Modulation of ornithine decarboxylase activity and ornithine decarboxylase-antizyme complex in rat heart by hormone and putrescine treatment

Ornithine decarboxylase was present in a cryptic, complexed form in an amount approximately equivalent to that of free ornithine decarboxylase activity in adult rat heart. Addition of isoproterenol (10 mg/kg) caused a notable rise in ornithine decarboxylase activity and a simultaneous decrease in the amount of the complexed enzyme. During the period of ornithine decarboxylase decay, when cardiac putrescine content had reached high values, the level of the complex increased above that of the control. Administration of putrescine (1.5 mmol/kg, twice) or dexamethasone (4 mg/kg) produced a decrease of heart ornithine decarboxylase activity, while it did not remarkably affect the level of complexed ornithine decarboxylase, therefore raising significantly the ratio of bound to total ornithine decarboxylase. Putrescine also elicited the appearance of free antizyme, concomitantly with the disappearance of free ornithine decarboxylase activity after 3-4 h of treatment. These results indicate that a significant amount of ornithine decarboxylase occurs in an inactive form in the heart under physiological conditions and that its absolute and relative levels may vary following stimuli which affect heart ornithine decarboxylase activity.     

Immunoreactive luliberin in the visceral organs of rats]

The distribution of luliberin (luteinizing hormone-releasing hormone, LH-RH) was studied. LH-RH-like factor showing analogous immunochemical and chromatographic properties was detected in the liver, kidneys, duodenum, pancreas, adrenal glands and heart of rats. The concentration of immunoreactive LH-RH in the liver, kidneys, duodenum, pancreas and adrenal glands was nearly equal (5 to 7 pg per mg of methanol extract obtained from acetic acid extract of acetone powder), its concentration in the heart being somewhat lower (2 pg per mg of extract). Only minute amounts of this factor were present in blood cells. Immunoreactive LH-RH found in visceral organs may be of hypothalamic origin or may be synthesized in these organs (in situ).     

Effect of oxygen radicals and hyperoxia on rat heart ornithine decarboxylase activity

Rat heart ornithine decarboxylase activity from isoproterenol-treated rats was inactivated in vitro by reactive species of oxygen generated by the reaction xanthine/xanthine oxidase. Reduced glutathione, dithiothreitol and superoxide dismutase had a protective effect in homogenates and in partially purified ornithine decarboxylase exposed to the xanthine/xanthine oxidase reaction, while diethyldithiocarbamate, which is an inhibitor of superoxide dismutase, potentiated the damage induced by O₂^? on enzyme activity. Dithiothreitol at concentrations above 1.25 mM had an inhibitory effect oupon supernatant ornithine decarboxylase activity, while at 2.5 mM it was most effective in the recovery of ornithine decarboxylase activity, after the purification of the enzyme by the ammonium sulphate precipitation procedure. The ornithine decarboxylase inactivated by the xanthine/xanthine oxidase reaction showed a higher value of K_m and a reduction of V_max with respect to control activity. The exposure of rates to 100% oxygen for 3 h reduced significantly the isoproterenol-induced heart ornithine decarboxylase activity. The injection with diethyldithiocarbamate 1 h before hyperoxic exposure further reduced heart ornithine decarboxylase activity.     

Effect of alterations in membrane lipid unsaturation on the properties of the insulin receptor of Ehrlich ascites cells

Rat heart ornithine decarboxylate activity from isoproterenol-treated rats was inactivated in vitro by reactive species of oxygen generated by the reaction xanthine/xanthine oxidase. Reduced glutathione, dithiothreitol and superoxide dismutase has a protective effect in homogenates and in partially purified ornithine decarboxylase exposed to the xanthine/xanthine oxidase reaction, while diethyldithiocarbamate, which is an inhibitor of superoxide dismutase, potentiated the damage induced by O2- on enzyme activity. Dithiothreitol at concentrations above 1.25 mM had an inhibitory effect upon supernatant ornithine decarboxylase activity, while at 2.5 mM it was most effective in the recovery of ornithine decarboxylase activity, after the purification of the enzyme by the ammonium sulphate precipitation procedure. The ornithine decarboxylase inactivated by the xanthine/xanthine oxidase reaction showed a higher value of Km and a reduction of Vmax with respect to control activity. The exposure of rats to 100% oxygen for 3 h reduced significantly the isoproterenol-induced heart ornithine decarboxylase activity. The injection with diethyldithiocarbamate 1 h before hyperoxic exposure further reduced heart ornithine decarboxylase activity.     

