Increased Polyamine Uptake Research Articles

ATP13A3 variants promote pulmonary arterial hypertension by disrupting polyamine transport.

Potential loss-of-function variants of ATP13A3, the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in patients with pulmonary arterial hypertension (PAH). ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. In this study, we sought to determine the biological function of ATP13A3 in vascular endothelial cells (ECs) and how PAH-associated variants may contribute to disease pathogenesis. We studied the impact of ATP13A3 deficiency and overexpression in EC models [human pulmonary ECs, blood outgrowth ECs (BOECs), and human microvascular EC 1], including a PAH patient-derived BOEC line harbouring an ATP13A3 variant (LK726X). We also generated mice harbouring an Atp13a3 variant analogous to a human disease-associated variant to establish whether these mice develop PAH. ATP13A3 localized to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation, and increased monolayer permeability to thrombin. The assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, and L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germline Atp13a3 frameshift variant representing a human variant spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling, and muscularization of pulmonary vessels. We identify ATP13A3 as a polyamine transporter controlling polyamine homeostasis in ECs, a deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to alleviate the imbalances in polyamine homeostasis and EC dysfunction in PAH.

ATP13A4 Upregulation Drives the Elevated Polyamine Transport System in the Breast Cancer Cell Line MCF7.

Polyamine homeostasis is disturbed in several human diseases, including cancer, which is hallmarked by increased intracellular polyamine levels and an upregulated polyamine transport system (PTS). Thus far, the polyamine transporters contributing to the elevated levels of polyamines in cancer cells have not yet been described, despite the fact that polyamine transport inhibitors are considered for cancer therapy. Here, we tested whether the upregulation of candidate polyamine transporters of the P5B transport ATPase family is responsible for the increased PTS in the well-studied breast cancer cell line MCF7 compared to the non-tumorigenic epithelial breast cell line MCF10A. We found that MCF7 cells presented elevated expression of a previously uncharacterized P5B-ATPase, ATP13A4, which was responsible for the elevated polyamine uptake activity. Furthermore, MCF7 cells were more sensitive to polyamine cytotoxicity, as demonstrated by cell viability, cell death and clonogenic assays. Importantly, the overexpression of ATP13A4 WT in MCF10A cells induced a MCF7 polyamine phenotype, with significantly higher uptake of BODIPY-labeled polyamines and increased sensitivity to polyamine toxicity. In conclusion, we established ATP13A4 as a new polyamine transporter in the human PTS and showed that ATP13A4 may play a major role in the increased polyamine uptake of breast cancer cells. ATP13A4 therefore emerges as a candidate therapeutic target for anticancer drugs that block the PTS.

Difluoromethylornithine (DFMO) and AMXT 1501 inhibit capsule biosynthesis in pneumococci

Polyamines are small cationic molecules that have been linked to various cellular processes including replication, translation, stress response and recently, capsule regulation in Streptococcus pneumoniae (Spn, pneumococcus). Pneumococcal-associated diseases such as pneumonia, meningitis, and sepsis are some of the leading causes of death worldwide and capsule remains the principal virulence factor of this versatile pathogen. α-Difluoromethyl-ornithine (DFMO) is an irreversible inhibitor of the polyamine biosynthesis pathway catalyzed by ornithine decarboxylase and has a long history in modulating cell growth, polyamine levels, and disease outcomes in eukaryotic systems. Recent evidence shows that DFMO can also target arginine decarboxylation. Interestingly, DFMO-treated cells often escape polyamine depletion via increased polyamine uptake from extracellular sources. Here, we examined the potential capsule-crippling ability of DFMO and the possible synergistic effects of the polyamine transport inhibitor, AMXT 1501, on pneumococci. We characterized the changes in pneumococcal metabolites in response to DFMO and AMXT 1501, and also measured the impact of DFMO on amino acid decarboxylase activities. Our findings show that DFMO inhibited pneumococcal polyamine and capsule biosynthesis as well as decarboxylase activities, albeit, at a high concentration. AMXT 1501 at physiologically relevant concentration could inhibit both polyamine and capsule biosynthesis, however, in a serotype-dependent manner. In summary, this study demonstrates the utility of targeting polyamine biosynthesis and transport for pneumococcal capsule inhibition. Since targeting capsule biosynthesis is a promising way for the eradication of the diverse and pathogenic pneumococcal strains, future work will identify small molecules similar to DFMO/AMXT 1501, which act in a serotype-independent manner.

