Oili Hietala

Ornithine decarboxylase and adenosylmethionine decarboxylase in mouse brain--effect of electrical stimulation.

THE POLYAMINES. spermidine and spermine, and the diamine. putrescine, have received considerable attention in recent years as they have been implicated in various growth processes and in cellular differentiation (RAINA & JANNE, 1975). They occur in high concentrations in neural tissue (SHASKAN eta/., 1973; SEILER & SCHMIDT-GLENEWINKEL, 1975; SHAW & PATEMAN. 1973; HARIK & SNYDER, 1974) and show a distinct developmental pattern and age dependency (JANNE el al., 1964; KREMZNER et al., 1970). Ornithine decarboxylase (EC 4.1.1.17) catalyzes the conversion of L-ornithine to the diamine putrescine, the first and perhaps rate-limiting step in polyamine biosynthesis. S-Adenosyl-~-methionine decarboxylase (EC 4. I . 1.50) forms S-methyladenosylhomocysteamine from adenosylmethionine and spermidine synthase transfers the propylamino group from decarboxylated adenosylmethionine to putrescine. The spermine synthase reaction is analogous, transferring the propylamino group from decarboxylated adenosylmethionine to spcrmidine, forming spermine. The synthesis of polyamines has been extensively studied during past 10 years, but relatively little is known about their catabolism. Especially relevant to the function of the nervous system there is an important route to catabolize putrescine to GABA and further either to COz via the tricarboxylic cycle (SEILER et a)., 1971) or homocarnosine (KALYAKKER & MEISTER. 1959; KONISHI et a/., 1977). In this paper we have investigated the eficct of electrical stimulation of mouse brain on the activity of ODC and SAM-DC.

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. Immuno‐reactive 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.

Expression of ODC and its regulatory protein antizyme in the adult rat brain

Ornithine decarboxylase and its inhibitor protein, antizyme are key regulators of polyamine biosynthesis. We examined their expression in the adult rat brain using in situ hybridization and immunocytochemistry. Both genes were widely expressed and their expression patterns were mostly overlapping and relatively similar. The levels of antizyme mRNA were always higher than those of ornithine decarboxylase mRNA. The highest expression for both genes was detected in the cerebellar cortex, hippocampus, hypothalamic paraventricular and supraoptic nuclei, locus coeruleus, olfactory bulb, piriform cortex and pontine nuclei. Ornithine decarboxylase and antizyme mRNAs appeared to be localized in the nerve cells. ODC antibody displayed mainly cytoplasmic staining in all brain areas. Antizyme antibody staining was mainly cytoplasmic in the most brain areas, although predominantly nuclear staining was detected in some areas, most notably in the cerebellar cortex, anterior olfactory nucleus and frontal cortex. Our study is the first detailed and comparative analysis of ornithine decarboxylase and antizyme expression in the adult mammalian brain. J. Neurosci. Res. 62:675–685, 2000.

THE EFFECT OF dl‐ALLYLGLYCINE ON POLYAMINE AND GABA METABOLISM IN MOUSE BRAIN

Abstract— dl‐Allylglycine, a potent inhibitor of glutamate decarboxylase in vivo when given intraperitoneally, causes a marked decrease in brain GABA concentration and at the same time a dramatic increase in l‐ornithine decarboxylase activity and a simultaneous decrease in S‐adenosyl‐l‐methionine decarboxylase activity followed by putrescine accumulation. It does not, however, alter the degree of GABA formation from putrescine. The timing of the recovery of glutamate decarboxylase activity after the injection of dl‐allylglycine is concomitant with that of the GABA concentration, indicating that it is probably glutamate decarboxylase that is solely responsible for making up the GABA deficit caused by dl‐allylglycine, and that the changes in polyamine metabolism are associated in some indirect way with the recovery process.

Reindeer BMP extract in the healing of critical-size bone defects in the radius of the rabbit.

Background Native BMP extracts from reindeer effectively induce ectopic new bone formation in vivo, but their bone healing properties have not yet been evaluated. We investigated the effect of reindeer BMP extracts on the healing of long bone defects. Methods The implants tested contained 5 mg or 10 mg of unsterilized BMP extract from reindeer and 10 mg of gamma-sterilized BMP extract administered with collagen carrier (Lyostypt, B. Braun, Germany). 70 μg of rhBMP-2 with collagen carrier (InductOs; Wyeth Europa) served as positive control, and collagen implants (Lyostypt) and untreated defects served as negative controls. New Zealand White rabbits with 1.5 cm of critical-size radius bone defects were used, with 8 weeks of follow-up. Results Radiographic analysis showed bone formation (BF) to be higher in all groups containing BMPs than in the untreated controls. BF was also higher in the rhBMP-2 group, and marginally higher in the group treated with 10 mg of unsterilized reindeer BMP extract (p = 0.06) as compared to the collagen controls. Bone union (BU) was better in the unsterilized BMP extract groups and rhBMP-2 group than in the untreated controls. BU was also better in the implants with 10 mg of unsterilized reindeer BMP extract and rhBMP-2 than in the collagen-treated implants. The mean area of new bone at the site of the defect proved to be higher in all implants containing BMP than in the untreated defects. It was also higher in the groups with 10 mg of unsterilized reindeer BMP extract and rhBMP-2 than in the collagen-treated controls. Mechanical tests showed torsional stiffness of the bones to be higher in the group with 10 mg of unsterilized BMP extract than in the collagen group. The mean cross-sectional bone area measured by pQCT densitometry was higher in the rhBMP-2 group than in the collagen group. The mean bone density at the defect area was higher in the group with 10 mg of unsterilized BMP than in the rhBMP-2 group. Interpretation We conclude that both reindeer BMP extract and rhBMP-2 induced improved healing of the rabbit radius bone defects at the doses used. Gamma sterilization of reindeer BMP extract reduced osteoinductivity slightly, but not significantly.

