Increase In Ornithine Decarboxylase mRNA Research Articles

Effects of Ganoderma lucidum polysaccharides on IEC-6 cell proliferation, migration and morphology of differentiation benefiting intestinal epithelium healing in vitro

Restoration of epithelial continuity in the intestinal surface after extensive destruction is important since intestinal epithelial cells stand as a boundary between the body's internal and external environment. Polysaccharides from Ganoderma lucidum (Gl-PS) may benefit intestinal epithelial wound healing in different aspects, which awaits clarification. To identify potential effects, a non-transformed small-intestinal epithelial cell line, IEC-6 cells, was used. Effects on epithelial cell proliferation, migration, morphology of differentiation and transforming growth factor beta (TGF-β) protein expression, as well as the cellular ornithine decarboxylase (ODC) mRNA and c-Myc mRNA expression, were assessed, respectively, by MTT assay, wound model in vitro, observation under a microscope after hematoxylin and eosin staining, enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction assays. It was shown that Gl-PS stimulated IEC-6 cell proliferation and migration significantly in a dose-dependent manner; 10 µg/ml Gl-PS improved the morphology of differentiation in IEC-6 cells. Inefficacy in expression of TGF-β in IEC-6 cells indicated a possible TGF-β independent action of Gl-PS. However, Gl-PS increased ODC mRNA and c-Myc mRNA expression in a dose-dependent manner, indicating, at least partially possible involvement of ODC and c-Myc gene expression in improvement of intestinal wound healing. These results suggest the potential usefulness of Gl-PS to cure intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.

Hypoxia‐induced proliferation via epidermal growth factor receptor signaling in pulmonary endothelial cells

Arginase activity is involved in cellular proliferation via ornithine production and its subsequent metabolism to proline and/or polyamines. Ornithine decarboxylase (ODC) is the rate‐controlling enzyme in polyamine biosynthesis. We have previously shown that epidermal growth factor receptor (EGFR) is important in hypoxia‐induced cellular proliferation in endothelial cells. We hypothesized that hypoxia would activate EGFR which would in turn up‐regulate arginase expression and thereby promote cellular proliferation. We studied human pulmonary microvascular endothelial cells (hPMVEC) kept in either normoxia (20% O2 , 5% CO2) or hypoxia (1% O2, 5% CO2) for 24 hours. Arginase II protein expression was ~3 fold greater in hypoxia than in normoxia, and this hypoxia‐induced increase was attenuated by inhibition of EGFR using AG1478 (30nM). Urea production was also greater in hypoxia than in normoxia, and this hypoxia‐induced increase was completely prevented by EGFR inhibition. The mRNA levels of ODC were ~2.5 fold greater in hypoxia than in normoxia, and treatment with AG1478 prevented the hypoxia‐induced increase in ODC mRNA. Pharmacological inhibition of either arginase or ODC prevented hypoxia‐induced cellular proliferation. Our data demonstrate that EGFR tyrosine kinase activity is critical for hypoxia‐induced hPMVEC proliferation via up‐regulation of arginase and ODC.

Influence of testosterone on regulation of ODC, antizyme, and N1-SSAT gene expression in mouse kidney.

Polyamines are involved in the control of the cell cycle and cell growth. In murine kidney, testosterone enhances gene expression of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis. In this study, we document the time course effect of testosterone on 1) gene expression of ODC, antizyme 1 (AZ1), and spermidine/spermine-N1-acetyltransferase (N1-SSAT); 2) ODC activity in proximal convoluted tubules (PCT) and cortical proximal straight tubules (CPST); and 3) renal polyamine levels. Female mice were treated with testosterone for a period of 1, 2, 3, and 5 consecutive days. ODC gene expression was extremely low in kidneys of untreated female mice compared with that of males. Consequently, the renal putrescine level was sevenfold lower in females than in males, whereas spermidine and spermine levels did not differ between sexes. In female kidneys, testosterone treatment sharply increased ODC mRNA and protein levels as well as ODC activity. Testosterone increased the expression of ODC in PCT and CPST over different time courses, which suggests that ODC activity is differentially regulated in distinct tubules. The expression of AZ1 and N1-SSAT mRNA was similar in male and female mouse kidneys. Testosterone treatment enhanced AZ1 and N1-SSAT mRNA levels in a time-dependent manner by unknown molecular mechanisms. Putrescine and spermidine levels increased after testosterone treatment in female kidneys. Surprisingly, although ODC protein and activity were undetectable in female kidneys, the levels of AZ1 mRNA and protein were similar to those in males. Therefore, one may propose that ODC protein could be continuously degraded by AZ1 in female kidneys.

