Increase In Ornithine Decarboxylase Activity Research Articles

Structure and Enzymatic Activity of an Intellectual Disability-Associated Ornithine Decarboxylase Variant, G84R.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are required for proliferation, and increased ODC activity is associated with cancer and neural over-proliferation. ODC levels and activity are therefore tightly regulated, including through the ODC-specific inhibitor, antizyme AZ1. Recently, ODC G84R has been reported as a partial loss-of-function variant that is associated with intellectual disability and seizures. However, G84 is distant from both the catalytic center and the ODC homodimerization interface. To understand how G84R modulates ODC activity, we have determined the crystal structure of ODC G84R in both the presence and the absence of the cofactor pyridoxal 5-phosphate. The structures show that the replacement of G84 by arginine leads to hydrogen bond formation of R84 with F420, the last residue of the ODC C-terminal helix, a structural element that is involved in the AZ1-mediated proteasomal degradation of ODC. In contrast, the catalytic center is essentially indistinguishable from that of wildtype ODC. We therefore reanalyzed the catalytic activity of ODC G84R and found that it is rescued when the protein is purified in the presence of a reducing agent to mimic the reducing environment of the cytoplasm. This suggests that R84 may exert its neurological effects not through reducing ODC catalytic activity but through misregulation of its AZ1-mediated proteasomal degradation.

A Tear Metabolomic Profile Showing Increased Ornithine Decarboxylase Activity and Spermine Synthesis in Thyroid-Associated Orbitopathy.

About half of patients with Graves’ disease develop an orbitopathy related to an inflammatory expansion of the periorbital adipose tissue and muscles. We used a targeted metabolomic approach measuring 188 metabolites by mass spectrometry to compare the metabolic composition of tears in patients with active (n = 21) versus inactive (n = 24) thyroid-associated orbitopathy. Among the 44 metabolites accurately measured, 8 showed a significant alteration of their concentrations between the two groups. Two short-chain acylcarnitines, propionylcarnitine and butyrylcarnitine, and spermine showed increased concentrations in the tears of patients with active orbitopathy, whereas ornithine, glycine, serine, citrulline and histidine showed decreased concentrations in this group. In addition, the ratio putrescine/ornithine, representing the activity of ornithine decarboxylase, was significantly increased in patients with active compared to inactive orbitopathy (p = 0.0011, fold change 3.75). The specificity of this candidate biomarker was maintained when compared to a control group with unclassified dry eye disease. Our results suggest that the stimulation of ornithine decarboxylase by TSH receptor autoantibodies in orbital fibroblasts could lead to increased synthesis of spermine, through the increased activity of ornithine decarboxylase, that may contribute to periorbital expansion in Graves’ ophthalmopathy.

904P The role of arginine/citrulline cycle disorders in the liver injury pathogenesis in acute myeloid leukemia patients with concomitant obesity

Against the manifest of acute myeloid leukemia (AML) in patients with concomitant obesity there is a high risk of liver injury development, that requires a detailed study of its pathogenesis. The aim - to investigate the effect of Arginine/Citrulline cycle disorders on the liver injury development in obese AML patients. We examined 20 patients with newly diagnosed AML, including 10 (50%) men and 10 (50%) women, aged 18-77 years. The body mass index (BMI) was calculated. The study included AML patients with BMI above 25. The average BMI was 29.35±0.93 kg/m2. The biochemical blood test was assessed (alanine and asparagine (AST) aminotransferases, total bilirubin, alkaline phosphatase (AP), gammaglutamyltranspeptidase (GGTP), urea). Arginase, ornithine decarboxylase (ODC) activity, arginine and citrulline content were measured in the blood serum before induction chemotherapy. Viral hepatitis B and C were excluded. Statistical analysis was performed. In AML patients with concomitant obesity before chemotherapy AST was 1.7-fold higher (p=0.03), AP – 1.7-fold (p=0.002), GGTP – 2.4-fold (p=0.001), the content of total bilirubin – 1.8-fold (p=0.002) and urea - 2.9-fold (p=0.0003) higher than normal ranges. The study results showed a 3-fold decrease in arginine content (p<0.0001) compared to normal. At the same time there was an increase in arginase activity in 3.9 times (p<0.0001), ODC – in 1.9 times (p=0.0007), citrulline content – in 7.6 times (p=0.0001) compared with control. There was a strong inverse correlation between the concentration of citrulline and ODC activity in the blood serum of obese AML patients (r=-0.61; p<0.05), a moderate direct correlation between the ODC activity and AP in these patients was also revealed (r=+0.44; р<0.05). Consequently, the liver injury with cytolytic and cholestatic syndromes in AML patients with concomitant obesity may be caused by the disorders in the Arginine/Citrulline cycle, which were associated with an increased ODC activity in the blood serum. Excess weight and obesity affect serum arginine level, leading to potential drug-induced hepatotoxicity development during induction chemotherapy.

