Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells

We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by alpha-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages. DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48+/-5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production. Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10 mM) to potentiate LPS-induced NO synthesis. Metabolism of L-[3H]arginine to L-[3H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1-10 mM) or by putrescine (0.001-1 mM), spermidine (0.001-1 mM) or spermine (0.001-1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70+/-0.07%) by L-NMMA (100 microM). Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (approximately 2 fold) iNOS protein expression. These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.     

Inhibitory effects of nitric oxide donors on nitric oxide synthesis in rat gastric myenteric plexus

We investigated whether nitric oxide (NO) exerts an inhibition on its own synthesis in the gastric myenteric plexus in rats. Nonadrenergic, noncholinergic relaxations in response to transmural electrical stimulation (TS) were markedly antagonized by NG-nitro-L-arginine methyl ester, (10(-4) M) and abolished by tetrodotoxin (10(-6) M). Pretreatment with various NO donors (3-morpholino-sydnonymide [SIN-1 (3 x 10(-7) to 3 x 10(-6) M)], S-nitroso-N-acetylpenicillamine (10(-6) to 10(-5) M), sodium nitroprusside (10(-8) to 3 x 10(-8) M) and 8-bromoquanosine 3', 5'-cyclic monophosphate [8-bromo-cGMP (10(-6) to 3 x 10(-6) M)]) significantly inhibited TS-evoked nonadrenergic, noncholinergic relaxations in a dose-dependent manner. In contrast, vasoactive intestinal polypeptide (10(-8) M)-induced relaxations were not affected by SIN-1 or 8-bromo-cGMP. TS evoked a significant increase in 3H-citrulline formation, which was completely abolished by calcium-free medium, NG-nitro-L-arginine methyl ester, (10(-4) M) and tetrodotoxin (10(-6) M). 3H-citrulline formation evoked by TS was significantly inhibited by SIN-1 (10(-7) to 10(-5) M) and 8-bromo-cGMP (10(-7) to 10(-5) M) in a dose-dependent manner. The inhibitory effect of SIN-1 was partially prevented by 1H-[1,2, 4]oxadiazolo[3,4-a]quinoxalin-1-one (10(-5) M), a guanylate cyclase inhibitor. We conclude that NO synthesis in the gastric myenteric plexus is negatively regulated by NO and cGMP. This suggests an autoregulatory feedback mechanism of NO synthesis in the gastric myenteric plexus.     

Association between high levels of ornithine decarboxylase activity and favorable prognosis in human colorectal carcinoma

Several studies have documented increased expression of ornithine decarboxylase (ODC) in neoplastic colorectal tissue versus normal-appearing colonic mucosa. The present study was undertaken to determine whether there is an association between the degree of overexpression of ODC in colorectal carcinomas and survival in a series of 74 patients. A high level of tumor ODC expression was found to be significantly associated with greater survival in our patient series. Patients with tumor ODC activities greater than the median and especially in the highest quartile experienced a more favorable outcome than those patients with ODC values below the median or in the lowest quartile (P = 0.03 and 0.02, respectively). The presence of a GTP-activatable isoform of ODC was also significantly associated with a favorable prognosis but only in tumors of the right colon (P = 0.01). There was no association found between ODC activity and tumor grade, tumor size, or patient age, sex, or race. Our results demonstrate that high levels of ODC expression (and presence of a GTP-activatable isoform for right-sided colon tumors) predict a favorable prognosis in human colorectal carcinoma. Knowledge of a patient's ODC status at the time of surgery may be useful in decisions regarding adjuvant therapy. Understanding the mechanism(s) involved should lead to new therapeutic approaches for advanced colorectal carcinoma.     

Differential activity of nitric oxide synthase in human nasal mucosa and polyps

A quantification method suitable for determination of individual oligosaccharide compounds from human milk has been established. The crude milk oligosaccharide fraction was separated into acidic oligosaccharides, neutral oligosaccharides, and lactose by gel permeation chromatography. After this separation step neutral and acidic oligosaccharides were analyzed separately by high-pH anion-exchange chromatography with pulsed amperometric detection. The concentrations of 14 neutral oligosaccharides, of 6 acidic oligosaccharides, and of N-acetylneuraminic acid were determined on the internal standards stachyose and galacturonic acid, respectively. Thus, previously applied quantification methods for milk oligosaccharides based on gel permeation chromatography have been decisively improved.     