Novel Chemical Probes targeting the Polyamine Regulatory Circuit

Polyamines are vital for cellular life. Polyamines such as putrescine, spermidine, and spermine regulate the essential cellular process, including gene expression and cell proliferation. Given its cellular role, polyamine concentration in cells is tightly controlled. However, aberrant polyamine metabolism is associated with numerous diseases, including cancer. Growing evidence suggests that elevated polyamine levels are the absolute requirement for tumor growth and progression. The concentrations of cellular polyamines are controlled by the polyamine regulatory circuit comprising three different proteins: Ornithine Decarboxylase (ODC), Ornithine Decarboxylase Antizyme (OAZ), and Antizyme Inhibitor (AZIN). While ODC is directly involved in polyamine biosynthesis, OAZ and AZIN regulate the ODC activity via protein‐protein interactions. The dysregulation of ODC, OAZ, and AZIN leads to elevated polyamines in numerous pathologies, making them attractive targets to control polyamine levels. Besides regulating polyamine synthesis, OAZ is believed to modulate polyamine transport via multiple pathways. However, the precise mechanism of OAZ‐mediated polyamine regulation outside of the polyamine biosynthetic pathway remains elusive. We hypothesize that OAZ sequestration results in increased polyamine uptake. To provide evidence to our hypothesis, we generated covalent OAZ‐mimetic inactivators targeting both ODC and AZIN. Using biochemical experiments, we demonstrate that these OAZ‐mimetics inhibit the activity of ODC and prevent the sequestration of OAZ by AZIN. Using Mass spectrometric experiments, we determine the covalent binding of OAZ‐mimetics to both ODC and AZIN. We employ a novel gel assay to assess the rate of covalent modification of ODC and AZIN. Finally, using computational modeling, we determine the mode of binding of OAZ‐mimetics to both ODC and AZIN to provide insights into inhibition mechanisms.

Three Microbial Musketeers of the Seas: Shewanella baltica, Aliivibrio fischeri and Vibrio harveyi, and Their Adaptation to Different Salinity Probed by a Proteomic Approach.

Osmotic changes are common challenges for marine microorganisms. Bacteria have developed numerous ways of dealing with this stress, including reprogramming of global cellular processes. However, specific molecular adaptation mechanisms to osmotic stress have mainly been investigated in terrestrial model bacteria. In this work, we aimed to elucidate the basis of adjustment to prolonged salinity challenges at the proteome level in marine bacteria. The objects of our studies were three representatives of bacteria inhabiting various marine environments, Shewanella baltica, Vibrio harveyi and Aliivibrio fischeri. The proteomic studies were performed with bacteria cultivated in increased and decreased salinity, followed by proteolytic digestion of samples which were then subjected to liquid chromatography with tandem mass spectrometry analysis. We show that bacteria adjust at all levels of their biological processes, from DNA topology through gene expression regulation and proteasome assembly, to transport and cellular metabolism. The finding that many similar adaptation strategies were observed for both low- and high-salinity conditions is particularly striking. The results show that adaptation to salinity challenge involves the accumulation of DNA-binding proteins and increased polyamine uptake. We hypothesize that their function is to coat and protect the nucleoid to counteract adverse changes in DNA topology due to ionic shifts.