Ornithine decarboxylase, the rate-limiting enzyme of polyamine synthesis, modifies brain pathology in a mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a rare autosomal dominant neurodevelopmental disorder characterized by variable expressivity. TSC results from inactivating variants within the TSC1 or TSC2 genes, leading to constitutive activation of mechanistic target of rapamycin complex 1 signaling. Using a mouse model of TSC (Tsc2-RG) in which the Tsc2 gene is deleted in radial glial precursors and their neuronal and glial descendants, we observed increased ornithine decarboxylase (ODC) enzymatic activity and concentration of its product, putrescine. To test if increased ODC activity and dysregulated polyamine metabolism contribute to the neurodevelopmental defects of Tsc2-RG mice, we used pharmacologic and genetic approaches to reduce ODC activity in Tsc2-RG mice, followed by histologic assessment of brain development. We observed that decreasing ODC activity and putrescine levels in Tsc2-RG mice worsened many of the neurodevelopmental phenotypes, including brain growth and neuronal migration defects, astrogliosis and oxidative stress. These data suggest a protective effect of increased ODC activity and elevated putrescine that modify the phenotype in this developmental Tsc2-RG model.

Ornithine decarboxylase is involved in methyl jasmonate-regulated postharvest quality retention in button mushrooms (Agaricus bisporus).

In the present study, we investigated the role of ornithine decarboxylase (ODC) in the methyl jasmonate (MeJA)-regulated postharvest quality maintenance of Agaricus bisporus (J. E. Kange) Imbach button mushrooms by pretreating mushrooms with a specific irreversible inhibitor called α-difluoromethylornithine (DFMO) before exposure to MeJA vapor. Mushrooms were treated with 0 or 100 µmol L-1 MeJA or a combination of 120 µmol L-1 DFMO and 100 µmol L-1 MeJA, respectively, before storage at 4 °C for 21 days. Treatment with MeJA alone induced the increase in ODC activity whereas this effect was greatly suppressed by pretreatment with DFMO. α-Difluoromethylornithine strongly attenuated the effect of MeJA on decreasing cap opening, slowing the decline rate of soluble protein and total sugar, and accumulating total phenolics and flavonoids. α-Difluoromethylornithine pretreatment also counteracted the ability of MeJA to inhibit polyphenol oxidase and lipoxygenase activities, and malondialdehyde production, and to stimulate superoxide dismutase and catalase activities. It also largely downregulated MeJA-induced accumulation of free putrescine (Put). These results reveal that ODC is involved in MeJA-regulated postharvest quality retention of button mushrooms, and this involvement is likely to be associated with Put levels. © 2018 Society of Chemical Industry.

Activation of ornithine decarboxylase protects against methylmercury toxicity by increasing putrescine

We previously reported significantly increased level of putrescine, a polyamine, in the brains of mice administered methylmercury. Moreover, addition of putrescine to culture medium reduced methylmercury toxicity in C17.2 mouse neural stem cells. In this study, the role of ornithine decarboxylase (ODC), an enzyme involved in putrescine synthesis, in response to methylmercury toxicity was investigated. Methylmercury increased ODC activity in mouse cerebrum and cerebellum, but this increase was hardly observed in the kidney and liver, where methylmercury accumulated at a high concentration. In the cerebrum and cerebellum, increased putrescine was observed with methylmercury administration. Methylmercury increased ODC activity in C17.2 cells, but this was almost completely abolished in the presence of an ODC inhibitor. Methylmercury also increased the level of ODC protein in mouse brain and C17.2 cells. In addition, C17.2 cells pretreated with ODC inhibitor showed higher methylmercury sensitivity than control cells. These results suggest that the increased ODC activity by methylmercury is involved in the increase in putrescine level, and ODC plays an important role in the reduction of methylmercury toxicity. This is the first study to provide evidence that increased ODC activity may be a protective response against methylmercury-induced neurotoxicity.

