Developmental changes in mouse brain polyamine metabolism

Mouse brain ornithine decarboxylase (ODC) activity is high at the time of birth, whereas S-adenosyl-l-methionine decarboxylase (SAM-DC) activity is low. ODC activity, and putrescine, spermidine and spermine concentrations decline rapidly during postnatal development to the low level characteristic of mature brains, while SAM-DC activity behaves in the opposite manner. The fluctuations in mouse brain polyamine metabolism are in accord with those found in the rat. The apparentK _m values of ODC and SAM-DC for their substrates decline parallel with the decrease of substrate and product concentrations during ontogeny suggesting substrate and/or product dependent regulation of polyamine synthesis in the developing brain.     

Maturation of growth hormone stimulation of kidney ornithine decarboxylase in the rat

The effect of ovine growth hormone (GH) on kidney ornithine decarboxylase (ODC) was studied in newborn, preweanling and young adult rats. Basal kidney ODC activity was very low from 4 to 22 days after birth but rose 20-fold by day 25; it remained elevated through day 45. GH failed to stimulate ODC in the first two weeks after birth. GH did however stimulate ODC markedly from 20 through 45 days. Kidney ODC was stimulated in the neonate by vasopressin and by isoproterenol, but not angiotensin II. Liver ODC remained relatively low and stable during development, and was responsive to GH at all ages studied. We conclude that a) the pattern of development of basal kidney ODC appears to be unique to this tissue and may be related to the postnatal maturation of renal morphology and/or function, b) neonatal kidney ODC is unresponsive to certain hormones but is not completely refractory to stimulation. These findings may have implications for the role of hormones in the maturation of the kidney and in the regulation of early renal function.     

Repeated maternal separation in the neonatal rat: cellular mechanisms contributing to brain growth sparing.

Separation of neonates from their dam has been shown to evoke acutely a variety of biochemical responses, typified by depression of ornithine decarboxylase (ODC) activity. In the current study where rat pups were removed from their nursing dams for 6 h, ODC activities in the liver, heart, kidney and lung were markedly suppressed, but the enzyme in the brain was not altered during the early postnatal ages. These data suggest that the brain was protected from maternal separation insults, a homeostatic response mediated in part, by an increase of circulating corticosterone and glycogen mobilization from peripheral tissues, particularly the liver. In addition, we examined whether these responses were extended to pups who were subject to repeated episodes of maternal deprivation, and whether this stress paradigm might be associated with corresponding changes of cellular growth and maturation. Pups were removed from their dams for 6 h daily beginning at 4 days of age until weaning at 21 days. Plasma corticosterone levels of the deprived pups were elevated significantly at the end of each stress episode but returned to basal (control) levels subsequently. The repeating stress paradigm did not influence the magnitude of this hormonal response at the ensuing ages. Consistent with findings observed in the single episodes of stress, ODC activities in the peripheral tissues were significantly depressed in pups subject to repeated maternal deprivation, but the enzyme appeared to recover to control levels 18 h after each insult. In contrast, brain ODC activity did not exhibit any change throughout the period examined. Moreover, while ontogenetic gains of DNA and protein in the peripheral tissues of the deprived rats lagged slightly but consistently behind those of controls, these macromolecules in the brain were not affected appreciably. These results thus suggest that brain growth was, by and large, spared from insults associated with repeated maternal separation; but this stressful paradigm did produce marked, though reversible biochemical and physiological responses in the peripheral tissues of neonates, which cumulatively led to a lag of cellular development.     

Developmental regulation of ornithine decarboxylase (ODC) in rat testis: comparison of changes in ODC activity with changes in ODC mRNA levels during testicular maturation.

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)     

Changes of malic enzyme activity in the developing rat brain are due to both the increase of mitochondrial protein content and the increase of specific activity.