High molecular protein of Helicobacter pylori responsible for inhibition of ornithine decarboxylase activity of human gastric cultured cells.

Ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis, mediates epithelial cell proliferation and plays a critical role in the optimal repair of gastric mucosal damage. Several studies have shown that Helicobacter pylori inhibits the growth and proliferation of gastric cells in vitro. To test whether H. pylori extract affects ODC mRNA expression and its enzyme activity in gastric cells and to examine the partial characterization of the molecule responsible for this effect. Human gastric cells (MKN-45) were used. Bacterial extracts from various E. coli or H. pylori strains, namely (1) cagA+, vacA+, CagA+, VacA+; (2) cagA+, vacA+, CagA+ VacA-; or (3) cagA-, vacA+, CagA-, VacA- were added to the cells. Cell proliferation was assessed by [3H]-thymidine incorporation, viability by MTT assay and LDH release test, ODC enzyme activity by 14CO2 counts from L-[1(14)C]ornithine, and ODC mRNA by Northern blotting. H. pylori and E. coli extract did not affect viability of gastric cells. H. pylori extract, especially extracts containing a protein greater than 50 kDa, significantly inhibited proliferation and ODC activity of gastric cells while E. coli extract had no effect. Inhibition of ODC activity was found in extracts of all H. pylori strains, irrespective of CagA and VacA protein expression. Serum stimulation induces an increase in ODC mRNA while H. pylori extract did not affect ODC mRNA expression. High molecular weight (greater than 50 kDa) proteins of H. pylori extract without CagA or VacA protein inhibited proliferation and ODC activity of human gastric cells, but did not affect ODC mRNA expression, suggesting that inhibition of ODC activity is regulated at the post-transcriptional level.

Proliferation of intestinal crypt cells by gastrin-induced ornithine decarboxylase.

To determine whether the gastrin stimulated intestinal crypt cell (IEC-6) proliferation by induction of ornithine decarboxylase (ODC). IEC-6 cells were grown in DMEM containing 50 mL x L(-1) dialyzed fetal bovine serum for 24h and then were treated with gastrin. The proliferative capability of the cells was monitored subsequently on d 1, 2, 3, and 4 after treatment with MTT assay at aborbance 570 nm.The cellular ODC mRNA expression, ODC activity, and putrescine content were examined by RT-PCR method, radiometric technique and high-performance liquid chromatography(HPLC) analysis respectively after 12h of treatment. On d1 after exposure of IEC-6 cells to pentagastrin, the proliferation increased initially and reached a peak on d3 at 250 microg x L(-1) concentration. Pentagastrin 500microg x L(-1) increased cell proliferation on day 1 and day 2, and then decreased. Compared with control group, pentagastrin 250 microg x L(-1) increased ODC mRNA level by 1.09-fold (P<0.05), ODC activity by 1.71-fold(P<0.01), and putrescine content 5.30-fold (P<0.01), respectively. Similarly, pentagastrin of 500 microg x L(-1) also increased ODC mRNA level by 1.16-fold (P<0.05), ODC activity 1.63-fold(P<0.05), and putrescine content 4.41-fold (P<0.01), respectively. But there was not significant difference between them. Gastrin is an agent which promotes IEC-6 cell proliferation involved in regulating ODC activity mechanism.

Polyamine- and insulin-like growth factor-I-mediated proliferation of porcine uterine endometrial cells: a potential role for spermidine/spermine N(1)-acetyltransferase during peri-implantation.