Harnessing the polyamine transport system to treat BRAF inhibitor-resistant melanoma

ABSTRACT BRAF mutations are present in over half of all melanoma tumors. Although BRAF inhibitors significantly improve survival of patients with metastatic melanoma, recurrences occur within several months. We previously reported that BRAF mutant melanoma cells are more sensitive to a novel arylmethyl-polyamine ( AP ) compound that exploits their increased polyamine uptake compared to that of BRAF wildtype cells. Using an animal model of BRAF inhibitor-resistant melanoma, we show that co-treatment with the BRAF inhibitor, PLX4720, and AP significantly delays the recurrence of PLX4720-resistant melanoma tumors and decreases tumor-promoting macrophages. Development of BRAF inhibitor-resistance enriches for metastatic cancer stem cells (CSC) and increases tumor-promoting macrophages. In vitro studies demonstrated that CD304+, CXCR4+ spheroid cultures of BRAF mutant melanoma cells are resistant to PLX4720 but are more sensitive to AP compared to monolayer cultures of the same cells. AP significantly inhibited YUMM1.7 melanoma cell invasiveness across a Matrigel-coated filter using the CXCR4 ligand, SDF-1α, as the chemoattractant. AP also blocked the chemotactic effect of SDF-1α on CXCR4+ macrophages and inhibited M2 polarization of macrophages. In melanoma-macrophage co-cultures, AP prevented the PLX4720-induced release of pro-tumorigenic growth factors, such as VEGF, from macrophages and prevented the macrophage rescue of BRAF mutant melanoma cells treated with PLX4720. Our study offers a novel therapy ( AP ) to treat chemo-resistant melanoma. AP is unique because it targets the polyamine transport system in BRAF inhibitor-resistant CSCs and also blocks CXCR4 signaling in invasive melanoma cells and pro-tumorigenic macrophages.

Vascular smooth muscle cell proliferation depends on caveolin-1-regulated polyamine uptake.

Much evidence highlights the importance of polyamines for VSMC (vascular smooth muscle cell) proliferation and migration. Cav-1 (caveolin-1) was recently reported to regulate polyamine uptake in intestinal epithelial cells. The aim of the present study was to assess the importance of Cav-1 for VSMC polyamine uptake and its impact on cell proliferation and migration. Cav-1 KO (knockout) mouse aortic cells showed increased polyamine uptake and elevated proliferation and migration compared with WT (wild-type) cells. Both Cav-1 KO and WT cells expressed the smooth muscle differentiation markers SM22 and calponin. Cell-cycle phase distribution analysis revealed a higher proportion of Cav-1 KO than WT cells in the S phase. Cav-1 KO cells were hyper-proliferative in the presence but not in the absence of extracellular polyamines, and, moreover, supplementation with exogenous polyamines promoted proliferation in Cav-1 KO but not in WT cells. Expression of the solute carrier transporters Slc7a1 and Slc43a1 was higher in Cav-1 KO than in WT cells. ODC (ornithine decarboxylase) protein and mRNA expression as well as ODC activity were similar in Cav-1 KO and WT cells showing unaltered synthesis of polyamines in Cav-1 KO cells. Cav-1 was reduced in migrating cells in vitro and in carotid lesions in vivo. Our data show that Cav-1 negatively regulates VSMC polyamine uptake and that the proliferative advantage of Cav-1 KO cells is critically dependent on polyamine uptake. We provide proof-of-principle for targeting Cav-1-regulated polyamine uptake as a strategy to fight unwanted VSMC proliferation as observed in restenosis.

P0200 Use of polyamine transport system by polyamine anthracene conjugates in human leukaemic cells

