboxylase (ODC). The elevation of cellular polyamine levels on the other hand accelerates the induction of ornithine decarboxylase antizyme (antizyme), which is involved not only in ODC-degradation, but in the negative regulation of polyamine transport. Taking advantage of these characteristics of antizyme, the potential of antizyme as a factor having anti-cell growth and anti-tumor activity was investigated. We show that antizyme can induce cell death associated with a rapid decline of intracellular polyamine contents. The possible anti-tumor activities of ectopically expressed antizyme were tested in p21H-ras (Val 12)-transformed NIH3T3 cells and several human malignant cell lines including a line with loss of p53 expression, and they were shown to be as sensitive as nontransformed NIH3T3 cells in vitro. The in vivo anti-tumor activity was also tested using nude mice inoculated with H-ras transformed NIH3T3 cells that had been transfected with inducible antizyme expression vector and the results showed that antizyme expression in vivo blocks tumor formation in these mice. These results suggest that ectopic antizyme expression is of possible therapeutic benefit in the treatment of cancer, which is mediated by ODC inactivation and intracellular polyamine depletion.

Effects of methylacetylenic putrescine, an ornithine decarboxylase inhibitor and potential novel anticancer agent, on human and mouse cancer cell lines.

Sensitivity of several human and mouse cancer cell lines to methylacetylenic putrescine (MAP) was evaluated using clonogenic, sulforhodamine B and cell counting assays. The effects of MAP on cell morphology, cell cycle phase distribution and changes in polyamine metabolism of xenografted MCF-7 and MDA-MB-231 human mammary tumor cells were also investigated. On the basis of IC50 values, BHT-101 human thyroid carcinoma cells were the most sensitive (9 micrograms/ml), followed by P388 mouse lymphoma (32 micrograms/ml), MCF-7 (48 micrograms/ml) and MDA-MB-231 (110 micrograms/ml) human breast carcinoma cell lines. MAP treatment led to accumulation of P388 cells in G1 phase. At higher doses, the cytoplasm of the cells became vacuolated followed by apoptosis. The foamy cytoplasm may suggest a rare type of cell death (Clarke III type) called non-apoptotic programmed cell death. MAP treatment resulted in a total inhibition of ornithine decarboxylase (ODC) activity with a concomitant decrease of intracellular polyamine (mostly putrescine and spermidine) content in the breast cancer cells, whilst the spermine concentration was shown to increase. MAP proved at least 10 times more potent than the formerly studied DL-alpha-difluoromethylornithine making it an attractive candidate for clinical testing.

Effect of Retinoic Acid on Proliferation and Polyamine Metabolism in Cultured Bovine Retinal Pigment Epithelial Cells

Reports regarding the effect of all-trans-retinoic acid (RA) on the cell growth of retinal pigment epithelial cells (RPE) have been contradictory. The aims of this study are to clarify the in vitro effect of RA on RPE cells and to examine polyamine metabolism after RA stimulation. A 4-day incubation of fetal-calf-serum (FCS)-stimulated RPE cells with 10 or 25 µM RA significantly increased both cell number and [³H]thymidine incorporation. RPE cells grown over an extended period for 8 days also increased in number and reached full confluency. However, if the incubation was further extended to 12 days, no further increase in cell number was detected. RA treatment of FCS-stimulated RPE cells shifted the peak of ornithine decarboxylase (ODC) activity from 16 to 4 h. S-adenosylmethionine decarboxylase (SAMDC) activity and spermidine/spermine N¹-acetyltransferase (SAT) activity of RA-treated RPE cells were significantly greater until 8 and 16 h after incubation, respectively. The putrescine content was significantly increased in RA-treated RPE cells up until 24 h, while spermidine, spermine and N¹-acetylspermidine contents were significantly increased until 16 h. Our findings suggest that RA treatment increases the intracellular polyamine concentration of RPE cells via activation of ODC, SAMDC and SAT and that this results in the promotion of RPE cell growth until the cells reach full confluency.

Differential Changes in the Photosynthetic Pigments and Polyamine Content during Photoadaptation and Photoinhibition in the Unicellular Green Alga Scenedesmus obliquus

In the unicellular green alga Scenedesmus obliquus the level of photoinhibition and the recovery of the cells after reversal to the initial light conditions in relation to the pre-photoadaptation of the culture to low, medium and high light intensity was determined. The changes in the photosynthetic pigment content and in the intracellular polyamine concentration allowed to distinguish between photoadaptation and photoinhibition. In particular, the level of chlorophylls, xanthophylls and carotenoids decreased inversely proportional to the light intensity applied during photoadaptation, whereas their concentrations remained constant during photoinhibition. The violaxanthin/zeaxanthin and the loroxanthin/lutein cycle work only under photoinhibitory conditions, but not under photoadaptive premises. Changes in the level of these carotenoids in relation to the changes in the photosynthetic apparatus during photoadaptation are discussed. In addition, it was found that the intracellular polyamine level increased only under stress conditions, i. e. during photoinhibition, and decreased during recovery of the cells after reversal to the initial light conditions. The increase of the putrescine level during photoinhibition is inversely proportional to the light intensity used for pre-adaptation. This rise of the polyamine level in the cells photoadapted to high light conditions is an additional indication for the finding that photoadaptation and photoinhibition are different phenomena which are clearly distinguishable from each other. Finally, the changes of the chlorophyll, violaxanthin, zeaxanthin, loroxanthin, lutein and polyamine levels under photoadaptation in high light intensity (50 W m -2) in relation to the range of photoadaptation in Scenedesmus obliquus are discussed.