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The polyamines putrescine, spermidine and spermine were analyzed in chick embryos during the first 2 days of development. A rapid increase in the activity of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine synthesis, was observed immediately after onset of incubation. Peak activities were found at 15 and 23 h of incubation. The first peak coincides with gastrulation and the second peak with early neurulation in the embryo. All three polyamines varied in a similar manner as did ODC, with putrescine and spermidine at about the same level and spermine at a lower level. The ODC activity was blocked by alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor. The inhibitor was administered to embryos in ovo at 5 h of incubation, i.e. prior to the first major increase in ODC activity. This block prevented the accumulation of the polyamines and inhibited development as gastrulation, suggesting a decisive role for polyamines in this developmental event.

A role for antizyme inhibitor in cell proliferation.

The polyamines are important for a variety of cellular functions, including cell growth. Their intracellular concentrations are controlled by a complex network of regulatory mechanisms, in which antizyme (Az) has a key role. Az reduces the cellular polyamine content by down-regulating both the enzyme catalysing polyamine biosynthesis, ornithine decarboxylase (ODC), and the uptake of polyamines. The activity of Az is repressed by the binding of a protein, named Az inhibitor (AzI), which is an enzymatically inactive homologue of ODC. Two forms of AzI have been described: AzI1, which is ubiquitous, and AzI2 which is expressed in brain and testis. In the present study, we have investigated the role of AzI1 in polyamine homeostasis and cell proliferation in breast cancer cells. The results obtained showed that the cellular content of AzI increased transiently after induction of cell proliferation by diluting cells in fresh medium. Inhibition of polyamine biosynthesis induced an even larger increase in the cellular AzI content, which remained significantly elevated during the 7-day experimental period. However, this increase was not a consequence of changes in cell cycle progression, as demonstrated by flow cytometry. Instead, the increase appeared to correlate with the cellular depletion of polyamines. Moreover, induced overexpression of AzI resulted in an increased cell proliferation with a concomitant increase in ODC activity and putrescine content. During mitosis, AzI1 was localised in a pattern that resembled that of the two centrosomes, confirming earlier observations. Taken together, the results indicate that AzI fulfils an essential regulatory function in polyamine homeostasis and cell proliferation.

Spermidine/spermine N1-acetyltransferase activity associates with white blood cell count in myeloid leukemias

The metabolism of polyamines, the cationic small molecules essential for cell proliferation and differentiation, is altered in cancer cells and can be exploited in cancer diagnosis and therapy. Spermidine/spermine N(1)-acetyltransferase (SSAT), which regulates intracellular levels of polyamines by catabolizing spermidine and spermine, has a controversial role in the development of cancers. In this study, the polyamine metabolism and function of SSAT were characterized in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoid leukemia patient samples. Also, mice overexpressing SSAT and having a myeloproliferative phenotype were analyzed for their response to decitabine and histone deacetylase inhibitor trichostatin A. The presence of epigenetic factors in the bone marrow cells of SSAT mice was analyzed. Elevated levels of spermidine and spermine, as well as increased activity of SSAT, were detected in AML, CML, and acute lymphoid leukemia patients compared with the controls. However, we found SSAT activity to be associated with white blood cell count only in AML and CML patients. Decitabine treatment brought the peripheral blood and bone marrow cell counts of SSAT mice to the level of wild-type mice. Spermidine/spermine N(1)-acetyltransferase mice had increased histone methylation and an increased level of histone deacetylase 1 in their bone marrow cells. The study suggests that SSAT influences the development of myeloid malignancies, and epigenetic factors partly contribute to the SSAT overexpression-induced myeloproliferative disease in mice.

Phasic study of intestinal homeostasis disruption in experimental intestinal obstruction.