Insulin-like growth factor-I (IGF-I) and the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) are progesterone-regulated genes with maximal expression at peri-implantation in the porcine uterine endometrium. However, while IGF-I stimulates cell proliferation, SSAT, by acetylating the naturally occurring polyamines (PA) spermine (SPM) and spermidine (SPD), typically functions as a cell growth inhibitor. The present study examined the functional relationships of IGF-I, SSAT, and PA in the control of endometrial cell proliferation. Northern blot analysis indicated that SSAT mRNA levels change with distinct pregnancy stages, in contrast to those for the PA biosynthetic enzyme ornithine decarboxylase (ODC). Primary cultures of luminal and glandular epithelial (LE, GE) and stromal (ST) cells isolated from Day 12 pregnant pig endometrium had IGF-I mRNA levels for ST > LE > GE cells. The mRNA levels for SSAT and ODC were transiently diminished by IGF-I treatment, but only in GE cells. By contrast, SPM and SPD increased SSAT mRNA levels in GE and ST cells, but increased ODC mRNA levels only in GE cells. IGF-I, putrescine (PUT), and SPM individually increased cellular DNA synthesis as measured by tritiated thymidine incorporation in GE and ST cells, while SPD had an effect only in ST cells. IGF-I enhanced the proliferative effect of each PA in GE cells, but only of SPD in ST cells. The mitogen-activated protein kinase inhibitor, PD98059, inhibited the induction by SPM of GE cell DNA synthesis but not that of IGF-I. Wortmannin, a phosphatidylinositol-3-kinase inhibitor had no effect on either IGF-I or SPM induction of GE cell DNA synthesis. The relative concentrations of SPM, SPD, and PUT in uterine luminal fluids differed, with the levels for each PA higher at pregnancy Day 12 than at 11.5. These results suggest that IGF-I and PA act through distinct signaling pathways to mediate cell-type-specific growth of early pregnancy pig uterine endometrium. Further, SSAT, through its control of intracellular PA levels, likely plays a modulatory role in the establishment of an optimal uterine environment for successful embryo attachment.

Synergistic induction of ornithine decarboxylase by asparagine and gut peptides in intestinal crypt cells.

The objective of this study was to determine whether the amino acid asparagine stimulated the activity of ornithine decarboxylase (ODC) synergistically with epidermal growth factor (EGF) or gastrin in IEC-6 cells, a line of normal rat small intestinal crypt cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum, and serum was deprived for 24 h before experiments. Exposure to EGF or gastrin alone increased ODC activity 4.5- to 6-fold. Asparagine alone increased the enzyme activity 10- to 13-fold in IEC-6 cells. Simultaneous addition of asparagine and EGF or gastrin, however, increased ODC activity more than 40-fold. In contrast, there was no synergistic induction of ODC activity when gastrin and EGF were added together. Increased ODC activity in cells treated with asparagine and EGF or gastrin was associated with an increase in ODC mRNA and protein levels. The rate of transcription of the ODC gene was significantly increased by exposure to EGF or gastrin. Asparagine alone had little or no effect on the rate of transcription of the ODC gene. When given together with EGF or gastrin, asparagine also had no additional effect on the transcription rate of the ODC gene. The half-life of mRNA for ODC in unstimulated IEC-6 cells was approximately 30 min and increased to more than 2 h in cells exposed to asparagine, although neither gastrin nor EGF prolonged the stability of ODC mRNA. The half-life of mRNA for ODC after combined addition of asparagine and EGF or gastrin was extended to approximately 2 h, similar to asparagine alone. Combined addition of asparagine and EGF or gastrin also significantly increased DNA synthesis compared with cells exposed to each of the three agents alone. In conclusion, 1) simultaneous addition of asparagine and EGF or gastrin increases ODC activity in a synergistic manner and 2) asparagine increases ODC mRNA levels through completely distinct mechanisms from EGF or gastrin. EGF or gastrin specifically stimulates transcription of the ODC gene, whereas asparagine affects a posttranscriptional process.

Expression of the ornithine decarboxylase gene in response to asparagine in intestinal epithelial cells.