The effects of injection of water or hypertonic saline on ornithine decarboxylase activity of neonatal rat heart and brain

Intraperitoneal injection of deionized water (0.25ml/10 g body wt) produced a large increase in ornithine decarboxylase activity in cerebral cortex and heart of 6 day old rats, but had no effect on those activities in the 20 day old rat. Injection of the same dose of hypertonic (1.8%) saline caused a marked decline in the activity of this enzyme in both cerebral cortex and heart of the 6 day old rat and in the heart of 20 day old rats. In neonatal rats, the increase in heart ornithine decarboxylase elicited by the injection of water and the decline in activity which follows the injection of hypertonic saline were both evident within 30 minutes after injection; both effects were maximal two hours post-injection and both persisted for longer than four hours after injection. A decline in enzyme activity observed after injection of hypertonic saline was also found following the injection of hypertonic glucose, suggesting that osmotic effects, rather than specific ion effects, were mediating the loss of activity. The K_M of ornithine decarboxylase in neonatal heart decreased following hypertonic saline injection, whereas that of cerebral cortex did not, supporting previous suggestions that the ornithine decarboxylase in heart may have unique regulatory controls.     

Phosphorylation of rat heart ornithine decarboxylase by type-2 casein kinase

Highly purified preparations of rat heart ornithine decarboxylase are readily phosphorylated by rat liver type-2 casein kinase-TS at the same 54 KDa protein band which is also radiolabeled by 3H-DFMO. The reaction, which is stimulated by polylysine leads to the incorporation of up to 0.8 mol P/mol ornithine decarboxylase at seryl residue(s) included in a single 8.6 KDa CNBr fragment. Partially purified preparations of ornithine decarboxylase contain a type-2 casein kinase which promotes the phosphorylation of ornithine decarboxylase at the same CNBr fragment affected by rat liver casein kinase-TS.     

Ornithine decarboxylase activity in insulin-deficient states

The activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis, was determined in tissues of normal control rats and rats made diabetic with streptozotocin. In untreated diabetic rats fed ad libitum, ornithine decarboxylase activity was markedly diminished in liver, skeletal muscle, heart and thymus. Ornithine decarboxylase was not diminished in a comparable group of diabetic rats maintained on insulin. Starvation for 48h decreased ornithine decarboxylase activity to very low values in tissues of both normal and diabetic rats. In the normal group, refeeding caused a biphasic increase in liver ornithine decarboxylase; there was a 20-fold increase in activity at 3h followed by a decrease in activity, and a second peak between 9 and 24h. Increases in ornithine decarboxylase in skeletal muscle, heart and thymus were not evident until after 24–48h of refeeding, and only a single increase occurred. The increase in liver ornithine decarboxylase in diabetic rats was greater than in normal rats after 3h of refeeding, but there was no second peak. In peripheral tissues, the increase in ornithine decarboxylase with refeeding was diminished. Skeletal-muscle ornithine decarboxylase is induced more rapidly when meal-fed rats are refed after a period without food. Refeeding these rats after a 48h period without food caused a 5-fold increase in ornithine decarboxylase in skeletal muscle at 3h in control rats but failed to increase activity in diabetic rats. When insulin was administered alone or together with food to the diabetic rats, muscle ornithine decarboxylase increased to activities even higher than in the refed controls. In conclusion, these findings indicate that the regulation of ornithine decarboxylase in many tissues is grossly impaired in diabetes and starvation. They also suggest that polyamine formation in vivo is an integral component of the growth-promoting effect of insulin or some factor dependent on insulin.     

