Regulation of ornithine decarboxylase activity in rat ovarian cells in vitro.

Incubation of rat ovarian cell suspension with human choriogonadotropin (hCG) caused a marked enhancement of ornithine decarboxylase (EC 4.1.1.17) activity after a lag period of several hours. Even though ovarian ornithine decarboxylase could be induced in minimum essential medium by the hormone alone, supplementation of the medium with various sera greatly enhanced the stimulation of the enzyme activity. All the sera tested (human, fetal calf and horse) were able to stimulate ornithine decarboxylase activity even in the absence of hCG. Maximum stimulation of the enzyme activity by hCG and/or serum occurred in ovarian cell suspensions prepared from 30 to 33-day-old rats. There was a close correlation between the stimulation of ornithine decarboxylase activity and the accumulation fo cyclic AMP in response to the administration of the hormone (in the presence or absence of serum). However, while various sera alone markedly enhanced ovarian ornithine decarboxylase activity in vitro they, if anything, only marginally stimulated the accumulation of cyclic AMP and the secretion of progesterone in ovarian cells in the absence of gonadotropin. A similar dissociation of the stimulation of ornithine decarboxylase activity from the production of cyclic AMP and progesterone was likewise found when the ovarian cells were incubated in an enriched medium (M199) supplemented with albumin and lactalbumin hydrolysate in the absence of the hormone. Under these culture conditions ornithine decarboxylase activity was strikingly enhanced, greatly exceeding the stimulation obtained with various sera, while the accumulation of cyclic AMP and the secretion of progesterone remained virtually unchanged. Specific inhibition (up to 90%) of gonadotropin-induced ornithine decarboxylase activity by difluoromethyl ornithine or 1,3-diamino-2-propanol had little effect on the ability of the ovarian cells to respond to the hormone with increasing production of cyclic AMP and progesterone. While showing that rat ovarian ornithine decarboxylase can be induced in vitro by choriogonadotropin or various sera, our results indicate that the activation of the enzyme involves at least two different mechanisms: (i) One (in response to gonadotropin) involving a prior stimulation of cyclic AMP production, and (ii) another (in response to serum) that is not associated with increases in the accumulation of the cyclic nucleotide.     

Prostaglandin stimulation of ornithine decarboxylase activity in mammary gland explants from mid-pregnant mice

The effects of various prostaglandins on ornithine decarboxylase (ODC) activity in mammary gland explants from mid-pregnant mice have been tested. PGE1, E2 and I2 elicit a concentration-dependent stimulation of ODC activity. The minimally effective concentrations are 0.5 ug/ml for PGE1 and E2, and 50 ug/ml for PGF2 alpha and 6-keto-PGF1 alpha. The PGE1 effect had a time course identical to that of prolactin. The prolactin action on ODC activity was attenuated by indomethacin, an inhibitor of prostaglandin biosynthesis. Arachidonic acid stimulated ODC activity and its effect was abolished by indomethacin. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, potentiated the PGE1 effect on ODC activity. The results suggest that the prostaglandins may modulate prolactin's action on ODC activity via a cAMP dependent mechanism.     

Prostaglandin stimulation of ornithine decarboxylase activity in mammary gland explants from mid-pregnant mice

The effects of various prostaglandins on ornithine decarboxylase (ODC) activity in mammary gland explants from mid-pregnant mice have been tested. PGE₁, E₂ and I₂ elicit a concentration-dependent stimulation of ODC activity. The minimally effective concentrations are 0.5 ug/ml for PGE₁ and E₂, and 50 ug/ml for PGF_2α and 6-keto-PGF_1α. The PGE₁ effect had a time course identical to that of prolactin. The prolactin action on ODC activity was attentuated by indomethacin, an inhibitor of prostaglandin biosynthesis. Arachidonic acid stimulation ODC activity and its effect was abolished by indomethacin. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, potentiated the PGE₁ effect on ODC activity. The results suggest that the prostaglandins may modulate prolactin's action of ODC activity via a cAMP dependent mechanism.     