Refeeding fasted rats significantly stimulates mucosal growth and ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, but the exact mechanism responsible for induction of ODC at the molecular level is unknown. Of normal dietary constituents, the amino acid asparagine markedly increases ODC activity and mucosal growth when administered intragastrically. The current study examined the expression of the ODC gene in IEC-6 cells (a line of normal rat small intestinal crypt cells) after exposure to asparagine. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum. They were deprived of serum for 24 h before experiments. Exposure to asparagine at the dose of 10 mM resulted in the rapid increase in ODC mRNA levels. The increased expression of the ODC gene began 1 h after and peaked between 3 and 5 h after treatment with asparagine. Maximum increases in ODC mRNA levels were approximately fivefold the normal value. Increased levels of ODC mRNA in cells exposed to asparagine were paralleled by increases in ODC protein and enzyme activity and cellular polyamine levels. The half-life of mRNA for ODC in unstimulated IEC-6 cells was approximately 30 min and increased to > 2 h in cells exposed to 10 mM asparagine. The half-life of ODC activity also was increased in asparagine-treated cells. When cellular protein synthesis was inhibited by cycloheximide, asparagine superinduced ODC mRNA levels. Furthermore, asparagine also significantly stimulated DNA synthesis in IEC-6 cells. These results indicate that 1) asparagine stimulates ODC in IEC-6 cells through multiple pathways and 2) increased ODC mRNA levels result partly from a delay in the rate of degradation. These findings suggest that luminal amino acids stimulate gut mucosal growth in association with their ability to regulate ODC gene expression.

Post-translational cooperativity of ornithine decarboxylase induction by estrogens and peptide growth factors in human breast cancer cells

Mutual interactions between 17β-estradiol (E 2) and insulin or insulin-like growth factor-I (IGF-1) in the regulation of ornithine decarboxylase (ODC) expression were examined in estrogen-responsive MCF-7 human breast cancer cells. Whereas E 2 only retarded the rapid decay of ODC activity observed upon mitogen withdrawal, both insulin and IGF-1 led to a rapid (< 4 h), net increase in ODC activity that was mediated, at least in part, through their cognate receptors. E 2 synergistically potentiated the induction of ODC by IGF-1, resulting in a 170-fold elevation of enzyme activity after 48 h, as compared with 23- and 70-fold increases caused by E 2 and IGF-1 alone, respectively. Cooperativity was more pronounced at suboptimal peptide concentrations due to a decrease in the half-maximal concentration of insulin or IGF-1 required for ODC induction. Phorbol-12-myristate-13-acetate (PMA) also strongly induced ODC activity in a transient manner, and additively to the effect of IGF-1. IGF-1-and PMA additively increased ODC mRNA level, whereas E 2 alone had no effect on ODC mRNA abundance. IGF-1 increased the half-life of ODC activity by 60%, whereas E 2 or PMA alone had no significant effect on enzyme stability. On the other hand, the simultaneous addition of IGF-1 and either E 2 or PMA cooperatively reduced ODC turnover, resulting in 3.5- and 2-fold increases, respectively, in the half-life of ODC activity. Thus, ODC expression in breast cancer cells is primarily regulated by tyrosine kinase- and protein kinase C-dependent pathways, whereas estrogens increase ODC activity through a novel type of synergistic interaction with growth factors that results in a decreased rate of enzyme turnover.

Blockade of ornithine decarboxylase enzyme protects against ischemic brain damage.

Polyamines are derived from ornithine by the actions of ornithine decarboxylase (ODC), which is the rate-limiting step in this pathway. Polyamines play a role in cell growth, neoplasia, differentiation, and response to injury. We have shown that transient cerebral ischemia gives rise to increased ODC mRNA and enzyme activity in the gerbil brain. ODC and polyamines are thought to be important in the generation of edema and the neuronal cell loss associated with cerebral ischemia. To test this theory, we examined the ODC activity, putrescine levels, and neuronal density in the CA1 region of the hippocampus following ischemia and reperfusion injury in the absence and presence of an inhibitor of ODC activity, alpha-difluoromethylornithine (DFMO). Pretreatment of animals with DFMO resulted in attenuation of the ODC activity following 5 min of ischemia and 4 h of reperfusion. In addition, DFMO prevented the increase in polyamine levels, as determined by measurement of putrescine in the ischemic brain. These alterations were not due to changes in ODC mRNA level. Further analysis revealed that DFMO treatment blocked the delayed neuronal cell death in the CA1 region of the hippocampus that accompanies ischemia and reperfusion injury. Administration of DFMO resulted in a dose-dependent beneficial effect upon neuronal cell survival. These results suggest that ODC enzyme activity and the production of polyamines play a significant role in the response of the brain to ischemic injury.

