Separate mechanisms of induction for ornithine decarboxylase by triiodothyronine and aminophylline

A single dose of aminophylline (200 μmol/kg, i.p.) or triiodothyronine (T₃, 300 μg/kg, i.p.) resulted in the induction of ornithine decarboxylase (ODC) in rat liver with maximal activity 10-fold and 6-fold above controls, respectively, 4 hr after the administration of the drug or hormone. After either agent, the induction of ODC was blocked by either cycloheximide or actinomycin D. The same concentrations of aminophylline and T₃ administered simultaneously produced an additive 16-fold increase in ODC activity. After T₃ administration, the cyclic AMP-dependent protein kinase activity ratio was unaltered at all times measured. After aminophylline, the protein kinase activity ratio was elevated by 15 min and remained elevated for 2 hr. Somatostatin administration (50 μg/100 g), which lowers plasma growth hormone to 30% of control, had no effect on the ability of T₃ to induce ODC. These data suggest separate routes of induction of ODC in response to aminophylline and T₃. Aminophylline induction occurs via cycyclic AMP-mediated event whereas T₃ does not involve ccyclic AMP but results from a direct nuclear interaction.     

Calcium stimulates ornithine decarboxylase activity in cultured mammalian epithelial cells

Ornithine decarboxylase (ODC) activity usually rises to a peak a few hours after a trophic stimulus. The stimulation of ODC has been shown to depend on extracellular calcium in several in vitro eukaryotic systems. We have investigated the effect of calcium concentration on ODC activity and have found that ODC is stimulated when CaCl2 alone is added to calcium-deprived cells. Epithelial cells from calf esophagus were cultured and grown until stratified. Replacement of medium with fresh serum-free medium resulted in stimulation of ODC activity, which peaked at 4 hours and declined to basal level by 10 hours. Subsequent depletion of Ca2+ either by addition of ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or by replacement of medium with Ca2+-free medium, resulted in obliteration of ODC activity 4 hours later. Conversely, cultures in which medium was replaced with Ca2+-free medium and at 10 hours were repleted with Ca2+ (either by addition of CaCl2 or by replacement of medium with Ca2+-containing medium) exhibited a pronounced elevation of ODC activity 4 hours later. ODC activity peaked at 6 hours after the addition of CaCl2 and declined by 8 hours. The effect was elicited by a wide range of concentrations of added Ca2+ from 0.1 mM to 4.0 mM, but was maximal at 1.0 mM. ODC activity was totally abolished if either cycloheximide (10 micrograms/ml) or putrescine (10 mM) was added to cultures immediately prior to Ca2+ addition. Actinomycin D (2, 5, or 10 micrograms/ml) added 30 minutes before Ca2+ did not prevent the stimulation of ODC by added Ca2+. Stimulation by Ca2+ is dependent on (1) absence of Ca2+ during the initial 10-hour incubation and (2) duration of incubation in Ca2+-free medium prior to Ca2+ replenishment. The results indicate that Ca2+ can increase ODC in epithelial cells exposed to Ca2+-depleted medium and that the increase in ODC depends on protein synthesis but is not inhibited by actinomycin D.     

Effects of two hexachlorobiphenyl isomers on ornithine decarboxylase induction: inhibitory effects correlate with aryl hydrocarbon hydroxylase activity

The effects of 2,2',4,4',5,5'-hexachlorobiphenyl (2,4,5-HCB) or 3,3',4,4',5,5'-hexachlorobiphenyl (3,4,5-HCB) on hepatic ornithine decarboxylase (ODC) induction by dexamethasone were investigated. At one week after a single i.p. dose of corn oil or 2,4,5,-HCB and 4 h after administration of dexamethasone, rats exhibited 50- to 60-fold increases of ODC activity. However, rats that had received 3,4,5-HCB in place of 2,4,5-HCB exhibited only a 8-fold increase in ODC activity in response to dexamethasone administration. 2,4,5-HCB administration resulted in increased hepatic aryl hydrocarbon hydroxylase (AHH) activity. Administration of 3,4,5-HCB produced increased AHH activity and decreased N-demethylase activity. It is suggested that the ODC-inhibitory effects may have resulted from Ah-receptor-mediated events.     