Prognostic influence on survival of increased ornithine decarboxylase activity in human breast cancer

Although considerable experimental evidence suggests an important role of polyamines in breast cancer biology, compelling supportive data in patients are lacking. To address this issue, we measured ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase, and spermidine/spermine acetyltransferase (the three key polyamine metabolic enzymes) in a cohort of 50 primary human breast cancers and related their levels of activity to disease-free survival and overall survival. The major finding of our study was that ODC activity level was a negative independent prognostic factor for both end points. With regard to overall survival, the adverse influence of ODC expression was superior even to that provided by the number of positive nodes. Furthermore, the statistical significance of the ODC effect on survival was enhanced when breast cancer-specific mortality was included in the analysis as opposed to death from any cause. In addition, high tumor ODC activity may predict a shorter time from recurrence to death, although this effect was of only borderline statistical significance. In summary, these results provide the first concrete evidence supporting the prognostic role of ODC in human breast cancer.     

Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport

Various authors have suggested that nitric oxide (.NO) exerts cytotoxic effects through the inhibition of cellular respiration. Indeed, in intact cells .NO inhibits glutamate-malate (complex I) as well as succinate (complex II)-supported mitochondrial electron transport, without affecting TMPD/ascorbate (complex IV)-dependent respiration. However, experiments in our lab using isolated rat heart mitochondria indicated that authentic .NO inhibited electron transport mostly by reversible binding to the terminal oxidase, cytochrome a3, having a less significant effect on complex II- and no effect on complex I-electron transport components. The inhibitory action of .NO was more profound at lower oxygen tensions and resulted in differential spectra similar to that observed in dithionite-treated mitochondria. On the other hand, continuous fluxes of .NO plus superoxide (O.(2)(-)), which lead to formation of micromolar steady-state levels of peroxynitrite anion (ONOO-), caused a strong inhibition of complex I- and complex II-dependent mitochondrial oxygen consumption and significantly inhibited the activities of succinate dehydrogenase and ATPase, without affecting complex IV-dependent respiration and cytochrome c oxidase activity. In conclusion, even though nitric oxide can directly cause a transient inhibition of electron transport, the inhibition pattern of mitochondrial respiration observed in the presence of peroxynitrite is the one that closely resembles that found secondary to .NO interactions with intact cells and strongly points to peroxynitrite as the ultimate reactive intermediate accounting for nitric oxide-dependent inactivation of electron transport components and ATPase in living cells and tissues.     

Effects of nitric oxide from exogenous nitric oxide donors on osteoblastic metabolism

We examined the effects of nitric oxide (NO) on the differentiation and mineralization of newborn rat calvarial osteoblastic cells (ROB cells) using exogenous NO donors, sodium nitroprusside, 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamin e (NOC-7) and 2,2'-(hydroxynitrosoydrazino)bis-ethanamine (NOC-18). Sodium nitroprusside and NOC-7 dose-dependently enhanced the rate of production of intracellular cGMP in ROB cells and the rat clonal osteogenic cell line ROB-C26. We used NOC (NOC is the trade name for NO complex manufactured by Dojindo, Kumamoto, Japan) as an NO donor in our experiments because sodium nitroprusside exhibited a marked cytotoxicity. Northern blot analysis revealed that the level of mRNA for osteocalcin, one of the osteoblastic differentiation markers, was enhanced in the ROB cells, which was continuously treated by NOC-18. NOC-18, however, did not affect the level of mRNA for alkaline phosphatase and the activity of alkaline phosphatase. Both the number and the total area of mineralized nodules that are a model of in vitro bone formation were shown to be increased by 10(-5) M NOC-18. Our data suggest that NO might act as a local regulator of the metabolism of osteoblastic cells.     

Functional expression of three isoforms of human nitric oxide synthase in baculovirus-infected insect cells

Complementary DNAs encoding three human isoforms (neuronal, inducible, and endothelial) of nitric oxide synthase were cloned into the baculovirus expression vector pVL1392/1393. Transfection of Sf-9 insect cells with the recombinant baculovirus resulted in the expression of high levels of nitric oxide synthases. The expressed proteins of neuronal and inducible nitric oxide synthase were predominantly soluble, whereas the endothelial enzyme was for the most part, particulate. Recombinant enzymes were purified with 2',5'-ADP Sepharose affinity chromatography. The effects of reference enzymatic inhibitors (NG-methyl-L-arginine, NG-nitro-L-arginine and N-iminoethyl-L-ornithine) on recombinant expressed proteins were not significantly different from native nitric oxide synthase enzyme preparations. L-aminoguanidine was found to be much less potent in inhibiting recombinant or native human inducible nitric oxide synthase compared to the murine isoform. These findings indicate previously unappreciated interspecies differences in the action of nitric oxide synthase enzymatic inhibitors. The functional expression of human nitric oxide synthase isoforms in a heterologous expression system allowed screening of novel inhibitors. Studies indicated that S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine were potent novel inhibitors of human nitric oxide synthases.     