Heterogeneous expression of ornithine decarboxylase gene in the proximal tubule of the mouse kidney following testosterone treatment.

The expression of the ornithine decarboxylase (ODC) gene in the mouse kidney following testosterone treatment was examined using in situ hybridization histochemistry. Testosterone (n = 5) or vehicle (n = 5) was subcutaneously injected (1 mg/animal) into male BALB/c mice (8 weeks in age) 14 h before sacrifice. Animals were sacrificed under ether anesthesia, their kidneys were removed and immediately frozen in liquid nitrogen. Frozen sections (10-microns-thick) were cut on a cryostat. Sections were hybridized with 35S-labeled sense or antisense RNA probe. The hybridization continued for 24 h at 50 degrees C and emulsion autoradiography was subsequently performed. A marked increase in ODC mRNA was exclusively detected in the proximal tubule of the renal cortex in the testosterone-treated animals. The hybridization signal was greater in the outer portion of the proximal tubule than in the inner portion. No significant hybridization signal was detected either in the distal tubule, renal corpuscle or peritubular tissues. These results indicate that testosterone induces the expression of the ODC gene in the proximal tubule of the renal cortex, leading to the increase in ODC activity in the same region.

Ornithine decarboxylase activity in fetal and newborn rat brain: responses to hypoxic and carbon monoxide hypoxia

In response to acute maternal hypoxia, ornithine decarboxylase (ODC) activity increased significantly in fetal rat brain, peaking at 4 h. This was associated with increased ODC mRNA and elevated polyamine concentrations. To correlate this response with development, we measured ODC activity in the rat from gestational day E 17 to postnatal day P 10. We also examined to what extent hypoxia induces increased ODC activity in adult rat brains and whether the response to chronic hypoxia differed from that to acute hypoxia. To test the hypothesis that this increased activity is due to hypoxic hypoxia per se, we subjected pregnant dams to inspired carbon monoxide concentrations ranging from 150 to 1000 ppm and assayed ODC activity in the fetal brain 4 h later. In the fetus, ODC activity was elevated on E 17 in the cerebrum and cerebellum. It declined gradually to about one-tenth E 17 levels by E 21 and remained low thereafter except for a postnatal elevation in the cerebellum on P 3. In response to 10.5% O 2, in the 3-day-old rat, ODC activity peaked between 2 and 3 h of hypoxia, increasing 3-fold in the hippocampus and 2-fold in cerebellum. Similar increases were seen in the hypoxic adult rat brain. In inspired oxygen dose-response studies, exposure of P 3 rat pups to 13.25% O 2 for 2.5 h produced a 1.5-fold increase in ODC activity; 10.5% O 2 produced a 2–3-fold increase while in response to 9% O 2, ODC activity remained at baseline levels. With maternal CO-hypoxia, ODC activity increased in the fetal brain at 4 h, as seen with hypoxic-hypoxia. For example, in hippocampus, ODC activity doubled at 500 ppm and tripled at 600 ppm. We conclude: (1) apparently, the ability to respond thus is not lost as the animal ages and may represent an important cellular response to acute hypoxia; (2) the increase in hypoxic-induced ODC activity is relative to the already elevated activity seen from E 17 to E 20; a vast reserve for the induction of fetal ODC activity probably exists and may indicate the importance of this enzyme during this time frame for differentiation and growth promotion; and (3) the CO-hypoxia studies suggest that some aspects of the cellular responses to CO- and hypoxic-hypoxia are similar.

Regulation of ovarian ornithine decarboxylase activity and its mRNA by gonadotropins in the immature rat

Previous studies have demonstrated that gonadotropin increases ornithine decarboxylase (ODC) activity in the rat ovary. The increase of ODC activity following gonadotropin stimulation is localized primarily to proliferating granulosa cells. The present studies were undertaken to characterize the mechanism of this hormone-induced increase of ODC. A single intraperitoneal injection of PMSG resulted in a 15-fold increase in ODC activity ( p < 0.0001). Activity was maximal 1 day following PMSG administration, and thereafter declined with time. PMSG also induced an increase in ODC mRNA levels ( p < 0.0001). However, ODC mRNA increased only 2-fold, as compared with a 15-fold increase in ODC activity. Actin mRNA levels were unaffected by PMSG. These results demonstrate that PMSG-induced increase in ODC activity cannot be fully explained by an increase in ODC mRNA, thus indicating that part of the regulation of ODC by PMSG is occurring at the translational and/or posttranslational level. Such regulation is likely a necessary dimension of the fine control of the levels/activity of this important enzyme which has a very short half-life.

