Stimulation Of Ornithine Decarboxylase Activity Research Articles

The Increase in Liver Polyamine Concentration Contributes to the Tryptophan‐induced Stimulation of Hepatic Protein Synthesis

Tryptophan has a unique role as a nutritional signaling molecule that regulates protein synthesis in mice and rat liver. However, the mechanism of the stimulating actions of tryptophan on hepatic protein synthesis has not been clearly elucidated. Tryptophan has been suggested to stimulate protein synthesis at the translational level, because polysome aggregation was observed in a short time after oral administration of tryptophan. Therefore, we focused on the acute effects of tryptophan on protein synthesis. In the present study, proteomic and metabolomic analysis was performed to identify candidate proteins and metabolites, which are likely to play a role in stimulation of protein synthesis by tryptophan. Eighteen hours fasted Wistar rats were divided into 3 groups. One group of animals was sacrificed without any treatment as a control. Remaining two groups of animals were administered 135mg/100g body weight L‐tryptophan by oral gavage and then sacrificed after 1h or 3h after administration. With two‐dimensional electrophoresis and image analysis, four differentially expressed protein spots were detected at 3h after administration of tryptophan, and one of which was identified as key enzyme in the biosynthetic pathway of ornithine, ornithine aminotransferase (OAT) precursor. OAT is the main catabolic enzyme of ornithine and its expression was significantly decreased by tryptophan administration. The concentration of ornithine was increased in both serum and liver at 3h after administration of tryptophan. Ornithine is a precursor for polyamine biosynthesis. Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway forming putrescine, which is then converted into the polyamines spermidine and spermine. It has been reported that oral administration of tryptophan to overnight‐fasted rats exhibited increase in the hepatic ODC activity. Significantly increased concentrations of polyamines, as well as ornithine were found in the liver at 3h after administration of tryptophan. Based on these results, we speculate that the increase in ornithine level through suppression of OAT expression and stimulation of ODC activity by tryptophan administration may lead to accelerated polyamine synthesis and thereby promote protein synthesis in the liver.

Regulation of intestinal mucosal growth by amino acids

Amino acids, especially glutamine (GLN) have been known for many years to stimulate the growth of small intestinal mucosa. Polyamines are also required for optimal mucosal growth, and the inhibition of ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, blocks growth. Certain amino acids, primarily asparagine (ASN) and GLN stimulate ODC activity in a solution of physiological salts. More importantly, their presence is also required before growth factors and hormones such as epidermal growth factor and insulin are able to increase ODC activity. ODC activity is inhibited by antizyme-1 (AZ) whose synthesis is stimulated by polyamines, thus, providing a negative feedback regulation of the enzyme. In the absence of amino acids mammalian target of rapamycin complex 1 (mTORC1) is inhibited, whereas, mTORC2 is stimulated leading to the inhibition of global protein synthesis but increasing the synthesis of AZ via a cap-independent mechanism. These data, therefore, explain why ASN or GLN is essential for the activation of ODC. Interestingly, in a number of papers, AZ has been shown to inhibit cell proliferation, stimulate apoptosis, or increase autophagy. Each of these activities results in decreased cellular growth. AZ binds to and accelerates the degradation of ODC and other proteins shown to regulate proliferation and cell death, such as Aurora-A, Cyclin D1, and Smad1. The correlation between the stimulation of ODC activity and the absence of AZ as influenced by amino acids is high. Not only do amino acids such as ASN and GLN stimulate ODC while inhibiting AZ synthesis, but also amino acids such as lysine, valine, and ornithine, which inhibit ODC activity, increase the synthesis of AZ. The question remaining to be answered is whether AZ inhibits growth directly or whether it acts by decreasing the availability of polyamines to the dividing cells. In either case, evidence strongly suggests that the regulation of AZ synthesis is the mechanism through which amino acids influence the growth of intestinal mucosa. This brief article reviews the experiments leading to the information presented above. We also present evidence from the literature that AZ acts directly to inhibit cell proliferation and increase the rate of apoptosis. Finally, we discuss future experiments that will determine the role of AZ in the regulation of intestinal mucosal growth by amino acids.

