Rat Liver Ornithine Decarboxylase Research Articles

Effect of different thyroid state on ornithine decarboxylase activity and receptors of T3 in rat liver.

The effects of thyreoidectomy, of the administration of 3,5,3'-triiodothyronine (T3), of thyroxine (T4) together with the inhibitor of iodothyronine 5'-deiodinase activity 6-propyl-2-thiouracil (PTU) and of PTU alone on rat liver ornithine decarboxylase (ODC) activity was studied. 28 days after thyroidectomy there was a decrease in ODC activity, whereas T3 treatment of normal rats increased enzyme activity. PTU decreased ODC activity in intact rats and inhibited ODC activity in animals treated with T4. Neither thyroidectomy T3, T4 given in vivo nor the polyamines spermine and spermidine added in vitro influenced the binding of 125I-T3 to its receptor in liver nuclei. Only addition of PTU increased Ka significantly (P less than 0.01). It is concluded that ODC activity in the liver is directly correlated with the thyroid state but not to parameters of thyroid hormone receptor in liver nuclei.

Characterization of Arginine Decarboxylase in Rat Brain and Liver

We compared the properties of mammalian arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in rat liver and brain. Mammalian ADC is thermally unstable and associated with mitochondrial membranes. ADC decarboxylates both arginine (Km = 0.75 mM) and ornithine (Km = 0.25 mM), a reaction not inhibited by the specific ODC inhibitor, difluoromethylomithine. ADC activity is inhibited by Ca2+, Co2+, and polyamines, is present in many organs being highest in aorta and lowest in testis, and is not recognized by a specific monoclonal antibody to ODC. In contrast, ODC is thermally stable, cytosolic, and mitochondrial and is expressed at low levels in most organs except testis. Although ADC and ODC are expressed in cultured rat C6 glioma cells, the patterns of expression during growth and confluence are very different. We conclude that mammalian ADC differs from ADC isoforms expressed in plants, bacteria, or Caenorhabditis elegans and is distinct from ODC. ADC serves to synthesize agmatine in proximity to mitochondria, an organelle also harboring agmatine's degradative enzyme, agmatinase, and a class of imidazoline receptor (I2) to which agmatine binds with high affinity.

Arsenite, but not cadmium, induces ornithine decarboxylase and heme oxygenase activity in rat liver: relevance to arsenic carcinogenesis

Sodium arsenite and cadmium chloride, were administered orally to adult female rats at 21 and 4 h prior to sacrifice. Liver, lung, skin and urinary bladder were the tissues studied. DNA damage, cytochrome P450, glutathione content (GSH), ornithine decarboxylase (ODC), serum alanine aminotransferase and heme oxygenase activity were measured. Sodium arsenite increased rat hepatic ODC activity at 1.6 and 24.6 mg/kg and hepatic heme oxygenase activity at 8.2 and 24.6 mg/kg, but did not cause any DNA damage. Cadmium chloride did not affect any of the six parameters tested. These findings suggest that sodium arsenite may be a promoter rather than an initiator of carcinogenesis.

Arsenite, but not cadmium, induces ornithine decarboxylase and heme oxygenase activity in rat liver: relevance to arsenic carcinogenesis

Sodium arsenite and cadmium chloride, were administered orally to adult female rats at 21 and 4 h prior to sacrifice. Liver, lung, skin and urinary bladder were the tissues studied. DNA damage, cytochrome P450, glutathione content (GSH), ornithine decarboxylase (ODC), serum alanine aminotransferase and heme oxygenase activity were measured. Sodium arsenite increased rat hepatic ODC activity at 1.6 and 24.6 mg/kg and hepatic heme oxygenase activity at 8.2 and 24.6 mg/kg, but did not cause any DNA damage. Cadmium chloride did not affect any of the six parameters tested. These findings suggest that sodium arsenite may be a promoter rather than an initiator of carcinogenesis.

2-substituted 3-(aminooxy)propanamines as inhibitors of ornithine decarboxylase: synthesis and biological activity.

1-Amino-3-(aminooxy)-2-propanol (6a) has been synthesized and found to inhibit rat liver ornithine decarboxylase (ODC) with an IC50 in the nanomolar range. Compound 6a served as a basis for the design of new enzyme inhibitors, which led to the identification of 3-(aminooxy)-2-fluoropropanamine (15) as a new powerful enzyme blocker. Compound 15 inhibited ODC at 3 times lower concentrations than 6a and 3-(aminooxy)propanamine (APA), and it was superior to APA as an antiproliferative agent in inhibiting the growth of human T24 bladder carcinoma cells in vitro.

