Ornithine Decarboxylase Induction by Glucocorticoids in Brain and Liver of Adrenalectomized Rats

Abstract: Ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, was measured in the brain and the liver of adrenalectomized rats after an acute S.C. treatment with glucocorticoids. The effects of corticosterone and dexamethasone were compared in three brain areas, the cerebral cortex, hippocampus, and cerebellum. These structures have similar concentrations of cytosolic glucocorticoid receptor, as measured by an in vitro exchange assay using a specific glucocorticoid ligand, [³H]RU 26988, but contain different amounts of mineralocorticoid receptor. Corticosterone and dexamethasone increased ODC activity in the liver and brain areas in a dose dependent manner, dexamethasone being more active than corticosterone in all tissues. Moreover, estradiol, progesterone, and testosterone were inactive. Aldosterone, at high doses, increased brain ODC activity. Glucocorticoids, selected for their weak binding, or lack of binding to the mineralocorticoid receptor, were tested and found to be highly active in inducing brain and liver ODC, thus showing that ODC induction by steroids is specific for glucocorticoids. These results are among the first to suggest biochemically a central action of glucocorticoids following an acute treatment and confirm that the brain is a glucocorticoid target organ.     

Detection of Ornithine Decarboxylase Antizyme in Mouse Brain

Ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, is known to be regulated by a macromolecular inhibitor, termed antizyme, in a number of cellular systems. The present results show that the antizyme is also a functional component of polyamine metabolism in the brain. It could be demonstrated both in normal randomly selected mice and in animals which had been subjected either to intracerebroventricular injection of saline, which is known to cause a transient activation of ornithine decarboxylase, or to 1,3-diamino-2-propanol, an antizyme-inducing agent. When compared to tissues or cell systems studied so far, the cytosol fraction from mouse brain homogenate appeared to contain an exceptionally high amount of antizyme, that was bound to some material other than active ornithine decarboxylase. This feature was seen in all the animal groups studied, being most prominent after saline injection, when the amount of dissociable antizyme exceeded 14-fold the corresponding released ornithine decarboxylase activity. In untreated animals the excess was about eightfold and after 1,3-diamino-2-propanol about fivefold.     

Involvement of an "Antizyme'' in the Inactivation of Ornithine Decarboxylase

DL-Allylglycine causes a marked increase in mouse brain ornithine decarboxylase (ODC) activity. The amount of immunoreactive enzyme protein increases concomitantly with the activity, but the enzyme protein decreases more slowly than that of the activity. The amount of immunoreactive ODC in brain is many hundred times that of the catalytically active enzyme. The fact that mouse brain cytosol contains high amounts of dissociable antizyme (an inactivating protein) indicates the existence of an inactive, immunoreactive ODC-antizyme pool. The total antizyme content does not change markedly, but instead there are significant changes in different antizyme pools. Putrescine concentrations start to increase 8 h after treatment with allylglycine and concomitantly with this increase, antizyme is released to inhibit enzyme activity. These results indicate the involvement of antizyme in the inactivation process of ODC.     

Transcription-Dependent and -Independent Induction of Cerebral Ornithine Decarboxylase

Ornithine decarboxylase (ODC; EC 4.1.1.17) is a highly inducible, rate-limiting enzyme of the polyamine pathway. We have studied the mechanisms that lead to the induction of ODC activity in response to electrical stimulation in three brain regions. Hippocampal ODC activity was found to exhibit much larger elevations than that of the neocortex and the cerebellum. The levels of ODC gene expression were also followed to examine its relationship to the existing regional differences in ODC activity. In the neocortex, there was an elevation of both the ODC mRNA and enzyme activity. However, the hippocampal ODC mRNA level was not increased by electroconvulsive shock. Furthermore, the effects of hormonal changes and seizures on these regional differences in ODC induction were also examined. Adrenalectomy did not affect ODC activity, but pretreatment with the anticonvulsant MK-801 caused a depression of the induced levels of enzyme activity. Our data suggest that ODC activity in all the brain regions studied is directly elevated by electrically stimulated seizures. However, this induced ODC activity may or may not involve enhanced gene expression.     

