THE DISTRIBUTION AND METABOLISM OF PUTRESCINE, SPERMIDINE AND SPERMINE INJECTED INTO THE CEREBRAL VENTRICLES OF RABBITS

Following the intracerebroventricular injection into rabbits of spermidine or spermine the highest concentrations were initially found in the caudate nucleus, hypothalamus and medulla. Subsequently there was a rapid decline in the amounts present in the caudate nucleus and hypothalamus and, particularly in the case of spermidine, an increase in the conccntration in the lower brain stem and cervical cord. This pattern of changes is consistent with the amines being redistributed by passage in CSF. Intraventricularly injected putrescine followed the same initial distribution pattern but within 2 days it had been largely converted to spermidine and spermine. Synthesized polyamines accumulated in all the regions examined. The time course of synthesis indicated that spermidine was the precursor of spermine. Spermine was also formed from injected spermidine and vice-versa. These findings concur with the pharmacological and neurotoxic actions of putrescine, spermidine and spermine.     

ACETYL-COENZYME A: 1,4-DIAMINOBUTANE N-ACETYLTRANSFERASE: ACTIVITY IN RAT BRAIN DURING DEVELOPMENT, IN EXPERIMENTAL BRAIN TUMOURS AND IN BRAINS OF FISH OF DIFFERENT METABOLIC ACTIVITY

—Acetyl-CoA: 1,4-diaminobutane N-acetyltransferase catalyses the first step of putrescine catabolism in mammalian brain. It may be important in putrescine degradation of other tissues as well. Its specific activity is higher in homogenates of immature than of mature rat brains. A steady decline of putrescine acetylase activity is observed from birth until approx adult levels are reached at day 30. Microsomes and purified nuclei from brains of 2-day-old rats show considerably higher putrescine acetylase activities than the corresponding subcellular organelles from adult brains. Increased putrescine acetylase activities were found in nitroso-ethylurea-induced gliomas, together with a dramatic increase of putrescine concentration. High tissue concentrations of putrescine are, however, not necessarily correlated with enhanced putrescine acetylase activities. In trout brains a linear increase of acetyl-CoA: 1,4-diaminobutane N-acetyltransferase activity was observed together with a decrease of putrescine concentration after adaptation of the animals to increased water temperature.     

POLYAMINE METABOLISM IN THE BRAIN AND LIVER OF THE DEVELOPING MONKEY

Abstract— The concentrations of putrescine and the polyamines, spermidine and spermine, along with the specific activities of the enzymes involved in their biosynthesis, ornithine decarboxylase, S -adenosylmethionine decarboxylase and spermidine synthase have been measured in brain and liver of the developing Rhesus monkey from mid-gestation, through birth and neonatal life to maturity. The results suggest that it is an increased concentration of putrescine and an increased specific activity of ornithine decarboxylase which are associated with the rapid growth of fetal brain during the middle of gestation. By the end of two-thirds of gestation, both of these parameters have attained values similar to those found in mature brain. The concentration of spermidine in brain and the specific activities of S -adenosylmethionine decarboxylase and spermidine synthase are lower in fetal brain than adult brain and increase slowly after birth to reach values similar to those of the adult only after several months. These results provide additional evidence that in the mature brain spermidine serves some function other than one associated with rapid growth.
Fetal liver at mid-gestation was characterized by increased concentrations of both putrescine and spermidine and increased specific activities of the enzymes which synthesize them. By two-thirds of gestation, values similar to those found in adult liver had been attained. Liver has thus reached maturity with regard to polyamine metabolism by this time.     

CHANGES IN BLOOD FLOW IN THE COMPONENT STRUCTURES OF THE DOG BRAIN DURING POSTNATAL MATURATION

Abstract— Blood flow was measured quantitatively in 35 structures of the brains of dogs of various ages from birth to maturity. In general, values were low at birth and rose to maximal levels between 3 and 7 weeks of postnatal age; declines from the peak levels then followed until values characteristic of maturity were attained by 13 weeks of postnatal age. From relatively uniform perfusion rates throughout the brain at birth there gradually emerged a marked heterogeneity, in parallel with the structural and functional maturation and differentiation known to occur in the brain during this period of life. Our observations may reflect the summation of the changes in energy demands associated on the one hand with biosynthetic processes essential for growth and development and with the support for progressively increasing functional activities on the other.     