小腸上皮細胞IEC-6におけるinsulin-like growth factor-Iによるオルニチン脱炭酸酵素の活性化機構とブドウ糖濃度による影響

小腸上皮細胞IEC-6におけるinsulin-like growth factor-Iによるオルニチン脱炭酸酵素の活性化機構とブドウ糖濃度による影響

ODCp, a brain- and testis-specific ornithine decarboxylase paralogue, functions as an antizyme inhibitor, although less efficiently than AzI1

ODC (ornithine decarboxylase), the first enzyme in the polyamine biosynthesis pathway in mammalian cells, is a labile protein. ODC degradation is stimulated by Az (antizyme), a polyamine-induced protein, which in turn is regulated by an ODC-related protein termed AzI (Az inhibitor). Recently, another ODCp (ODC paralogue) was suggested to function as AzI, on the basis of its ability to increase ODC activity and inhibit Az-stimulated ODC degradation in vitro. We show in the present study that ODCp is indeed capable of negating Az functions, as reflected by its ability to increase ODC activity and polyamine uptake and by its ability to provide growth advantage in stably transfected cells. However, ODCp is less potent than AzI1 in stimulating ODC activity, polyamine uptake and growth rate. The superiority of AzI1 to ODCp in inhibiting the Az-stimulated ODC degradation is also demonstrated using an in vitro degradation assay. We show that the basis for the inferiority of ODCp as an AzI is its lower affinity towards Az (Az1 and Az3). Further, we show here that ODCp, like AzI, is degraded in a ubiquitin-dependent manner, in a reaction that does not require either interaction with Az or the integrity of its C-terminus. Interaction with Az actually stabilizes ODCp by interfering with its ubiquitination. This results in sequestration of Az into a stable complex with ODCp, which is the central feature contributing to the ability of ODCp to function as AzI.

Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: Crosstalk between ODC and BCL-2

Prolactin has more than 300 separate functions including affecting mammary growth, differentiation, secretion and anti-apoptosis. In the previous studies, prolactin induced Bcl-2 expression to prevent apoptosis and also provoked the activity of ornithine decarboxylase (ODC). Our previous data showed that ODC overexpression upregulates Bcl-2 and prevents tumor necrosis factor alpha (TNF-alpha)- and methotrexate (MTX)-induced apoptosis. Here, we further investigate whether prolactin prevents MTX-induced apoptosis through inducing ODC activity and the relationship between ODC and Bcl-2 upon prolactin stimulation. Prolactin prevented MTX-induced apoptosis in a dose-dependent manner in HL-60 cells. Following prolactin stimulation, ODC enzyme activity also shows an increase in a dose-dependent manner, expressing its maximum level at 3 h, and rapidly declining thereafter. Prolactin-induced ODC activity is completely blocked by a protein kinase C delta (PKCdelta) inhibitor, rottlerin. However, there are no changes in the expressions of ODC mRNA and protein level after prolactin stimulus. It indicates that prolactin may induce ODC activity through the PCKdelta pathway. Besides, Bcl-2 expresses within 1 h of prolactin treatment and this initiating effect of prolactin is not inhibited by alpha-difluoromethylornithine (DFMO). However, Bcl-2 is further enhanced following prolactin stimulation for 4 h and this enhancement is blocked by DFMO. Bcl-2 has no effect on ODC activity and protein levels, but ODC upregulates Bcl-2, which is inhibited by DFMO. Overall, there are two different forms of prolactin effect, it induces Bcl-2 primarily, and following this it stimulates ODC activity. Consequently induced ODC activity further enhances the expression of Bcl-2. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, ODC activity is one basis for the anti-apoptotic mechanisms of prolactin. A Bcl-2 inhibitor, HA14-1, together with DFMO, completely blocks the anti-apoptotic effects of prolactin. These results suggest that increasing ODC activity is another way of prolactin preventing MTX-induced apoptosis and that this induction of ODC activity enhances the expression of Bcl-2 strongly enough to bring about the anti-apoptotic function.

Metabolic Stability of α-Methylated Polyamine Derivatives and Their Use as Substitutes for the Natural Polyamines

Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of alpha-methylspermidine, alpha-methylspermine, and bis-alpha-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase. alpha-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, alpha-methylspermine was readily converted to alpha-methylspermidine and spermidine; similarly, bis-alpha-methylspermine was converted to alpha-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N(1), N(2)-bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N(1)-acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both alpha-methylspermidine and bis-alpha-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.