Differential regulation of endothelial cell adhesion molecule expression by nitric oxide donors and antioxidants

Although nitric oxide (NO) and antioxidants inhibit adhesion molecule expression, their inhibitory effects on nuclear factor kappaB (NF-kappaB) activation may differ. The NO donors, but not 8-bromo-cGMP, decreased tumor necrosis factor alpha (TNF-alpha)-induced VCAM-1, ICAM-1, and E-selectin expression by 11-70%. In contrast, NAC completely abolished VCAM-1 and E-selectin expression and decreased ICAM-1 expression by 56%. Gel shift assays demonstrate that NF-kappaB activation was inhibited by both NO and antioxidants. The activation of NF-kappaB involves the phosphorylation and degradation of its cytoplasmic inhibitor IkappaB-alpha by 26S proteasomes. The 26S proteasome inhibitor MG132 prevented the degradation of phosphorylated IkappaB-alpha. NAC inhibited IkappaB kinase (IKK) activity and prevented IkappaB-alpha phosphorylation and degradation. In contrast, NO did not inhibit IKK activity, IkappaB-alpha phosphorylation, or IkappaB-alpha degradation. However, NO, but not antioxidants, induced IkappaB-alpha promoter activity. The inhibitory effects of NO on adhesion molecule expression, therefore, differs from that of antioxidants in terms of the mechanism by which NF-kappaB is inactivated.     

Effect of haloperidol on adrenal ornithine decarboxylase activity of the rat

The administration of the dopamine antagonist haloperidol (HLP) to rats produced a temporary increase in adrenomedullary and cortical ornithine decarboxylase (ODC) activity. The time-course of stimulation of ODC activity by HLP showed different patterns in both structures. Medullary ODC activity was highest at 2.5 h, decreasing at later times; cortical ODC activity was not affected by the drug at 2.5 h, but then increased up to at least 6.5 h. The medullary increase observed at 2.5 h was dose-related and could be prevented by splanchnicotomy. Hypophysectomized rats, on the contrary, showed an enhanced response to HLP. The results suggest that haloperidol-induced increase of adrenomedullary ODC activity is caused by a reflex increase in preganglionic nerve activity, and that the pituitary gland can modulate this response. Cortical ODC response to HLP, as previously demonstrated, is mediated entirely by the hypophysis.     

Acute effects of cocaine on ornithine decarboxylase activity in fetal and neonatal rat heart: evidence for cardiotoxicity

Fetal exposure to cocaine is associated with increased perinatal cardiac risk. In the current study, we examined the effects of acute cocaine administration on ornithine decarboxylase (ODC) activity in fetal and neonatal rat heart. ODC is a key regulatory enzyme in the control of cell differentiation and growth, and rapid changes in ODC are associated with the response to cell injury. Administration of 30 mg/kg s.c. of cocaine to pregnant rats on the 20th day of gestation caused acute elevation of fetal cardiac ODC that persisted throughout the ensuing 24 h. In contrast, the same dose given directly to neonatal rats the day after birth evoked only a short-term (1-h) stimulation of ODC that was reversed by 4 h after treatment. By 4 days of age and subsequently, cocaine was unable to elicit acute stimulation of heart ODC and only evoked inhibition of enzyme activity. Elevated progesterone levels during pregnancy have been shown to sensitize the maternal myocardium to cocaine-induced catecholaminergic effects; the greater sensitivity of fetal heart ODC to cocaine, as compared to neonatal heart, supports the hypothesis that similar enhancement of fetal cardiac irritability can contribute to cocaine-induced cell damage.     

Action of prolactin on ornithine decarboxylase activity in mammary gland explants of mice

Prolactin stimulated ornithine decarboxylase activity in mammary gland explants from midpregnant mice. The enhanced enzyme activity occurred in explants which were preincubated for 1 day in medium containing insulin, hydrocortisone, insulin plus hydrocortisone, or in medium containing no hormones. The largest prolactin effect was observed in tissues which were pretreated with insulin plus hydrocortisone; a greater than ten-fold increase in ornithine decarboxylase activity was observed when these tissues were incubated with prolactin for 2 hours. An effect of prolactin on ornithine decarboxylase activity was also observed in explants prepared from lactating mouse mammary glands.     