Divergent effects of arginine vasopressin and angiotensin II on proliferation and expression of the immediate early genes c-fos, c-jun and Egr-1 in cultured rat glomerular mesangial cells

The vasoactive peptides arginine vasopressin (AVP) and angiotensin II (Ang II) induce similar second messengers in cultured glomerular mesangial cells, as shown by the rise in intracellular calcium and the activation of phospholipases C and A2. In contrast, AVP is a strong mitogen for cultured rat mesangial cells while Ang II is not. To elucidate the level of signal divergence, we examined the effects of AVP and Ang II on the expression of the immediate early genes c-fos, c-jun and Egr-1, which have been associated with cell growth. We also tested the effect of AVP and Ang II on the induction of the ornithine decarboxylase gene and enzyme in order to examine a process that is induced in cell activation, e.g. it has been associated with the G1 phase after mitogen-receptor binding. Cellular effects of Ang II and AVP were studied in rat mesangial cells in conventional two-dimensional culture. Proliferation of the mesangial cells was measured by cell-counting and by [3H]-thymidine uptake. The presence of receptors for Ang II and AVP was assessed by measurement of prostaglandin E2 by a radioimmunoassay following stimulation with vasoactive peptides and a receptor-binding assay for Ang II. Messenger (m)RNA levels of c-fos, c-jun Egr-1 and ornithine decarboxylase were determined by Northern blot analysis. Ornithine decarboxylase activity was measured by an enzyme substrate assay. AVP (10(-7) mol/l) stimulated [3H]-thymidine uptake by 3.7-fold after 48 h and increased mesangial cell counts by 42% (P < 0.05), while Ang II was not mitogenic. Stimulation of resting mesangial cells with AVP (10(-9) to 10(-6) mol/l) caused maximal expression of the immediate early genes after 0.5-1 h, which disappeared after 2-4 h. The relative increases were: c-fos, 15-fold; c-jun, 12-fold; Egr-1, sixfold. Ang II (10(-9) to 10(-6) mol/l) did not induce these genes at any time. In contrast, ornithine decarboxylase mRNA and enzyme activity were induced by both AVP and Ang II. The results demonstrate that AVP, but not Ang II, is a strong inducer of the immediate early genes c-fos, c-jun and Egr-1 in cultured mesangial cells. We conclude that signalling pathways activated by AVP and Ang II in mesangial cells diverge within 30 min after ligand-receptor binding and proximal to the transient expression of immediate early genes. While mitogenesis of cultured mesangial cells, as effected by AVP, involves the expression of immediate early genes, Ang II-induced non-mitogenic changes in the mesangial cell phenotype do not. The increase in ornithine decarboxylase mRNA and enzyme activity following stimulation with both AVP and Ang II indicates that its induction can occur independently of the immediate early gene expression and mitogenesis.

Function and Origin of Multiple Ionic Forms of Hormone-Induced Ornithine Decarboxylase

Ornithine decarboxylase (ODC) occurs in liver and kidney of control and hormone-treated rats as two ionic forms, A and B in order of elution, separable by ion-exchange chromatography of cell-free extracts. These studies were designed to investigate the origin of the multiple forms and their role in the hormonal stimulation of ODC activity. The percentage of activity present in A and B was determined in liver (73/27) and kidney (50/ 50) of control animals. The proportion of activity present as form B had increased by approximately 15% at 5 h after treatment with dexamethasone, prolactin, or growth hormone. In tissues of control rats, the half-life of both A and B was short, in the range of 7-11 min. The half-life of form A was unchanged after hormone treatment, but the half-life of B increased 2- to 3-fold in liver and 5- to 15-fold in kidney. When hormone-treated rats received LiCl 0.5-1 h before sacrifice, ODC activity was less than that after hormone treatment alone, the proportion of activity in A and B reverted to that present in control animal tissues, and the half-life of B reverted to the range of 7-10 min. Thus the production or maintenance of the enhanced stability of B is disrupted by lithium. A post-translational modification of A could be responsible for producing the more acidic and more stable B in the hormone-treated rats. A 3- to 5-fold increase in ODC mRNA occurred in kidney of rats treated with dexamethasone or prolactin, and was largely reversed by LiCl. Stimulation of ODC in kidney by growth hormone did not involve a major change in ODC mRNA, nor was the amount affected by LiCl.