Premature stimulation of rat sucrase-isomaltase (SI) by exogenous insulin and the analog B-Asp10 is regulated by a receptor-mediated signal triggering SI gene transcription.

The mechanism(s) by which insulin enhance prematurely the activity of brush border membrane (BBM) hydrolases in rat immature intestine is unknown. Therefore, we have compared the responses of four BBM enzymes [sucrase-isomaltase (SI), maltase, lactase-phloridzine hydrolase (LPH), and aminopeptidase] with exogenous insulin, the analog B-Asp10, IGF-I, and antireceptor MAb [insulin-receptor (IR) MAb] given to preweaning pups. Low doses of insulin caused a precocious induction of SI and of SI mRNA and stimulated maltase activity without effect on LPH nor on aminopeptidase activities. IGF-I given at the same dose as that of insulin had no detectable effect on these enzymes. Administration to sucklings of IR MAb prevented the effect of endogenous insulin by inhibiting the expression of SI and maltase without effect on LPH activity. B-Asp10, an insulin analogue that exhibits in vitro a 3.5-fold increase in receptor affinity with sustained signaling of the receptor tyrosine kinase, caused an overexpression of SI by 3.5-fold and of maltase by 1.5-fold compared with equivalent doses of normal insulin. The premature increases in SI activity, SI mRNA, and maltase activity in response to insulin were dose-dependent and were associated with dose-dependent increases in intracellular spermine and spermidine concentrations. In conclusion, these data suggest that the premature induction of SI by insulin is mediated by a dose-dependent signal initiated by binding of the hormone to its intestinal receptor, which after transduction into the cell indirectly triggers the transcription of the SI gene, possibly by changes in intracellular polyamine concentrations.

Polyamine Uptake Across the Basolateral Membrane of the Enterocyte Is Mediated by a High-Affinity Carrier

Background/Aims: Polyamine uptake from the circulation plays an important role in the maintenance of the intracellular polyamine content during extensive proliferation in intestinal mucosal cells. Methods: Isolated basolateral membrane vesicles of the rabbit enterocyte were used to characterize polyamine transport across the basolateral side of the intestinal epithelium. Incorporation of spermidine and spermine into the basolateral membrane was rapid, although 30–60% of polyamines were initially bound to the basolateral membrane. In order to avoid the influence of binding on the actual uptake into the vesicles, polyamine incorporation was measured at 37 and 4°C, and kinetic parameters were calculated from the difference in polyamine incorporation rates at these temperatures. Results: Uptake kinetics was saturable, with K_m values of 13.34 and 12.35 µmol/l and V_max of 159 and 105 pmol/mg protein/min for spermidine and spermine, respectively. It was also temperature dependent, with Q₁₀ values (calculated between uptake velocities at 37 and 25°C) of 2.56 for spermidine and 1.90 for spermine. At physiological pH, polyamine uptake was at its highest. Since at this pH polyamines are fully charged, charge might be essential for polyamines to be taken up across the basolateral membrane. Polyamine uptake was inhibited by di-, tri- and tetracations, and there was no evidence for sodium cotransport. Transport of putrescine was not inhibited by spermine and spermidine, although spermidine inhibited spermine uptake in a competitive manner, with K_i of 127 µmol/l. Conclusion: These results imply that a saturable high-affinity transport system for polyamine does exist at the basolateral side of the enterocyte. Such a transport system may be responsible for the active transport of polyamine into rapidly proliferating enterocytes.

The antiproliferative effect of HGF on hepatoma cells involves induction of apoptosis with increase in intracellular polyamine concentration levels.

Hepatocyte growth factor (HGF) induced apoptosis and decreased the DNA synthesis in Hep G2 cells. In the HGF group interleukin-1 converting enzyme, ornithine decarboxylase (ODC) activity and intracellular polyamine concentrations were increased compared to those of the control group. Administration of the ODC inhibitor decreased polyamine concentration, and inhibited apoptotic changes in the cells. These changes were reversed by exogenous addition of polyamine. These findings suggest that one of the mechanisms by which HGF exerts its antiproliferative effect is induction of apoptosis and that increase in intracellular polyamine concentration may be one of the triggers of cell death.

Ability of bisbenzylputrescine and propanediamine analogs to modulate the intracellular polyamine pool of P388D1 cells.