Blockade of α-and β-adrenergic receptors can prevent stimulation of liver ornithine decarboxylase activity by glucocorticoid or laparatomy

Rat liver ornithine decarboxylase induction by dexamethasone or laparatomy, which is dramatically impaired by catecholamine depletion, is not affected by α-and β-adrenergic blockers administered simultaneously 1 h prior to steroid injection or operation. However, if blockade is maintained for 24 h, an effect comparable to that of catecholamine depletion is obtained. Reciprocally, the response of the decarboxylase to catecholamines is severely compromised in adrenalectomized rats. Under the same conditions, induction of tyrosine aminotransferase by dexamethasone is not significantly affected by catecholamine availability, which altogether demonstrates that rat liver ornithine decarboxylase activity is specifically governed by the interaction between glucocorticoids and catecholamines.

Mirex induces ornithine decarboxylase in female rat liver

Ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, was significantly induced in female rat liver following oral administration of the pesticide mirex. After dual oral exposure (120 mg/kg of mirex; 21 and 4 hr prior to sacrifice), ornithine decarboxylase activity in rat liver cytosol was 70-fold higher than control values. A single oral dose of mirex (180 mg/kg) induced hepatic ornithine decarboxylase activity 55-fold over controls. After a single oral dose of mirex the maximal induction of ODC activity occurred at 36 hr. Mirex is an unusually potent and long-lasting inducer of rat hepatic ornithine decarboxylase activity.

Monoclonal Antibody Studies on the Properties and Regulation of Murine Ornithine Decarboxylase Antizymes1

Seven monoclonal antibodies were prepared against rat liver ornithine decarboxylase antizyme, a unique regulatory protein of the enzyme induced by its products. The monoclonal antibodies reacted with antizymes from all the rat and mouse tissues examined, indicating that these antizymes share similar structural features, though some size heterogeneity has been reported for rat antizymes. A sandwich enzyme immunoassay which could detect 0.1 ng of antizyme was developed using these antibodies. The amount of antizyme protein in rat liver, analyzed by the immunoassay, increased on putrescine treatment in parallel with antizyme activity, indicating that the changes in antizyme activity were attributable to the changes in the amount of its protein. The putrescine-induced increase of antizyme protein, as well as that of its activity, was completely inhibited by cycloheximide but not by actinomycin D, confirming the importance of post-transcriptional regulation in antizyme induction.

Purification and Some Properties of Rat Intestinal Ornithine Decarboxylase

Ornithine decarboxylase (ODC) was induced in rat small intestine by treatment with hypotonic solution in vitro and purified by two procedures, a conventional procedure and an immunoaffinity procedure. SDS-polyacrylamide gel electrophoresis showed that the molecular weight of the preparation purified by the immunoaffinity procedure (Mr = 53,000) was slightly larger than that of the preparation obtained by the conventional procedure (Mr = 52,000). Values for the Km for L-ornithine (0.1 mM), the isoelectric point (5.4), and the final specific activity (5.1-5.5 x 10(5) nmol CO2/mg protein/30 min) of the two preparations were similar to those reported for the rat liver ODC. Addition of a protease inhibitor (limabean trypsin inhibitor) to the crude extract prevented the appearance of the smaller enzyme (Mr = 52,000) obtained by the conventional purification procedure. Our result indicates that the large enzyme is native ODC and the smaller one is a partial proteolysis product of native ODC.

Induction of Ornithine Decarboxylase in Rat Liver by Phorbol Ester Is Mediated by Prostanoids from Kupffer Cells

Administration of phorbol 12-myristate 13-acetate (PMA) to rats in vivo resulted in the induction of ornithine decarboxylase activity in the liver which could be blocked by preinjection of indomethacin, a cyclooxygenase inhibitor. In vitro administration of PMA to primary cultures of rat parenchymal cells did not lead to an induction of ornithine decarboxylase activity. It was investigated to what extent non-parenchymal liver cells could play an intermediary role in the expression of the PMA effect on ornithine decarboxylase activity in parenchymal liver cells. Addition of conditioned medium from PMA-activated Kupffer cells to cultured parenchymal cells led to the induction of ornithine decarboxylase activity in parenchymal cells. This effect was not observed with conditioned medium from untreated Kupffer cells or from Kupffer cells treated with PMA plus indomethacin. Conditioned media from PMA-treated or untreated endothelial liver cells were ineffective in the induction of ornithine decarboxylase activity in parenchymal liver cells. Prostaglandin D2, the main eicosanoid produced by Kupffer cells, was able to stimulate the synthesis of ornithine decarboxylase in parenchymal liver cells (up to 40-fold) in a dose-dependent way. Prostaglandin (PG) D2 appeared to be a more potent inducer of ornithine decarboxylase activity in parenchymal cells than PGE1 and PGE2. It is concluded that intercellular communication inside the liver mediated by prostaglandins derived from activated Kupffer cells may form a mechanism to induce synthesis of specific proteins in parenchymal cells.