Inhibitory effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced increase in ornithine decarboxylase mRNA in mouse epidermis.

Application of 5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) to the skin of female CD-1 mice led to a rapid increase in the concentration of epidermal ornithine decarboxylase (ODC) mRNA from an undetectable level in control mice to a high maximum level at 4-5 h after TPA administration. The concentration of epidermal ODC mRNA then decreased rapidly during the next 5 h. The time course for TPA-induced increases in epidermal ODC enzyme activity paralleled very closely the time course for TPA-induced increases in ODC mRNA. Topical administration of 1, 3 or 10 mumol curcumin together with 5 nmol TPA inhibited by 66, 81 and 91% respectively TPA-induced increases in epidermal ODC enzyme activity measured 5 h later. In a parallel study, TPA-induced increases in the concentration of epidermal ODC mRNA was inhibited by 54, 85 and 82% respectively. Intraperitoneal injection of 10 or 30 mumol curcumin 1h before topical application of 5 nmol TPA inhibited TPA-induced increases in epidermal ODC enzyme activity by 75 or 89% respectively. In a parallel study, the induction of epidermal ODC mRNA was inhibited by 53 and 65% respectively. The results indicate that curcumin inhibits TPA-induced increases in epidermal ODC enzyme activity by inhibiting the synthesis and/or enhancing the breakdown of ODC mRNA.

Effects of vasoactive intestinal peptide on adenosine 3',5'-monophosphate, ornithine decarboxylase, and cell growth in a human colon cell line.

Vasoactive intestinal peptide (VIP) is a widely distributed neuropeptide that has been considered a potential regulator of cell growth and differentiation in various tissues, including the gut. To examine this idea, we used a human colon carcinoma cell line (LoVo) as a model system and measured ornithine decarboxylase (ODC), because this is the rate-limiting enzyme for the formation of polyamines, which are thought to be key factors in regulating cell growth. LoVo cells, grown to about 80% confluence in F-12 medium containing 10% fetal bovine serum, were preincubated for 5 h in low serum medium (1% fetal bovine serum in F-12), and ODC activity was determined by measuring 14CO2 liberated from 14C-labeled ornithine. VIP caused a dose-related biphasic change in ODC, with activity increased at 10 pM, maximal (5-fold increase) at 10 nM, and decreased toward basal at 100 nM to 1 microM. Incubation of cells for 6 days with VIP in low serum medium showed similar changes in cell numbers, with growth being increased by doses in the 1 pM to 100 nM range and decreased at higher doses (greater than or equal to 100 nM). Exposure of cells to 5 mM alpha-difluoromethylornithine blocked both the VIP-induced increase in cell number and the VIP-induced increase in ODC activity. Increased ODC mRNA was detected after 2 h of exposure to VIP, a time at which ODC activity peaked after treatment, and the increase in ODC mRNA caused by VIP was dose-dependent. In related experiments LoVo cells were found to have high affinity VIP receptors (Kd = 0.4 nM), as assessed by examination of [125I]VIP binding in the presence of varying concentrations of unlabeled VIP. Studies of intracellular cAMP revealed a dose-related increase in cAMP in response to VIP (ED50 = 11 pM), and the adenylate cyclase activator forskolin increased both ODC activity and ODC mRNA. The findings support the idea that LoVo cells have VIP receptors linked to cAMP which can stimulate cell growth at least in part by increasing ODC synthesis and activity, thereby altering the production of polyamines. The decreased growth and ODC activity observed with high doses of VIP may involve a second messenger other than cAMP.