Tissue specific compartmental analysis of gonadotropin stimulation of ovarian ornithine decarboxylase

Luteinizing hormone is known to stimulate the enzyme ornithine decarboxylase in the ovary. Highly purified human follicle stimulating hormone that is devoid of significant biologically active luteinizing hormone can also induce ornithine decarboxylase activity in intact immature rats with a time course of induction similar to that reported for luteinizing hormone. A maximum of 8–10-fold stimulation above controls was observed 4 h following intravenous administration of human follicle stimulating hormone. This stimulation followed a strict dose response relationship. Ovine luteinizing hormone and human chorionic gonadotropin always induced more ovarian ornithine decarboxylase activity than that achieved by maximally effective doses of follicle stimulating hormone. This could not be attributed solely to the ability of specific cell population to respond to the respective gonadotropins. Although granulosa cells contained little receptor for luteinizing hormone/human chorionic gonadotropin and the residual tissue contained little receptor for follicle stimulating hormone, each tissue responded to these gonadotropins in a manner suggestive of the mediation by one or more diffusable factors. A relationship between gonadotropin induced 3’5’-cyclic adenosine monophosphate (cyclic adenosine monophosphate) concentration and ornithine decarboxylase activity suggests that the mediation of gonadotropin stimulated ovarian ornithine decarboxylase is not solely through cyclic adenosine monophosphate, indicating the presence of other factors in the induction of gonadotropin increased ornithine decarboxylase activity.     

Indomethacin blocks gonadotropin stimulation of ovarian ornithine decarboxylase activity by inhibiting protein synthesis

Both gonadotropins and prostaglandins stimulate the ornithine decarboxylase (ODC) activity of porcine granulosa cells (1,2). To investigate a possible intermediary role of prostaglandins in this gonadotropin action, the effects of indomethacin on gonadotropin-induced ODC activity were studied. Indomethacin had no effect at concentrations lower than 10⁻⁵ M; at higher concentrations indomethacin exerted a dose-dependent suppression of LH-stimulated ODC activity which was essentially complete at 5 × 10⁻⁴ M. The effects of PGE₂ and 8-Bromo-cAMP, potent stimulators of ODC, were also blocked by indomethacin (5 × 10⁻⁴ M). This effect did not represent direct inhibition of enzyme activity, but appeared to be due to inhibition of protein synthesis by the drug. Thus, incorporation of ¹⁴C-leucine into proteins by these cells was blocked by indomethacin with a dose-response curve similar to that for ODC suppression. This distal effect of indomethacin may complicate the interpretation of some experiments if the inhibitor is assumed to act only at the prostaglandin synthetase step.     

Beta-2-Adrenergic stimulation of ornithine decarboxylase activity in porcine granulosa cells in vitro

Epinephrine, norepinephrine, and isoproterenol produced dose-dependent stimulation of ornithine decarboxylase (EC 4.1.1.17) activity in isolated porcine granulosa cells maintained under defined conditions in vitro. beta- but not alpha-receptor-blocking agents prevented enzyme stimulation by catecholamines. Application of preferential beta-1 and beta-2-receptor antagonists and agonists localized the epinephrine effect to beta-2-adrenergic mediation. Epinephrine action was enhanced by the phosphodiesterase inhibitor, 1-methyl-3-isobutyl-xanthine, but not by saturating concentrations of the cyclic AMP analogue, 8-bromocyclic AMP, of follicle-stimulating hormone, or of prostaglandin E2. However, stimulation by epinephrine was additive to that of luteinizing hormone. Follicular fluid obtained from immature Graafian follicles contained concentrations of norepinephrine and epinephrine active in vitro. Thus, catecholamines may participate in the regulation of ornithine decarboxylase activity in the ovary. Catecholamine effects may be mediated by beta-2-receptors linked to the adenylate cyclase system.     