Ornithine Decarboxylase Activity in Brain Regulated by a Specific Macromolecul, the Antizyme

Mouse brain ornithine decarboxylase activity is about 70-fold higher at the time of birth compared with that of adult mice. Enzyme activity declines rapidly after birth and reaches the adult level by 3 weeks. Immunoreactive enzyme concentration parallels very closely the decrease of enzyme activity during the first postnatal week, remaining constant thereafter. The content of brain antizyme, the macromolecular inhibitor to ornithine decarboxylase, in turn is very low during the first 7 days and starts then to increase and at the age of 3 weeks it is about six times the level of that in newborn mice. This may explain the decrease in enzyme activity during brain maturation, and suggests the regulation of polyamine biosynthesis by an antizyme-mediated mechanism in adult brain.     

Ornithine Decarboxylase Activity During Development of Cerebellar Granule Neurons

Abstract: Ornithine decarboxylase (ODC), the key enzyme for polyamine biosynthesis, dramatically decreases in activity during normal cerebellar development, in parallel with the progressive differentiation of granule neurons. We have studied whether a similar pattern is displayed by cerebellar granule neurons during survival and differentiation in culture. We report that when granule cells were kept in vitro under trophic conditions (high K⁺ concentration), ODC activity progressively decreased in parallel with neuronal differentiation. Under nontrophic conditions (cultures kept in low K⁺ concentration), the enzymatic activity dropped quickly in parallel with an increased apoptotic elimination of cells. Cultures kept in high K⁺ but chronically exposed to 10 m M lithium showed both an increased rate of apoptotic cell death at 2 and 4 days in vitro and a quicker drop of ODC activity and immunocytochemical staining. A short chronic treatment of rat pups with lithium also resulted in transient decrease of cerebellar ODC activity and increased programmed cell death, as revealed by in situ detection of apoptotic granule neurons. The present data indicate that a sustained ODC activity is associated with the phase of survival and differentiation of granule neurons and that, conversely, conditions that favor their apoptotic elimination are accompanied by a down-regulation of the enzymatic activity.     

Ornithine Decarboxylase Activity and Edema Formation in Cerebral Ischemia of Conscious Gerbils

Abstract: General anesthetic agents often affect the biochemical and physiologic changes triggered by cerebral ischemia. This study examined the regional activities of ornithine decarboxylase (ODC) in gerbils subjected to 5 min of bilateral carotid occlusion without anesthesia. At 2, 4, and 6 h of reperfusion, significant ODC activity was observed in both the cortex and the hippocampus. Pretreatment with α-difluoromethylornithine (DFMO) significantly blocked the ODC activity at 2, 4, and 6 h. Significant edema formation was found at 2, 4, and 6 h. At 2 h, edema formation was unaffected by administration of DFMO. However, DFMO treatment reduced later edema formation at 4 and 6 h. These results demonstrate that ODC activity and edema formation are delayed in gerbils after the induction of transient ischemia even with the removal of anesthetic agents and their potentially protective effects. These findings suggest that ODC activity and its induction of delayed cerebral edema are specific to cerebral ischemia and not to an anesthetic effect. DFMO treatment reduced both the ODC activity and edema formation, indicating a role for polyamines in postischemic edema formation.     

人鸟氨酸脱羧酶抗酶1突变基因的克隆与原核表达

克隆人鸟氨酸脱羧酶抗酶1(Homo sapiensornithine decarboxylase antizyme 1,HOAZ1)开放性阅读框+1核糖体移码位点缺失的突变基因,构建突变基因的原核表达质粒,分离纯化其原核表达的重组蛋白。采用巢式-PCR和重叠延伸-PCR技术,从人非小细胞肺癌细胞株A549的cDNA中获得人类鸟氨酸脱羧酶抗酶1开放性阅读框+1核糖体移码(+1RF)位点缺失突变的基因序列(DM-HOAZ1)。将该序列克隆到原核表达载体pET-28a(+)后,转化表达菌Rosseta(DE3)感受态细胞。阳性克隆用IPTG诱导重组蛋白表达,然后在尿素变性条件下经Ni-NTA树脂亲和层析纯化重组HOAZ1。原核表达和纯化的HOAZ1重组蛋白用Western Blot鉴定。结果显示,成功获得HOAZ1开放阅读框中+1RF位点缺失的突变基因和该突变基因的原核表达质粒pET-28a(+)/DM-HOAZ1;用pET-28a(+)/DM-HOAZ1转化大肠杆菌后,HOAZ-1可被IPTG诱导性高表达,且表达量随诱导时间延长递增;原核表达的HOAZ1可用Ni-NTA树脂亲和层析有效纯化。建立了原核表达和分离纯化HOAZ1蛋白的试验方法,为进一步研究HOAZ1的功能和临床应用奠定了基础。     