METABOLISM OF POLYAMINES IN NORMAL AND SCRAPIE-AFFECTED MOUSE BRAIN AND SPLEEN

Abstract— Following intracerebral inoculation of mouse adapted scrapie agent into mice, polyamine concentration in the brain decreases to about 75 per cent of the normal level during the first 2 months after intracerebral inoculation of the agent. Between 2 and 4 months after infection thelevel of spermidine and spermineincreased by 80 and 40 percent respectively to reach concentrations of 25 and 20 per cent higher than controls of the same age. During the same period the rate of incorporation of [¹⁴C]putrescine into spermidine is increased four-fold as compared with controls. The changes in polyamine levels correlate well with the pattern of astrocyte hypertrophy and are similar to those reported for human brain tumours. The concentration of polyamines in spleen increases soon after inoculation. Whilst changes in brain polyamines might be referred to the hypertrophic growth of astrocytes those in spleen are perhaps due to an increased metabolic activity of spleen cells associated with the replication of the agent. These results are derived from experimental mouse scrapie and not naturally occurring disease in sheep.     

REGIONAL CHANGES IN RAT BRAIN CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE ACTIVITY RESULTING FROM UNDERNUTRITION IMPOSED DURING DIFFERENT PERIODS OF DEVELOPMENT1

Abstract— The severity of mental changes in malnourished children is related to both the period of development when the malnutrition occurs and the amount of environmental stimulation. In the present study the effect of imposing protein undernutrition during the period of gestation or postweaning period, and protein-energy undernutrition during the suckling period on cholinergic enzyme activity was investigated in the rat. Six different dietary treatments were given and the activity of ChAc, ChE, and AChE determined in the forebrain, brainstem, and cerebellum of male rats on day 49. Undernutrition imposed during gestation, suckling or postweaning all resulted in changes in cholinergic enzyme activity. The direction and degree of change of enzyme activity depended on the period when undernutrition was imposed as well as the brain region. In the forebrain ChE and AChE activities were altered, in the brainstem, ChAc, ChE and AChE activities were altered, and in the cerebellum ChAc activity was altered. The effect on the activity of the individual cholinergic enzymes was complex and was not the same in the different regions of the brain or even for the same brain region exposed to undernutrition during different periods of development. These results along with earlier work indicate that cholinergic enzyme activity in brain of undernourished rats can be altered by both the period of development when undernutrition is imposed and the amount of environmental stimulation.     

INTERRELATIONS BETWEEN POLYAMINES AND NUCLEIC ACIDS: CHANGES OF POLYAMINE AND NUCLEIC ACID CONCENTRATIONS IN THE DEVELOPING RAT BRAIN

The rat brain concentrations of protein, RNA, DNA, putrescine, and of the polyamines spermidine and spermine, were studied during development. Putrescine formation is apparently controlled by ornithine decarboxylase. Spermidine and spermine concentrations change in inverse directions to their anabolic enzymes. It has been presumed, therefore, that the low concentrations of the polyamine-synthesizing enzymes in immature brain are compensated for, by high putrescine and S-adenosylmethionine concentrations. In agreement with previous findings for fish brain, the changes in RNA and spermidine concentrations were most closely correlated. The functions of DNA: spermine are directly correlated only during the periods of brain maturation, after cell proliferation has nearly ceased.     

Association of putrescine,spermidine, spermine,and GABA with structural elements of brain cells