1. The pattern of NADP-linked malic enzyme activity estimated in the whole brain homogenate did not parallel that found in liver of developing rat. 2. Studies on intracellular distribution of malic enzyme in brain showed that the mitochondrial enzyme increased about three-fold between 10th and 40th day of life. Thereafter, a slow gradual increase to the adult level was observed. 3. The extramitochondrial malic enzyme from brain, like the liver enzyme, increased at the time of weaning, although to a lesser extent. At day 5 the brain malic enzyme was equally distributed between mitochondria and cytosol. 4. During the postnatal development, the contribution of the mitochondrial malic enzyme in the total activity was increasing, reaching the value approx. 80% at day 150 after birth. 5. The increase with age of the malic enzyme specific activity was observed in both synaptosomal and non-synaptosomal mitochondria, the changes in the last fraction being more pronounced. 6. The activity of citrate synthase developed markedly between 10-40 postnatal days, increasing about five-fold, while the specific activity of the enzyme did change neither in the synaptosomal nor in non-synaptosomal mitochondria at this period. 7. We conclude that the changes in malic enzyme activity in the developing rat brain are mainly due both to the increase of mitochondrial protein content and to the increase of specific activity of the mitochondrial malic enzyme.     

Chronic neonatal blockade of NMDA receptor does not affect developmental polyamine metabolism but results in altered response to the excitotoxic induction of ornithine decarboxylase

Neonatal rats were subjected to chronic blockade of the (NMDA) receptor through daily systemic administration of increasing doses of the competitive antagonist CGP 39551 from postnatal days 1–22. Treatment did not result in any significant alteration of the levels of putrescine, spermidine and spermine or in the constitutively expressed activity of the key enzyme for polyamine biosynthesis, ornithine decarboxylase (ODC), as evaluated at 10 and 20 days of age. However, in 30-day-old rats significant differences were observed in the process of excitotoxic ODC induction in the olfactory cortex and the hippocampus of chronically-treated rats: the increase of ODC activity caused by systemic administration of kainic acid took place more rapidly but it was shorter and apparently reached a smaller peak in treated animals as compared to controls. This result, in conjunction with previous data on neurochemistry and locomotor activity of similarly treated rats, strengthens the suggestion that functional alterations of some brain circuits may be the consequence of the blockade of NMDA receptor during the critical neonatal period of brain maturation.     

Expression of ornithine decarboxylase in pancreatic development

We examined the relationship between ornithine decarboxylase (ODC) and growth and differentiation in the developing rat exocrine pancreas. The ODC activity profile showed 2 distinct stages of increases with the first occurred at 14–16 days of age, and a second at 21–23 days of age. Growth parameters evaluated as gains in tissue mass, protein and DNA content in the pancreas indicated a low growth rate soon after birth with a transition to a much more rapid growth rate around the age of 20–21 days, a time corresponded to the second rise in ODC activity. Differentiation parameters evaluated as the accumulation of trypsinogen, amylase and lipase showed different temporal changes. While the rate of accumulation of all three enzymes was relatively low following birth, a rapid rate of accumulation of trypsinogen and amylase started around 15–16 days, a time corresponding to the first rise in ODC activity. Lipase, however, did not show an increase in its accumulation until around age 20 days. These results indicate that a rise in ODC activity is closely associated with growth and differentiation in the developing rat pancreas. To further examine this issue, the steady state levels of ODC mRNA in developing rats were evaluated by Northern blots probed with an ODC cDNA. The developmental profile of ODC mRNA showed a broad peak with a pronounced shoulder occurring at 10 days of age. A higher peak was reached around 20 days of age, then dropped precipitously to a very low level at the age of 24 days. This temporal changes in the level of ODC mRNA show good relationship to the changes in ODC activity suggesting that the control of ODC expression occurs at least in part at the pre-translational level.     

Regulation of succinyl-CoA:3-oxoacid CoA-transferase in developing rat brain: responsiveness associated with prenatal but not postnatal hyperketonemia

Activities of ketone body-metabolizing enzymes in rat brain rise 3- to 5-fold during the suckling period, then fall more than 50% after weaning. Our purpose was to determine the mechanism of the developmental changes in activity of 3-oxoacid CoA-transferase in rat brain and to study its regulation by dietary modification. Purified rat brain 3-oxoacid CoA-transferase was used to generate specific antibody. Immunotitrations of the enzyme from brains of 4-, 24-, and 90-day-old rats indicated that changes in 3-oxoacid CoA-transferase activity during development are due to changes in content of the enzyme protein. Pulse-labeling studies showed that changes in enzyme specific activity reflected changes in its relative rate of synthesis, which increased 2.5-fold between the nineteenth day of gestation and the third postnatal day, remained at this high level until the twelfth postnatal day, and declined thereafter, returning by Day 38 to the level observed in utero. The enzyme is apparently degraded very slowly during early postnatal life. Fetal hyperketonemia induced by feeding pregnant rats a high-fat diet was associated with an increase in the relative rate of synthesis of 3-oxoacid CoA-transferase in brains of 19-day-old fetuses and newborn rats and with an increase in the specific activity of the enzyme at birth. To examine the role of postnatal hyperketonemia in the development of the enzyme in brains of suckling rats, neonates received intragastric cannulas and were fed, for up to 13 days, a modified milk formula low in fat. Postnatal hyperketonemia was abolished but cerebral 3-oxoacid CoA-transferase specific activity on Days 10 and 17 was not significantly affected. Thus, the physiological hyperketonemia caused by the high fat content of rat milk is not required for the normal development of 3-oxoacid CoA-transferase in rat brain.     