To investigate the phasic alteration of intestinal homeostasis in an experimental model of intestinal obstruction. A rabbit model of intestinal obstruction was established by transforming parts of an infusion set into an in vivo pulled-type locking clamp and creating a uniform controllable loop obstruction in the mesenteric non-avascular zone 8 cm from the distal end of the ileum. The phasic alteration of intestinal homeostasis was studied after intestinal obstruction. The changes in goblet cells, intraepithelial lymphocytes, lamina propria lymphocytes, and intestinal epithelium were quantified from periodic acid-Schiff-stained sections. Ornithine decarboxylase (ODC) activity and serum citrulline levels were measured by high-performance liquid chromatography. Claudin 1 mRNA expression was examined by real-time polymerase chain reaction analysis. Intestinal microorganisms, wet/dry weight ratios, pH values, and endotoxin levels were determined at multiple points after intestinal obstruction. Furthermore, the number and ratio of CD3(+), CD4(+) and CD8(+) T cells were determined by flow cytometry, and secretory IgA levels were measured with an enzyme-linked immunosorbent assay. A suitable controllable rabbit model of intestinal obstruction was established. Intestinal obstruction induced goblet cell damage and reduced cell number. Further indicators of epithelial cell damage were observed as reduced serum citrulline levels and claudin 1 gene expression, and a transient increase in ODC activity. In addition, the wet/dry weight ratio and pH of the intestinal lumen were also dramatically altered. The ratio of Bacillus bifidus and enterobacteria was reversed following intestinal obstruction. The number and area of Peyer's patches first increased then sharply decreased after the intestinal obstruction, along with an alteration in the ratio of CD4/CD8(+) T cells, driven by an increase in CD3(+) and CD8(+) T cells and a decrease in CD4(+) T cells. The number of lamina propria lymphocytes also gradually decreased with prolonged obstruction. Intestinal obstruction can induce disruption of intestinal homeostasis.

Regulation of intestinal mucosal growth by amino acids

Amino acids, especially glutamine (GLN) have been known for many years to stimulate the growth of small intestinal mucosa. Polyamines are also required for optimal mucosal growth, and the inhibition of ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, blocks growth. Certain amino acids, primarily asparagine (ASN) and GLN stimulate ODC activity in a solution of physiological salts. More importantly, their presence is also required before growth factors and hormones such as epidermal growth factor and insulin are able to increase ODC activity. ODC activity is inhibited by antizyme-1 (AZ) whose synthesis is stimulated by polyamines, thus, providing a negative feedback regulation of the enzyme. In the absence of amino acids mammalian target of rapamycin complex 1 (mTORC1) is inhibited, whereas, mTORC2 is stimulated leading to the inhibition of global protein synthesis but increasing the synthesis of AZ via a cap-independent mechanism. These data, therefore, explain why ASN or GLN is essential for the activation of ODC. Interestingly, in a number of papers, AZ has been shown to inhibit cell proliferation, stimulate apoptosis, or increase autophagy. Each of these activities results in decreased cellular growth. AZ binds to and accelerates the degradation of ODC and other proteins shown to regulate proliferation and cell death, such as Aurora-A, Cyclin D1, and Smad1. The correlation between the stimulation of ODC activity and the absence of AZ as influenced by amino acids is high. Not only do amino acids such as ASN and GLN stimulate ODC while inhibiting AZ synthesis, but also amino acids such as lysine, valine, and ornithine, which inhibit ODC activity, increase the synthesis of AZ. The question remaining to be answered is whether AZ inhibits growth directly or whether it acts by decreasing the availability of polyamines to the dividing cells. In either case, evidence strongly suggests that the regulation of AZ synthesis is the mechanism through which amino acids influence the growth of intestinal mucosa. This brief article reviews the experiments leading to the information presented above. We also present evidence from the literature that AZ acts directly to inhibit cell proliferation and increase the rate of apoptosis. Finally, we discuss future experiments that will determine the role of AZ in the regulation of intestinal mucosal growth by amino acids.

Elevated ornithine decarboxylase activity promotes skin tumorigenesis by stimulating the recruitment of bulge stem cells but not via toxic polyamine catabolic metabolites