Effect of experimental diabetes on ornithine decarboxylase activity of rat tissues

Measurements have been made of the activity of ornithine decarboxylase of liver, heart, kidney and brain in alloxan-diabetic and control rats. In all these tissues this enzyme had decreased markedly at four weeks after induction of diabetes. These results are discussed in relation to the hormonal control and cyclic nucleotide regulation of ornithine decarboxylase.     

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.     

Transgenic mice aberrantly expressing human ornithine decarboxylase gene.

We have generated transgenic mice carrying human ornithine decarboxylase gene. Two different transgene constructs were used: (i) a 5'-truncated human ornithine decarboxylase gene and (ii) an intact human ornithine decarboxylase gene. Transgenic mice carrying the 5'-truncated gene did not express human ornithine decarboxylase-specific mRNA. Transgenic mice carrying the intact human ornithine decarboxylase gene expressed human-specific ornithine decarboxylase mRNA in all tissues studied. However, as indicated by actual enzyme assays, the expression pattern was highly unusual. In comparison with their wild-type littermates, the transgenic mice exhibited greatly elevated enzyme activity in almost every tissue studied. Ornithine decarboxylase activity was moderately elevated in parenchymal organs such as liver, kidney, and spleen. Tissues like heart, muscle, lung, thymus, testis, and brain displayed an enzyme activity that was 20 to 80 times higher than that in the respective tissues of nontransgenic animals. The offspring of the first transgenic male founder animal did not show any overt abnormalities, yet their reproductive performance was reduced. The second transgenic founder animal, showing similar aberrant expression of ornithine decarboxylase in all tissues studied, including an extremely high activity in testis, was found to be infertile. Histological examination of the tissues of the latter animal revealed marked changes in testicular morphology. The germinal epithelium was hypoplastic, and the spermatogenesis was virtually totally shut off. Similar examination of male members of the first transgenic mouse line revealed comparable, yet less severe, histological changes in testis.     

Absence of inactivation or phosphorylation of ornithine decarboxylase by nuclear protein kinase NII and of immunological cross-reactivity between RNA polymerase I and ornithine decarboxylase

Incubation with protein kinase NII did not result in phosphorylation or inactivation of mouse kidney ornithine decarboxylase. Partially purified ornithine decarboxylase preparations contained a protein kinase activity and stimulated the activity of RNA polymerase I. However, these properties were due to contaminating protein(s) since further purification reduced the kinase activity and removal of the ornithine decarboxylase with a specific antiserum did not abolish the ability to stimulate RNA polymerase I. Antibodies to RNA polymerase I did not interact with ornithine decarboxylase and antibodies to ornithine decarboxylase did not interact with RNA polymerase I. These results indicate that: a) mammalian ornithine decarboxylase activity is not regulated by phosphorylation by protein kinase NII or the contaminating kinase, and b) the ability of impure preparations of ornithine decarboxylase to stimulate RNA polymerase I is due to a contaminating unrelated protein.     

Subcellular localization of ornithine decarboxylase in liver of control and growth-hormone-treated rats.

1. Ornithine-2-oxo acid aminotransferase activity was inhibited by amino-oxyacetate (10(-5) M). This permitted the measurement of ornithine decarboxylase in the presence of mitochondria by using the 14CO2-trapping technique. 2. Subcellular fractionation of rat liver by differential centrifugation, followed by the assay of ornithine decarboxylase in the presence of amino oxyacetate and of marker enzymes for each fraction, demonstrated that ornithine decarboxylase was located in the cytosol. 3. The greatly increased ornithine decarboxylase activity observed after growth-hormone administration was also found to be localized in the cytosol. 4. The Km of ornithine decarboxylase from rat liver for ornithine was 28 muM. Administration of growth hormone 4 h before death did not affect the apparent affinity of ornithine decarboxylase for ornithine.     

Regulation of the activity of ornithine decarboxylase and S-adenosylmethionine decarboxylase in mammary gland and liver of lactating rats. Effects of starvation, prolactin and insulin deficiency.