The present experiments are the first survey of the association of endogenous and exogenous putrescine, spermidine, and spermine with subcellular structures of rat brain cortex. The differences of distribution in subfractions obtained from salt-free and salt-containing density gradients were studied, with the following results: (1) In contrast with liver preparation, putrescine and the polyamines spermidine and spermine are not distributed in parallel with RNA. (2) In salt-containing media, putrescine and the polyamines were preferentially associated with synaptosomes and with synaptosomal membranes. Significant association with myelin constituents was observed only in salt-free media. (3) Exogenous putrescine and the polyamines were less firmly attached to synaptosomes and to synaptosomal membrane fractions than the endogenous amines. There is good evidence for similar subcellular localizations of putrescine and GABA. Putrescine seems to be entrapped in the nerve endings. (4) Uptake studies with crude mitochondria under conditions of high-affinity uptake showed no temperature-sensitive component of polyamine accumulation in synaptosomes, in contrast with GABA, monoacetylputrescine, and ornithine. (5) Polyamines bound to myelin constituents or mitochondria could be displaced by a 200-fold concentration of nonradioactive amines; this was not the case with polyamines bound to synaptosomes. Mg²⁺ did not effectively compete with spermine for binding sites at synaptic regions. (6) Electrical stimulation and stimulation by mono- and bivalent cations did not change the concentrations of the polyamines and GABA in guinea pig cortex. (7) There is no evidence for a neurotransmitter role of putrescine, spermidine, or spermine, although these compounds might function as modulators of neurotransmission.     

Changes in Polyamine Concentrations in Amygdaloid-Kindled Rats

Concentrations of the polyamines putrescine, spermidine, and spermine were investigated in the left and right amygdala and in the remaining cerebrum, in which kindling was induced by repeated application of electrical stimulation of the left amygdala of rats. In kindled rats, the concentrations of spermidine and spermine increased slightly, but elevations did not reach significant levels in any brain regions. The most profound increase was detected in the putrescine concentration in all parts of the cerebrum 1-8 h after the final stimulation. These results suggest that the increases in concentrations of polyamines, particularly of putrescine, are involved in the pathogenesis of amygdaloid kindling.     

INTERRELATIONSHIPS BETWEEN POLYAMINES AND NUCLEIC ACIDS. CHANGES OF POLYAMINE AND NUCLEIC ACID CONCENTRATIONS IN THE GROWING FISH BRAIN (SALMO IRIDEUS GIBB.)

Abstract— In contrast to mouse brain, the content of putrescine in fish brain considerably exceeds that of spermine and spermidine. While we observed constant protein, RNA and spermidine concentrations in fish brains of weights between 60 and 800 mg, DNA and spermine concentrations diminished with increasing brain weight, the content of spermine per cell being constant throughout life. It can be concluded from our results that growth of fish brain results both from cell enlargement and cell proliferation. The concomitant changes of spermine and DNA concentrations in the growing fish brain are the first example of a direct quantitative relationship between these cell constituents and provides evidence on their possible functional relationship in the cell nucleus.     

Tyrosine Hydroxylase, Tryptophan Hydroxylase, Biopterin, and Neopterin in the Brains of Normal Controls and Patients with Senile Dementia of Alzheimer Type

The activities of tyrosine hydroxylase and tryptophan hydroxylase, and the concentrations of the biopterin cofactor and the precursor neopterin were measured in 14 regions of postmortem brains from four histologically verified patients of senile dementia of the Alzheimer type (SDAT) and eight histologically normal controls. Neopterin concentrations were measured in the human brain for the first time. The activities of tyrosine hydroxylase and tryptophan hydroxylase in the brains of patients with SDAT were significantly reduced in the substantia nigra and in the lateral segment of the globus pallidus, locus ceruleus, and substantia nigra, respectively. The concentrations of total biopterin in the brains of patients with SDAT were significantly reduced in the putamen and substantia nigra, but the total neopterin concentrations did not change significantly. These results suggest that the reduction in biogenic amines in SDAT might be related to reductions in biosynthetic enzymes associated with biogenic amines, due to destruction of monoaminergic neurons.     

RNA,proteins and polyamines during gametophytic and androgenetic development of pollen in Nicotiana tabacum and Datura innoxia

Variations in polyamines, proteins and RNA during in vivo gametogenesis and in vitro androgenesis in Datura innoxia and in Nicotiana tabacum were studied. Spermidine was the major polyamine during gametogenesis in both species. Marked differences in proteins, RNA and polyamines were evident during meiosis and at the first haploid mitosis. In Nicotiana an unknown amine (X₆₀) appears at the beginning of the first haploid mitosis. At the same time a rapid increase in the concentrations of RNA and proteins is observed. In Datura, at the time of the first haploid mitosis there is large increase in amine and RNA levels followed by an arginine peak. During androgenesis, putrescine and spermidine were the major polyamines in both species. In Nicotiana during androgenesis an unknown amine (X₈₁) was observed together with putrescine and spermidine. This unknown compound peaks during the developmental stages of embryogenesis. In Datura androgenic induction was marked by an arginine peak followed by an increase in the putrescine and spermidine levels associated with maximum RNA. These biochemical events are tentatively correlated with structural changes during pollen development. The significance of these results is discussed in relation to the role of polyamines during gametogenesis and androgenesis.     

