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.     

Effects of cyclic nucleotides on ornithine decarboxylase activity in mammary gland explants from mid-pregnant mice

Dibutyryl cAMP and prolactin stimulated ornithine decarboxylase activity in mouse mammary gland explants which had been preincubated with insulin and cortisol for 1 day; maximally stimulatory concentrations of dibutyryl cAMP and prolactin produced a response which was greater than the sum of the responses of prolactin and dibutyryl cAMP when tested alone. 8-Bromo-cGMP inhibited ornithine decarboxylase activity whereas other derivatives of cyclic nucleotides were without effect. Cortisol concentrations were found to be important for optimizing the dibutyryl cAMP and prolactin responses. Optimal prolactin responses were obtained with cortisol concentrations greater than 10(-7) M, whereas optimal dibutyryl cAMP responses were observed with cortisol concentrations less than 10(-7) M. Despite the differing optimal cortisol concentrations for the prolactin and dibutyryl cAMP responses, it is concluded that prolactin and dibutyryl cAMP probably stimulate ornithine decarboxylase activity in the mammary gland via the same mechanism.     

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.     

Effects of mezerein and diglycerides on ornithine decarboxylase activity in mouse mammary gland explants

One of the most rapid actions of prolactin in mouse mammary gland explants is the stimulation of ornithine decarboxylase (ODC) activity. Several protein kinase C activators including mezerein, dicaprin, diolein, and 1-oleoyl-2-acetyl-rac-glycerol were found to stimulate ODC activity as does prolactin. Both mezerein and the diglycerides produced nonadditive responses when tested with maximum stimulatory concentrations of prolactin. The results of these studies therefore provide further evidence that the prolactin stimulation of ODC activation in the mammary gland may involve an activation of protein kinase C.     

Prostaglandin stimulation of ovarian ornithine decarboxylase in vitro.

Prostaglandins E₁ and E₂ caused a 5–10 fold stimulation of ornithine decarboxylase activity in granulosa cells isolated from porcine ovarian follicles. The minimally effective concentration of prostaglandin E₂ was 10 ng/ml and the plateau of activity was reached at 500 ng/ml. Prostaglandin F_2α was ineffective. 1-Methyl,3-isobutyl-xanthine, a phosphodiesterase inhibitor, potentiated the effect of both submaximal and maximal effective doses of prostaglandin E₂, suggesting that the effect of prostaglandin E₂ is mediated by cAMP. The effect of prostaglandin E₂ was similar to that of luteinizing hormone and a cAMP analogue, 8-Bromo-cAMP.     

Prolactin stimulation of ornithine decarboxylase activity in the mammary gland may involve an activation of protein kinase C

Phorbol myristate acetate (TPA), a protein kinase C activator, stimulates ornithine decarboxylase (ODC) activity in mammary gland explants derived from 12-14 day pregnant mice. The calcium ionophore A23187 similarly stimulates ODC activity. Maximally stimulatory concentrations of TPA and A-23187 produce additive responses. The prolactin (PRL) stimulation of ODC activity is nonadditive to that caused by TPA, A23187 or TPA plus A23187. These observations are compatible with the thesis that the stimulation of ODC activity by PRL may occur via an activation of protein kinase C.     

Effect of various concentrations of prolactin and growth hormone on the magnitude of stimulation of RNA synthesis, casein synthesis and ornithine decarboxylase activity in mouse mammary gland explants

The effects of various concentrations of prolactin and growth hormone on the rates of [3H]-uridine incorporation into RNA, [3H]-leucine incorporation into casein, and ornithine decarboxylase (ODC) activity were determined in mouse mammary gland explants. The lowest concentrations of prolactin which produced significant responses were between 5 and 25 ng/ml. Growth hormone, in contrast, produced significant response at concentrations between 250 and 1,000 ng/ml. The prolactin actions on RNA and casein synthesis were essentially all-or-none type responses, i.e. the magnitude of the responses were maximal at about 10 ng/ml prolactin. The action of prolactin on ODC activity was quite different; a concentration-response relationship was observed with prolactin at concentrations from 10 t 250 ng/ml. It is apparent from these studies that different concentrations of prolactin are required to produce optimal actions on different biochemical parameters in cultured mammary tissues.     

Hormonal regulation of the number and activity of ribosomes in mammary gland explants of mice

Possible effects of insulin, hydrocortisone and prolactin on the number and activity of ribosomes enganged in protein biosynthesis in mammary gland explants were explored. The rate and extent of [3H]-puromycin attachment to nascent peptides was used to assess, respectively, the activity and number of ribosomes engaged in protein biosynthesis. None of the hormones altered the number of ribosomes engaged in protein biosynthesis. In addition, of the hormones tested, only insulin appeared to accelerate the rate at which ribosomes carried out the translocation process. The early (1 hr) effect of insulin on protein biosynthesis in the mammary gland would therefore appear to occur via an activation of ribosomal activity. In contrast, the early (6 hr.) effect of prolactin on protein biosynthesis would appear to be exclusively via an RNA-DNA dependent mechanism.     