1. Starvation caused a marked decrease in the activity of ornithine decarboxylase in mammary gland, together with a lesser decrease in the activity of S-adenosylmethionine decarboxylase and a marked fall in milk production. Liver ornithine decarboxylase and S-adenosylmethionine decarboxylase activities were unaffected. 2. Refeeding for 2.5 h was without effect on ornithine decarboxylase in mammary gland, but it returned the S-adenosylmethionine decarboxylase activity in mammary gland to control values and elevated both ornithine decarboxylase and S-adenosylmethionine decarboxylase in liver. 3. Refeeding for 5 h returned the activity of ornithine decarboxylase in mammary gland to fed-state values and resulted in further increases in S-adenosylmethionine decarboxylase in mammary gland and liver and in ornithine decarboxylase in liver. 4. Prolactin deficiency in fed rats resulted in decreased milk production and decreased activity of ornithine decarboxylase in mammary gland. The increase in ornithine decarboxylase activity normally seen after refeeding starved rats for 5 h was completely blocked by prolactin deficiency. 5. In fed rats, injection of streptozotocin 2.5 h before death caused a decrease in the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase in mammary gland, which could be reversed by simultaneous injection of insulin. Insulin deficiency also prevented the increase in S-adenosylmethionine decarboxylase in liver and mammary gland normally observed after refeeding starved rats for 2.5 h.     

Effects of Ca2+ ionophore A23187, 1,2-dioctanoylglycerol, and dibutyryl cAMP on the activity and expression of ornithine decarboxylase in guinea pig lymphocytes

The Ca2+ ionophore A23187 induced small increases in ornithine decarboxylase activity and ornithine decarboxylase mRNA in guinea pig lymphocytes. 1,2-Dioctanoylglycerol potentiated the A23187-induced ornithine decarboxylase activity and the accumulation of mRNA for this enzyme. Dibutyryl cAMP also potentiated the enzyme activity, but had little effect on the accumulation of mRNA. 1,2-Dioctanoylglycerol and 12-O-tetradecanoylphorbol-13-acetate potentiated ornithine decarboxylase activity that had been increased by treatment with both A23187 and dibutyryl cAMP with a consistent increase in the ornithine decarboxylase mRNA. However, dibutyryl cAMP augmented ornithine decarboxylase activity that had been increased by the combination of A23187 and 1,2-dioctanoylglycerol without affecting the ornithine decarboxylase mRNA level. These results suggest that the protein kinase C and cyclic AMP pathways are involved in the enhancement of ornithine decarboxylase activity in guinea pig lymphocytes, but that the mechanisms of the enhancement differ for each pathway, the former increasing the ornithine decarboxylase mRNA level, but not the latter.     

Decarboxylation of ornithine and lysine in rat tissues.

The possibility that arginine and lysine might be decarboxylated by rat tissues was investigated. No evidence for decarboxylation of arginine could be found. Lysine decarbosylase (L-lysine carboxy-lyase, EC 4.1.1.18) activity producing CO2 and cadaverine was detected in extracts from rat ventral prostate, androgen-stimulated mouse kidney, regenerating rat liver and livers from rats pretreated with thioacetamide. These tissues all have high ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activities. Lysine and ornithine decarboxylase activities were lost to similar extents on inhibition of protein synthesis by cycloheximide and on exposure to alpha-difluoromethylornithine. A highly purified ornithine decarboxylase preparation was able to decarboxylate lysine and the ratio of ornithine to lysine decarboxylase activities was constant throughout purification. Kinetic studies of the purified preparation showed that the V for ornithine was about 4-fold greater than for lysine, but the Km for lysine (9 mM) was 100-times greater than that for ornithine (0.09 mM). These experiments indicate that all of the detectable lysine decarboxylase activity in rat and mouse tissues was due to the action of ornithine decarboxylase and that significant cadaverine production in vivo would occur only when ornithine decarboxylase activity is high and lysine concentrations substantially exceed those of ornithine.     

Induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol 13-acetate in hamster fibroblasts. Relationship between levels of enzyme activity, immunoreactive protein, and RNA during the induction process

Phorbol ester tumor promoters and growth factors rapidly stimulate ornithine decarboxylase activity in the transformed hamster fibroblast line HE68BP. We report here a close correspondence between the time courses and magnitudes of induction of ornithine decarboxylase activity and immunoreactive ornithine decarboxylase protein following treatment of HE68BP cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) and/or refeeding with fresh medium. Cycloheximide addition to induced cells caused a rapid fall in the levels of both ornithine decarboxylase activity and ornithine decarboxylase protein. Northern blot analysis of RNA isolated from HE68BP cells indicated that treatment with TPA and fresh medium increased the amount of two species of mRNA of lengths 2.4 and 2.1 kilobase. This increased accumulation of ornithine decarboxylase mRNA corresponded temporally to that observed at the protein level, with a 15-fold maximal induction 7 h after treatment followed by a rapid decline in hybridizable RNA. These data indicate that stimulation of ornithine decarboxylase activity by TPA or refeeding involves changes in levels of ornithine decarboxylase mRNA as well as changes in the rate of synthesis of ornithine decarboxylase protein.     

Non-classic effects of luliberin confirming the pleiotropy of neuropeptides

The effects of synthetic neuropeptide LH-RH and its analogues were studied in experiments on 174 white male rats. The influence of the substances was shown on instrumental avoidance learning in Y-shaped maze. Convulsive and anticonvulsive effects of the preparations were studied on experimental model of corasol seizures. The analgetic effect of the substances was evaluated by behavioural pain reaction to electrical stimulation of the tail root. Analgetic, anticonvulsive, and psychostimulating LH-RH properties confirm polyfunctionality and "pleiotropy" of neuropeptides as a class of new endogenous informational compounds.     

The dual action of the non-physiological diamines 1,3 diaminopropane and cadaverine on ornithine decarboxylase of HTC cells

In rat hepatoma tumor (HTC) cells 1,3 diaminopropane and cadaverine induced the ornithine decarboxylase antizyme as well as the end product of the ornithine decarboxylase reaction putrescine. Although at equal exogenous concentrations (10^?3M) the two non-physiological diamines penetrated the cells as effectively as putrescine; they decreased cellular ornithine decarboxylase considerably less rapidly than the naturally present diamine. Cell extracts treated with high concentrations of 1,3 diaminopropane and putrescine, and which as a result had a high specific activity of ornithine decarboxylase antizyme, were chromatographed on a superfine Sephadex G-75 column in the presence of 250 mM NaCl. No ornithine decarboxylase-antizyme complex could be detected indicating the original decrease of ornithine decarboxylase in the cells was likely due to some mechanism other than antizyme. These results indicate that 1,3 diaminopropane and cadaverine probably can act on ornithine decarboxylase, like putrescine, by two distinct regulatory mechanisms.     

Purification and characterization of antizyme inhibitor of ornithine decarboxylase from rat liver

A protein inhibiting a protein inhibitor (antizyme) to ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (ODC), antizyme inhibitor, was purified from the liver cytosol of thioacetamide-treated rats by procedures including antizyme affinity chromatography. Overall purification was roughly estimated to be about 17,000,000-fold and recovery was about 2.4%. The purified preparation showed one major protein band and a faint band corresponding in mobility to molecular weights of 51,000 and 53,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Judging from the ornithine decarboxylase activity of the final preparation, the faint band may be ornithine decarboxylase. The apparent molecular weight of antizyme inhibitor estimated by gel filtration on Sephacryl S-200 was approx. 62,000, indicating that antizyme inhibitor may be composed of a single polypeptide chain. In order to examine the question of whether antizyme inhibitor is a protein derived from ornithine decarboxylase, an inactive ornithine decarboxylase, in an immunotitration study and analysis of the binding to antizyme were investigated. The results indicate that antizyme inhibitor may be a protein distinct from ornithine decarboxylase.