Effect of dexamethasone on the activity and expression of ornithine decarboxylase in rat liver and thymus

A single intraperitoneal injection of the synthetic glucocorticoid dexamethasone into rats resulted in a marked stimulation (more than 60-fold) of hepatic ornithine decarboxylase (ODC) at 4 h after the injection, whereas the enzyme activity in thymus was almost totally (about 95%) depressed at the same time. The stimulation of ODC activity in liver was in all likelihood attributable to a greatly enhanced accumulation of mRNA species for the enzyme as revealed by Northern blot and dot-blot hybridization analyses. ODC activity in thymus, in response to dexamethasone, was only 5% of that found in control animals, but this decrease was apparently not accompanied by similar reductions of the levels of ODC message, which was in fact decreased only by 50% at the maximum. In addition to two mRNA species (2.1 and 2.6 kilobases; kb), typical to mouse cells, rat tissues seemed to contain a third hybridizable message for ODC, smaller (1.6 kb) than the above-mentioned species and not seen in samples obtained from mouse or human cells. Interestingly, these smaller poly(A) + RNA sequences, hybridizable with cDNA complementary to mouse ODC mRNA, were apparently constitutively expressed, as the treatment with glucocorticoid altered the amount of these sequences only slightly.

Identical catalytic-centre activity for mouse kidney and rat liver ornithine decarboxylases as determined with antizyme and affinity labelling.

Since the catalytic-centre activity of mouse kidney ornithine decarboxylase (ODC) has been assumed to be twice as high as that of rat liver ODC, we compared relative catalytic-centre activity of the two enzymes by titration with antizyme, which inhibits ODC by stoichiometric binding. In either a crude or a purified state, both enzymes were inhibited by rat liver antizyme to the same extent, indicating that they have nearly identical catalytic-centre activities. This conclusion was supported by comparison of affinity labelling of the enzymes with alpha-difluoromethyl[14C]ornithine.

Kinetic study of the inhibition of rat liver ornithine decarboxylase by diamines; considerations on the mechanism of interaction between enzyme and inhibitor

1. 1. Partially purified rat liver ornithine decarboxylase is inhibited by several diamines including putrescine, 1,3-diaminopropane, cadaverine and p-phenylenediamine. 2. 2. The inhibition is dependent on pH, being strong at pH above 8 and negligible below pH 6.5. 3. 3. The kinetic study of the inhibition showed that while the aromatic diamine behaved as a simple competitive inhibitor, the aliphatic diamines presented a more complex pattern of inhibition in which two molecules of inhibitor might bind to the enzyme active site. 4. 4. The K I values for the different inhibitors were calculated and the degree of affinity for the enzyme was p-phenylenediamine > putrescine > cadaverine > 1,3-diaminopropane. 5. 5. A molecular mechanism explaining how one or two molecules of inhibitor can bind to the enzyme is proposed.

Interaction of alkylputrescines with ornithine decarboxylase from rat liver and Escherichia coli: an in vitro and in vivo study