The effects of a series of bisbenzyldiamine analogs have been tested on P388D1 cell line in vitro. Their effects on cell growth, polyamine oxidase (PAO) activity and intracellular polyamine content were determined. The cytotoxicity tests were performed in culture medium supplemented with 100 micromol/L aminoguanidine (I), 100 micromol/L aminoguanidine and 100 micromol/L N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527) (II), and finally 100 micromol/L aminoguanidine and 200 micromol/L D,L-difluoromethylornithine (DFMO) (III). The IC50 values under conditions I and III were similar, suggesting that inhibition of ornithine decarboxylase by DFMO did not affect the biological effect of our derivatives. Spermine and spermidine remained nontoxic in conditions I and III. However in the condition II, the toxicity of all tested compounds (excepted spermidine) was increased, suggesting that the inhibition of cellular PAO increased their toxicity. The enzymatic test of PAO showed that at high doses inhibition of this enzyme by putrescine analogs occurred, while the N-methylated propanediamine derivative increased the enzyme activity; however, these results do not correlate with cytotoxicity tests. When these derivatives were incubated for 48 h with the cells, all of them increased the cell content in putrescine (approximately 160%) and spermine (approximately 145%) and decreased the spermidine content (approximately 75%) without any modification of the total amount of polyamine. The correlation between the cytotoxic results and the intracellular polyamine determination shows that the increase in spermine content along with the inhibition of retroconverting PAO enzyme increases the toxic effect of tested compounds (including spermine), suggesting that spermine toxicity is more important in the absence of intracellular oxidation processes.

Glutamine depletion and increased gut permeability in nonanorectic, non-weight-losing tumor-bearing rats

BACKGROUND & AIMS: Glutamine is an essential amino acid for rapidly dividing cells such as enterocytes. The progress of cancer is associated with a decrease of arterial and muscle glutamine concentrations. The aim of this study was to test whether increasing tumor loads affect gut intracellular glutamine handling, protein turnover, and gut absorptive and barrier function. METHODS: Methylcholantrene-induced tumor-bearing rats were studied with a subcutaneous tumor load of 5%-15% or 15%-30% of body weight. Portal drained visceral net uptake or release of energy substrates, amino acids, and intestinal protein turnover were studied. Gut absorptive capacity and permeability was assessed by the urinary recovery of 3-O- methyl-D-glucose or lactulose-rhamnose ratio after an oral gavage. RESULTS: In tumor-bearing rats, the net uptake of energy substrates (ketones and glutamine) and net protein synthesis increased across the portal drained viscera, whereas mucosal glutamine concentrations decreased. Absorptive capacity remained unchanged in both tumor-bearing groups. However, the lactulose-rhamnose ratio increased with increasing tumor load, indicating loss of gut barrier function. This was not related to changes in villus height, crypt depth, or changes in mucosal cell populations but to decreased intracellular polyamine concentrations. CONCLUSIONS: The presence of a methylcholantrene tumor leads to altered mucosal glutamine metabolism and loss of gut barrier function possibly related to disturbed proliferation or differentiation of enterocytes. (Gastroenterology 1997 Jan;112(1):118-26)

Regulation by Spermine of Native Inward Rectifier K+ Channels in RBL-1 Cells

Polyamines have been shown to participate in the rectification of cloned inwardly rectifying potassium channels, a class of potassium channel proteins that conducts inward current more readily than outward current. Here, basophil leukemia cells were used to determine the effects of polyamines on a native, inwardly rectifying potassium current. Rat basophil leukemia cells were cultured in the presence of two different polyamine biosynthesis inhibitors, and both the electrophysiological properties and the polyamine levels were monitored. Treatment with alpha-difluoromethylornithine, a specific ornithine decarboxylase inhibitor, resulted in no significant change of electrophysiological properties. In contrast, treatment with 5'-[(Z)-4-amino-2-butenyl]- methyl-amino-5'-deoxyadenosine (MDL73811), an inhibitor of S-adenosylmethionine decarboxylase, resulted in increased outward currents through inwardly rectifying potassium channels while intracellular putrescine was markedly increased and spermidine and spermine levels were decreased. Fluctuations of intracellular polyamine concentrations as imposed by MDL73811 were directly translated in an altered cell excitability. Based on these results we conclude that the rectification properties of native inwardly rectifying potassium channels are largely controlled by intracellular spermine.

Regulation of polyamine synthesis and transport by fibroblast growth factor in aortic smooth muscle cells.

Basic-FGF (FGF2) is implicated as a regulator of smooth muscle cell proliferation that develops after arterial injury. Polyamines are essential for cell growth and differentiation and may mediate some of the FGF2-elicited responses. To examine this possibility, the effect of FGF2 on polyamine synthesis and uptake was tested on rat arterial smooth muscle cells. Exposure of cells to FGF2 for 24 and 48 h resulted in increased intracellular polyamine content. Ornithine decarboxylase (ODC) activity increased in FGF2-treated cells after 6 h of treatment, whereas no increases were detected in ODC mRNA steady-state levels. Basic-FGF increased maximal polyamine transport rate without changes in Km. Treatment with actinomycin D decreased polyamine transport. The effect of cyclohexamide on polyamine uptake was dose dependent. These studies indicate that treatment of vascular smooth muscle cells with FGF2 results in increases in intracellular polyamine content, polyamine synthetic activity, and polyamine transport.