β-2-adrenergic stimulation of ornithine decarboxylase activity in porcine granulosa cells in vitro

Epinephrine, norepinephrine, and isoproterenol produced dose-dependent stimulation of ornithine decarboxylase (EC 4.1.1.7) activity in isolated porcine granulosa cells maintained under defined conditions in vitro. β- but not α-receptor-blocking agents prevented enzyme stimulation by catecholamines. Application of preferential β-1 and β-2-receptor antagonists and agonists localized the epinephrine effect to β-2-adrenergic mediation. Epinephrine action was enhanced by the phosphodiesterase inhibitor, 1-methyl-3-isobutyl-xanthine, but not by saturating concentrations of the cyclic AMP analogue, 8-bromocyclic AMP, of follicle-stimulating hormone, or of prostaglandin E₂. However, stimulation by epinephrine was additive to that of luteinizing hormone. Follicular fluid obtained from immature Graafian follicles contined concentrations of norepinephrine and epinephrine active in vitro.Thus, catecholamines may participate in the regulation of ornithine decarboxylase activity in the ovary. Catecholamine effects may be mediated by β-2-receptors linked to the adenylate cyclase system.     

L-ornithine-induced inactivation of mammalian ornithine decarboxylase in vitro

Partially purified ornithine decarboxylase, isolated from the liver of thioacetamide-treated rats, is stable in the absence of added low-molecular-mass thiols or other reducing agents. However, under these conditions, the enzyme is rapidly inactivated upon incubation with L-ornithine or L-2-methylornithine. The inactivation process follows first-order kinetics, and saturation kinetics are observed. Rapid recovery of activity is observed after subsequent addition of dithiothreitol. As distinct from L-ornithine, D-ornithine, putrescine, spermidine, or spermine do not produce inactivation of ornithine decarboxylase. Very similar results are obtained with pure ornithine decarboxylase isolated from androgen-stimulated mouse kidney, stabilized with a rat liver extract.     

Androgen and estrogen stimulation of ornithine decarboxylase activity in mouse kidney

In the present work, the activity of mouse renal ornithine decarboxylase (ODC) from CBA female mice was used as a biological marker to detect (anti)androgenic activity of different groups of endocrine disruptors and steroids. Daily injections of testosterone or dihydrotestosterone (DHT) into 60 day old female mice for 4 days increased renal ODC activity in a dose-dependent manner that reached up to 100-fold (testosterone) or 250-fold (DHT) above the baseline when the highest dose, 200 microg/mouse, was used. Administration of flutamide concurrently with testosterone (75 microg/mouse) caused a potent decrease of ODC induction in a dose-dependent manner, suppressing the enzyme activity at the doses of 0.1 and 0.5 mg/mouse by about 88 and 95%, respectively. In contrast, estradiol at the doses of 0.5 and 1 mg/mouse induced a significant stimulation of renal ODC activity in a dose-dependent manner when it was given alone or in combination with testosterone. Using a sensitive increase in ODC activity in response to androgens as an end point, we did not detect an antiandrogenic effect of several antiandrogens, such as cyproterone acetate, spironolactone, p,p'DDE and vinclozolin. Also, none of these antiandrogens were able to change the basal level of renal ODC activity, with the exception of cyproterone acetate that at a dose of 0.1 mg/mouse stimulated ODC activity. The data obtained suggest that mouse renal ODC from CBA females is not strictly androgen-specific and cannot be used for estimation of antiandrogenic effects of compounds having an affinity to different types of receptors.     

Studies on the mechanisms of ornithine decarboxylase in vitro inactivation

Hydrocortisone-induced rat liver ornithine decarboxylase appears quite stable in the soluble fraction of the homogenate incubated at 37 degrees C. In contrast, the incubation of the whole homogenate causes a rapid loss of activity. The ornithine decarboxylase-inactivating capacity appears mainly bound to microsomes. Lysosomes seem to play a role only after the microsome-induced inactivation. Different reducing agents (dithiothreitol, NADPH, NADH, GSH) are effective both in preventing and in reversing ornithine decarboxylase inactivation. NADPH is peculiar in that it can reactivate the enzyme at very low concentrations. Oxidized glutathione potentiates the inactivating effect of microsomes. On the basis of present results it is suggested that ornithine decarboxylase may be reversibly inactivated through microsome-catalyzed formation of mixed or enzyme-enzyme disulfides and that NADPH plays a crucial role in ornithine decarboxylase reactivation, probably by cytosolic reductase(s).     