Elevated expression of ornithine decarboxylase (ODC), the regulatory enzyme in polyamine biosynthesis, targeted to the epidermis is sufficient to promote skin tumor development following a single subthreshold dose of dimethylbenz(a)anthracene (DMBA). Since skin tumor promotion involves recruitment of hair follicle bulge stem cells harboring genetic lesions, we assessed the effect of increased epidermal ODC on recruitment of bulge stem cells in ODC-ER transgenic mice in which ODC activity is induced de novo in adult skin with 4-hydroxytamoxifen (4OHT). Bromodeoxyuridine-pulse labeling and use of K15.CrePR1;R26R;ODC-ER triple transgenic mice demonstrated that induction of ODC activity is sufficient to recruit bulge stem cells in quiescent skin. Because increased ODC activity not only stimulates proliferation but also increases reactive oxygen species (ROS) generation via subsequent induction of polyamine catabolic oxidases, we used an inhibitor of polyamine catabolic oxidase activity, MDL72527, to investigate whether ROS generation by polyamine catabolic oxidases contributes to skin tumorigenesis in DMBA-initiated ODC-ER transgenic skin. Newborn ODC-ER transgenic mice and their normal littermates were initiated with a single topical dose of DMBA. To assess tumor development originating from dormant bulge stem cells that possess DMBA-initiated mutations, epidermal ODC activity was induced in ODC-ER mice with 4OHT 5weeks after DMBA initiation followed by MDL72527 treatment. MDL72527 treatment resulted in a shorter tumor latency time, increased tumor burden, increased conversion to carcinomas, and lower tumor levels of p53. Thus, elevated epidermal ODC activity promotes tumorigenesis by stimulating the recruitment of bulge stem cells but not via ROS generation by polyamine catabolic oxidases.

The induction of cardiac ornithine decarboxylase by β2‐adrenergic agents is associated with calcium channels and phosphorylation of ERK1/2

The role that the induction of cardiac ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by beta-adrenergic agents may have in heart hypertrophy is a controversial issue. Besides, the signaling pathways related to cardiac ODC regulation have not been fully elucidated. Here we show that in Balb C mice the stimulation of cardiac ODC activity by adrenergic agents was mainly mediated by β2 -adrenergic receptors, and that this induction was lower in the hypertrophic heart. Interestingly, this stimulation was abolished by the L-calcium channel antagonists verapamil and nifedipine. In addition, whereas the treatment with β2 -adrenergic agents was associated to both the increases in ODC, ODC-antizyme inhibitor 1 (AZIN1), c-fos and c-myc mRNA levels and the phosphorylation of CREB and MAP kinases ERK1 and ERK2 (ERK1/2), the co-treatment with L-calcium channel blockers differentially prevented most of these changes. These results suggest that the stimulation of cardiac ODC by β2 -adrenergic agents is associated with the activation of MAP kinases through the participation of L-calcium channels, and that by itself p-CREB does not appear to be sufficient for the transcriptional activation of ODC. In addition, post-translational mechanisms related with the induction of AZIN1 appear to be related to the increase of cardiac ODC activity.

EFFECT OF PEA APHID INFESTATION ON ACTIVITY OF AMINO ACID DECARBOXYLASES IN PEA TISSUES

The study examined changes in lysine decarboxylase (LDC), ornithine decarboxylase (ODC) and tyrosine decarboxylase (TyDC) activity in tissues of pea (Pisum sativum L.) infested by the pea aphid (Acyrthosiphon pisum Harris). The aphid induced increased ODC activity after one day and at two weeks. The effect was clearly systemic. TyDC activity increased after one day and at one week at feeding sites (aerial parts), while LDC activity increased only after one day of infestation and then decreased. Attack by aphids also affected enzyme activity in root tissues not directly damaged by the herbivores. The mechanisms of the response induced by pea aphid infestation in pea are discussed.

Evidence that AMP-activated protein kinase can negatively modulate Ornithine decarboxylase activity in cardiac myoblasts

The responses of AMP-activated protein kinase (AMPK) and Ornithine decarboxylase (ODC) to isoproterenol have been examined in H9c2 cardiomyoblasts, AMPK represents the link between cell growth and energy availability whereas ODC, the key enzyme in polyamine biosynthesis, is essential for all growth processes and it is thought to have a role in the development of cardiac hypertrophy. Isoproterenol rapidly induced ODC activity in H9c2 cardiomyoblasts by promoting the synthesis of the enzyme protein and this effect was counteracted by inhibitors of the PI3K/Akt pathway. The increase in enzyme activity became significant between 15 and 30min after the treatment. At the same time, isoproterenol stimulated the phosphorylation of AMPKα catalytic subunits (Thr172), that was associated to an increase in acetyl coenzyme A carboxylase (Ser72) phosphorylation. Downregulation of both α1 and α2 isoforms of the AMPK catalytic subunit by siRNA to knockdown AMPK enzymatic activity, led to superinduction of ODC in isoproterenol-treated cardiomyoblasts. Downregulation of AMPKα increased ODC activity even in cells treated with other adrenergic agonists and in control cells. Analogue results were obtained in SH-SY5Y neuroblastoma cells transfected with a shRNA construct against AMPKα. In conclusion, isoproterenol quickly activates in H9c2 cardiomyoblasts two events that seem to contrast one another. The first one, an increase in ODC activity, is linked to cell growth, whereas the second, AMPK activation, is a homeostatic mechanism that negatively modulates the first. The modulation of ODC activity by AMPK represents a mechanism that may contribute to control cell growth processes.