Background It is well known that cancer cells have increased polyamine concentrations compared with normal cells. This relation is due to a combination of increased polyamine uptake via the polyamine transport system (PTS) as well as increased biosynthesis. Thus, we may be able to exploit the PTS for selective delivery of cytotoxic agents to cancer cells by attaching them to polyamine vectors. Such an approach should reduce non-specific toxicities and therefore decrease the side-effects associated with chemotherapy. This concept is also supported by the nature of PTS, which can accommodate a wide range of substrates. Three compounds combining the DNA intercalator, anthracene, with a polyamine side chain have been synthesised: Ant 4, Ant 44, and Ant 444.The aim of this study was to investigate the mode of cell death induced by anthracene conjugates in HL-60 cell lines. Methods We used a variety of techniques to study the response of HL-60 cells to Ant 4, Ant 44, and Ant 444. Findings Deconvolution microscopy analysis revealed that the conjugates entered the cells. Quantitative analysis using high-performance liquid chromatography showed that the amount of Ant 4 in cells increased 30% from 6 h to 12 h. Ant 44 showed the highest level in cells after 6 h of exposure. Ant 444 showed a gradual increase between 0 h to 48 h. The indirect evidence using pre-treated cells with 5 mmol difluromethylornithine (DFMO) showed a significant decrease of IC 50 compared with no DFMO pretreatment for Ant 4, Ant 44, and Ant 444. The uptake study using radiolabelled polyamines showed there is a competition uptake between radiolabelled polyamines [ 14 C] putrescine, spermidine, or spermine with Ant 4, Ant 44, and Ant 444. Interpretation Ant 4, Ant 44, and Ant 444 gain entry into HL-60 cells via the PTS. The idea of selectively targeting cancer cells via the PTS shows promise. In the future, the concept of selective targeting of cancer cells via the PTS could lead to the development of novel and effective anticancer therapies.

The mechanisms by which polyamines accelerate tumor spread

Increased polyamine concentrations in the blood and urine of cancer patients reflect the enhanced levels of polyamine synthesis in cancer tissues arising from increased activity of enzymes responsible for polyamine synthesis. In addition to their de novo polyamine synthesis, cells can take up polyamines from extracellular sources, such as cancer tissues, food, and intestinal microbiota. Because polyamines are indispensable for cell growth, increased polyamine availability enhances cell growth. However, the malignant potential of cancer is determined by its capability to invade to surrounding tissues and metastasize to distant organs. The mechanisms by which increased polyamine levels enhance the malignant potential of cancer cells and decrease anti-tumor immunity are reviewed. Cancer cells with a greater capability to synthesize polyamines are associated with increased production of proteinases, such as serine proteinase, matrix metalloproteinases, cathepsins, and plasminogen activator, which can degrade surrounding tissues. Although cancer tissues produce vascular growth factors, their deregulated growth induces hypoxia, which in turn enhances polyamine uptake by cancer cells to further augment cell migration and suppress CD44 expression. Increased polyamine uptake by immune cells also results in reduced cytokine production needed for anti-tumor activities and decreases expression of adhesion molecules involved in anti-tumor immunity, such as CD11a and CD56. Immune cells in an environment with increased polyamine levels lose anti-tumor immune functions, such as lymphokine activated killer activities. Recent investigations revealed that increased polyamine availability enhances the capability of cancer cells to invade and metastasize to new tissues while diminishing immune cells' anti-tumor immune functions.

Polyamine transport is mediated by both endocytic and solute carrier transport mechanisms in the gastrointestinal tract.

The polyamines spermidine and spermine, and their precursor putrescine, are required for cell growth and cellular functions. The high levels of tissue polyamines are implicated in carcinogenesis. The major sources of exogenous polyamines are diet and intestinal luminal bacteria in gastrointestinal (GI) tissues. Both endocytic and solute carrier-dependent mechanisms have been described for polyamine uptake. Knocking down of caveolin-1 protein increased polyamine uptake in colon cancer-derived HCT116 cells. Dietary supplied putrescine was accumulated in GI tissues and liver in caveolin-1 knockout mice more than wild-type mice. Knocking out of nitric oxide synthase (NOS2), which has been implicated in the release of exogenous polyamines from internalized vesicles, abolished the accumulation of dietary putrescine in GI tissues. Under conditions of reduced endogenous tissue putrescine contents, caused by treatment with the polyamine synthesis inhibitor difluoromethylornithine (DFMO), small intestinal and colonic mucosal polyamine contents increased with dietary putrescine levels, even in mice lacking NOS2. Knocking down the solute carrier transporter SLC3A2 in HCT116-derived Hkh2 cells reduced the accumulation of exogenous putrescine and total polyamine contents in DFMO treated cells, relative to non-DFMO-treated cells. These data demonstrate that exogenous putrescine is transported into GI tissues by caveolin-1- and NOS2-dependent mechanisms, but that the solute carrier transporter SLC3A2 can function bidirectionally to import putrescine under conditions of low tissue polyamines.