Developmental Regulation of Ornithine Decarboxylase (ODC) in Rat Testis: Comparison of Changes in ODC Activity with Changes in ODC mRNA Levels during Testicular Maturation1

These studies were undertaken to analyze the changes in testicular ornithine decarboxylase (ODC) mRNA levels and ODC activity in rats from birth to maturity. Levels of ODC mRNA were initially low in animals aged 10-17 days. Beginning at 21 days, ODC mRNA levels began to rise, reaching maximal levels by 40 days (p less than 0.01). The size of the 2.2- and 2.6-kb ODC mRNAs did not appear to change with age, as determined by Northern blot analysis. The increase in ODC mRNA that began at 21 days paralleled the increase in testis weight. This increase in ODC mRNA preceded the appearance of rat protamine-1 mRNA, a germ cell-specific mRNA found in round spermatids, which was first detected on Day 40. In contrast, levels of sulfated glycoprotein-2 mRNA, which, in the testis, is found exclusively in Sertoli cells, were highest at Day 17 and thereafter declined gradually with age. Unlike the increase in ODC mRNA levels, ODC activity was highest in 10-day-old animals and thereafter declined steadily with age, reaching minimal levels by 40 days (p less than 0.01). Thus, the increase in testicular ODC mRNA levels was in marked contrast to the decrease in testicular ODC activity. Incubation of cytosolic extract from 40-day-old animals with that from 10- or 17-day-old animals inhibited ODC activity approximately 50%, when compared to cytosols from 10- or 17-day-old animals. Dialysis of cytosol from 40-day-old animals prior to incubation with cytosol from 10-day-old animals relieved this inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of Ornithine Decarboxylase mRNA following Transient Ischemia in the Gerbil Brain

Ornithine decarboxylase (ODC) is the rate-limiting enzyme that catalyzes the synthesis of polyamines from ornithine and is thought to be involved in the cellular response to growth, differentiation, and stress. Previous studies have demonstrated that transient cerebral ischemia results in an increase in ODC activity and polyamine synthesis. We have used the Mongolian gerbil as a model system to test the hypothesis that the cellular response to ischemia induces a distinct pattern of ODC gene expression. Our results indicate that transient ischemia, induced by bilateral carotid occlusion, elevates ODC mRNA within 1-4 h after reperfusion, which correlates with increased ODC activity and polyamine synthesis. Increased ODC mRNA can be detected in the forebrain, striatum, hippocampus, and midbrain but not the cerebellum, which is not subject to ischemic injury. In contrast, c-fos mRNA increased by 15 min after reperfusion and actin mRNA did not demonstrate alterations in level after ischemia. Pentobarbital prevented the increase in ODC mRNA, whereas the glutamate antagonist MK-801 had no effect on the elevation of ODC gene expression after ischemia. We conclude that the ischemia-induced increase in ODC enzyme activity may be attributed in part to transcriptional activation of the ODC gene.

Role of ornithine decarboxylase for proliferation of mesangial cells in culture

To elucidate the role of polyamine metabolism in the regulation of mesangial cell growth, we examined the involvement of ornithine decarboxylase (ODC), the rate limiting enzyme for polyamine synthesis, in the mitogenesis of cultured rat mesangial cells (MCs). Resting MCs, stimulated with fetal calf serum (FCS 10%), showed an induction of ODC activity from undetectable values in resting cells to mean = 5035 nmol CO2/10(10) cells.hr (range 3157 to 7154, N = 5), which is 25-fold above the detection limit. We found a single peak of ODC activity eight to ten hours after stimulation, declining to 22 to 34% of peak levels after 24 hours. 3H-thymidine (TdR) uptake, an S-phase marker of MC replication, peaked at 24 hours, reaching 10.7-fold values of resting MCs. ODC mRNA levels were low in resting cells. After serum stimulation there was a two- to 10-fold increase in ODC mRNA with a maximum after six hours. ODC activity with similar kinetics but lower peak levels was also induced by incubating MCs with mitogens, such as platelet-derived growth factor (PDGF-AB 20 ng/ml), arginine vasopressin (AVP 10(-7) M), phorbol myristate acetate (PMA 10(-7) M), interleukin 1 alpha and beta (IL-1 alpha 10 U/ml, IL-1 beta 10 U/ml). In the presence of alpha-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, the growth rate of MCs, assessed by cell counts and by 3H-TdR uptake, was markedly reduced by 62 to 100%. This antiproliferative effect of DFMO could be reversed by addition of putrescine, the reaction product of ODC.(ABSTRACT TRUNCATED AT 250 WORDS)