Hepatic cyclic AMP generation and ornithine decarboxylase induction by glucagon and beta adrenergic agonists

The relationship of hepatic ornithine decarboxylase (ODC) activity to cyclic AMP levels and nutritional status was studied in the pre-weanling rat. Previous studies demonstrated that 2 hr without food causes a loss of hepatic ODC induction after glucagon or catecholamine injection. Isoproterenol or glucagon administration produced increased hepatic cyclic AMP and tyrosine aminotransferase activity which were not prevented by nutritional deprivation. Blockade of hepatic beta 2 receptors by the selective antagonist ICI 118,551 prevented increased cAMP levels and ODC activity after isoproterenol administration. Blockade of beta 1 receptors by atenolol did not prevent increased cAMP levels or ODC induction by isoproterenol although it did block activation of cardiac ODC. The phosphodiesterase inhibitor RO20-1724 increased hepatic cAMP levels as well as ODC and TAT activities, although the increase in ODC activity was attenuated by nutritional deprivation. RO20-1724 also potentiated the induction of hepatic ODC after glucagon or isoproterenol administration. Administration of 8-bromo cAMP elevated hepatic ODC activity regardless of nutritional status but also elevated serum levels of growth hormone and corticosterone. Hepatic ODC induction by glucagon or beta 2 agonists can be dissociated from changes in cAMP levels during nutritional deprivation.     

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.     

Cell-free synthesis of ornithine decarboxylase. Changes in mRNA activity in the liver of thioacetamide-treated rats

Ornithine decarboxylase (ODC)mRNA associated with free polysomes of rat liver was translated in a reticulocyte lysate cell-free system. Newly synthesized ODC protein was identified by specific immunoprecipitation, molecular size as determined by polyacrylamide gel electrophoresis with sodium dodecyl sulfate, and competition by excess unlabeled ODC in the immunoprecipitation. A single injection of thioacetamide was found to cause several fold increases in both immunotitratable ODC protein and polysomal ODC-mRNA activity, while it provoked a much larger increase in ODC activity in rat liver. The results indicate that the induction of hepatic ODC activity by thioacetamide treatment is due not only to an increase in the activity of polysomal ODC-mRNA but also to a translational and/or posttranslational control.     

Induction of intestinal ornithine decarboxylase by single amino acid feeding

Intestinal and hepatic ornithine decarboxylase (ODC) activities increased to a peak 4 h after administration of a diet containing casein or an amino acid mixture simulating that of casein to rats starved for 12 h. All amino acids except cysteine with a two or three carbon skeleton, including those with a D-configuration, and alpha-amino-isobutyric acid (AIB) strongly induced intestinal ODC when given in the diet or administered intragastrically. Amino acids with a four carbon skeleton were far less effective as inducers and other amino acids did not induce intestinal ODC at all. The amino acids that induced hepatic ODC showed no particular structural characteristics: glycine and cysteine were very effective, threonine, tryptophan, methionine, and phenylalanine were less effective, and serine, valine, isoleucine, and histidine were only slightly effective. Elevation of ODC activity after amino acid administration was not due to stabilization of the enzyme protein with the amino acids. Intestinal ODC was induced by intragastric but not intraperitoneal injection of glycine, although these treatments resulted in similar increases in the tissue concentration of glycine. On the contrary, hepatic ODC was induced by glycine regardless of the administration route. Intestinal ODC was also induced only in the segment of the intestine perfused with a solution of an amino acid with which the activity increased in the feeding experiment. These results suggest that the accumulation of an amino acid per se is not a trigger for induction of intestinal ODC and that an amino acid must act on the mucosal surface to induce the enzyme.     

Effect of diaminobutene and diaminopropane on diet-stimulated polyamine synthesis and cell proliferation in rat liver.

The effect of two putrescine analogs were studied on hepatic polyamine synthesis and cell proliferation, both of which were stimulated by food intake. Trans-1, 4-diamino-2-butene (diaminobutene), which is a potent competitive inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), repressed the induction of ODC and effectively inhibited the accumulation of putrescine in rat liver which was induced by the feeding of dietary protein. Unexpectedly, diaminobutene did not suppress DNA synthesis and mitotic activity in rat liver, suggesting that it can mimic the role of putrescine in cell proliferation. 1,3-Diaminopropane effectively repressed the induction of ODC caused by food intake and also suppressed DNA synthesis and mitotic activity without affecting the accumulation of RNA or protein. The suppression of mitotic activity by 1,3-diaminopropane was reversed by a single injection of putrescine, spermidine, spermine, or diaminobutene. It was concluded that rapid accumulation of polyamines, especially putrescine, was a prerequisite for the later enhancement of DNA synthesis and cell proliferation in rat liver caused by food intake.     