Maternal dietary protein deficiency decreases nitric oxide synthase and ornithine decarboxylase activities in placenta and endometrium of pigs during early gestation

Little is known about the mechanism responsible for retarded placental and fetal growth induced by maternal dietary protein malnutrition. On the basis of the recent finding that nitric oxide (NO) and polyamines (products of L-arginine) play an important role in embryonic and placental development, the present study was designed to determine whether protein deficiency decreases placental and endometrial activities of NO synthase (NOS) and ornithine decarboxylase (ODC) (the first and key regulatory enzyme in polyamine synthesis). Primiparous gilts selected genetically for low or high plasma total cholesterol concentrations (low line and high line, respectively) were mated and then fed 1.8 kg/d of isocaloric diets containing 13% or 0.5% crude protein. At d 40 or 60 of gestation, they were hysterectomized, and placenta and endometrium were obtained for incubations, NOS and ODC assays, and measurements of free amino acids and polyamines. Maternal dietary protein restriction decreased arginine and ornithine concentrations, constitutive and inducible NOS activities and NO production, as well as ODC activity and polyamine concentrations in placenta and endometrium of both lines of gilts. Placental NO synthase activity and NO generation were lower in high line gilts than in low line gilts. ODC activities and polyamine concentrations in placenta and endometrium were decreased at d 60 compared with d 40 of gestation. These changes in placental and endometrial synthesis of NO and polyamines during early gestation may be a mechanism responsible for reduced placental and fetal growth in protein-deficient gilts and for altered conceptus development in high line gilts.     

Selective expression of inducible nitric oxide synthase in human prostate carcinoma

BACKGROUND: Nitric oxide (NO) is synthesized by inducible nitric oxide synthase (iNOS) and plays an important role in tumor growth and angiogenesis. NO generation by iNOS also influences the cytotoxicity of macrophages and tumor-induced immunosuppression. Before now, the expression of iNOS in prostate carcinoma tissue had not been determined. METHODS: In this study, tissue sections from 16 patients with prostate carcinoma were studied immunohistochemically and compared with tissue specimens from 10 patients with benign hyperplasia. RESULTS: Positive iNOS immunostaining was detected in all sections from patients with prostate carcinoma. The malignant epithelial cells were highly positive. The antibody against iNOS also marked round cells, which had the same cell shape as that observed for macrophages. These cells were located in stroma and epithelium adjacent to tumor islets. However, round cells in benign tissue stained negative for iNOS. None of the benign hyperplasia specimens stained positive for iNOS immunohistochemically. CONCLUSIONS: Prostate carcinoma tissue had a high iNOS content, whereas benign tissue did not. The authors suggest that epithelial iNOS expression can be used as a specific immunohistochemical marker for prostate carcinoma. NO generation by iNOS may play multiple roles in the development of this disease.     

Relationship between structure and inhibitory effect of arginine analogues on neuronal nitric oxide synthase activity

2,3,5-Trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504), an inhibitor of 5-lipoxygenase and thromboxane A2 synthase and a scavenger of active oxygen species, has been shown to exhibit profound anti-tumour activity against three established murine adenocarcinomas (MACs) that are generally refractory to standard cytotoxic agents. For the cachexia-inducing MAC16 tumour, optimal anti-tumour activity was seen at dose levels of 10 and 25 mg kg-1 day-1, together with a reversal of cachexia and a doubling of the time to sacrifice of the animals through cachexia from 8 days to 17 days. The remaining tumour fragments showed extensive necrosis in regions distal from the blood supply. Growth of the MAC13 tumour was also effectively suppressed at dose levels between 5 and 50 mg kg-1 day-1, resulting in a specific growth delay between 1.0 and 1.2. Growth of the MAC26 tumour was also inhibited a concentration-related manner, with doses of 25-50 mg kg-1 day-1 being optimal. Anti-tumour activity towards all three tumours at low dose levels of CV-6504 was effectively suppressed by concurrent administration of linoleic acid (1 g kg-1 day-1), suggesting that inhibition of linoleate metabolism was responsible for the anti-tumour effect. Tumour sensitivity may be correlated with increased DT-diaphorase that are required to metabolise CV-6504 to the active hydroquinone, which inhibits 5-lipoxygenase activity.     