Immunocytochemical detection of ornithine decarboxylase.

Ornithine decarboxylase (ODC), a regulatory enzyme of polyamine biosynthesis, is involved in cell growth and differentiation. Lack of information about the exact cellular and subcellular localization of ODC is one of the main obstacles to precise interpretation of the biological roles of the ODC/polyamine system. Here we describe the development and optimization of an immunocytochemical method to detect ODC in cells and tissues. For this purpose a monoclonal antibody (MP16-2) against a defined epitope of ODC protein was developed. Specificity of the antibody for ODC was substantiated by Western blotting and ELISA analysis using cell and tissue homogenates. In cultured cells, optimal staining results were obtained after fixation with crosslinking fixatives followed by permeabilization with methanol. In rat tissues, ODC immunoreactivity was best preserved in paraffin sections fixed with Bouin's fixative. Antigen retrieval using SDS and citrate buffer substantially increased ODC immunostaining and decreased background staining. Localization studies of ODC in different cell lines showed that strongest staining for ODC was found in the nucleoplasm of mitotic cells, whereas confluent cells showed moderate perinuclear staining. Immunocytochemical studies of various rat tissues showed high cytoplasmic immunostaining of ODC in epithelial cells of kidney, prostate, and adrenal medulla of testosterone-treated rats, in glandular epithelium of small intestine, and in pancreas of neonatal and adult rats. (J Histochem Cytochem 47:1395-1404, 1999)     

Feedback control of ornithine decarboxylase expression by polyamines. Analysis of ornithine decarboxylase mRNA distribution in polysome profiles and of translation of this mRNA in vitro.

Cell growth and differentiation require the presence of optimal concentrations of polyamines. Ornithine decarboxylase (ODC) catalyses the first and rate-controlling step in polyamine synthesis. In studies using cultures of Ehrlich ascites-tumour cells, we have shown that the expression of ODC is subject to feedback regulation by the polyamines. A decrease in the cellular polyamine concentration results in a compensatory increase in the synthesis of ODC, whereas an increase in polyamine concentration results in suppression of ODC synthesis. These changes in ODC synthesis were attributed to changes in the efficiency of ODC mRNA translation, because the steady-state amount of ODC mRNA remained constant. We now show that the number of ribosomes associated with ODC mRNA is low, and that the increase in ODC mRNA translation takes place without a shift in the distribution of ODC mRNA towards larger polysomes. This finding indicates that the polyamines regulate the efficiency of ODC mRNA translation by co-ordinately affecting the rates of initiation and elongation. By analysing ODC mRNA translation in vitro, using a rabbit reticulocyte lysate, polyadenylated RNA from a cell line with an amplified ODC gene, and a monospecific anti-ODC antibody, we also show that spermidine, but not putrescine, exerts a direct regulatory effect on ODC synthesis.     

Saponin effects of prolactin-like stimulation of ornithine decarboxylase activity in mouse mammary gland explants.

Saponin, a naturally occurring plant glycoside, was found to elicit a prolactin-like stimulation of ornithine decarboxylase (ODC) activity in mouse mammary gland explants. A dose-response activation of ODC was observed with saponin at concentrations between 2 and 10 micrograms/ml. At concentrations of 10 and 15 micrograms/ml, saponin effected a response similar to that of PRL; when tested in concert, PRL and saponin caused a nonadditive response. The time-course of the saponin and PRL effects on ODC activation were not different; a maximum response occurred 2-4 hours after addition of saponin. The saponin and PRL responses were abolished by antibiotics (puromycin and cyclohexamide) that inhibit protein synthesis, but not by actinomycin D which inhibits RNA synthesis. Finally, saponin, by itself, did not affect the rate of milk product formation, but at higher concentrations (above 0.5 microgram/ml) impaired the PRL stimulation of lipid and casein synthesis.