Stimulation of Ornithine Decarboxylase Activity in Neural Cell Culture: Potential Role of Insulin

Abstract: Age-dependent decreases in the levels of ornithine decarboxylase activity were observed in the optic lobes, cerebral hemispheres, and midbrain-diencephalon of 6–17-day-old chick embryos. In dissociated cell cultures from chick embryonic brains a similar pattern of declining ornithine decarboxylase activity with time in culture was observed. Ornithine decarboxylase activity in the dissociated brain cell cultures was stimulated by changing the culture medium. The peak stimulatory effect was shown to occur 12 h after changing the medium. Although serum-free medium stimulated ornithine decarboxylase activity slightly, the presence of serum in the medium was the primary stimulatory factor. Both fetal calf serum and heat-inactivated fetal calf serum produced dose-dependent stimulation of ornithine decarboxylase activity. Dialyzed fetal calf sera stimulated ornithine decarboxylase, but to a lower level than that produced by nondialyzed sera. Insulin (0.5–10 μg/ml) stimulated ornithine decarboxylase activity in a dose-dependent manner in serum free medium. In addition, 10² M-L-asparagine stimulated ornithine decarboxylase activity in serum-free medium.     

Transcription-Independent Activation of Ornithine Decarboxylase Activity by Heparin in Cloned Cerebral Endothelial Cells

Abstract: Heparin, a highly sulfated glycosaminoglycan, is known to be obligatory for long-term endothelial cell cultures; it potentiates the mitogenic activities of endothelial cell growth factors and prolongs the replicative life span of the cells. Here we have shown that besides its growth factor-supportive role, heparin exerts a specific action on cerebral capillary endothelial cells (cECs), unrelated to serum or growth factors, by increasing activity of ornithine decarboxylase (ODC; EC 4.1.1.17) in these cells. For our experiments we have used two different types of cloned cECs: type I cECs, grown in the presence of endothelial cell growth factor and heparin, and type II cECs, usually cultivated without growth factors. Heparin action on ODC activity was shown to be dose dependent within the range of 1–100 μg/ml. Increasing concentrations of or depletion of endothelial cell growth factor from type I cultures had no effect on ODC activity. The increase in enzyme activity was highest after 30 min to 1 h of heparin treatment. As evidenced by northern analysis, the heparin-mediated enhancement of ODC activity was not accompanied by changes of ODC mRNA levels. Studies of DNA replication revealed that in the absence of heparin-binding growth factors, heparin did not affect the proliferative activity of cloned cECs.     

Induction of Ornithine Decarboxylase by N-Methyl-D-Aspartate Receptor Activation Is Unrelated to Potentiation of Glutamate Excitotoxicity by Polyamines in Cerebellar Granule Neurons

Abstract: Polyamines positively modulate the activity of the N -methyl- d -aspartate (NMDA)-sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)—the key regulatory enzyme in polyamine synthesis—and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate-induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate-triggered neuronal death, but the glutamate-induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.     

Induction of Ornithine Decarboxylase in Cerebral Cortex by Excitotoxin Lesion of Nucleus Basalis: Association with Postsynaptic Responsiveness and N-Methyl-d-Aspartate Receptor Activation

The major cholinergic innervation of the rat cerebral cortex arises from the nucleus basalis in the basal forebrain. Introduction of the excitotoxins kainate or ibotenate into the nucleus basalis by stereotaxic injection results in degeneration of the cholinergic cells. We have investigated the effect of this excitotoxic action on ornithine decarboxylase (ODC) activity and cholinergic responsiveness in the cerebral cortex. A massive and rapid induction of ODC activity was seen in ipsilateral cortex after injection of excitotoxin. A maximal increase in ODC activity of 268 times the control value was seen in ipsilateral cerebral cortex 8 h after lesioning. Thereafter, ODC activity declined but remained significantly greater than control levels for 32 h. Pretreatment of animals with the irreversible ODC inhibitor difluoromethylornithine prevented the induction of ODC by kainate. Tissue content of the ODC product putrescine showed a marked increase in cerebral cortex ipsilateral to the lesion, increasing sevenfold at 24 h, the maximal concentration reached. After 24 h, the level of putrescine decreased but remained significantly elevated above control values for 5 days. Levels of the polyamines spermidine and spermine were unaffected by lesioning. Increases on ODC activity of much smaller magnitude were also seen in brain regions not directly innervated from the ipsilateral nucleus basalis. However, the response in ipsilateral cortex was found to be dependent on an intact projection from nucleus basalis to cortex. The induction of ODC was shown to be prevented by treatment of rats with MK-801, a result indicating the involvement of N-methyl-D-aspartate (NMDA) receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamines, Ornithine Decarboxylase, and Diamine Oxidase in the Substantia Nigra and Striatum of the Male Rat After Hemitransection