Ornithine Decarboxylase Activity in Tissues from Rats Exposed to 60 Hz Magnetic Fields, Including Harmonic and Transient Field Characteristics

Ornithine decarboxylase (ODC) activity is used widely as a biomarker for tumor promotion in animal model systems. Several previous studies have reported increases in ODC activity in tissues of rats exposed to 60 Hz magnetic fields. The goals of this study were to confirm these findings and to determine whether ODC activity is increased in tissues of animals exposed to magnetic fields containing complex metrics. Three experiments were conducted in male F344 rats. Each study included a sham control group and a group exposed to pure continuous 60 Hz fields (0.2 mT). Additional groups included animals exposed to randomly time-varying 60 Hz fields (range of 0.02 to 0.2 mT); intermittent 60 Hz fields (2 mT) with on-off cycles ranging from 5 s to 5 min; pure continuous 180 Hz fields (2 mT); 60 Hz fields with a superimposed 3rd harmonic (total field strength, 2 mT); 60 Hz fields with superimposed third, fifth, and seventh harmonics (total field strength, 2 mT); 60 Hz fields (2 mT) with superimposed transients; and randomly time-varying 60 Hz fields (range of 0.02 to 0.2 mT) with superimposed transients. After 4 weeks of exposure (18.5 h/day), eight animals per group were euthanized within 1 h of magnetic field deactivation. Homogenates of liver, kidneys, spleen, and brain were prepared from each animal, quick-frozen, and shipped for analysis by four independent laboratories. No consistent pattern of differences in the ODC activity among experimental groups was found either within a laboratory or among laboratories. The results do not support the hypothesis that exposure to extremely low frequency magnetic fields stimulates ODC activity.

Regulation of ornithine decarboxylase in B16 mouse melanoma cells: synergistic activation of melanogenesis by αMSH and ornithine decarboxylase inhibition

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationic compounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression of genes regulating cell growth and/or differentiation can be controlled by α-melanocyte-stimulating hormone (αMSH) and other melanogenesis modulating agents. In the B16 mouse melanoma model, αMSH stimulates melanogenesis by upmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibits melanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenic pathway. Treatment of B16 cells with TPA or αMSH rapidly stimulated ODC activity. The effect was stronger for TPA and appeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor α-difluoromethylornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMO stimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatment with αMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic response of B16 cells to αMSH. Rather, increased intracellular concentrations of polyamines following ODC induction might constitute a feedback mechanism to limit melanogenesis activation by αMSH.

Effect of Leishmania donovani lipophosphoglycan on ornithine decarboxylase activity in macrophages.

Lipophosphoglycan (LPG), a major surface molecule from Leishmania donovani, stimulated ornithine decarboxylase (ODC) activity in macrophages in a dose- and time-dependent manner. LPG stimulated the rapid increase in ODC activity within 30 min after exposure, suggesting that the interaction of LPG with its receptor stimulated a specific signal transduction pathway. However, LPG-induced ODC activity was a transient event because 3 hr after exposure to LPG, no stimulation of ODC activity was detectable. ODC activity appeared to be coupled to the activation of protein kinase C (PKC) in macrophages, as activators of PKC caused a rapid increase in the ODC activity. Macrophages pretreated with LPG for 1 hr became unresponsive to subsequent stimulation by the PKC activators 1-oleoyl-2-acetyl-glycerol and the calcium ionophore A23187. In contrast, the ability of macrophages to express ODC activity in response to the cyclic AMP analogue dibutyryl cyclic AMP was not impaired by LPG.