Polyamine concentrations in the brain of vitamin B12-deficient rats

To study the pathophysiology of the neuronal degeneration in vitamin B12 deficiency, we investigated the concentrations of the polyamines putrescine, spermidine, and spermine in brain regions and liver using high-performance liquid chromatography with fluorescence detection. Male Wistar rats were fed either a control or vitamin B12-deficient diet for 20 weeks. No remarkable behavioral changes were observed. Serum vitamin B12 and hepatic methionine concentrations were significantly lower and hepatic homocysteine was elevated in rats fed vitamin B12-deficient diet than in controls. Vitamin B12 deficiency was associated with decreased concentrations of spermidine, spermidine in liver and some regions of brain, although there were no observed abnormalities in behavior. These results suggest that vitamin B12 deficiency may play a role in neuronal degeneration through the disturbance of polyamine concentrations in rat brain.     

Nuclear Magnetic Resonance Spectroscopy Study on Energy Metabolism, Intracellular pH, and Free Mg2+ Concentration in the Brain of Transgenic Mice Overexpressing Human Ornithine Decarboxylase Gene

We have generated a transgenic mouse line strikingly overexpressing the human ornithine decarboxylase (ODC) gene in their brain. Brain ODC activity was increased in the transgenic animals by a factor of 70 in comparison with their nontransgenic littermates. The content of brain putrescine, the product of ODC, was greater than 60 mumol/g of tissue in the transgenic mice, whereas in the normal animals it was below the level that could be detected by an HPLC method. The concentrations of the higher polyamines (spermidine and spermine) were not significantly different from control values. 31P nuclear magnetic resonance (31P NMR) spectroscopy analyses revealed a significantly reduced (40%) free Mg2+ concentration as calculated from the chemical shift differences of the nucleoside triphosphate alpha and beta peaks in the brains of the transgenic animals. The lower free Mg2+ concentration in the brains of ODC transgenic mice was not a consequence of altered intracellular pH or changes in cellular high-energy metabolites. 1H NMR showed no differences in brain choline/N-acetylaspartate and total creatine/N-acetylaspartate ratios between the two animal groups. These ODC transgenic animals may serve as models in vivo for studies on cerebral postischemic events and on epilepsy, as polyamines are supposed to be involved in these processes.     

Correlation of changes in transglutaminase activity and polyamine content of neoplastic tissue during the metastatic process

Transglutaminase activity and the levels of the polyamines putrescine, spermidine and spermine were measured in two transplantable rat sarcomata: P8 which metastasises consistently to the lung, and P7 which metastasises infrequently. With the P7 sarcoma no metastases were detected following implantation; similarly, no significant changes occurred in the levels of transglutaminase activity, putrescine, spermidine or spermine during tumour growth. However, with the P8 sarcoma at approx. 30 days after implantation there was a marked decrease in transglutaminase activity, mirrored exactly by a 20-fold increase in the levels of acid-soluble putrescine. Measurement of covalently-bound polyamines in the P8 sarcoma indicated a significant and corresponding decrease in the levels of bound putrescine. The timing of these changes coincided with the time at which the P8 sarcoma was shown to have metastasised, and suggests that the changes observed may be related to this phenomenon.     

Changes in endogenous polyamines during flower development in two diverse species of rose

Changes in the concentrations of endogenous free, conjugated and bound polyamine were determined in petals of two different species of rose, viz. Rosa damascena and Rosa bourboniana, from small bud (stage 1) till full bloom (stage 8). High free putrescine and spermidine concentrations were associated with early stages of flower development and then decreased in R. damascena. At full bloom, the concentration of free putrescine was higher than rest of the polyamines measured. A steady increase in conjugated putrescine, spermidine and spermine was observed during entire period of flower development with predominance of conjugated putrescine at full bloom. In R. damascena the bound spermine was higher than rest of the polyamines during full bloom. In R. bourboniana, during the early stages of flower development, similar situation was observed, however, at full bloom, free spermidine concentration was higher than rest of the polyamines. In this species, the concentration of conjugated and bound spermine was higher than rest of the polyamines during full bloom. Polyamine concentrations were generally lower in the petals of R. bourboniana than R. damascena which may be due to genotypic differences. The possible roles of the observed polyamines are discussed in relation to flower development.IHBT Communication no, 0345.     