Organophosphorus insecticides affect normal polyamine metabolism in amphibian embryogenesis

The objective of the present study was to evaluate the concentration- and time-dependent effects of the organophosphorus insecticides malathion and azinphos-methyl on polyamine metabolism, and relate them to normal and altered embryonic development of the common toad Rhinella arenarum. Control embryos showed that the higher polyamines spermidine and spermine acquired importance with respect to the diamine putrescine as embryonic development progressed. The activity of ornithine decarboxylase significantly decreased in complete operculum embryos. Continuous exposure to malathion caused a decrease in polyamine levels during embryonic development. However, there was an increase in putrescine levels in complete operculum embryos exposed to a sublethal concentration of the insecticide. Embryos exposed to malathion displayed a decrease in fresh weight and size, along with an increase in the number of malformed individuals. R. arenarum embryos exposed to a lethal concentration of azinphos-methyl showed an increase in putrescine levels and a decrease in spermidine and spermine levels, accompanied by an increase in ornithine decarboxylase activity. In conclusion, as the embryonic development of the toad R. arenarum progresses, polyamine metabolism shifts to higher polyamine levels with a more preponderant contribution of spermidine and spermine with respect to putrescine and involves a dramatic change in ornithine decarboxylase activity, one of the key regulatory enzymes of the pathway. Organophosphorus insecticides are capable of altering polyamine metabolism, slowing embryo development in parallel with a reduction in spermidine and spermine levels. An increase in the oxidative degradation of polyamines might be involved in the toxic action of organophosphorus insecticides and might also be related to other effects such as teratogenesis.

Role of peripheral polyamines in the development of inflammatory pain

Polyamines (putrescine, spermidine and spermine) are aliphatic amines that are produced by the action of ornithine decarboxylase (ODC) in a rate-limiting and protein kinase C (PKC)-regulated step. Because high levels of polyamines are found in the synovial fluid of arthritic patients, the aim of the present study was to identify the role of peripherally produced polyamines in a model of inflammatory pain induced by adjuvant. The subcutaneous injection of Complete Freund's adjuvant (CFA, 50μL/paw) caused the development of mechanical allodynia and edema. Moreover, it increased ODC expression and activity and PKC activation. Administration of the selective ODC inhibitor DFMO (10μmol/paw) attenuated the development of allodynia and edema and decreased ODC activity in both control and CFA-treated animals. Furthermore, administration of the PKC inhibitor GF109203X (1nmol/paw) reduced allodynia and ODC activity in animals injected with CFA. A subcutaneous injection of putrescine (10μmol/paw), spermidine (3–10μmol/paw) or spermine (0.3–3μmol/paw) into the rat paw also caused mechanical allodynia and edema. The present results suggest that endogenously synthesized polyamines are involved in the development of nociception and edema caused by an adjuvant. Moreover, polyamine production in inflammatory sites seems to be related to an increase in ODC activity stimulated by PKC activation. Thus, controlling polyamine synthesis and action could be a method of controlling inflammatory pain.