Interleukin-2 regulates the activity of ornithine decarboxylase in a cloned murine T lymphocytic cell line: evidence for a protein kinase C-dependent pathway

The role of protein kinase C (PKC) in the regulation of ornithine decarboxylase (ODC) activity during interleukin-2 (IL-2)-dependent cell growth was investigated. A large biphasic increase in the activity of ODC was observed after treatment of IL-2-deprived CTLL-2 cells with recombinant human IL-2 (rec IL-2). The PKC activators phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD), but not the inactive analog 4 alpha-PDD, induced ODC activity in exponentially growing cultures. Unlike IL-2, however, phorbol esters were poor inducers of IL-2-depleted cultures. H-7, a potent inhibitor of PKC and cyclic nucleotide-dependent protein kinases (CN-PK), suppressed the IL-2-induced ODC activity, while HA1004, a more potent inhibitor of CN-PK than of PKC, had opposite effects depending on its concentration. The results suggest that activation of PKC is involved in but is not the sole mechanism for the induction of ODC by rec IL-2. At concentrations which suppressed the induction of ODC activity by IL-2, H-7 inhibited DNA synthesis and HA1004 did not.     

Inhibition of the 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p′DDT)- and estradiol-mediated induction of rat uterine ornithine decarboxylase by prior treatment with o,p′DDT,estradiol, and tamoxifen

This study was designed to determine whether prior administration of inducers of rat uterine ornithine decarboxylase (ODC), such as 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p′DDT), estradiol-17β (E₂), or tamoxifen, inhibits the elevation of ODC by subsequently administered o,p′ DDT or estradiol-17β. o,p′ DDT (10 mg/day) was injected for 2 days to ovariectomized rats. One or two days later, when the levels of ODC returned to basal levels, o,p′ DDT (10 mg) and E₂ (0.05 μg) were administered intraperitoneally and, 6 or 5 h after these injections, uterine ODC was analyzed. The pretreatment with o,p′ DDT almost entirely blocked the induction of ODC by E₂ or o,p′ DDT. In another experiment, pretreatment with o,p′ DDT for 5 or 6 days eliminated the induction of ODC after injection of o,p′ DDT on Day 7. Similarly, the treatment of rats with the antiestrogen-tamoxifen (0.1 mg/day) for 4 days completely inhibited the subsequent elevation of ODC by either E₂ or o,p′ DDT administered on the fifth day. However, attempts to block the E₂-mediated elevation of ODC by prior treatment with E₂ yielded variable results. Two possibilities were considered to attempt to explain the mechanism of inhibition of induction of ODC by o,p′ DDT and tamoxifen: (a) induction of hepatic monooxygenase by these compounds, resulting in increased metabolism of the subsequently administered o,p′ DDT and E₂ into biologically less active components; (b) involvement of putrescine, the product of ODC action, in inhibiting ODC formation at the pretranslational level or at the post-translational level. It appears unlikely that the o,p′ DDT- and tamoxifen-mediated inhibition of ODC induction was due to an increase in hepatic biotransformation of o,p′ DDT and E₂. Pretreatment with tamoxifen or E₂ did not appear to induce the formation of hepatic microsomal cytochrome P-450, a component of the monooxygenase system. Furthermore, pretreatment with 2,2-bis-(p-chlorophenyl)-1,1-dichloroethylene (a compound with a structure similar to o,p′ DDT), which is not estrogenic but like o,p′ DDT elevates hepatic monooxygenase activity, did not inhibit E₂-or o,p′ DDT-mediated induction of uterine ODC. Concerning the possibility of putrescine inhibitory action, our observations that uterine diamine oxidase activity is negligible and that o,p′ DDT administration has no effect on this enzymatic activity suggested that elevation of ODC may result in higher levels of uterine putrescine or spermidine and spermine. The finding that the administration of putrescine to ovariectomized rats inhibited uterine ODC induction by o, p′ DDT supports the treatise that inhibition of ODC elevation after initial induction of ODC by antiestrogens and o,p′ DDT is due to putrescine- or polyamine-mediated inhibition of ODC. The possible mechanism of such product inhibition of ODC is disucssed.     