Polyamine profile in the paralytic shellfish poison-producing alga Alexandrium minutum

The polyamine profiles of the toxic dinoflagellate Alexandriumminutum during different growth stages were measured by high-performanceliquid chromatography. Both free and conjugated polyamines werefound, including putrescine, cadaverine, spermine, spermidineand norspermidine. During the growth cycle of A. minutum T1,the levels of norspermidine and putrescine in the free polyamineswere the highest after 3 days culture. Putrescine and cadaverinewere the major components in the conjugated polyamines. Theamount of conjugated amines was higher than that of free aminesduring the exponential phase of A. minutum T1.     

Involvement of Putrescine in the Development of Kindled Seizure in Rats

During the development of kindling by daily electrical stimulations applied to the left amygdala of rats, concentrations of the polyamines putrescine, spermidine, and spermine were measured in the left amygdala and the remainder of the cerebrum. A significant increase of putrescine concentration appeared first at the left amygdala in prekindled rats and then propagated to the remainder of the cerebrum with the development of kindling. This increase in putrescine concentration in the left amygdala was higher in prekindled rats than in fully kindled rats and lasted for at least 24 h after the final kindling stimulation. The concentrations of spermidine and spermine were slightly increased in a fully kindled state. To clarify the role of putrescine in kindling, the development of amygdaloid kindling was examined in rats after microinjections of alpha-difluoromethylornithine, a specific inhibitor of polyamine synthesis, and putrescine into the ipsilateral amygdala. Pretreatment with alpha-difluoromethylornithine (50 nmol) for 10 days accelerated both the development of behavioral kindling and the propagation of the afterdischarge from the left amygdala to the frontal cortex. In contrast, pretreatment with putrescine (200 nmol) for 10 days retarded the development of kindling. These results suggest that the increase in putrescine concentration in the kindled brain has an inhibitory effect on the development of kindling.     

MEMBRANE PROTEINS OF RAT BRAIN: COMPOSITIONAL CHANGES DURING POSTNATAL DEVELOPMENT

Abstract— Membrane fractions from forebrain of rat were isolated at ages ranging from 5 to 93 days. Among these fractions were total membranes, three fractions isolated by density gradient centrifugation, and three subfractions which consisted of purified myelin and of two supernatant fractions. All membrane fractions showed an increase in protein content during the first postnatal month; however, only the myelin fraction and one of its supernatant fractions showed a prolonged accumulation. Myelin protein increased continually from 0.17 mg/g brain at 15 days to 8.3 mg/g brain at 93 days.
All fractions were analysed for protein composition by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Characteristic changes in protein composition were noted during postnatal development, most of which were pronounced up to the age of 20 days. Among others was a decrease in histones as compared to other proteins, with a concomitant shift in preponderance from the slow- to the fast-migrating histone band. In parallel, other proteins of high molecular weight became more prominent. No myelin could be isolated at 5 and 10 days. The deposition of myelin proteins was parallelled by the appearance of the Wolfgram protein which points to a close correlation of the Wolfgram protein to the process of myelination.     

Polyamines: developmental alterations in regional disposition and metabolism in rat brain

The polyamines spermidine and spermine and the activity of the polyamine synthesizing enzyme, S-adenosyl-L-methionine (SAM) decarboxylase, were measured in regions of adult rat brains and during postnatal development. In the adult, although spermidine levels tended to correlate with the relative amounts of white matter in some areas, there were striking exceptions. SAM decarboxylase activity of the adult brain was higher than in most other mammalian tissues, although brain levels of polyamines were among the lowest. SAM decarboxylase activity appeared to be localized to cellular cytoplasm. Its activity increased with age in contrast to the levels of spermine, spermidine, DNA and RNA which decreased during postnatal development.