Partial hemitransection at the mesodiencephalic junction in the rat increased striatal and nigral putrescine concentrations on the lesioned side for at least 168 h, with maximal increases between 24 and 48 h. Spermidine and spermine levels declined at 24 h in the striatum, rising above control values at 48 h and further at 168 h. In the substantia nigra, they remained unchanged for the first 48 h and then increased by 168 h. Cadaverine in the striatum also increased at 48 h. On the intact side putrescine increased but to a much lesser extent (at 48 h in the striatum and at 24 and 48 h in the substantia nigra). Ornithine decarboxylase and diamine oxidase activities showed maximal increases at 24 h in the striatum of the lesioned side, whereas in the substantia nigra ornithine decarboxylase attained a very high value as early as 4 h after the operation and diamine oxidase activity peaked at 48 h. The enzyme activities returned toward the basal values at 168 h. On the intact side, ornithine decarboxylase showed a small increase starting at 4 h and diamine oxidase was enhanced at 48 h. These results indicate that the stimulation of biosynthetic and degradative enzymes of polyamine metabolism accompanied by marked and prolonged increases in putrescine may be essential events in the early phases of neuronal response to mechanical injury in the CNS.     

Reciprocal Regulation of Ornithine Decarboxylase and Choline Kinase Activities by Their Respective Reaction Products in the Developing Rat Cerebellar Cortex

Changes in the activity of choline kinase were measured in the cerebellum during development. Early transient increase was found in the enzyme activity just prior to and during birth. This period of increase did not coincide with the periods of transient elevation in ornithine decarboxylase and choline acetyltransferase previously observed in the developing cerebellum. The effects of the naturally occurring polyamines (putrescine, spermidine, and spermine) on choline kinase and choline acetyltransferase activities, and of phosphorylcholine (the product of the reaction catalyzed by choline kinase) on ornithine decarboxylase and choline acetyltransferase activities, were also examined. Choline acetyltransferase activity was not influenced by either polyamines or phosphorylcholine. However, choline kinase activity from 7-day-old, but not from adult, cerebellum was increased 25% in the presence of 4 mM spermine. In contrast, low spermidine concentrations (less than 2 mM) inhibited choline kinase activity selectively in 7-day-old cerebellum. Ornithine decarboxylase activity from 7-day-old cerebellum was inhibited in a concentration-dependent manner by phosphorylcholine. The present data together with other previous reports suggest that: (a) polyamines may play a role in choline utilization during development via their regulation of choline kinase activity, on the one hand, and of acetylcholinesterase activity on the other; and (b) during development, a reciprocal regulation of choline kinase and ornithine decarboxylase activities by their respective reaction products may exist, whereby choline kinase activity is regulated in a complex manner by polyamines and, in turn, ornithine decarboxylase is inhibited by phosphorylcholine.     

Effect of Oxotremorine on Ornithine Decarboxylase Activity of the Adrenal Gland in Rat

Abstract: In this work we have studied the mechanism for the increase of adrenal ODC (ornithine decarboxylase, EC 4.1.1.17) activity provoked by oxotremorine, a muscarinic agonist. 1. Oxotremorine increased medullary ODC activity maximally at 2 h. Cortical enzyme responded much more slowly. 2. Blockade of peripheral muscarinic receptors with methylatropine partially reduced the response to oxotremorine in the medulla, but not cortex. 3. Hy-pophysectomy abolished the cortical, but not the medullary, responses to oxotremorine. Methylatropine reduced the effect of oxotremorine on medullary ODC in hypophysectomized rats. 4. In unilaterally splanchnicotomized rats oxotremorine caused an increase of ODC activity of the denervated adrenal gland relative to control value; activities in both medulla and cortex were significantly lower than those observed in the innervated gland. Evidence was obtained for a compensatory increase of ODC activity of the adrenal cortex (but not medulla) on the intact side of unilaterally operated rats. 5. Surgical intervention, in the form of a sham operation for transection of the spinal cord, leads to an increase of ODC activity in both parts of the adrenal gland. Transection of the cord attenuates these increases. 6. The additional increase of medullary ODC activity owing to the administration of oxotremorine to sham-operated rats is partially reduced in the adrenal medulla by muscarinic blockade, and completely in the cortex. This effect of methylatropine in regard to cortical ODC activity was not apparent in the other experiments with intact or unilaterally splanchnicotomized (unoperated side) rats. The results with unilaterally splanchnicotomized rats and those with transected spinal cord suggest that oxotremorine-induced modifications of adrenal ODC activity are centrally mediated, above the level of origin of the splanchnic nerves in the spinal cord (T_8–10). Experiments with hypophysectomized rats show that the response of the adrenal cortex to oxotremorine is entirely mediated by the hypophysis.     