Polyamine biosynthesis and DNA synthesis in cultured mammary gland explants from virgin mice

The mammary cells in virgin mice are essentially non-proliferative, but they can be induced to undergo DNA synthesis in vitro in the presence of insulin. Time course studies on polyamine biosynthesis and DNA synthesis showed that insulin elicits sequential stimulation of the activity of the polyamine biosynthetic enzymes, ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase (SAMDC) and spermidine synthase, and an increase in the concentration of spermidine prior to the augmentation of DNA synthesis. At 48 to 72 hours of culture when DNA synthesis is maximal, the concentration of spermidine increased 2? to 3-fold, whereas the level of spermine remained unchanged. Addition of methyl glyoxal bis(guanylhydrazone) (5—10 μM), a potent inhibitor of SAMDC, to the medium at the onset of culture resulted in inhibition of spermidine formation and DNA synthesis, but when added at 24 hours or 48 hours of culture, the inhibitory effect on DNA synthesis was greatly reduced. The drug, however, produced little inhibition of RNA and protein synthesis. Inhibition of DNA synthesis by the drug can be reversed by addition of spermidine or other polyamines such as putrescine, cadaverine and spermine to the culture. Spermidine is, however, the only polyamine that is effective at physiological concentrations (100～150 pmoles/mg tissue). These results suggest a possibility that spermidine may play a key role in the regulation of mammary cell proliferation.     

Effect of 3-isobutyl-1-methylxanthine on prolactin actions on RNA synthesis, casein synthesis, lipid synthesis and ornithine decarboxylase activity in mouse mammary gland explants

The effect of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, was tested on several actions of prolactin in cultured mouse mammary tissues. At concentrations of 0.5 mM and above, IBMX abolished the actions of prolactin on RNA and casein synthesis. IBMX by itself, stimulated ornithine decarboxylase (ODC) activity in a dose-response fashion; but the IBMX at concentrations up to 1 mM had no effect on the magnitude of the prolactin-stimulated ODC activity. IBMX inhibited in a dose-response fashion the rate of [14C]-acetate incorporation into lipids; however, prolactin stimulated lipid biosynthesis in the presence of IBMX concentrations of up to 1 mM.     

Changes in ornithine decarboxylase activity in the developing parotid salivary gland

Ornithine decarboxylase activity in the rat parotid salivary gland was estimated during the early postnatal development. Regular alterations of enzymatic activity were found in different periods of gland development. The data obtained were compared with the evidence on the establishment of protein synthesis circahoralian rhythm of this organ in ontogenesis. The ornithine decarboxylase activity, being rather high in the parotid gland as long as up to the 20th-21st days, decreases during the termination of differentiation in this organ. It rises simultaneously with the emergence of circahoralian rhythm of protein synthesis.     

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.     

Alterations in ornithine decarboxylase activity in the rat mammary gland after different periods of 50 Hz magnetic field exposure.

In a series of experiments with the chemical carcinogen DMBA (7, 12-dimethyl[a]anthracene), we recently found that exposure of female Sprague-Dawley rats in 50 Hz magnetic fields (MF) in the microtesla range significantly facilitates the development and growth of mammary tumors. One possible explanation for this finding would be enhanced proliferation of breast epithelial stem cells by MF exposure, thereby increasing the sensitivity of these cells to chemical carcinogens. In line with this possibility, we previously determined that 50 Hz, 50 microT MF exposure induces increases in ornithine decarboxylase (ODC), i.e., a key enzyme in cell proliferation, in the mammary gland of female Sprague-Dawley rats. In the present study, we examined the time course of this effect, by using different periods of exposure to a 50 Hz, 100 microT MF. Furthermore, we determined ODC in different mammary complexes of the rat mammary gland to evaluate whether differences in response to MF exist over the anterior-posterior extension of this organ. Exposure of young female Sprague-Dawley rats induced marked increases in ODC in the mammary gland that were similar to ODC increases seen in "positive control" experiments with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). However, this effect of MF critically depended on the duration of MF exposure, with no effect, or at least no consistent effect, for short (<1 week) or long (8 weeks and above) exposure periods, but a robust and reproducible enhancing effect on ODC activity after 2 weeks of exposure. Furthermore, we found that the effect of MF exposure depends on the part of the mammary complexes examined, the cranial thoracic (or cervical) complexes being particularly sensitive to ODC alterations in response to MF. This is in line with recent DMBA experiments of our group in which MF-induced increases in tumor development and growth were predominantly seen in this large cranial/cervical part of the mammary gland. The most likely explanation for the observed ODC changes after MF exposure is the "melatonin hypothesis," although other cellular and molecular effects of MF might be involved as well.     

Juvenile hormone stimulation of ornithine decarboxylase activity during vitellogenesis in Drosophila melanogaster

Summary The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, becomes elevated in intact female Drosophila melanogaster shortly after adult eclosion. This activity reaches a peak at 24 h following eclosion, and then drops to lower levels by 48 h. This pattern is not observed in males, consistent with the hypothesis that polyamine synthesis is involved in ovarian maturation in Drosophila. Abdomens isolated within 2 h of adult eclosion do not display elevated ODC activity or ovarian maturation. However, a 250-ng dose of the juvenile hormone analog methoprene (ZR-515) applied in acetone to these abdomens, recovers ovarian maturation and causes a 5–10 fold increase in enzyme activity over controls treated with acetone alone. The same dose of the inactive precursor methyl farnesoate caused no such increase, whereas a 500-ng dose of the newly discovered natural Drosophila JHB₃ stimulated a four-fold response. The response to methoprene was dose-dependent, showing stimulatory activity at a dose as low as 10 ng. This stimulation by JHA is rapid, occurring between 1 and 3 h following hormone treatment, reminiscent of JH induction of fat body vitellogenin synthesis in Drosophila. Elevated ODC activity appeared to be localized in the adult fat body. During embryogenesis, ODC activity remained undetectable until just prior to hatching, when a large increase was detected. We postulate that JH may, either directly or indirectly, regulate polyamine biosynthesis in vivo, and that this synthesis may be required for the production of macromolecules during Drosophila vitellogenesis or embryogenesis.Abbreviations JH juvenile hormone - JHA juvenile hormone analog - ODC ornithine decarboxylase - SAMDC S-adenosyl-methionine decarboxylase - JHB ₃ juvenile hormone III bisepoxide