The inhibitory effect of a series of 2-alkylputrescines on rat liver and Escherichia coli ornithine decarboxylase ( l-ornithine carboxy-lyase, EC 4.1.1.17) was examined. At 2.5 mM concentrations, 2-methyl-, 2-propyl-, 2-butyl-, 2-pentyl- and 2-hexylputrescines were stronger inhibitors of the mammalian enzyme than putrescine. Only the higher homologues (from 2-propyl- to 2-hexylputrescine) were inhibitors of the E. coli enzyme. An analysis of the effect of increasing concentrations of the 2-alkylputrescines showed that the main difference in the behaviour of the mammalian and E. coli decarboxylases toward 2-alkylputrescines was that the former was strongly inhibited by 2-methylputrescine whereas the latter was not. 2-Alkylputrescines were found to be competitive inhibitors of both the bacterial and mammalian enzyme. The smallest K i values (0.1 and 0.5 mM) were found for the 2-hexyl- and 2-pentylputrescines. N-Methyl-, N-ethyl-, N-propyl- and N-butylputrescines (50 μmol per 100 g body weight) were assayed as inhibitors of thioacetamide-induced rat liver ornithine decarboxylase. N-Propylputrescine was found to be the most inhibitory (66% inhibition) and although the N-alkylputrescines were taken up by the liver, they did not inhibit the liver polyamine pools. Both putrescine and N-methylputrescine were found to stabilize the thioacetamide-induced ornithine decarboxylase at the onset of the enzyme's degradation, while 2-alkylputrescines were inhibitory under similar conditions. N-Methylputrescine induced antizyme in thioacetamide-treated rats. In thioacetamide- or dexamethasone-treated rats, 2-methylputrescine was found to be the strongest in vivo inhibitor of the liver decarboxylase. Although 2-alkylputrescines were efficiently taken up by the liver, they did not noticeably inhibit its polyamine pools. 2-Methylputrescine decreased the putrescine concentration of the liver, but not its spermidine and spermine content. No induction of ornithine decarboxylase antizyme by 2-methylputrescine could be detected. The intrahepatic concentration of the latter decreased with time, very likely due to its degradation by a diamine oxidase, since the decrease was inhibited by aminoguanidine.

Induction of ornithine decarboxylase by aromatic amines in rat liver

The induction of ornithine decarboxylase (ODC) in rat liver by the carcinogen 2-acetylaminofluorene (2AAF), the strong liver tumor initiator trans-4-acetylaminostilbene (AAS), the non-carcinogen 4-acetylaminofluorene (4AAF) and, as a control, 3-methylcholanthrene (MC) has been examined. MC and all aromatic amines increased the ODC activity of rat liver from 3.6-fold (MC) to maximal 5.7-fold (AAS). The time course of the maximal induction differs from 2.5 h (2AAF) to 10 h (MC). The cytosolic concentrations of the unchanged compounds parallel the ODC activity. These data indicate that ODC induction in rat liver shows no correlation with tumor promoting properties of the model compounds. It is concluded that the unchanged compound is responsible for the ODC induction.

Selenium and poly amine metabolism: Different effect of selenite on liver and bursa of Fabricius ornithine decarboxylase activity

1. 1. When injected i.p., sodium selenite promoted a marked increase of rat liver ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities; when administered with the diet for 6 weeks, a less marked increase in liver ODC was observed, whereas SAMDC was not significantly changed. 2. 2. Protein synthesis was involved in the observed modifications. The rate of ODC inactivation was also changed, 3. 3. ODC increase was accompanied by an enhanced putrescine concentration in liver. 4. 4. A marked increase of ODC, accompanied by an enhancement of putrescine, was promoted by selenite (i.p.) also in chicken liver, together with an enhancement of glutathione concentration. Spermidine acetyltransferase (SAT) was also increased. 5. 5. In the bursa of Fabricius, SAT activity was also increased, whereas ODC was decreased. However the expected modifications in polyamine concentration were not observed. 6. 6. Decrease of ODC activity in the bursa was not due to an antizyme. 7. 7. In vitro, selenite concentrations known to inhibit cell proliferation ( > 1 μg/ml) inhibited both ODC and SAT activities; at lower concentration, SAT activity was enhanced.

Effect of N-alkyl and C-alkylputrescines on the activity of ornithine decarboxylase from rat liver and E. coli