Properties and fluctuations in vivo of rat liver antizyme inhibitor.

Antizyme inhibitor was highly purified from rat liver by using affinity chromatography. It has some structural resemblance to ornithine decarboxylase (ODC), as judged from Mr, immunoreactivity and reversible binding with antizyme. However, unlike hepatic amounts of ODC and ODC-antizyme complex, that of antizyme inhibitor did not show much fluctuation upon putrescine treatment, whereas it decreased as rapidly as ODC decay in the presence of cycloheximide. These results suggested that antizyme inhibitor is an independent regulatory protein rather than a derivative of ODC. Changes in hepatic amounts of antizyme inhibitor, antizyme and ODC upon feeding suggested that antizyme inhibitor may play a role in ODC regulation by trapping antizyme and thereby suppressing ODC degradation. A monoclonal antibody to rat liver antizyme inhibitor was obtained. This antibody was shown to be utilizable for a simple assay of antizyme-inhibitor activity in tissue extracts.     

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.     

Synergistic induction of rat hepatic ornithine decarboxylase by multiple doses of cobalt chloride

The level of rat hepatic ornithine decarboxylase (ODC) induced by repetitive administration of Co2+ was determined by affinity labeling with [3H]difluoromethylornithine. Such a treatment with Co2+ ion induced ODC level to a 10-fold greater extent than single dose of the metal ion or well-known inducers of the enzyme, such as thioacetamide or carbon tetrachloride. The half life of ODC activity induced by repetitive treatment with Co2+ (95 min) was substantially increased to about 10-fold over the value obtained from the enzyme induced by single treatment with the metal ion (10 min). ODC activity induced by repetitive treatment with Co2+ was separated into two peaks by DEAE-Sepharose column chromatography. The two independently collected fractions of ODC peaks exhibited different affinity for pyridoxal 5'-phosphate in vitro and sensitivity to cycloheximide in vivo.     

Decrease in hepatic cytochrome P-450 and catalase following allylisopropylacetamide: The effect of concomitant hemin administration

It is known that administration of allylisopropylacetamide (AIA) to rats increases δ-aminolevulinic acid synthetase (ALA-S), urine ascorbic acid excretion and decreases hepatic hemoproteins cytochrome P-450 and catalase. Hemin has been shown to inhibit induction of ALA-S by AIA. To investigate further the regulatory role of hemin, AIA was administered to rats over a 5 day period with and without hemin. Hemin did not prevent the decrease in P-450 or catalase caused by AIA but partially inhibited induction of ALA-S and the increase in ascorbic acid excretion. It is unlikely that the hemin is replacing endogenous heme in hemoprotein synthesis and we conclude that the role of hemin in suppressing induction phenomena that follow AIA treatment reflects a regulatory function not related to a role in hemoprotein synthesis.     

Induction of ornithine decarboxylase in cultured mouse L cells. I. Effects of cellular growth, hormones, and actinomycin D.

The activity of ornithine decarboxylase [EC 4.1.1.17] (ODC) in mouse L cells in the confluent state was induced within 4 hr by cyclic AMP (cAMP) or by insulin. During growth of L cells the concentration of cAMP increased first, then induction of ODC occurred and finally the cell number increased: the levels of cAMP and ODC increased only transitorily and returned to the basal levels when the cells become confluent. In growing cultures, however, the presence of cAMP reduced induction of ODC and cell growth. These results suggest that cAMP is involved in induction of ODC and that its concentration may be important for enzyme induction as well as for cell growth. Actinomycin D with or without these inducers stimulated induction of ODC in L cells, whereas cycloheximide inhibited it, suggesting that these hormones affect the translational level of ODC synthesis. The effect of actinomycin D on induction of ODC was much greater in non-growing cells than in growing cells. It was also found that the half life of ODC was 81 min in non-growing cells and 112 min in growing cells. This suggests that turnover of the enzyme is more rapid in the non-growing than in the growing state and that there may be an RNA fraction which controls its turnover and which also has a very short half life.     