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 apparentKm 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.

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 l,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.

Effect of gamma irradiation on the osteoinductivity of morphogenetic protein extract from reindeer bone

Background Bone morphogenetic proteins (BMPs), which are capable of stimulating the production of new bone, must be sterilized before preclinical and clinical use to reduce the risk of infections and associated complications. In this study, we investigated the effects of gamma sterilization on the osteoinductivity of native reindeer BMP extract in the Balb/C mouse thigh muscle pouch model. Methods 5 mg of native reindeer BMP extract and 5 mg of bovine serum albumin were administered separately either in gelatine capsules or mixed with gelatine as injections. The dose of gamma irradiation was 4.1 MRad. Unsterile capsules and injections served as controls. New bone formation was evaluated based on the incorporation of Ca45and also radiographically 3 weeks after implantation. Results Albumin-containing implants and injections did not induce new bone formation, as monitored in radiographs. Gamma sterilization did not reduce the osteoinductivity of native BMP extract in capsules, but a significant decrease in osteoinductivity-measured as area (50%) and Ca45incorporation of new bone (27%)-was seen after injection. Gamma sterilization had no effect on the optical density of new bone induced by native BMP extract administered in capsules or by injection. Interpretation We conclude that, as gamma irradiation did not reduce the osteoinductivity of reindeer BMP extract in gelatine capsules, this method appears to be suitable for sterilization of BMPs to be given in capsule form. Native reindeer BMP extract was more sensitive to irradiation in soluble collagen (gelatine) than BMP in gelatine capsules. This finding must be given serious consideration regarding treatment of patients, but the remaining activity may be sufficient for the induction of bone formation in preclinical and clinical situations.

THe inverse changes of mouse brain ornithine and S-adenosylmethionine decarboxylase activities by chlorpromazine and imipramine: Dependence of ornithine decarboxylase induction on β-adrenoceptors

Intraperitoneal injection of chlorpromazine and imipramine increases mouse brain ornithine decarboxylase but decreases S-adenosyl-L-methionine decarboxylase activity. Maximal effect was obtained 6-8 hr after treatment at which time single dose of chlorpromazine (50 mg/kg) stimulated ornithine decarboxylase activity 7-fold and decreased S-adenosylmethionine decarboxylase activity to 50% from the control level. Correspondingly, ornithine decarboxylase activity was 5.5 times higher than the control value and S-adenosylmethionine decarboxylase activity about 40% from that after imipramine injection (80 mg/kg). The possible dependence of the enzyme responses on adrenergic receptors was studied using alpha-adrenoceptor antagonist, phentolamine, and beta-adrenoceptor antagonist, propranolol, concurrently with chlorpromazine and imipramine. The stimulation of ornithine decarboxylase but not the inhibition of S-adenosylmethionine decarboxylase could be abolished by propranolol (10 mg/kg), whereas phentolamine (10 mg/kg) slightly increased ornithine decarboxylase activity even when given alone. This suggests that beta- but not alpha-adrenergic mediation is involved in the stimulation of mouse brain ornithine decarboxylase activity and that brain ornithine and S-adenosylmethionine decarboxylase activities are independently regulated. When chlorpromazine and imipramine were tested in vitro, both of them turned out to have an inhibitory effect on S-adenosylmethionine decarboxylase. The former caused 50% inhibition at a concentration of 1 mM and the latter at 2 mM. Preliminary tests suggest that the type of inhibition is noncompetitive for both of them.

The effect of polyamines on the enzymes of the 4-aminobutyric acid metabolism in mouse brain in vitro

4Aminobutyric acid (GABA) functions in mammalian brain as an inhibitory neurotransmitter. The synthesis of GABA is catalysed by L-glutamate decarboxylase (EC 4.1 .l .I 5, GAD) and it is degraded by the consecutive action of 4-aminobutyrate-2-oxoglutarate aminotr~sferase (EC 2.6.1.19, GABA-T) and succinic semialdehyde dehydrogenase (EC 1.2.1.16, SSA-DH). The activities of the GABA shunt enzymes listed above have been reported to be affected by monovalent and bivalent cations in vitro [l--6]. The aliphatic amines putrescine, spermidine and spermine, commonly referred to as polyamines, are present in relatively high concentrations in mammalian nervous tissue. The polyamines influence numerous biochemical functions, in which they have been regarded as acting as cations (reviewed [7-lo]). In vivo studies [ 1 l-131 suggest that there may be regulatory interrelations between polyamines and GABA in the mammalian nervous tissue. Here we investigate the effects of the 3 polyamines on the enzymes of the GABA shunt in vitro in order to obtain information on their possible roles in regulating the activities of these enzymes. We report a significant inhibition of SSA-DH by spermidine and to a lesser degree by spermine. The reversible inhibition by spermidine was hyperbolic and non-competitive in respect of both of the substrates, succinic semialdehyde (SSA) and NAD. The app. Ki of spermidine was