Opposite effects of nitric oxide donors on DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide

1. The effects of three different NO donors on tert-butylhydroperoxide (tB-OOH)-induced DNA cleavage and toxicity were investigated in U937 cells. 2. Treatment with S-nitroso-N-acetyl-penicillamine (SNAP, 1-30 microM), while not in itself DNA-damaging, potentiated the DNA strand scission induced by 200 microM tB-OOH in a concentration-dependent fashion. The enhancing effects of SNAP were observed with two different techniques for the assessment of DNA damage. Decomposed SNAP was inactive. S-nitrosoglutathione (GSNO, 300 microM) and (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NO, 1 mM) also increased DNA cleavage generated by tB-OOH and these responses, as well as that mediated by SNAP, were prevented by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (PTIO). 3. SNAP neither inhibited catalase activity nor increased the formation of DNA lesions in cells exposed to H2O2. Furthermore, SNAP did not affect the rate of rejoining of the DNA single strand breaks generated by tB-OOH. 4. Under the conditions utilized in the DNA damage experiments, treatment with tB-OOH alone or associated with SNAP did not cause cell death. However, SNAP as well as GSNO markedly reduced the lethal response promoted by millimolar concentrations of tB-OOH and these effects were abolished by PTIO. Decomposed SNAP was inactive. 5. It is concluded that low levels of NO donors, which probably release physiological concentrations of NO, enhance the accumulation of DNA single strand breaks in U937 cells exposed to tB-OOH. This NO-mediated effect appears to (a) not depend on inhibition of either DNA repair (which would increase the net accumulation of DNA lesions by preventing DNA single strand break removal) or catalase activity (which would also enhance the net accumulation of DNA lesions since H2O2 is one of the species mediating the tB-OOH-induced DNA cleavage) and (b) be caused by enforced formation of tB-OOH-derived DNA-damaging species. In contrast to these results, similar concentrations of NO prevented cell death caused by millimolar concentrations of tB-OOH. Hence, DNA single strand breakage generated by tB-OOH in the absence or presence of NO does not represent a lethal event.     

Alpha-difluoromethylornithine (DFMO) as a potent arginase activity inhibitor in human colon carcinoma cells

Alpha-difluoromethylornithine (DFMO) is commonly used as a specific ornithine decarboxylase (ODC, EC4.1.1.17) irreversible inhibitor. ODC is the enzyme responsible for polyamine biosynthesis, which has been shown to be strictly necessary for cell proliferation. In HT-29 Glc-/+ cells, L-arginine is the major precursor of these molecules through the sequential actions of arginase, which leads to L-ornithine generation and ODC. L-ornithine, a substrate for ODC, retroinhibits arginase. Since DFMO is an ornithine analogue, we searched for a direct effect of this agent upon arginase. The flux of L-arginine through arginase in intact cells was inhibited by 51+/-11% by 10 mM of DFMO whereas 10 mM of L-valine, a known potent arginase inhibitor, inhibited this flux by 73+/-6%. DFMO equilibrated between extracellular and intercellular spaces and, when used at 10-mM concentration, was without effect on L-arginine net uptake. Measurement of arginase activity in HT-29 cell homogenates with increasing concentrations of DFMO and L-arginine led to an inhibition with a calculated Ki (inhibitory constant) equal to 3.9+/-1.0 mM. L-ornithine was less effective than DFMO in inhibiting arginase activity. Bovine liver arginase, used as another source of the enzyme, was also severely inhibited by DFMO. The inhibitory effect of DFMO upon arginase, one step upstream of the ODC reaction in the metabolic conversion of L-arginine to polyamines, is of potential physiological importance, since it could alter the production of ornithine and thus its metabolism in pathways other than the ODC pathway.     

Mutation of aspartate-233 to valine in mouse ornithine decarboxylase reduces enzyme activity

Ornithine decarboxylase is the first and key enzyme in mammalian polyamine biosynthesis. All eukaryotic ornithine decarboxylases contain several highly conserved regions and the amino acid residues 232-238 form one of the most highly conserved sequences. This region contains a glycine-rich sequence typically found in a number of pyridoxal 5'-phosphate-dependent or nucleotide-binding proteins. We mutated aspartate-233 which is the only acidic residue within this region to valine. This mutation causes striking sequence similarity with the guanine nucleotide binding domain of c-H-ras. Mutated ornithine decarboxylase cDNA with a mouse mammary tumor virus long terminal repeat promoter has been transfected for stable expression into ornithine decarboxylase-deficient C55.7 cells. Ornithine decarboxylase activity of the mutated enzyme was about 20% of wild-type ornithine decarboxylase activity and it was not activated by guanosine triphosphate like the ornithine decarboxylase isoform found in some tumors and rat brain. The mutation caused an increase in K(m) value of about 20-fold both for the substrate L-ornithine and for the cofactor pyridoxal 5'-phosphate. The Ki value for the irreversible inhibitor alpha-difluoromethylornithine was also increased, whereas the half-life of the enzyme was shortened. These results suggest that the region containing aspartate-233 is essential for binding of the cofactor and thus forms part of enzymatic active site, and the mutation of aspartate-233 to valine cannot, at least alone, cause the activation of ornithine decarboxylase by guanosine triphosphate (230).