Increases in ornithine decarboxylase activity in the positive inotropism induced by androgens in isolated left atrium of the rat

It is well established that the intracellular receptors of androgens act as transcription factors upon their activation by androgen binding. However, a growing number of studies have associated androgens with rapid biological responses independent of their classical action mechanism. In this sense, 5α- and 5β-dihydrotestosterone elicited a rapid positive inotropism in the isolated left atrium of the rat via cAMP-dependent mechanisms that may involve genomic effects. In addition, polyamines are mediators of several biological actions including those acute and long-term effects induced by androgens in the heart. The present study analyzed the role of polyamine synthesis in the cardiotonic effect of androgens in the left atrium of male Wistar rats, electrically stimulated (0.5 Hz, 5 ms and supramaximal voltage) and placed in an organ bath in 10 ml of Tyrode's solution. Incubation in the organ bath with an inhibitor of ornithine decarboxylase activity, α-difluoromethylornithine 10 mM, significantly decreased the positive inotropism induced by 5α- and 5β-dihydrotestosterone (0.1–100 μM). This suggests that ornithine decarboxylase seems to be involved in androgen-induced positive inotropism. Furthermore, 6-min exposure to 5α- or 5β-dihydrotestosterone significantly increased the activity of ornithine decarboxylase from 61.81±7.53 (control) to 93.28±9.45 and 80.28±12 pmol/h/mg of protein, respectively. Northern blot analysis showed that 5α- and 5β-dihydrotestosterone did not modify the level of expression of the ornithine decarboxylase gene. Therefore, our results suggest that polyamine synthesis might be involved in the positive inotropism elicited by androgens through the stimulation of ornithine decarboxylase activity without changes in the expression of the ornithine decarboxylase gene.

Androgen and estrogen stimulation of ornithine decarboxylase activity in mouse kidney

In the present work, the activity of mouse renal ornithine decarboxilase (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 μg/mouse, was used. Administration of flutamide concurrently with testosterone (75 μg/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.

Epigenetic events during the process of cell transformation induced by carcinogens (review).

Recent studies clearly demonstrate that several environmental carcinogens lack the ability to initially induce genetic damage. In that view, multistage chemical carcinogenesis may be processed under the control of a variety of epigenetic events in addition to genotoxic impacts. The understanding of this mechanism as reviewed in this report requires knowledge of early changes induced by carcinogens in target cells, biochemical, biological and molecular reactions closely related to both sides of the growth equation: cell proliferation and programmed death. Among several cell transformation models, the most suitable for carcinogen detection and mechanistic study is the Syrian hamster embryo (SHE) cell transformation assay. This closely mimics the multistage carcinogenesis and we can examine, in a relatively short time (8 days), the mechanisms by which genotoxic and non-genotoxic agents may increase the frequency of cell transformation as a preneoplastic end-point. The mode of action of hundred of compounds, carcinogens and non-carcinogens, has been explored so far using one-stage and two-stage treatment protocols. In general, with the two-stage protocol, all carcinogens, irrespective of their genotoxic or non-genotoxic potential, give unambiguous positive results. Since perturbations of cell proliferation and death are considered essential events in the process of carcinogenesis, studies have been conducted on the dysregulation of two specific parameters, the induction of ornithine decarboxylase (ODC) an enzyme related to cell proliferation, and the apoptosis rate, when SHE cells are exposed to carcinogens. In one-stage treatment (5 h-24 h), only the promoter TPA induces ODC activity, while other carcinogens do not increase this activity. Using the two-stage exposure protocol (1 h xenobiotic/5 h TPA), all carcinogens both genotoxic and non-genotoxic, are able to stimulate ODC activity above the level obtained with TPA alone. Based on the two-stage treatment with carcinogens a close relationship can be obtained between the ODC superinduction and the increase of morphological cell transformation frequency. In cancer development, it is postulated that the inhibition of apoptosis may help altered cells to escape cell death and acquire a tumorigenic phenotype. Two-stage treatment carcinogen/TPA, effectively decreases the apoptotic rate. This is accompanied by an upregulation of the Bcl-2 oncoprotein, a well-known apoptotic inhibitor. However, treatment with a non-carcinogen phthalic anhydride, also inhibits apoptosis while it does not superinduce ODC activity. Although inhibition of apoptosis is not specific to the carcinogenic compound, both superinduction of ODC activity and inhibition of apoptosis via Bcl-2 upregulation may cooperate during the early stages of the carcinogenic process. In a long-term stage transformation assay, the rate of transformed colonies is relatively low (2-8%) bringing about the slow evolution of tumoral disease in humans and tumoral induction in rodents. This could be the consequence of the activation of various cellular repair mechanisms during the exposure time. Experimental data reported so far point out that genotoxic and non-genotoxic carcinogens, thought to be more active in the initiation or in the promotion stage, must share the same stage pathway leading to cancer development.