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.     

ACCELERATED POSTNATAL DEVELOPMENT OF d(-)-β- HYDROXYBUTYRATE DEHYDROGENASE (EC 1.1.1.30) ACTIVITY IN THE BRAIN IN HYPERTHYROIDISM

Abstract— The activity of d (-)-β-hydroxybutyrate dehydrogenase, a mitochondrial enzyme involved in ketone body metabolism, was found to be low in rat brain at birth, to rise. progressively to a peak during the first 3 weeks of postnatal life, and to decline after weaning to the low levels characteristic of the mature brain. Hyperthyroidism, induced from birth by administration of exogenous thyroxine, accelerated the postnatal development of the enzymic activity in brain and shifted the entire pattern of maturation to approximately 2 days earlier. The effects on the activity of the enzyme were the same with excessive doses of thyroxine which exaggerated the catabolic effects of the hormone and retarded brain and body growth or with lesser doses which had no apparent effects on brain and body growth or on the contents of nucleic acids and proteins in the brain. The accumulation of proteolipid protein in brain was also enhanced in hyperthyroidism. These results suggest that biochemical maturation of the brain is accelerated in hyperthyroidism.     

Polyamine metabolism in enterocytes isolated from newborn pigs.

In the pig, the growth of intestinal mucosa is very intense after birth. Since the polyamines are key elements affecting cell proliferation and differentiation, the present work was undertaken in order to know whether this hypertrophy is associated with an adaptation of polyamine metabolism. Villus enterocytes isolated from pig immediately after birth or 2 days later were found to contain similar amounts of putrescine, spermidine and spermine, i.e., 0.23; 0.41 and 1.24 nmol/10(6) cells, respectively. At birth, despite a relatively high ODC activity, putrescine synthesis from 1 mM L-arginine or 2 mM L-glutamine was very low in isolated enterocytes (6.4 +/- 3.8 pmol/10(6) cells per 30 min), while spermidine and spermine production were not detectable. This could be explained by a very low L-ornithine generation from both amino acids and to an inhibitory effect of polyamines on ODC activity. Two days later, polyamine synthesis from L-arginine remained undetectable despite a higher L-ornithine generation. This was concomitant with a dramatic fall in ODC activity. At both stages, enterocytes were able to take up polyamines from the extracellular medium in a temperature-dependent manner. It is concluded that de-novo synthesis of polyamines from L-arginine or L-glutamine does not play a significant role in the control of polyamine content of pig enterocytes during the postnatal period. In contrast, polyamine uptake by enterocytes would contribute to maintain a steady-state polyamine content during this period.     

Changes in ornithine decarboxylase and antizyme activities in developing mouse brain.

A macromolecular inhibitor to ornithine decarboxylase (ODC) present in mouse brain was identified as ODC antizyme [Fong, Heller & Canellakis (1976) Biochim. Biophys. Acta 428, 456-465; Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1858-1862] on the basis of kinetic properties, Mr and reversal of its inhibition by antizyme inhibitor. The brain antizyme, however, did not cross-react immunochemically with any of seven monoclonal antibodies to rat liver antizyme. ODC activity in mouse brain rapidly decreased after birth, in parallel with putrescine content, and almost disappeared by 3 weeks of age. Free antizyme activity appeared shortly after birth and increased gradually, whereas ODC-antizyme complex already existed at birth and then gradually decreased. Thus total amount of antizyme remained about the same throughout the developmental period in mouse brain. In addition to ODC-antizyme complex, inactive ODC protein was detected by radioimmunoassay in about the same level as the complex at 3 weeks of age. Upon cycloheximide treatment, both free ODC activity and ODC-antizyme complex rapidly disappeared, although free antizyme and the inactive ODC protein were both quite stable.     