Induction of Brain Ornithine Decarboxylase During Recovery from Metabolic, Mechanical, Thermal, or Chemical Injury

Metabolic, mechanical, thermal, and chemical injury induced ornithine decarboxylase (ODC) activity in rat brain. A two- to sixfold increase in ODC activity was measured at 5-9 h after different modes of injury to the brain. During the early phase of recovery from transient ischemia, when average protein synthesis was less than 50% of control, ODC activity was increased nearly fivefold. The rise in activity could be blocked by anisomycin, or reduced by intracerebral injections of actinomycin D. Drilling burr holes into the skull, injection of the vehicle for actinomycin D, hyperthermia, and freezing lesions all caused increased ODC activity. Neurotoxic chemicals (ammonia, methionine sulfoximine, acrylamide, carbon tetrachloride, and anisomycin) also increased brain ODC activity, whereas other chemicals (mannitol and valine) did not. Treatments known to stimulate the synthesis of heat shock proteins (carotid occlusion, hyperthermia, Cd2+, canavanine, and ethanol) induced ODC activity in the liver, whereas only hyperthermia and ethanol caused significant increases in spleen ODC activity. All increases in ODC activity were blocked by difluoromethylornithine, an irreversible inhibitor of ODC. The cellular response to noxious or stressful stimuli includes the synthesis of a small number of proteins of unknown functions; ODC may be one of these "heat shock" or "trauma" proteins.     

Insulin induction of ornithine decarboxylase. Importance of mRNA secondary structure and phosphorylation of eucaryotic initiation factors eIF-4B and eIF-4E

We investigated the possibility that insulin could stimulate translation of ornithine decarboxylase (ODC) mRNA in a murine fibroblast cell line that expresses large numbers of human insulin receptors (HIR 3.5 cells). Within 3 h after exposure to 70 nM insulin, ODC enzyme activity increased approximately 50-fold and mRNA accumulation 3-fold in the HIR 3.5 cells but not in normal fibroblasts. Pretreatment of cells with cycloheximide completely inhibited insulin-stimulated ODC expression; actinomycin D partially inhibited this effect. To determine the influence of the 5' untranslated region (5'UTR) of ODC mRNA on insulin-regulated ODC expression, plasmids were constructed which contained sequences from the 5'UTR of a rat ODC mRNA interposed between the ferritin promoter and the coding region of the human growth hormone gene. These constructions were then expressed transiently in HIR 3.5 cells. Insulin stimulated a 2-4-fold change in growth hormone accumulation in the medium of cells transiently expressing plasmids containing the entire 5'UTR of ODC mRNA or just the 5'-most 115 bases, a G/C-rich conserved sequence predicted to form a stem-loop structure and shown previously to be responsible for constitutive inhibition of translation. There was a direct correlation between the extent of insulin stimulation and the predicted secondary structure of the added 5'UTR sequences. To determine whether this effect might be due to insulin activation of initiation factors responsible for melting mRNA secondary structure, we examined the effect of insulin on the phosphorylation states of two such factors, eucaryotic initiation factors eIF-4B and eIF-4E. Insulin stimulated the phosphorylation of both initiation factors; this stimulation was evident at 15 min and maximal by 60 min. These results suggest a potential general mechanism by which insulin could preferentially stimulate translation of mRNAs whose 5'UTRs exhibit significant secondary structure by activating initiation factors involved in melting such secondary structures.     

Hormonal alteration of methotrexate and folate polyglutamate formation in cultured hepatoma cells

Glutamylation of the antifolate methotrexate in H35 hepatoma cells was stimulated by physiologic concentrations of insulin and dexamethasone. At saturating concentrations of the hormone a 2.7-fold stimulation could be obtained with insulin (65 nM, 16-h exposure) and a 1.8-fold stimulation with dexamethasone (100 nM, 16-h exposure). The increases in glutamylation caused by the hormones were not additive, and both were inhibited by actinomycin D and cycloheximide. N6,O2'-dibutyryl cAMP and theophylline caused a modest reduction of glutamylation in control and dexamethasone-treated cultures, but repressed the stimulation caused by insulin by approximately one-third. Enhancement of synthesis by dexamethasone and insulin was associated with increases in the tri-, tetra-, and pentaglutamate derivatives of methotrexate, with little change in intracellular methotrexate and methotrexate diglutamate. When the conversion of folinic acid into the folylpolyglutamate pool was examined in folate-depleted H35 cells, insulin and dexamethasone had similar effects. The results suggest that these hormones play a role in the glutamylation of the folate coenzymes in a liver-derived transformed cell line in culture and that these effects are also reflected in the interaction of the cells with antifolates such as methotrexate.     

Induction of hepatic and renal ornithine decarboxylase by cobalt and other metal ions in rats.