Early response of ornithine decarboxylase activity and energy metabolism to postsurgery refeeding in rat small intestine

Introduction: Enterocyte proliferation and cellular energy status are important to intestinalintegrity after starvation and trauma. The proliferative response to nutrients is expressed in the activity of ornithine decarboxylase (ODC), but ODC activity and ATP level in the intestinal mucosa the first hours after surgery and immediate refeeding are not known. Methods: Male Wistar rats (240–280 g) were starved for 48 h and submitted to laparotomy with distalileal transection, gastrostomy and jejunal instillation of either enteral formula or saline. The ODC activity and ATP content of the jejunal mucosa were analysed in samples taken at 1, 2, 4 and 6 h after surgery. Results: ODC activity increased and reached the highest peak at 2 h in the refed animals. ATP concentrationand energy charge of jejunal mucosa were significantly reduced 6 h after surgery compared to initial levels, but there were no differences between animals that were refed or not. Intestinal transection did not stimulate ODC activity. Conclusion: ATP levels in intestinal mucosa decreased after surgery, and early enteral feeding did not seem to prevent this decrease during the first 6 h. Refeeding immediately after surgery elicits an early but transient increase of ODC activity in rat jejunal mucosa.

Disruption of the Gene Encoding the Mitogen-regulated Translational Modulator PHAS-I in Mice

PHAS-I is the prototype of a group of eIF4E-binding proteins that can regulate mRNA translation in response to hormones and growth factors. To investigate the importance of PHAS-I in the physiology of the intact animal, we disrupted the PHAS-I gene in mice. Tissues and cells derived from the knockout mice contained no detectable PHAS-I protein. A related protein, PHAS-II, and eIF4E were readily detectable in tissues from these animals, but neither appeared to be changed in a compensatory manner. Mice lacking PHAS-I appeared normal at birth. However, male knockout mice weighed approximately 10% less than controls at all ages, whereas female weights were similar to those of controls. Both males and females were fertile. Tissues from adult animals appeared to be normal by routine histological staining techniques, as were routine blood cell counts and chemistries. Fibroblasts derived from PHAS-I-deficient mouse embryos exhibited normal rates of growth and overall protein synthesis, responded normally to serum stimulation of ornithine decarboxylase activity and cell growth, and rapamycin inhibition of cell growth. Under these experimental conditions, PHAS-I is apparently not required for the normal development and reproductive behavior of female mice, but is required for normal body weight in male mice; the mechanisms responsible for this phenotype remain to be determined.

Central administration of morphine inhibits brain and liver ornithine decarboxylase activity in neonatal rats: Involvement of transcription- and non-transcription-dependent mechanisms

This study examined whether the developmental deficits usually observed in infants born to opiate addicted mothers could involve effects on ornithine decarboxylase, a growth-controlling enzyme. Intracerebroventricular (i.c.v.) injection of a single dose of morphine (2 μg) to 6-day-old rats markedly decreased basal brain and liver ornithine decarboxylase activity as well as the increases in hepatic ornithine decarboxylase activity produced by subcutaneously (s.c.) administered insulin, an important trophic hormone. Centrally applied morphine acts supraspinally to downregulate peripheral ornithine decarboxylase activity, since s.c. administration of the same dose as used i.c.v. decreased neither basal liver ornithine decarboxylase levels nor tissue responsiveness to insulin. This does not imply that the opiate is unable to affect ornithine decarboxylase when applied systemically. In fact, a robust inhibition of both basal and induced liver ornithine decarboxylase activity was obtained in rat pups given 20 μg of morphine s.c. This larger dose is able to trigger the hepatic ornithine decarboxylase effects presumably by stimulating opiate receptors located at central sites after crossing the blood–brain barrier and penetrating into the brain. Concomitant administration of naloxone plus morphine i.c.v. prevented morphine from downregulating ornithine decarboxylase activity, confirming the participation of supraspinal opioid receptors in morphine ornithine decarboxylase actions. Finally, as was the case for insulin induced stimulation of ornithine decarboxylase activity, i.c.v. injection of morphine markedly diminished insulin induced stimulation of hepatic ornithine decarboxylase mRNA accumulation. In turn, contrary to the inhibition of basal ornithine decarboxylase activity, morphine did not lower basal hepatic ornithine decarboxylase mRNA levels when given alone. Thus, CNS morphine can apparently suppress tissue ornithine decarboxylase expression through both transcriptional and posttranscriptional mechanisms. The evidence obtained suggest that postnatal exposure to opiate drugs might detrimentally affect development by altering normal tissue ornithine decarboxylase ontogeny.