Pneumocystis Mediates Overexpression of Antizyme Inhibitor Resulting in Increased Polyamine Levels and Apoptosis in Alveolar Macrophages

Pneumocystis pneumonia (PcP) is the most common opportunistic disease in immunocompromised patients. Alveolar macrophages are responsible for the clearance of Pneumocystis organisms; however, they undergo a high rate of apoptosis during PcP due to increased intracellular polyamine levels. In this study, the sources of polyamines and mechanisms of polyamine increase and polyamine-induced apoptosis were investigated. The level of ornithine decarboxylase (ODC) was elevated in alveolar macrophages, and the number of alveolar macrophages that took up exogenous polyamines was increased 20-fold during PcP. Monocytes, B lymphocytes, and CD8+ T lymphocytes that were recruited into the lung during PcP expressed high levels of ornithine decarboxylase, suggesting that these cells are sources of polyamines. Both protein and mRNA levels of antizyme inhibitor (AZI) were increased in alveolar macrophages during PcP. This AZI overexpression correlated with increased polyamine uptake by alveolar macrophages, because AZI expression knockdown decreased the polyamine uptake ability of these cells. AZI expression knockdown also decreased the apoptosis rate of alveolar macrophages. Pneumocystis organisms and zymosan A were found to induce AZI overexpression in alveolar macrophages, suggesting that beta-glucan, which is the major component of the Pneumocystis cell wall, induces AZI overexpression. The levels of mRNA, protein, and activity of polyamine oxidase were increased in alveolar macrophages during PcP, indicating that the H(2)O(2) generated during polyamine catabolism caused alveolar macrophages to undergo apoptosis. Taken together, results of this study indicate that Pneumocystis organisms induce AZI overexpression in alveolar macrophages, leading to increased polyamine synthesis and uptake and apoptosis rate of these cells.

Activated K‐RAS increases polyamine uptake in human colon cancer cells through modulation of caveolar endocytosis

Endocytic pathways have been implicated in polyamine transport in mammalian cells, but specific mechanisms have not been described. We have shown that expression of a dominant negative (DN) form of the GTPase Dynamin, but not Eps15, diminished polyamine uptake in colon cancer cells indicating a caveolar and nonclathrin uptake mode. Polyamines co-sediment with lipid raft/caveolin-1 rich fractions, of the plasma membrane in a sucrose density gradient. Knock down of caveolin-1 significantly increased polyamine uptake. Conversely, ectopic expression of this protein resulted in diminished polyamine uptake. We also found that presence of an activated K-RAS oncogene significantly increased polyamine uptake by colon cancer cells. This effect is through an increase in caveolin-1 phosphorylation at tyrosine residue 14. Caveolin-1 is a negative regulator of caveolar endocytosis and phosphorylation in a K-RAS dependent manner leads to an increase in caveolar endocytosis. In cells expressing wild type K-RAS, addition of exogenous uPA was sufficient to stimulate caveolar endocytosis of polyamines. This effect was abrogated by the addition of a SRC kinase inhibitor. These data indicate that polyamine transport follows a dynamin-dependent and clathrin-independent endocytic uptake route, and this route is positively regulated by the oncogenic expression of K-RAS in a caveolin-1 dependent manner.

Putrescine transport in hypoxic rat main PASMCs is required for p38 MAP kinase activation.