Comparative studies of the effects of stilbene compounds on hepatic ornithine decarboxylase and S-adenosylmethionine decarboxylase induction in rats

Trans-Stilbene oxide (TSO, 2 mmol/kg, ip.) induced ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) to 60-fold and 5-fold of the controls, respectively, in the liver of rats. Parallel to ODC induction, there was a marked increase in putrescine content to 50-fold of the control levels. Cis-Stilbene oxide (CSO), a stereoisomer of TSO, also produced the induction of ODC and SAMDC and the increase in putrescine content. There was no difference in the ability to induce ODC and SAMDC between TSO and CSO with respect to the extents of induction and the time needed to reach maximal levels. Trans-Stilbene (TS), a mother compound of TSO, did not show such an effect on ODC, while cis-stilbene (CS) induced both ODC and SAMDC. Treatment with glutathione inhibited TSO- and CSO-mediated induction of ODC and SAMDC. These findings add new information concerning the abilities of TSO, CSO and CS on hepatic polyamine metabolism.     

Induction of ornithine decarboxylase and S-adenosylmethionine decarboxylase by sulfobromophthalein in rats

The administration of sulfobromophthalein (BSP, 0.5 mmol/kg, ip.) increased ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities to 30-fold and 5-fold, respectively, of the controls at 12 hr in the liver of rats. Parallel to the increase in ODC, there was an increase in hepatic putrescine content. However, spermine content tended to decrease. BSP increased ODC and SAMDC activities and putrescine content, but decreased spermine content, in a dose-dependent manner. Pretreatment of rats with actinomycin D and cycloheximide almost completely blocked the BSP-mediated increase of ODC and SAMDC activities. Pretreatment with glutathione (GSH) failed to inhibit BSP-mediated increase of ODC and SAMDC activities. In addition, the administration of BSP-GSH conjugate (0.5 mmol/kg, iv.) did not produce the increase of ODC and SAMDC activities. Pretreatment with phenobarbital and 3-methylcholanthrene did not inhibit BSP-mediated increase of ODC and SAMDC. The results indicate that BSP could cause changes in hepatic polyamine content due to the induction of ODC and SAMDC.     

Two distinct mechanisms for ornithine decarboxylase regulation by polyamines in rat hepatoma cells.

Exogenous diamines and polyamines added to rat hepatoma (HTC) cells in culture rapidly decrease ornithine decarboxylase (ODC) activity. Previous evidence has suggested that these amines set either at the level of blocking new enzyme synthesis or by the induction of a non-competitive protein inhibitor, termed antizyme, which complexes with ODC to form an inactive complex. Wth the use of HMOA cells, a recently cloned rat hepatoma cell line that has a greatly stabilized ODC, it has been possible to demonstrate that 10(-5) M of exogenous putrescine blocks the increase in ODC activity, but unlike in the parent HTC cell line, without induction of the antizyme or formation of any inactive ODC-antizyme complex. However, complete blockade of ODC at 10(-2) M putrescine is effected by induction of antizyme and formation of the ODC-antizyme complex, as now evidenced by the isolation of the active enzyme and antizyme components after Sephadex column chromatography in the presence of 250 mM NaCl. These findings indicate clearly that two polyamine-regulatory mechanisms for ODC exist and are separable in this cell line.     

Effect of hypotonic stress on ornithine decarboxylase mRNA expression in cultured cells.

This report examines the effect of hypotonic stress on ornithine decarboxylase (ODC) activity and ODC mRNA concentrations in LLC-PK1 cells. Earle's balanced salts solution minus glucose (EBSS-G) with decreasing concentrations of NaCl was utilized as the ODC induction medium. Hypotonic EBSS-G increased both the concentration of ODC mRNA and the specific activity of ODC in LLC-PK1 cells. Actinomycin D and cycloheximide prevented the increase in enzyme activity resulting from hypotonic stress. Actinomycin D was also a potent inhibitor of ODC mRNA expression resulting from hypotonic stress. Cycloheximide had very little effect on the induction of ODC mRNA in cells incubated in hypotonic EBSS-G. The magnitude of the increase in both ODC mRNA concentrations and enzyme activity was dependent on the incubation time in hypotonic media. The increase in ODC mRNA concentrations preceded the elevation in enzyme activity. ODC mRNA concentrations and the specific activity of ODC increased as a function of decreasing media osmolarity. The addition of putrescine, spermidine, and spermine to EBSS-G containing reduced NaCl suppressed the increase in LLC-PK1 ODC activity related to hypotonic stress. In contrast, these polyamines did not prevent the increase in ODC mRNA resulting from hypotonic shock. Furthermore, it was demonstrated that hypotonic stress increases ODC mRNA levels and enzyme activity in four additional cell lines from two different species. Based on these results it is suggested that one or more signal transducers associated with cell volume expansion enhance expression of the ODC gene.