Stimulation of ornithine decarboxylase by histamine or norepinephrine in brain regions of the developing rat: Evidence for biogenic amines as trophic agents in neonatal brain development

Ornithine decarboxylase (ODC) initiates the synthesis of polyamines which play key roles in regulation of cellular development. Intracisternal administration of histamine or norepinephrine to developing rats produced age-dependent stimulation of ODC in brain. In cerebral cortex and pons-medulla, stimulation by norepinephrine was demonstrable at postnatal day 7 and maximum stimulation occured at about day 9. In contrast, cerebellum showed no initial reactivity to norepinephrine but still developed a large peak of response capability by day 9. In all 3 regions, the response declined rapidly thereafter during the period of major synaptogenesis of noradrenergic pathways. With histamine, none of the regions displayed ODC reactivity at 7 days postnatally; stimulation appeared by day 9, peaked at about day 11 and then declined rapidly. Thus, the trophic effect of histamine or norepinephrine toward ODC activity is present or develops postnatally and appears to terminate with synaptogenesis and onset of neurotransmitter properties of the amines.     

Effects of Insulin on Cultured Rat Brain Cells: Stimulation of Ornithine Decarboxylase Activity

Abstract: Growth-promoting peptide hormones, including growth hormone and insulin, stimulate rat brain ornithine decarboxylase (ODC; EC 4.1.1.17) activity in vivo (Roger et al., 1974; Roger and Fellows, 1980). To determine if this is a result of a direct action on brain, we have investigated the effect of peptide hormones in primary cell cultures of brain from fetal rats of 20 days gestational age. Significant stimulation of ODC activity was observed 4 h after administration of porcine insulin and bovine growth hormone. On a molar basis, growth hormone was less potent than insulin. By contrast, glucagon, enkephalin, and angiotensin II did not stimulate ODC in this system. At 25 ng/ml, insulin stimulated ODC activity approximately threefold, with maximum stimulation of five- to sevenfold reached at 1 μg/ml. After a 1-h lag, insulin-stimulated ODC activity increased to a maximum between 5 h and 8 h and returned to basal levels by 24 h. The apparent K_m of ODC, 5.66 ± 1.16μM, was not significantly altered by insulin treatment, nor was any enzyme activator found in mediating insulin actions. Additional evidence suggests that insulin stimulation of ODC activity involves both de novo synthesis of the enzyme and a prolongation of ODC half-life by 50%. These findings, implicating insulin as a regulator of ODC activity in brain cells, suggest the possible involvement of insulin or an insulin-like peptide in the control of growth and development of the CNS.     

Postnatal Developmental Changes in Fucosyltransferase Activity in Rat Brain

Abstract: GDP-fucose:asialofetuin fucosyltransferase activity was studied during the postnatal development of rat brain. The enzymatic activity was very low during the first days of life and reached a maximum level around 21 days. This increase in enzymatic activity was characterized by two periods of rapid change. A rapid increase occurred between 3 and 7 days after birth, followed by a slow increase from 7 to 17 days, then a new rapid change from 17 to 21 days. Stimulation of the enzymatic activity by Triton X-100 increased with age. The developmental profiles of GDP-fucose pyrophosphatase and fucosidase did not change during this period.     

Cytosolic 3-hydroxy-3-methyl glutaryl coenzyme a synthase in rat brain: Properties and developmental change

The properties and developmental change in the activity of cytosolic 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) synthase in brain was examined and whether or not HMG-CoA lyase is present in cytosol and mitochondria from brain was determined. Although mitochondrial fractions contained significant HMG-CoA lyase activity, the enzyme activity was not detected in brain cytosol. The synthase activity was present in both mitochondrial and cytosolic fraction. The HMG-CoA synthesis by brain cytosol was optimal at pH 8.0 and did not require Mg²⁺ or exogenous acetoacetyl CoA. This indicates that brain cytosol can synthesize sufficient quantity of acetoacetyl CoA from acetyl CoA to be utilized for HMG-CoA synthesis. Our results also showed that the specific activity (nmol acetyl CoA incorporated/mg protein) of HMG-CoA synthase in brain cytosol was high (between 2–11 days of postnatal age) when the cholesterol content of brain is increasing rapidly, and the activity declined slowly thereafter. This suggests that in brain, cytosolic enzyme HMG-CoA synthase plays a role in the regulation of cholesterol synthesis.