Hypoxic pulmonary vascular remodeling in rats is associated with increased polyamine transport in pulmonary artery smooth muscle cells (PASMCs). We therefore defined constitutive and hypoxia-induced polyamine transport properties of rat cultured PASMCs and determined the impact of polyamine transport blockade on hypoxia-induced accumulation of p38 MAP kinase. PASMCs exhibited polyamine transport pathways that were characterized by Michaelis-Menten kinetics. RNA synthesis inhibition attenuated while inhibition of protein synthesis increased polyamine uptake, thus suggesting regulation by ornithine decarboxylase-antizyme. The presence of two transporters with overlapping selectivities, one for putrescine and another for all three polyamines, was inferred by cross-competition studies and by findings that only putrescine uptake was sodium dependent and that hypoxia caused a selective, time-dependent induction of putrescine transport. The pathophysiological significance of augmented putrescine import was suggested by the observation that polyamine transport inhibition suppressed hypoxia-induced p38 MAP kinase phosphorylation. These results indicate that rat PASMCs express two polyamine transporters and that a specific increase in the putrescine uptake pathway is necessary for hypoxia-induced activation of p38 MAP kinase.

Copper-dependent Autocleavage of Glypican-1 Heparan Sulfate by Nitric Oxide Derived from Intrinsic Nitrosothiols

Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 371-376). Increased polyamine uptake correlates with an increased number of positively charged N-unsubstituted glucosamine units in the otherwise polyanionic heparan sulfate chains of glypican-1. During intracellular recycling of glypican-1, there is an NO-dependent deaminative cleavage of heparan sulfate at these glucosamine units, which would eliminate the positive charges (Ding, K., Sandgren, S., Mani, K., Belting, M., and Fransson, L.-A. (2001) J. Biol. Chem. 276, 46779-46791). Here, using both biochemical and microscopic techniques, we have identified and isolated S-nitrosylated forms of glypican-1 as well as slightly charged glypican-1 glycoforms containing heparan sulfate chains rich in N-unsubstituted glucosamines. These glycoforms were converted to highly charged species upon treatment of cells with 1 mm l-ascorbate, which releases NO from nitrosothiols, resulting in deaminative cleavage of heparan sulfate at the N-unsubstituted glucosamines. S-Nitrosylation and subsequent deaminative cleavage were abrogated by inhibition of a Cu(2+)/Cu(+) redox cycle. Under cell-free conditions, purified S-nitrosylated glypican-1 was able to autocleave its heparan sulfate chains when NO release was triggered by l-ascorbate. The heparan sulfate fragments generated in cells during this autocatalytic process contained terminal anhydromannose residues. We conclude that the core protein of glypican-1 can slowly accumulate NO as nitrosothiols, whereas Cu(2+) is reduced to Cu(+). Subsequent release of NO results in efficient deaminative cleavage of the heparan sulfate chains attached to the same core protein, whereas Cu(+) is oxidized to Cu(2+).

Role of ATP and sodium in polyamine transport in bovine pulmonary artery smooth cells

Increased polyamine transport may be a key mechanism driving elevations in lung cell polyamine content necessary for the development of chronic hypoxic pulmonary hypertension. Bovine pulmonary artery smooth muscle cells (PASMCs) in culture exhibit two carriers for polyamines, a nonselective one shared by the three polyamines, putrescine (PUT), spermidine (SPD), and spermine (SPM), and another that is selective for SPD and SPM. Hypoxia appears to up-regulate both carriers. In this study, we examined the role of ATP and the Na + gradient in regulating polyamine transport in control PASMCs and in PASMCs with polyamine transport augmented by culture under hypoxic conditions (Po 2: 15–30 torr). Inhibition of ATP synthesis with dinitrophenol + iodoacetate profoundly reduced polyamine uptake in both control and hypoxic PASMCs. Putrescine uptake was somewhat more sensitive to iso-osmotic replacement of extracellular Na + with choline chloride or sucrose than were SPD or SPM in both hypoxic and standard cells, but under no conditions did Na + replacement substantially alter polyamine uptake. Treatment of PASMCs with ouabain, a Na +-K +ATPase inhibitor, or with gramicidin, a Na + ionophore, minimally attenuated polyamine transport, whereas the Na +/K + ionophore monensin increased polyamine uptake in standard, but not in hypoxic, cells. In general, the reduction in the extracellular Na + content or ionophore-induced increases in Na + permeability had a greater suppressive effect on polyamine transport in hypoxic cells than in standard cells, suggestive of the induction of Na +dependent polyamine carriers by hypoxia. These observations indicate that the activities of the two putative polyamine transport pathways in standard PASMCs, as well as their up- regulation by hypoxia, require ATP synthesis. In addition, it appears that polyamine transport in PASMCs is composed of two components: one a prominent sodium-independent transporter and the other a relatively minor component that is sodium dependent. The latter may be activated by hypoxic exposure in combination with the induction of new polyamine carriers.