N-Methyl, N-ethyl-, N-propyl-, N-butyl, N, N-dimethyl- and N, N′-dimethylputrescines were assayed as inhibitors of ornithine decarboxylase (EC 4.1.1.17) from rat liver and from Escherichia coli. They were found to be poor inhibitors, with the exception of N-propylputrescine and N, N-dimethylputrescine, which were inhibitory at 25 mM. A homologous series of 1-alkylputrescines ranging from 1-methylputrescine (1,4-diaminopentane) to 1-heptylputrescine (1,4-diaminoundecane) was assayed for effect on the activity of ornithine decarboxylase from the same sources. 1-methylputrescine (5 mM) inhibited the mammalian enzyme, while the higher homologues showed significantly less inhibitory activity. When assayed on the bacterial enzyme, 1-methylputrescine (5 mM) was not inhibitory, while the higher homologues showed inhibitory effects. At higher concentrations, 1-methylputrescine and 1-heptylputrescine, 2-methylputrescine, inhibitors of these series of rat liver ornithine decarboxylase. When 1-methylputrescine, 2-methylputrescine, 1,2-dimethylputrescine, 1,3-dimethylputrescine and 1,4-dimethylputrescine were assayed as inhibitors of the decarboxylase, 2-methylputrescine was found to be the best inhibitor of the rat liver enzyme, while 1,3-dimethylputrescine was the best inhibitor of the bacterial enzyme. 1,4-Dimethylputrescine (2,5-diaminohexane) did not inhibit the enzyme from either source. Both, 2-methylputrescine and 1-methylputrescine, as well as the 1,2- and 1,3-dimethylputrescines were competitive inhibitors of the enzyme, and a K i of 1 mM was obtained for 2-methylputrescine when the rat liver decarboxylase was used. N-Methyl, 1-methyl and 2-methylputrescines were found to inhibit in vivo the activity of rat liver ornithine decabroxylase which had been previously induced by thioacetamide treatment. 2-Methylputrescine (50 μmol/100 g body weight) was found to be the best in vivo inhibitor (93% inhibition), while putrescine under similar conditions inhibited 56% of the enzymatic activity.

Relation between induction of ornithine decarboxylase and specific gene expression in rat liver in response to the tumor promoter agent clofibrate.

The time course of changes in a number of biochemical parameters in rat liver was studied during 10 days of clofibrate administration. Ornithine decarboxylase (ODC) and putrescine levels began to increase within hours of the first dose and reached maxima at about 36 h (40 and 10 times control levels, respectively) and then returned to normal levels by 48 h. This ODC induction by clofibrate is different from that seen in compensatory liver hyperplasia or diethylnitrosamine administration in that it was not accompanied by elevations in cAMP or increased activation of cytoplasmic cAMP-dependent protein kinases, type I or II. Messenger RNA levels, notably of the species coding for the enzymes of the peroxisomal beta-oxidation pathway, increased in parallel with ODC and putrescine to reach a maximum also at 36 h. The enzymes of the peroxisomal beta-oxidation pathway, on the other hand, increased more gradually over time to reach a plateau at approximately 7 - 10 days. The magnitude of increase in mRNA (about 7-fold) was comparable to that of peroxisomal beta-oxidation as measured by cyanide-insensitive palmitoyl-CoA-dependent NAD+ reductase activity; comparable increases in the specific content of enoyl-CoA hydratase: beta-hydroxyacyl-CoA dehydrogenase and of peroxisomal thiolase were observed, as determined by SDS electrophoresis. A gradual increase in long-chain acyl-CoA (1.5-fold) followed the increase in beta-oxidation, whereas a 2-fold increase in acid-soluble CoA (free CoA and short-chain acyl-CoA) was seen as early as 36 h. This sequence of changes is at variance with proposals that increased levels of long-chain acyl-CoA mediate induction of peroxisomal beta-oxidation.

Effect on hepatic ornithine decarboxylase of some food additives and synthetic elastomers.

The food additives butylated hydroxytoluene, butylated hydroxyanisole and sodium cyclamate and the precursors for packaging materials acrylamide, acrylic acid, acrylonitrile and vinylpyrrolidone were investigated for their ability to induce hepatic ornithine decarboxylase (ODC) in vivo, in order to obtain indications about their possible tumour-promoting activities. It was shown that butylated hydroxyanisole, acrylonitrile, vinylpyrrolidone and acrylamide have the capacity to increase rat liver ODC activity, while butylated hydroxytoluene, acrylic acid and sodium cyclamate did not affect ODC activity.

Equilibrium between active and inactive forms of rat liver ornithine decarboxylase mediated by L-ornithine and salts

The mechanisms controlling the activity of ornithine decarboxylase (ODC) are complex and only partly understood. This study shows that ODC can exist as two different aggregation states, that differ in catalytic activity, the dimeric form being active and the monomeric form inactive. While L-ornithine shifts the association-dissociation equilibrium to the dimeric form, salts produce an opposite effect leading to subunit dissociation. α-DFMO, an enzyme-activated irreversible inhibitor of ODC, does not react with the monomeric form and therefore the influence of substrate and salts on the aggregation equilibrium must be taken into account in titration experiments with α-DFMO of the total amount of ODC in tissue preparations. Ornithine decarboxylase Polyamine Association equilibrium Rat liver Ionic strength