Myocardial ischemia and hypoxia exert opposite effects on the expression of amiloride binding protein 1 (Abp1)

The polyamine putrescine is required for normal cell growth and division. Putrescine formation is enhanced during myocardial ischemia presumably due to increased ornithine decarboxylase activity. The goal of this study was to assess the role of the diamine oxidase Abp1 in regulating putrescine degradation in the ischemic rat heart. At 24 h, 3 and 6 weeks after myocardial infarction Abp1 mRNA was significantly reduced compared to sham‐operation. Inhibition of Abp1 by amiloride prevented the in vitro differentiation of C2C12 myoblasts. Exposure of mice at 8% O2 (6 h) increased Abp1 mRNA in the hearts 11‐fold (P&lt;0.02, n=5). Abp1 mRNA was also increased in several cell lines at 1% O2 (24 h). Reporter gene assays revealed an approx. 3,5‐fold stimulation of the Abp1 promoter in transfected C2C12 myoblasts at 1% O2. Promoter activity was also significantly enhanced by the Wilms' tumor transcription factor (Wt1), which is activated in hypoxia and controls myocardial blood vessel formation. Abp1 transcripts were reduced 60% in the hearts of Wt1‐deficient vs. wild‐type murine embryos (P&lt;0.05, n=6). Silencing of Wt1 by RNAi reduced the Abp1 mRNA in M15 cells by more than 60%. In conclusion, myocardial ischemia and hypoxia exert opposite effects on Abp1 expression. A cooperative regulation of the Abp1 promoter by Wt1 and hypoxia may contribute to adequate putrescine levels in the developing and ischemic heart.

Influence of gender and sex hormones on 5α-dihydrotestosterone elicited effect in isolated left atria of rats: Role of β-adrenoceptors and ornithine decarboxylase activity

Androgens elicit an acute cardiotonic effect in cardiac preparations of rats. This effect is produced via an extracellular interaction that may be coupled to pertussis-sensitive G-proteins and is associated with an increase in cAMP, polyamine synthesis and intracellular calcium. The nature of the targets and the existence of a dimorphic effect in this nongenomic effect of androgens are unknown. The purpose of this study was to characterize a possible gender and sex hormone influence on the 5α-dihydrotestosterone-elicited cardiotonic effect, taking into account the possible role of the β-adrenoceptors and ornithine decarboxylase activity on this response. [Float1]Regarding this, the effect of 5α-dihydrotestosterone on isolated left atria from male, estrogenized female and gonadectomized male and female rats was studied. The results showed that 5α-dihydrotestosterone-elicited cardiotonic effect was preserved independent of gender and sex hormones, being higher in control males than in the rest of the groups. This correlated with the testosterone plasma levels, except in estrogenized females, suggesting that the androgens positively and the estrogens negatively regulated the response. In all groups, 5α-dihydrotestosterone produced an increase in cAMP levels, but only in control males did it produce an increase in ornithine decarboxylase activity. In the other groups, the absence of an effect on ornithine decarboxylase might limit the capability of the response to the androgen. Altogether, androgens may help to control cardiac performance by a direct interaction on the heart in both sexes. Gender and sex differences in the magnitude of inotropism being due mainly to changes in β-adrenoceptors and cAMP production and in intracellular polyamine synthesis.

Antizyme Inhibitor 2 (AZIN2/ODCp) Stimulates Polyamine Uptake in Mammalian Cells

One of the processes that regulate intracellular levels of polyamines in mammalian cells is polyamine uptake. We have measured polyamine uptake in COS7 cells for putrescine, spermidine, and spermine, obtaining K(m) values of 4.5, 1.0, and 0.8 mum, respectively. Treatment of nonconfluent cells with cycloheximide stimulated polyamine uptake and prevented the inhibitory effect found in cells preloaded with polyamines, suggesting the existence of a feedback repression mechanism mediated by antizymes. Transient transfected cells with mutated antizyme forms of AZ1, AZ2, and AZ3, which do not require frameshifting, showed a total blockade of polyamine uptake. Transfection of COS7 cells with mouse or human AZIN2, a novel member of the antizyme inhibitor family, recently characterized by our group, markedly stimulated polyamine uptake and counteracted the action of any of the three antizymes in co-transfected cells. The stimulatory effect of AZIN2 on polyamine uptake was abrogated when the putative antizyme binding sequence, formed by residues 117-140 in AZIN2, was deleted. Real time reverse transcription-PCR analysis of antizyme inhibitor transcripts revealed that in brain and testes AZIN2 is more expressed than AZIN1, especially in the testes where the relative expression was about 25-fold higher. Collectively, our results clearly indicate that AZIN2 affects polyamine homeostasis not only by increasing ornithine decarboxylase activity but also by stimulating polyamine uptake, through negating the inhibitory effect of the antizymes. This finding may have physiological relevance, mostly in testes where AZ3 and AZIN2 are mainly expressed.