Phorbol 12-myristate 13-acetate inhibits epidermal growth factor signalling in human keratinocytes, leading to decreased ornithine decarboxylase activity

Several studies have suggested that murine and human keratinocytes respond differently to phorbol 12-myristate 13-acetate (PMA). Using an in vitro assay, we found that in contrast to its effect on murine skin, PMA did not induce ornithine decarboxylase (ODC) activity in human skin biopsies. To explore the signalling induced by PMA and to determine whether an in vitro culture system could be used to predict biological activity of retinoids in human keratinocytes, we studied a simian virus 40 (SV40)-transformed human keratinocyte cell line. Epidermal growth factor (EGF) stimulates ODC activity and increases the steady-state level of ODC mRNA in a dose- and time-dependent manner in these cells [Prystowsky, Clevenger and Zheng (1993) Exp. Dermatol. 2, 125-132]. In this report, 10(-10) M-10(-7) M PMA induced ODC mRNA and enzyme synthesis at 7 h, but did not significantly induce ODC activity and inhibited the EGF induction of ODC activity. To explore the mechanism whereby PMA interfered with EGF signalling, the effect of PMA on EGF binding to its cell-surface receptor was studied; acute treatment with PMA (within 7 h) decreased EGF binding to 41-57% of the baseline level. In contrast, chronic treatment with PMA (24 h) increased EGF binding to 156% of the baseline level and was associated with an increase in quantity of EGF receptor protein. Protein kinase C (PKC) activation correlated with the acute decrease in EGF binding following PMA treatment. In summary, PMA induced ODC mRNA and ODC enzyme synthesis, while steady-state levels of immunoprecipitable ODC enzyme protein and ODC activity were not increased, demonstrating possible increased turnover of ODC enzyme protein. Additionally, PMA inhibited the induction of ODC by EGF through decreased EGF binding, possibly mediated by PKC activation. Finally treatment of the keratinocytes with retinoids including etretinate, Ro13-7410, etarotene, Ro40-8757, 13-cisretinoic acid, and acitretin blocked the PMA induction of ODC mRNA, suggesting this in vitro model could be a valuable screening assay for predicting biological activity in humans.

Hypoosmotic stimulation of ornithine decarboxylase activity in the brine shrimp Artemia franciscana

Hypoosmotic stimulation of ornithine decarboxylase activity in the brine shrimp Artemia franciscana

Hypoosmotic stimulation of ornithine decarboxylase activity in the brine shrimpArtemia franciscana

In the brine shrimp, long-term (several days) osmotic and ionic regulation is controlled principally by the activity of Na+K+ATPase. However, short-term cell volume regulation in Artemia and most euryhaline crustaceans is not understood. In this study, Artemia stage 1 nauplii reared at 32 ppt salinity and exposed to hypoosmotic conditions (12 or 4 ppt) exhibited an immediate increase in the activity of ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis. ODC activity increased within 15 min relative to control and reached a peak within 2 hr of hypoosmotic exposure. The level of increased enzyme activity was directly related to the degree of hypoosmotic exposure (a three- and sevenfold increase in activity at 12 and 4 ppt salinity, respectively). This increase in activity was apparently due to the synthesis of a new enzyme, based on cycloheximide administration. Activity decreased significantly by 4 hr but remained above that of the 32 ppt control throughout the 24-hr exposure period. Levels of putrescine increased significantly within 2 hr in organisms exposed to 4 ppt; spermidine and spermine levels were not affected. When nauplii were exposed to altered ion and/or osmotic concentrations it was determined that decreased total osmotic concentration was the proximate factor responsible for ODC induction. However, reduced ion concentration resulted in the induction/activation of DFMO-insensitive ODC activity, suggesting the production of a novel enzyme in polyamine metabolism during hypotonic exposure. Changes in ODC activity and polyamines preceded acclimatory changes normally associated with salinity acclimation, further suggesting that modulation of ODC activity and subsequent polyamines synthesis, degradation, and/or transport are mechanistically related to short-term regulation of osmotic and ionic balance. © 1996 Wiley-Liss, Inc.