Resolution and Brain Regional Distribution of Cysteine Sulfinate Decarboxylase Isoenzymes from Hog Brain

Abstract: Cysteine sulfinate decarboxylase (CSD; EC 4.1.1.29) activity from porcine brain was resolved into three peaks by hydroxylapatite chromatography. The first two peaks (I and II) did not decarboxylate and were not inhibited by glutamate. The third peak (III) cochromatographed with glutamate decarboxylase (GAD; EC 4.1.1.15) activity. The K_m values of cysteine sulfinate for peaks I, II, and III were 5.5 × 10⁻⁴ m , 1.3 × 10⁻⁴ m , and 4.5 × 10⁻³ m , respectively. The possibility that the same enzyme was responsible for peak III CSD and GAD activities was suggested by several findings: (1) Mutual competitive inhibition was observed between glutamate and cysteine sulfinate for these activities. (2) Similar first-order heat-inactivation curves were obtained for peak III CSD and GAD when incubated at 55xBOC. (3) Both activities were inhibited similarily by ATP and chloride ion. High concentrations of glutamate (0. l m ) inhibited peak III CSD activity more than 90% but had no effect on either peak I or II CSD activities. This difference in sensitivity of the isoenzymes to inhibition by glutamate was used to examine the relative regional distributions and the relative contributions to total activity of the glutamate-sensitive (peak III CSD, GAD) and glutamate-insensitive (peaks I and II CSD) isoenzymes. Glutamate-insensitive CSD activity contributed only part of the total activity in all brain regions tested (ranging from 23% in the superior colliculus to 64% in the pons). However, the specific activity of glutamate-insensitive CSD was more constant than the total or glutamate-sensitive specific activities among the brain regions tested. The results indicate that GAD is responsible for a significant proportion of the total CSD activity in porcine brain.     

The Response of Ornithine Decarboxylase During Neuronal Degeneration and Regeneration in Olfactory Epithelium

Mature olfactory neurons are continually replaced from a population of progenitor cells. Olfactory nerve section, bulbectomy, or treatment with certain chemicals induces degeneration of olfactory neurons followed in some cases by regeneration. Ornithine decarboxylase (ODC) activity was measured in mouse olfactory tissues as an indicator of cellular regeneration. ODC activity in olfactory tissue (0.2–0.4 nmol/mg protein/h) is 10-30 times higher than in a variety of other cerebral tissues. Within 3 h after unilateral olfactory nerve section, ODC activity in the epithelium declines to 50% of control followed by a slow return to basal activity by 6 days. In the same animals, ODC activity increases severalfold in bulb (1 day) with a gradual decline to normal (9 days). Except for an early transient increase, the effects of unilateral bulbectomy on epithelial ODC activity are similar to those seen after nerve section. The changes in ODC activity following intranasal irrigation with 10 mm -colchicine also closely mimic those seen after nerve section. The effects of intranasal irrigation on ODC activity with 0.5% Triton X-100 or 0.17 m -ZnSO₄ are more complex. Thus, when the mature neuronal population is degenerating after surgery or chemical treatments, ODC activity decreases in the epithelium. The subsequent increase of ODC activity prior to reconstitution of the mature neuronal population probably reflects the regeneration mechanism of the olfactory epithelium. The increase of ODC activity in the olfactory bulb after nerve section is best interpreted as a cellular injury response. These alterations in ODC activity in olfactory tissues after chemical and surgical treatments constitute the earliest biochemical events observed in these tissues in response to cellular damage.