Accumulation of exogenous polyamines in gerbil brain after ischemia.

Regionally selective delayed neuronal degeneration is a characteristic sequel of cerebral ischemia. Recent evidence indicates that changes in brain polyamine metabolism may be critical for nerve cell survival after ischemia. Within hours after ischemia, intracellular putrescine levels are greatly increased and remain elevated for days, whereas only minor changes are noted in the levels of the polyamines spermine and spermidine. In contrast, the extracellular levels of all polyamines are low after ischemia. Injections of polyamines following ischemia, however, can protect neurons in the gerbil brain from delayed cell death, with spermine being the most potent of the polyamines. In the present study, therefore, we sought to determine if increased polyamine uptake occurs in the brain after ischemia. In the hippocampal slice preparation, temperature-dependent uptake was unique for spermine, but not for spermidine or putrescine. Uptake of [14C]spermine was transiently increased after ischemia, peaking at 150% of control by 12-13 h and subsiding by 24 h. Intravenous injections of [3H]spermidine resulted in a postischemic accumulation of this polyamine throughout the forebrain parenchyma. We conclude that: 1. Active cellular uptake of spermine is transiently increased early after ischemia; 2. A nonspecific accumulation of exogenous polyamines occurs early after ischemia probably owing to a compromised blood-brain barrier, and 3. The findings indicate that exogenous polyamines can exert their effect directly in the brain after ischemia.

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.

Stretch-related changes in lung cAMP after partial pneumonectomy

In rats, left pneumonectomy (PNX) initiates rapid compensatory hyperplastic growth of the right lung. Previous work indicated that increased polyamine uptake associated with post-PNX distortion or "stretch" of the remaining tissue and altered adenosine 3',5'-cyclic monophosphate (cAMP) metabolism may play a role in the early phase of the growth response. Evidence was sought to link these observations with the goal of understanding control of compensatory growth. At day 1 or 3 after left PNX in vivo, increased right lung cAMP was associated with activation of the cAMP-dependent protein kinase (PKa). Total PKa activity was unaffected, but the PKa activity ratio (-cAMP/+cAMP) doubled, providing evidence for PNX-associated activation of the kinase in vivo. Neither cAMP nor PKa was altered in sham-operated control lungs. To determine whether increased distension of the right lung after PNX might initiate these changes, lungs of unoperated animals were perfused 40 min in vitro, either without ventilation or with a distending constant positive airway pressure (CPAP) of 20 cmH2O. CPAP rapidly increased uptake of the polyamine spermidine (SPD; 1.5 microM), tissue cAMP, and the PKa activity ratio, in a manner similar to that observed after PNX. Both tissue cAMP and the kinase activity ratio increased with 1 microM forskolin (FSK), as expected, but SPD uptake was unaffected by FSK. FSK did not diminish the CPAP-associated elevations of SPD uptake (P less than 0.01) or cAMP (P less than 0.05), but the kinase was not further activated by CPAP with FSK present.(ABSTRACT TRUNCATED AT 250 WORDS)