Vascular endothelial cell proliferation: regulation of cellular polyamines

The aims were to investigate the requirement for and regulation of cellular polyamines during vascular endothelial cell proliferation. Proliferation of cultured bovine pulmonary artery endothelial cells was determined after cellular polyamine depletion. In addition, polyamine synthesis and uptake mechanisms were examined in the presence and absence of trophic stimuli and density dependent inhibition of cell proliferation. Serum stimulated, subconfluent cells ceased cell division following the inhibition of ornithine decarboxylase (ODC), a rate limiting enzyme for polyamine biosynthesis. The addition of 10 microM putrescine, the product of the enzyme reaction catalysed by ODC, completely reversed the inhibition of cell growth. Serum deprivation reduced cytosolic ODC activity to near non-detectable levels. Readdition of 10% fetal bovine serum (FBS) resulted in transient increases in ODC activity which preceded DNA synthesis and mitosis. Basic fibroblast growth factor also stimulated ODC activity in a dose dependent manner with levels approximating the maximum obtainable with FBS. In contrast, platelet derived growth factor and epidermal growth factor did not stimulate ODC activity. Finally, mitogenic stimuli did not induce ODC activity in density arrested cells. The uptake of radiolabelled putrescine from the cell medium was time dependent and saturable. Kinetic studies from dividing cells revealed a single transport component for putrescine uptake [maximum rate of uptake (Vmax) = 11.2(SEM 2.0) pmol.10(5) cells-1.h-1; Michaelis constant (Km) = 1.1(0.3) microM]. Putrescine uptake by density arrested cells was characterised by a 57% decrease in Vmax with no change in Km. Readdition of FBS to serum deprived subconfluent cells, in the presence of ODC inhibitors, resulted in a rapid increase in the rate of putrescine uptake with Vmax increasing by 533% over FBS alone by 48 h. These data suggest that polyamines are essential for endothelial cell proliferation and that synthesis and uptake mechanisms are regulated according to cell growth.

Secretin inhibits induction of ornithine decarboxylase activity by gastrin in duodenal mucosa and IEC-6 cells

Ornithine decarboxylase (ODC) catalyzes the first rate-limiting step in polyamine biosynthesis, and increased ODC activity is one of the earliest biochemical events associated with the induction of cellular proliferation. The current study examines the regulation of ODC activity in rat duodenal mucosa and IEC-6 cells (a line of normal rat intestinal crypt cells) in response to the trophic hormone, gastrin, and its inhibitor, secretin. Rats were fasted 22 h before the various treatments, and ODC activity was measured in scraped duodenal mucosa. Gastrin significantly increased ODC activity within 3 h to 4.3 times control levels. The effect of gastrin was totally inhibited by 5 micrograms/kg secretin. In doses of 5 or 10 micrograms/kg, secretin had no effect on basal ODC. Epidermal growth factor (EGF) and refeeding fasted rats also significantly increased ODC activity in duodenal mucosa, but the effects of EGF and refeeding were not prevented by secretin. In cultured IEC-6 cells, ODC activity was significantly increased after exposure to gastrin, 5% dialyzed fetal bovine serum (FBS), EGF, and asparagine. Secretin in doses ranging from 10(-10) to 10(-6) M caused a linear and significant inhibition of the stimulation of ODC activity by gastrin. No dose of secretin affected basal ODC activity or enzyme activity stimulated by 5% dialyzed FBS, EGF, or asparagine in IEC-6 cells. The ODC mRNA levels in IEC-6 cells were also increased after exposure to gastrin. Administration of secretin significantly prevented the stimulated expression of the ODC gene in cells treated with gastrin.(ABSTRACT TRUNCATED AT 250 WORDS)