We previously showed that Cd2+ is able to induce hepatic and renal ornithine decarboxylase (ODC). In addition to Cd2+, the administration of Co2+ and other metal ions such as Se2+, Zn2+ and Cr2+ produced a significant increase of hepatic and/or renal ODC activity. Of the metal ions used in this study, Co2+ produced the greatest increase of ODC activity. The maximum increases in hepatic and renal ODC activity, to respectively 70 and 14 times the control values in male rats, were observed 6 h after the administration of Co2+. A similar response was seen in the liver, but not in the kidney, of female rats. Thereafter, ODC activity gradually returned to control values in the liver, but it was profoundly decreased to 7% of the control value at 24 h in the kidney. The pretreatment of animals with either actinomycin D or cycloheximide almost completely blocked the Co2+-mediated increase of ODC activity. Co2+ complexed with either cysteine or glutathione (GSH) failed to induce ODC. Depletion of hepatic GSH content by treatment of rats with diethyl maleate greatly enhanced the inducing effect of Co2+ on ODC. The inhibitors of ODC, 1,3-diaminopropane and alpha-difluoromethylornithine, were able to inhibit the induction of the enzyme, without affecting the induction of haem oxygenase by Co2+. Methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, significantly inhibited the Co2+-mediated induction of both ODC and haem oxygenase. It is suggested that the inducing effects of Co2+ on ODC and haem oxygenase are brought about in a similar manner.     

Ornithine decarboxylase induction and polyamine levels in the kidney of estradiol-treated castrated male rats

Ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and thymidine kinase (TK) activities and polyamine concentrations on the kidneys of male castrated rats were studied following sc injection of estradiol. Estradiol caused an 11-fold increase in ODC activity 24 hours after administration. SAMDC activity doubled but TK activity decreased by two-thirds 2 days after estradiol treatment. The concentrations of polyamines, especially putrescine, showed sharp elevations 2 days following estradiol treatment, 1 day after the peak of ODC activity. The increase in ODC activity was suppressed by cycloheximide and by actinomycin D. Estradiol and diethylstilbestrol (DES), but not progesterone increased ODC activity. Estradiol suppressed ODC activities of liver, thymus, adrenal glands, testes and prostate. A specific estradiol-binding protein was demonstrated in the rat kidney. The dissociation constant (Kd) was 1.64 × 10^?10 M and numbers of binding sites were 31 fmoles/mg protein. Correlation between the binding of estradiol to the cytosol protein and elevation of ODC by estradiol was observed.     

The induction of ornithine decarboxylase by tumor promoting phorbol ester analogues in Reuber H35 hepatoma cells

The induction of ornithine decarboxylase activity was studied in a rat hepatoma cell line (Reuber H35) incubated with a group of structurally-related phorbol ester analogues. A single application of 1.6 μM of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to H35 cells caused a dramatic increase in the activity of ornithine decarboxylase. The stimulation of the enzyme activity was rapid but transient, peaking at 4 to 5 hr with a value which was 116-fold greater than control and then declining to the basal level after 8 hr. In addition, the increase in ODC activity was dependent upon the concentration of TPA added to the culture medium and the EC₅₀ was estimated to be about 2.63 × 10^?7 M. Our studies of the effect of various phorbol ester analogues on the H35 ODC activity indicated an apparent correlation between the ability of phorbol ester derivatives to induce ODC activity in the H35 cells and their activity to promote papilloma formation in the mouse skin in that the various derivatives possessed the following relative abilities to increase ODC activity: TPA > PDB > PDA > 4 α-P > 4 α-PDD. Concurrent addition of either actinomycin D or cycloheximide abolished the increase in ODC activity after TPA treatment. Changes of intracellular concentrations of polyamines, particularly putrescine, were in good agreement with the increase in ODC activity in response to TPA: a 10-fold increase in putrescine over the control level was observed at 6 hr. Our data suggest that cultured Reuber H35 hepatoma cells exhibit a marked and specific response to the phorbol ester tumor promoters and may be of great value in studying the biochemical mechanism of ODC induction by these agents.     

Ornithine decarboxylase activity in foetal rat brain cells and in the mouse embryo fibroblasts C3H/10T1/2 CL8 cells: differences in response to medium change and to the tumor promoter TPA

12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced ornithine decarboxylase (ODC, EC 4.1.1.17) in normal, preneoplastic and malignant rat brain cells in culture, but treatment with phorbol, acetate or medium shift resulted in a similar response. Medium shift induced ODC activity in C3H/10T1/2 CL8 cells 4 and 12 hr after treatment. TPA induced only the 12 hr peak. ODC induction in C3H/10T1/2 CL8 cells was completely inhibited by cycloheximide and actinomycin D. Addition of alpha-amanitin abolished the 12 hr peak, but the TPA induced ODC activity was only partly inhibited. ODC induction by TPA was lower in C3H/10T1/2 CL8 cells initiated with 3-methyl-cholanthrene (MCA). ODC increased with TPA up to 10(-7) M and decreased at higher concentrations of TPA.     

Induction of cystathionase in human foetal liver (Short Communication)

Incubation of liver explants from second-trimester human foetuses with dexamethasone, glucagon or dibutyryl cyclic AMP (plus theophylline) increased the activity of liver cystathionase from unmeasurable or trace values to adult values. Simultaneous incubation with cycloheximide or actinomycin D inhibited this effect.